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Metal Ions in Life Sciences 13

Astrid Sigel
Helmut Sigel
Roland K.O. Sigel
Editors

Interrelations
between Essential
Metal Ions and
Human Diseases
Interrelations between Essential Metal Ions
and Human Diseases
Metal Ions in Life Sciences
Volume 13

Series Editors:
Astrid Sigel, Helmut Sigel, and Roland K.O. Sigel

For further volumes:


http://www.springer.com/series/8385 and http://www.mils-series.com
Astrid Sigel • Helmut Sigel • Roland K.O. Sigel
Editors

Interrelations between
Essential Metal Ions
and Human Diseases
Editors
Astrid Sigel Helmut Sigel
Department of Chemistry Department of Chemistry
Inorganic Chemistry Inorganic Chemistry
University of Basel University of Basel
Spitalstrasse 51 Spitalstrasse 51
CH-4056 Basel CH-4056 Basel
Switzerland Switzerland
astrid.sigel@unibas.ch helmut.sigel@unibas.ch

Roland K.O. Sigel


Institute of Inorganic Chemistry
University of Zürich
Winterthurerstrasse 190
CH-8057 Zürich
Switzerland
roland.sigel@aci.uzh.ch

ISSN 1559-0836 ISSN 1868-0402 (electronic)


ISBN 978-94-007-7499-5 ISBN 978-94-007-7500-8 (eBook)
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Historical Development and Perspectives
of the Series
Metal Ions in Life Sciences*

It is an old wisdom that metals are indispensable for life. Indeed, several of them,
like sodium, potassium, and calcium, are easily discovered in living matter. However,
the role of metals and their impact on life remained largely hidden until inorganic
chemistry and coordination chemistry experienced a pronounced revival in the
1950s. The experimental and theoretical tools created in this period and their appli-
cation to biochemical problems led to the development of the field or discipline now
known as Bioinorganic Chemistry, Inorganic Biochemistry, or more recently also
often addressed as Biological Inorganic Chemistry.
By 1970 Bioinorganic Chemistry was established and further promoted by the
book series Metal Ions in Biological Systems founded in 1973 (edited by H.S., who
was soon joined by A.S.) and published by Marcel Dekker, Inc., New York, for more
than 30 years. After this company ceased to be a family endeavor and its acquisition
by another company, we decided, after having edited 44 volumes of the MIBS series
(the last two together with R.K.O.S.) to launch a new and broader minded series to
cover today’s needs in the Life Sciences. Therefore, the Sigels new series is entitled

Metal Ions in Life Sciences.

After publication of the first four volumes (2006–2008) with John Wiley & Sons,
Ltd., Chichester, UK, and the next five volumes (2009–2011) with the Royal Society
of Chemistry, Cambridge, UK, we are happy to join forces now in this still new
endeavor with Springer Science & Business Media B.V., Dordrecht, The Netherlands;
a most experienced Publisher in the Sciences.

* Reproduced with some alterations by permission of John Wiley & Sons, Ltd., Chichester, UK
(copyright 2006) from pages v and vi of Volume 1 of the series Metal Ions in Life Sciences
(MILS-1).

v
vi Historical Development and Perspectives of the Series

The development of Biological Inorganic Chemistry during the past 40 years


was and still is driven by several factors; among these are (i) the attempts to reveal
the interplay between metal ions and peptides, nucleotides, hormones or vitamins,
etc., (ii) the efforts regarding the understanding of accumulation, transport, metabolism
and toxicity of metal ions, (iii) the development and application of metal-based
drugs, (iv) biomimetic syntheses with the aim to understand biological processes as
well as to create efficient catalysts, (v) the determination of high-resolution struc-
tures of proteins, nucleic acids, and other biomolecules, (vi) the utilization of
powerful spectroscopic tools allowing studies of structures and dynamics, and
(vii), more recently, the widespread use of macromolecular engineering to create
new biologically relevant structures at will. All this and more is and will be reflected
in the volumes of the series Metal Ions in Life Sciences.
The importance of metal ions to the vital functions of living organisms, hence, to
their health and well-being, is nowadays well accepted. However, in spite of all the
progress made, we are still only at the brink of understanding these processes.
Therefore, the series Metal Ions in Life Sciences will endeavor to link coordination
chemistry and biochemistry in their widest sense. Despite the evident expectation
that a great deal of future outstanding discoveries will be made in the interdisciplin-
ary areas of science, there are still “language” barriers between the historically
separate spheres of chemistry, biology, medicine, and physics. Thus, it is one of the
aims of this series to catalyze mutual “understanding”.
It is our hope that Metal Ions in Life Sciences proves a stimulus for new activities
in the fascinating “field” of Biological Inorganic Chemistry. If so, it will well serve
its purpose and be a rewarding result for the efforts spent by the authors.

Astrid Sigel and Helmut Sigel


Department of Chemistry, Inorganic Chemistry,
University of Basel, CH-4056 Basel, Switzerland

Roland K.O. Sigel


Institute of Inorganic Chemistry,
University of Zürich, CH-8057 Zürich, Switzerland

October 2005,
October 2008,
and August 2011
Preface to Volume 13

Interrelations Between Essential Metal Ions


and Human Diseases
Most of the 13 metals and 3 metalloids and their ions, which are covered in this
volume, have been proven to be essential for humans. Indeed, it is an old wisdom
that metal ions are indispensable for life. The main group metals, i.e., sodium,
potassium, magnesium, and calcium, belong to the so-called bulk elements, and
they occur in humans (70 kg) between about 20 g (Mg) and 1000 g (Ca) [H. Sigel,
A. Sigel, H. G. Seiler, in Handbook on Metals in Clinical and Analytical Chemistry,
Eds H. G. Seiler, A. Sigel, H. Sigel, Dekker, New York, 1994, pp. 1–12]. The
remaining 9 metals are transition elements, including zinc, and they all occur at
trace levels, though iron and zinc dominate in humans with about 4 and 2.5 g,
respectively. All the other metals, as well as the three metalloids (silicon, arsenic,
selenium), occur only at ultra-trace levels, e.g., manganese and cobalt with about 12
and 1 mg, respectively. They comprise the essential elements manganese, cobalt,
copper, molybdenum, and selenium; chromium, vanadium, nickel, silicon, and arse-
nic have been proposed as being essential in the second half of the last century.
However, it turned out that their essentiality is difficult to establish because, if at all,
they are certainly needed only in ultra-trace amounts, and because of their preva-
lence in the environment from natural and anthropomorphic sources, it has been
difficult to prove whether or not there is a requirement for them, though the likeli-
hood for vanadium and silicon as being essential appears to be high.
The introductory Chapter 1 presents an overview of the topic, metal ions and
infectious diseases, as seen from the clinic. The dilemma is that next to the bulk
elements, also the trace elements are required by both, humans and bacterial patho-
gens. Since these metal ions are both necessary for life, but toxic in excess, metal
homeostasis is tightly controlled by both bacteria and humans. Thus, pathogens
utilize a variety of strategies to sense, acquire, store, and export metal ions in/from
the vertebrate host.

vii
viii Preface to Volume 13

The bulk elements sodium, potassium, magnesium, and calcium are dealt with
in Chapters 2 to 4. All these elements are essential for human health and the chapters
summarize their basic physiological actions. For example, a proper cellular Mg2+
homeostasis is in all instances compulsory; deficiency or overload gives rise to dis-
eases, and these are described. Interestingly, evolution has thoroughly exploited the
chemical properties of Ca2+, i.e., its fast ligand-exchange rate and its reversible
binding to sites with an irregular geometry, and selected it as a carrier of cellular
signals.
The next chapters focus on the roles of the transition elements beginning in
Chapter 5 with vanadium: Since vanadate can be considered a close blueprint of
phosphate with respect to its built-up, it likely takes over a regulatory function in
metabolic processes depending on phosphate; e.g., phosphatases can be inhibited
and kinases activated, but its essentiality for humans has not been proven. Yet in
1982/83 the discovery of vanadate-dependent bromoperoxidase in the marine mac-
roalga Ascophyllum nodosum established that some forms of life need it. At com-
mon concentrations it is non-toxic for humans and this opens up a wide playground
for pharmacological applications. Similarly, is chromium essential, pharmacologi-
cally relevant or toxic? At present chromium cannot be considered as an essential
element because (i) nutritional data demonstrating chromium deficiency and
improvement in symptoms from chromium supplementation are lacking, and (ii) no
biomolecules have convincingly been demonstrated to bind chromium and to have
an essential function in the body.
Manganese, covered in Chapter 7, is important for human health. Though it is
absolutely necessary for development, metabolism, and the antioxidant system,
excessive exposure or intake may lead to manganism, a neurodegenerative disorder
that causes dopaminergic neuronal death and parkinson-like symptoms. The effects
of iron deficiency or overload are covered in great detail in Chapter 8. Iron is a
redox-active metal which is abundant in the Earth’s crust. It has played a key role in
the evolution of living systems and as such it is an essential element in a wide range
of biological phenomena, being critical for the function of an enormous array of
enzymes, energy transduction mechanisms, and oxygen carriers. Since the redox
nature of iron renders the metal toxic in excess, all biological organisms carefully
control iron levels. For example, low body iron levels are related to anemia, whereas
systemic iron overload results from, e.g., hyperabsorption, and can be treated by
iron-chelation therapy. Furthermore, iron chelators have been widely investigated
for the treatment of cancer, tuberculosis, and malaria.
Cobalt and its role in human health and disease is primarily defined by the func-
tioning of cobalamin (vitamin B12); it is dealt with in Chapter 9. Cobalamin acts in
humans as a cofactor for methylmalonyl-coenzyme A mutase and methionine syn-
thase, both enzymes being important for health. Especially the dysfunction of
methionine synthase causes disruption of many cellular processes and leads to dis-
ease. In contrast, so far no nickel-containing enzyme or cofactor is known in higher
animals. However, nickel has been included in the group of “possibly essential ele-
ments” for animals and humans already in the 1970s and its importance for plants,
bacteria, archaea, and unicellular eukaryotes is well documented. In this context
Preface to Volume 13 ix

Helicobacter pylori, a gram-negative bacterium, may be mentioned. This pathogen


colonizes the human gut, giving rise to acute and chronic gastric pathologies,
including peptic ulcer, and possibly also to gastric carcinomas and lymphomas. The
toxic effects of nickel can produce serious respiratory, cardiovascular, and kidney
diseases; they also alter the immune response giving rise to dermatitis, etc.
Copper, the metal of Chapter 11, represents in humans the 3rd most abundant
transition metal; it is essential but it can also harm cells due to its potential to cata-
lyze the generation of toxic reactive oxygen species. Therefore, the transport of
copper and the cellular copper content are tightly regulated. Nutritional copper defi-
ciency gives rise to anemia, to neuropathies, to impaired immune responses, etc.
Genetic copper deficiency leads to Menkes disease and distal hereditory peripheral
neuropathy. Genetic copper overload causes Wilson’s disease and infantile cirrho-
sis. Ingestion of high doses of copper gives rise to nausea, vomiting, headache,
diarrhea, hemolytic anemia, gastrointestinal hemorrhage, liver as well as kidney
failure and finally death may occur. Furthermore, alterations of copper homeostasis
have been associated with neurodegenerative diseases such as prion diseases,
Alzheimer’s disease, Parkinson’s disease or Huntington’s disease, etc., but the exact
role of copper in these important neurological disorders remains unclear.
Zinc is dealt with in Chapter 12: The total amount of zinc in a human (70 kg) is
2 to 3 g, i.e., there is nearly as much zinc as there is iron. Also the cellular Zn2+
concentrations are rather high, that is, nearly as high as those of major metabolites
like ATP. The vast knowledge of the physiological functions of zinc in at least 3000
proteins and the recent recognition of fundamental regulatory functions of Zn2+ ions
released from cells or within cells links this nutritionally essential metal ion to
numerous human diseases. It is not only the right amount of zinc in the diet that
maintains health, at least as important is the proper functioning of the dozens of
proteins that control cellular zinc homeostasis and regulate its intracellular traffic.
Zinc and its role in organ pathophysiology as well as in genetic, metabolic, chronic,
and infectious diseases are covered.
The essential trace element molybdenum, treated in Chapter 13, plays a crucial
role in human health and disease. Remarkably, it is the only metal of the 2nd transi-
tion row (4d) of the periodic table with a biological role for humans. Four mamma-
lian Mo-dependent enzymes are known, all of them harboring a pterin-based
molybdenum cofactor (Moco) in their active site. In the focus are the individual
pathways and the clinical and cellular consequences of their dysfunction. In all
these enzymes molybdenum catalyzes oxygen transfer reactions from or to sub-
strates using water as oxygen donor or acceptor, whereby it shuttles between the
oxidation states +IV and +VI. Especially important are the functions and deficien-
cies of xanthine dehydrogenase and sulfite oxidase. The underlying molecular basis
of Moco deficiency, possible treatment options, and links to other diseases includ-
ing neuropsychiatric disorders are discussed.
The metalloid silicon is the second most abundant element in the Earth’s crust
behind oxygen and has many industrial applications including its use as an additive
in the feed and beverage industry. Chapter 14 discusses the possible biological
potential of the metalloid, which is bioavailable as orthosilicic acid, and its potential
x Preface to Volume 13

beneficial effects on human health. Asbestos, its fibrous crystalline form, is a health
hazard promoting asbestosis and leading to significant impairment of lung function
and an increased cancer risk. Specific biochemical or physiological functions of
silicon, if any, are largely unknown, although generally thought to exist.
Can the toxic metalloid arsenic sustain life? Clearly, the biochemical and physi-
ological properties of arsenic are invariably linked with the toxicity of this element.
The aim of Chapter 15 is (i) to summarize the evidence for beneficial or sustaining
roles of arsenic in living organisms, including its substitution for phosphorus, and
(ii) to summarize its Janus-faced role in both causing and treating human disease.
Arsenic oxide, deadly at high doses, is also an approved and effective drug for the
treatment of acute promyelocytic leukemia. The well known toxicity of this element
and its ability to cause diseases, including cancer of the skin, lung, bladder, liver,
and kidney, make it a health hazard. So far it has not been recognized as being
essential for humans because it has been difficult to establish whether or not there is
a requirement for arsenic at ultra-trace levels considering its prevalence in the envi-
ronment from natural and anthropomorphic sources. In contrast, selenium is estab-
lished as an essential micronutrient for mammals, but it is also proven to be toxic in
excess, leading to selenosis. Selenium exerts its biological functions through sele-
noproteins which contain selenocysteine. In fact, 25 selenoproteins are encoded in
the human genome; most of their known functions are involved in redox systems
and signaling pathways.
Overall, this volume offers a wealth of information about human health and the
interrelations between essential, or possibly essential, metals or metalloids.

Astrid Sigel
Helmut Sigel
Roland K.O. Sigel
Contents

Historical Development and Perspectives of the Series ............................... v

Preface to Volume 13....................................................................................... vii

Contributors to Volume 13 ............................................................................. xvii

Titles of Volumes 1–44 in the Metal Ions in Biological Systems Series ........... xxi

Contents of Volumes in the Metal Ions in Life Sciences Series.................... xxiii

1 Metal Ions and Infectious Diseases. An Overview from the Clinic ...... 1
Peggy L. Carver
Abstract ....................................................................................................... 2
1 Introduction ........................................................................................... 3
2 Iron ........................................................................................................ 5
3 Zinc ....................................................................................................... 10
4 Selenium ............................................................................................... 14
5 Copper ................................................................................................... 18
6 Chromium ............................................................................................. 19
7 Manganese ............................................................................................ 20
8 Summary and Future Developments ..................................................... 22
References ................................................................................................... 23
2 Sodium and Potassium in Health and Disease ....................................... 29
Hana R. Pohl, John S. Wheeler, and H. Edward Murray
Abstract ....................................................................................................... 30
1 Introduction ........................................................................................... 30
2 Physiology of Sodium and Potassium in Humans ................................ 32
3 Pathology Associated with Sodium Levels ........................................... 38

xi
xii Contents

4 Pathology Associated with Potassium Levels ....................................... 41


5 Conclusion ............................................................................................ 45
References ................................................................................................... 46
3 Magnesium in Health and Disease........................................................... 49
Andrea M.P. Romani
Abstract ....................................................................................................... 50
1 Introduction ........................................................................................... 50
2 Cellular Magnesium Homeostasis ........................................................ 54
3 Magnesium in Disease .......................................................................... 55
4 Conclusions ........................................................................................... 73
References ................................................................................................... 75
4 Calcium in Health and Disease ................................................................ 81
Marisa Brini, Denis Ottolini, Tito Calì, and Ernesto Carafoli
Abstract ....................................................................................................... 82
1 Introduction ........................................................................................... 83
2 General Properties of Calcium as a Signaling Agent ............................ 88
3 Intracellular Calcium Handling............................................................. 93
4 Calcium as a Regulator of Biological Processes................................... 100
5 The Ambivalence of the Calcium Signal: Defects
of Calcium Regulation and Disease ...................................................... 116
6 Conclusions ........................................................................................... 126
References ................................................................................................... 130
5 Vanadium. Its Role for Humans .............................................................. 139
Dieter Rehder
Abstract ....................................................................................................... 139
1 Introduction ........................................................................................... 140
2 Distribution and Cycling of Vanadium ................................................. 142
3 The Aqueous Chemistry of Vanadium
and the Vanadate-Phosphate Antagonism ............................................. 147
4 The Medicinal Potential of Vanadium................................................... 152
5 Concluding Remarks and Prospects...................................................... 164
References ................................................................................................... 167

6 Chromium: Is It Essential, Pharmacologically


Relevant, or Toxic? .................................................................................... 171
John B. Vincent
Abstract ....................................................................................................... 172
1 Introduction ........................................................................................... 172
2 Is Chromium Essential? ........................................................................ 173
3 Is Chromium Pharmacologically Relevant?.......................................... 180
4 Is Chromium Toxic?.............................................................................. 191
5 Concluding Remarks and Future Direction .......................................... 192
References ................................................................................................... 194
Contents xiii

7 Manganese in Health and Disease ......................................................... 199


Daiana Silva Avila, Robson Luiz Puntel, and Michael Aschner
Abstract ..................................................................................................... 200
1 Introduction ....................................................................................... 200
2 Manganese Transport ........................................................................ 206
3 Manganism. A Neurodegenerative Disease ...................................... 210
4 Symptoms and Sensitive Populations ............................................... 211
5 Manganism versus Parkinson’s Disease ........................................... 211
6 Manganese in the Etiology of Other Neurodegenerative Disorders . 212
7 Molecular Mechanisms of Toxicity .................................................. 214
8 Genetic Susceptibility ....................................................................... 216
9 Treatment .......................................................................................... 217
10 General Conclusions ......................................................................... 218
References ................................................................................................. 220
8 Iron: Effect of Overload and Deficiency ............................................... 229
Robert C. Hider and Xiaole Kong
Abstract ..................................................................................................... 230
1 Introduction ....................................................................................... 231
2 Iron Deficiency and Anemia ............................................................. 248
3 Systemic Iron Overload .................................................................... 255
4 Iron-Selective Chelators with Therapeutic Potential ........................ 266
5 Neuropathology and Iron .................................................................. 277
6 The Role of Iron Chelation in Cancer Therapy................................. 281
7 Iron and Infection .............................................................................. 282
8 Overview and Future Developments ................................................. 284
References ................................................................................................. 286
9 Cobalt: Its Role in Health and Disease ................................................. 295
Kazuhiro Yamada
Abstract ..................................................................................................... 296
1 Introduction ....................................................................................... 296
2 Cobalamin, Vitamin B12................................................................... 297
3 Vitamin B12 Deficiency and Disease................................................ 310
4 Non-corrinoid Cobalt ........................................................................ 314
5 Implications and Future Development .............................................. 315
References ................................................................................................. 317
10 Nickel and Human Health ...................................................................... 321
Barbara Zambelli and Stefano Ciurli
Abstract ..................................................................................................... 322
1 Introduction: The Double Face of Nickel
in Biological Systems ....................................................................... 322
2 Nickel Hazard for Human Health ..................................................... 324
3 Nickel-Dependent Infectious Diseases ............................................. 336
xiv Contents

4 Nickel Essentiality in Animals and Humans ....................................... 348


5 Conclusions and Outlook .................................................................... 350
References ................................................................................................. 352
11 Copper: Effects of Deficiency and Overload ........................................ 359
Ivo Scheiber, Ralf Dringen, and Julian F.B. Mercer
Abstract ..................................................................................................... 360
1 Introduction ......................................................................................... 360
2 Copper Biochemistry and Homeostasis .............................................. 361
3 Copper Deficiency Disorders .............................................................. 371
4 Copper Overload Disorders ................................................................ 375
5 Neuropathology and Copper ............................................................... 376
6 Overview and Future Developments ................................................... 380
References ................................................................................................. 381
12 Zinc and Human Disease ........................................................................ 389
Wolfgang Maret
Abstract ..................................................................................................... 390
1 Introduction ......................................................................................... 390
2 Zinc Biochemistry............................................................................... 391
3 Zinc in Organ Pathophysiology .......................................................... 398
4 Zinc in Disease.................................................................................... 404
5 General Conclusions ........................................................................... 407
References ................................................................................................. 409
13 Molybdenum in Human Health and Disease ........................................ 415
Guenter Schwarz and Abdel A. Belaidi
Abstract ..................................................................................................... 416
1 Introduction ......................................................................................... 417
2 Deficiencies in Molybdenum Enzymes .............................................. 419
3 Molybdenum Cofactor Deficiencies ................................................... 426
4 Association of Molybdenum with Other Disorders ............................ 440
5 Concluding Remarks and Future Developments ................................ 442
References ................................................................................................. 444
14 Silicon: The Health Benefits of a Metalloid .......................................... 451
Keith R. Martin
Abstract ..................................................................................................... 452
1 Introduction ......................................................................................... 452
2 Silicon Biochemistry........................................................................... 453
3 Silicon and Its Potential Health Benefits ............................................ 457
4 Toxicology of Silicon and Silica ......................................................... 463
5 Potential Medicinal Uses of Silicon and Silicates .............................. 467
6 Summary and Future Directions ......................................................... 468
References ................................................................................................. 469
Contents xv

15 Arsenic. Can This Toxic Metalloid Sustain Life? ................................. 475


Dean E. Wilcox
Abstract ..................................................................................................... 476
1 Introduction ......................................................................................... 476
2 Toxicity ............................................................................................... 481
3 Sustaining Roles.................................................................................. 484
4 Beneficial Uses.................................................................................... 490
5 Summary ............................................................................................. 492
References ................................................................................................. 494
16 Selenium. Role of the Essential Metalloid in Health............................ 499
Suguru Kurokawa and Marla J. Berry
Abstract ..................................................................................................... 500
1 Introduction ......................................................................................... 501
2 Selenium in Biomolecules .................................................................. 502
3 Function of Selenoproteins ................................................................. 509
4 Selenium and Disease ......................................................................... 516
5 Health Benefits of Selenium in Humans ............................................. 520
6 General Conclusions ........................................................................... 525
References ................................................................................................. 527

Index ................................................................................................................. 535


Contributors to Volume 13

Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Michael Aschner Department of Pediatrics and Pharmacology, The Kennedy
Center for Research on Human Development and The Molecular Toxicology Center,
Nashville, TN 37232-0414, USA, michael.aschner@vanderbilt.edu (199)
Daiana Silva Avila Biochemistry Graduation Program, Universidade Federal do
Pampa, Uruguaiana, Rio Grande do Sul, Brazil, avilads1@gmail.com (199)
Abdel A. Belaidi Institute of Biochemistry, Department of Chemistry, Center for
Molecular Medicine, University of Cologne, Zuelpicher Str. 47, D-50674 Köln,
Germany (415)
Marla J. Berry Department of Cell & Molecular Biology, John A. Burns
School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA,
mberry@hawaii.edu (499)
Marisa Brini Department of Biology, University of Padova, Via U. Bassi 58/B,
I-35131 Padova, Italy, marisa.brini@unipd.it (81)
Tito Calì Department of Biology, University of Padova, Via U. Bassi 58/B, I-35131
Padova, Italy (81)
Ernesto Carafoli Venetian Institute of Molecular Medicine (VIMM), Via G. Orus
2, I-35129 Padova, Italy, ernesto.carafoli@unipd.it (81)
Peggy L. Carver University of Michigan College of Pharmacy, Department of
Clinical, Social, and Administrative Sciences, 428 Church St., Ann Arbor, MI
48109-1065, USA, peg@umich.edu (1)
Stefano Ciurli Laboratory of Bioinorganic Chemistry, Department of
Pharmacy and Biotechnology, University of Bologna, I-40127 Bologna, Italy, ste-
fano.ciurli@unibo.it (321)
Ralf Dringen Centre for Biomolecular Interactions Bremen, University of Bremen,
D-28334 Bremen, Germany (359)

xvii
xviii Contributors to Volume 13

Robert C. Hider Institute of Pharmaceutical Science, King’s College


London, Franklin-Wilkins Building, Stamford Street, London SE1 9NH, UK,
robert.hider@kcl.ac.uk (229)
Xiaole Kong Institute of Pharmaceutical Science, King’s College London, Franklin-
Wilkins Building, Stamford Street, London SE1 9NH, UK, k0839118@kcl.ac.uk (229)
Suguru Kurokawa Department of Cell & Molecular Biology, John A. Burns
School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA,
kurokawasuguru@gmail.com (499)
Wolfgang Maret King’s College London, School of Medicine, Diabetes and
Nutritional Sciences Division, Metal Metabolism Group, Franklin Wilkins Bldg,
150 Stamford St., London SE1 9NH, UK, wolfgang.maret@kcl.ac.uk (389)
Keith R. Martin School of Nutrition and Health Promotion, Healthy Lifestyles
Research Center, Arizona State University, 500 North 3rd Street, Phoenix, AZ
85004, USA, keith.r.martin13@gmail.com (451)
Julian F.B. Mercer Centre for Cellular and Molecular Biology, School of Life
and Environmental Sciences, Deakin University, Melbourne Campus at Burwood,
VIC 3125, Australia, jmercer@deakin.edu.au (359)
H. Edward Murray Agency for Toxic Substances and Disease Registry (ATSDR),
US Department of Health and Human Services, 1600 Clifton Road, Mailstop F-57,
Atlanta, GA 30333, USA (29)
Denis Ottolini Department of Biology, University of Padova, Via U. Bassi 58/B,
I-35131 Padova, Italy (81)
Hana R. Pohl Agency for Toxic Substances and Disease Registry (ATSDR), US
Department of Health and Human Services, 1600 Clifton Road, Mailstop F-57,
Atlanta, GA 30333, USA, hpohl@cdc.gov (29)
Robson Luiz Puntel Biochemistry Graduation Program, Universidade Federal do
Pampa, Uruguaiana, Rio Grande do Sul, Brazil, robsonunipampa@gmail.com (199)
Dieter Rehder Chemistry Department, University of Hamburg, D-20146
Hamburg, Germany, rehder@chemie.uni-hamburg.de (139)
Andrea M.P. Romani Department of Physiology and Biophysics, School of
Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH
44106-4970, USA, amr5@po.cwru.edu (49)
Ivo Scheiber Department of Parasitology, Faculty of Science, Charles University,
Prague, Czech Republic (359)
Guenter Schwarz Institute of Biochemistry, Department of Chemistry, Center for
Molecular Medicine, University of Cologne, Zuelpicher Str. 47, D-50674 Köln,
Germany, gschwarz@uni-koeln.de (415)
Contributors to Volume 13 xix

John B. Vincent Department of Chemistry, The University of Alabama, Tuscaloosa,


AL 35487-0336, USA, jvincent@bama.ua.edu (171)
John S. Wheeler Agency for Toxic Substances and Disease Registry (ATSDR),
US Department of Health and Human Services, 1600 Clifton Road, Mailstop F-57,
Atlanta, GA 30333, USA (29)
Dean E. Wilcox Department of Chemistry, Dartmouth College, Hanover, NH
03755, USA, dwilcox@dartmouth.edu (475)
Kazuhiro Yamada Department of Biochemistry, Uniformed Services University
of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA,
kazuhiro.yamada@usuhs.edu (295)
Barbara Zambelli Laboratory of Bioinorganic Chemistry, Department of
Pharmacy and Biotechnology, University of Bologna, I-40127 Bologna, Italy,
barbara.zambelli@unibo.it (321)
Titles of Volumes 1–44 in the
Metal Ions in Biological Systems Series

edited by the SIGELs


and published by Dekker/Taylor & Francis (1973–2005)

Volume 1: Simple Complexes


Volume 2: Mixed-Ligand Complexes
Volume 3: High Molecular Complexes
Volume 4: Metal Ions as Probes
Volume 5: Reactivity of Coordination Compounds
Volume 6: Biological Action of Metal Ions
Volume 7: Iron in Model and Natural Compounds
Volume 8: Nucleotides and Derivatives: Their Ligating Ambivalency
Volume 9: Amino Acids and Derivatives as Ambivalent Ligands
Volume 10: Carcinogenicity and Metal Ions
Volume 11: Metal Complexes as Anticancer Agents
Volume 12: Properties of Copper
Volume 13: Copper Proteins
Volume 14: Inorganic Drugs in Deficiency and Disease
Volume 15: Zinc and Its Role in Biology and Nutrition
Volume 16: Methods Involving Metal Ions and Complexes in Clinical Chemistry
Volume 17: Calcium and Its Role in Biology
Volume 18: Circulation of Metals in the Environment
Volume 19: Antibiotics and Their Complexes
Volume 20: Concepts on Metal Ion Toxicity
Volume 21: Applications of Nuclear Magnetic Resonance to Paramagnetic
Species
Volume 22: ENDOR, EPR, and Electron Spin Echo for Probing Coordination
Spheres
Volume 23: Nickel and Its Role in Biology
Volume 24: Aluminum and Its Role in Biology
Volume 25: Interrelations Among Metal Ions, Enzymes, and Gene Expression
Volume 26: Compendium on Magnesium and Its Role in Biology, Nutrition,
and Physiology
Volume 27: Electron Transfer Reactions in Metalloproteins

xxi
xxii Titles of Volumes 1–44 in the Metal Ions in Biological Systems Series

Volume 28: Degradation of Environmental Pollutants by Microorganisms


and Their Metalloenzymes
Volume 29: Biological Properties of Metal Alkyl Derivatives
Volume 30: Metalloenzymes Involving Amino Acid-Residue and Related
Radicals
Volume 31: Vanadium and Its Role for Life
Volume 32: Interactions of Metal Ions with Nucleotides, Nucleic Acids, and
Their Constituents
Volume 33: Probing Nucleic Acids by Metal Ion Complexes of Small Molecules
Volume 34: Mercury and Its Effects on Environment and Biology
Volume 35: Iron Transport and Storage in Microorganisms, Plants, and Animals
Volume 36: Interrelations Between Free Radicals and Metal Ions in Life
Processes
Volume 37: Manganese and Its Role in Biological Processes
Volume 38: Probing of Proteins by Metal Ions and Their Low-Molecular-
Weight Complexes
Volume 39: Molybdenum and Tungsten. Their Roles in Biological Processes
Volume 40: The Lanthanides and Their Interrelations with Biosystems
Volume 41: Metal Ions and Their Complexes in Medication
Volume 42: Metal Complexes in Tumor Diagnosis and as Anticancer Agents
Volume 43: Biogeochemical Cycles of Elements
Volume 44: Biogeochemistry, Availability, and Transport of Metals in the
Environment
Contents of Volumes in the
Metal Ions in Life Sciences Series

edited by the SIGELs

Volumes 1–4
published by John Wiley & Sons, Ltd., Chichester, UK (2006–2008)
<http://www.Wiley.com/go/mils>

Volume 5–9
by the Royal Society of Chemistry, Cambridge, UK (2009–2011)
<http://www.rsc.org/shop/metalionsinlifesciences>

and from Volume 10 on


by Springer Science & Business Media BV, Dordrecht, The Netherlands (since 2012)
<http://www.mils-series.com>

Volume 1 Neurodegenerative Diseases and Metal Ions

1 The Role of Metal Ions in Neurology. An Introduction


Dorothea Strozyk and Ashley I. Bush
2 Protein Folding, Misfolding, and Disease
Jennifer C. Lee, Judy E. Kim, Ekaterina V. Pletneva,
Jasmin Faraone-Mennella, Harry B. Gray, and Jay R. Winkler
3 Metal Ion Binding Properties of Proteins Related
to Neurodegeneration
Henryk Kozlowski, Marek Luczkowski, Daniela Valensin,
and Gianni Valensin
4 Metallic Prions: Mining the Core of Transmissible
Spongiform Encephalopathies
David R. Brown
5 The Role of Metal Ions in the Amyloid Precursor Protein
and in Alzheimer’s Disease
Thomas A. Bayer and Gerd Multhaup

xxiii
xxiv Contents of Volumes in the Metal Ions in Life Sciences Series

6 The Role of Iron in the Pathogenesis of Parkinson’s Disease


Manfred Gerlach, Kay L. Double, Mario E. Götz,
Moussa B.H. Youdim, and Peter Riederer
7 In Vivo Assessment of Iron in Huntington’s Disease
and Other Age-Related Neurodegenerative Brain Diseases
George Bartzokis, Po H. Lu, Todd A. Tishler, and Susan Perlman
8 Copper-Zinc Superoxide Dismutase and Familial
Amyotrophic Lateral Sclerosis
Lisa J. Whitson and P. John Hart
9 The Malfunctioning of Copper Transport in Wilson
and Menkes Diseases
Bibudhendra Sarkar
10 Iron and Its Role in Neurodegenerative Diseases
Roberta J. Ward and Robert R. Crichton
11 The Chemical Interplay between Catecholamines
and Metal Ions in Neurological Diseases
Wolfgang Linert, Guy N.L. Jameson, Reginald F. Jameson,
and Kurt A. Jellinger
12 Zinc Metalloneurochemistry: Physiology, Pathology, and Probes
Christopher J. Chang and Stephen J. Lippard
13 The Role of Aluminum in Neurotoxic and Neurodegenerative Processes
Tamás Kiss, Krisztina Gajda-Schrantz, and Paolo F. Zatta
14 Neurotoxicity of Cadmium, Lead, and Mercury
Hana R. Pohl, Henry G. Abadin, and John F. Risher
15 Neurodegerative Diseases and Metal Ions. A Concluding Overview
Dorothea Strozyk and Ashley I. Bush

Subject Index

Volume 2 Nickel and Its Surprising Impact in Nature

1 Biogeochemistry of Nickel and Its Release into the Environment


Tiina M. Nieminen, Liisa Ukonmaanaho, Nicole Rausch,
and William Shotyk
2 Nickel in the Environment and Its Role in the Metabolism
of Plants and Cyanobacteria
Hendrik Küpper and Peter M.H. Kroneck
3 Nickel Ion Complexes of Amino Acids and Peptides
Teresa Kowalik-Jankowska, Henryk Kozlowski, Etelka Farkas,
and Imre Sóvágó
Contents of Volumes in the Metal Ions in Life Sciences Series xxv

4 Complex Formation of Nickel(II) and Related Metal Ions


with Sugar Residues, Nucleobases, Phosphates, Nucleotides,
and Nucleic Acids
Roland K.O. Sigel and Helmut Sigel
5 Synthetic Models for the Active Sites of Nickel-Containing Enzymes
Jarl Ivar van der Vlugt and Franc Meyer
6 Urease: Recent Insights in the Role of Nickel
Stefano Ciurli
7 Nickel Iron Hydrogenases
Wolfgang Lubitz, Maurice van Gastel, and Wolfgang Gärtner
8 Methyl-Coenzyme M Reductase and Its Nickel Corphin
Coenzyme F430 in Methanogenic Archaea
Bernhard Jaun and Rudolf K. Thauer
9 Acetyl-Coenzyme A Synthases and Nickel-Containing
Carbon Monoxide Dehydrogenases
Paul A. Lindahl and David E. Graham
10 Nickel Superoxide Dismutase
Peter A. Bryngelson and Michael J. Maroney
11 Biochemistry of the Nickel-Dependent Glyoxylase I Enzymes
Nicole Sukdeo, Elisabeth Daub, and John F. Honek
12 Nickel in Acireductone Dioxygenase
Thomas C. Pochapsky, Tingting Ju, Marina Dang, Rachel Beaulieu,
Gina Pagani, and Bo OuYang
13 The Nickel-Regulated Peptidyl-Prolyl cis/trans
Isomerase SlyD
Frank Erdmann and Gunter Fischer
14 Chaperones of Nickel Metabolism
Soledad Quiroz, Jong K. Kim, Scott B. Mulrooney,
and Robert P. Hausinger
15 The Role of Nickel in Environmental Adaptation
of the Gastric Pathogen Helicobacter pylori
Florian D. Ernst, Arnoud H.M. van Vliet, Manfred Kist,
Johannes G. Kusters, and Stefan Bereswill
16 Nickel-Dependent Gene Expression
Konstantin Salnikow and Kazimierz S. Kasprzak
17 Nickel Toxicity and Carcinogenesis
Kazimierz S. Kasprzak and Konstantin Salnikow

Subject Index
xxvi Contents of Volumes in the Metal Ions in Life Sciences Series

Volume 3 The Ubiquitous Roles of Cytochrome P450 Proteins

1 Diversities and Similarities of P450 Systems: An Introduction


Mary A. Schuler and Stephen G. Sligar
2 Structural and Functional Mimics of Cytochromes P450
Wolf-D. Woggon
3 Structures of P450 Proteins and Their Molecular Phylogeny
Thomas L. Poulos and Yergalem T. Meharenna
4 Aquatic P450 Species
Mark J. Snyder
5 The Electrochemistry of Cytochrome P450
Alan M. Bond, Barry D. Fleming, and Lisandra L. Martin
6 P450 Electron Transfer Reactions
Andrew K. Udit, Stephen M. Contakes, and Harry B. Gray
7 Leakage in Cytochrome P450 Reactions in Relation
to Protein Structural Properties
Christiane Jung
8 Cytochromes P450. Structural Basis for Binding and Catalysis
Konstanze von König and Ilme Schlichting
9 Beyond Heme-Thiolate Interactions: Roles of the Secondary
Coordination Sphere in P450 Systems
Yi Lu and Thomas D. Pfister
10 Interactions of Cytochrome P450 with Nitric Oxide
and Related Ligands
Andrew W. Munro, Kirsty J. McLean, and Hazel M. Girvan
11 Cytochrome P450-Catalyzed Hydroxylations and Epoxidations
Roshan Perera, Shengxi Jin, Masanori Sono, and John H. Dawson
12 Cytochrome P450 and Steroid Hormone Biosynthesis
Rita Bernhardt and Michael R. Waterman
13 Carbon-Carbon Bond Cleavage by P450 Systems
James J. De Voss and Max J. Cryle
14 Design and Engineering of Cytochrome P450 Systems
Stephen G. Bell, Nicola Hoskins, Christopher J.C. Whitehouse,
and Luet L. Wong
15 Chemical Defense and Exploitation. Biotransformation
of Xenobiotics by Cytochrome P450 Enzymes
Elizabeth M.J. Gillam and Dominic J.B. Hunter
Contents of Volumes in the Metal Ions in Life Sciences Series xxvii

16 Drug Metabolism as Catalyzed by Human Cytochrome P450 Systems


F. Peter Guengerich
17 Cytochrome P450 Enzymes: Observations from the Clinic
Peggy L. Carver

Subject Index

Volume 4 Biomineralization. From Nature to Application

1 Crystals and Life: An Introduction


Arthur Veis
2 What Genes and Genomes Tell Us about Calcium Carbonate
Biomineralization
Fred H. Wilt and Christopher E. Killian
3 The Role of Enzymes in Biomineralization Processes
Ingrid M. Weiss and Frédéric Marin
4 Metal–Bacteria Interactions at Both the Planktonic
Cell and Biofilm Levels
Ryan C. Hunter and Terry J. Beveridge
5 Biomineralization of Calcium Carbonate. The Interplay
with Biosubstrates
Amir Berman
6 Sulfate-Containing Biominerals
Fabienne Bosselmann and Matthias Epple
7 Oxalate Biominerals
Enrique J. Baran and Paula V. Monje
8 Molecular Processes of Biosilicification in Diatoms
Aubrey K. Davis and Mark Hildebrand
9 Heavy Metals in the Jaws of Invertebrates
Helga C. Lichtenegger, Henrik Birkedal, and J. Herbert Waite
10 Ferritin. Biomineralization of Iron
Elizabeth C. Theil, Xiaofeng S. Liu, and Manolis Matzapetakis
11 Magnetism and Molecular Biology of Magnetic Iron
Minerals in Bacteria
Richard B. Frankel, Sabrina Schübbe, and Dennis A. Bazylinski
12 Biominerals. Recorders of the Past?
Danielle Fortin, Sean R. Langley, and Susan Glasauer
13 Dynamics of Biomineralization and Biodemineralization
Lijun Wang and George H. Nancollas
xxviii Contents of Volumes in the Metal Ions in Life Sciences Series

14 Mechanism of Mineralization of Collagen-Based


Connective Tissues
Adele L. Boskey
15 Mammalian Enamel Formation
Janet Moradian-Oldak and Michael L. Paine
16 Mechanical Design of Biomineralized Tissues. Bone
and Other Hierarchical Materials
Peter Fratzl
17 Bioinspired Growth of Mineralized Tissue
Darilis Suárez-González and William L. Murphy
18 Polymer-Controlled Biomimetic Mineralization
of Novel Inorganic Materials
Helmut Cölfen and Markus Antonietti

Subject Index

Volume 5 Metallothioneins and Related Chelators

1 Metallothioneins. Historical Development and Overview


Monica Nordberg and Gunnar F. Nordberg
2 Regulation of Metallothionein Gene Expression
Kuppusamy Balamurugan and Walter Schaffner
3 Bacterial Metallothioneins
Claudia A. Blindauer
4 Metallothioneins in Yeast and Fungi
Benedikt Dolderer, Hans-Jürgen Hartmann, and Ulrich Weser
5 Metallothioneins in Plants
Eva Freisinger
6 Metallothioneins in Diptera
Silvia Atrian
7 Earthworm and Nematode Metallothioneins
Stephen R. Stürzenbaum
8 Metallothioneins in Aquatic Organisms: Fish, Crustaceans,
Molluscs, and Echinoderms
Laura Vergani
9 Metal Detoxification in Freshwater Animals. Roles
of Metallothioneins
Peter G.C. Campbell and Landis Hare
Contents of Volumes in the Metal Ions in Life Sciences Series xxix

10 Structure and Function of Vertebrate Metallothioneins


Juan Hidalgo, Roger Chung, Milena Penkowa, and Milan Vašák
11 Metallothionein-3, Zinc, and Copper in the Central
Nervous System
Milan Vašák and Gabriele Meloni
12 Metallothionein Toxicology: Metal Ion Trafficking
and Cellular Protection
David H. Petering, Susan Krezoski, and Niloofar M. Tabatabai
13 Metallothionein in Inorganic Carcinogenesis
Michael P. Waalkes and Jie Liu
14 Thioredoxins and Glutaredoxins. Functions and Metal
Ion Interactions
Christopher Horst Lillig and Carsten Berndt
15 Metal Ion-Binding Properties of Phytochelatins
and Related Ligands
Aurélie Devez, Eric Achterberg, and Martha Gledhill

Subject Index

Volume 6 Metal-Carbon Bonds in Enzymes and Cofactors

1 Organometallic Chemistry of B12 Coenzymes


Bernhard Kräutler
2 Cobalamin- and Corrinoid-Dependent Enzymes
Rowena G. Matthews
3 Nickel-Alkyl Bond Formation in the Active Site
of Methyl-Coenzyme M Reductase
Bernhard Jaun and Rudolf K. Thauer
4 Nickel-Carbon Bonds in Acetyl-Coenzyme A Synthases/Carbon
Monoxide Dehydrogenases
Paul A. Lindahl
5 Structure and Function of [NiFe]-Hydrogenases
Juan C. Fontecilla-Camps
6 Carbon Monoxide and Cyanide Ligands in the Active
Site of [FeFe]-Hydrogenases
John W. Peters
7 Carbon Monoxide as Intrinsic Ligand to Iron in the Active
Site of [Fe]-Hydrogenase
Seigo Shima, Rudolf K. Thauer, and Ulrich Ermler
xxx Contents of Volumes in the Metal Ions in Life Sciences Series

8 The Dual Role of Heme as Cofactor and Substrate


in the Biosynthesis of Carbon Monoxide
Mario Rivera and Juan C. Rodriguez
9 Copper-Carbon Bonds in Mechanistic and Structural
Probing of Proteins as well as in Situations where Copper
Is a Catalytic or Receptor Site
Heather R. Lucas and Kenneth D. Karlin
10 Interaction of Cyanide with Enzymes Containing Vanadium
and Manganese, Non-Heme Iron, and Zinc
Martha E. Sosa-Torres and Peter M.H. Kroneck
11 The Reaction Mechanism of the Molybdenum Hydroxylase
Xanthine Oxidoreductase: Evidence against the Formation
of Intermediates Having Metal-Carbon Bonds
Russ Hille
12 Computational Studies of Bioorganometallic Enzymes and Cofactors
Matthew D. Liptak, Katherine M. Van Heuvelen, and Thomas C. Brunold

Subject Index

Author Index of MIBS-1 to MIBS-44 and MILS-1 to MILS-6

Volume 7 Organometallics in Environment and Toxicology

1 Roles of Organometal(loid) Compounds in Environmental Cycles


John S. Thayer
2 Analysis of Organometal(loid) Compounds in Environmental
and Biological Samples
Christopher F. Harrington, Daniel S. Vidler, and Richard O. Jenkins
3 Evidence for Organometallic Intermediates in Bacterial Methane
Formation Involving the Nickel Coenzyme F430
Mishtu Dey, Xianghui Li, Yuzhen Zhou, and Stephen W. Ragsdale
4 Organotins. Formation, Use, Speciation, and Toxicology
Tamas Gajda and Attila Jancsó
5 Alkyllead Compounds and Their Environmental Toxicology
Henry G. Abadin and Hana R. Pohl
6 Organoarsenicals: Distribution and Transformation
in the Environment
Kenneth J. Reimer, Iris Koch, and William R. Cullen
7 Organoarsenicals. Uptake, Metabolism, and Toxicity
Elke Dopp, Andrew D. Kligerman, and Roland A. Diaz-Bone
Contents of Volumes in the Metal Ions in Life Sciences Series xxxi

8 Alkyl Derivatives of Antimony in the Environment


Montserrat Filella
9 Alkyl Derivatives of Bismuth in Environmental
and Biological Media
Montserrat Filella
10 Formation, Occurrence and Significance of Organoselenium
and Organotellurium Compounds in the Environment
Dirk Wallschläger and Jörg Feldmann
11 Organomercurials. Their Formation and Pathways
in the Environment
Holger Hintelmann
12 Toxicology of Alkylmercury Compounds
Michael Aschner, Natalia Onishchenko, and Sandra Ceccatelli
13 Environmental Bioindication, Biomonitoring, and Bioremediation
of Organometal(loid)s
John S. Thayer
14 Methylated Metal(loid) Species in Humans
Alfred V. Hirner and Albert W. Rettenmeier

Subject Index

Volume 8 Metal Ions in Toxicology: Effects, Interactions, Interdependencies

1 Understanding Combined Effects for Metal Co-Exposure


in Ecotoxicology
Rolf Altenburger
2 Human Risk Assessment of Heavy Metals: Principles and Applications
Jean-Lou C.M. Dorne, George E.N. Kass, Luisa R. Bordajandi,
Billy Amzal, Ulla Bertelsen, Anna F. Castoldi, Claudia Heppner,
Mari Eskola, Stefan Fabiansson, Pietro Ferrari, Elena Scaravelli,
Eugenia Dogliotti, Peter Fuerst, Alan R. Boobis, and Philippe Verger
3 Mixtures and Their Risk Assessment in Toxicology
Moiz M. Mumtaz, Hugh Hansen, and Hana R. Pohl
4 Metal Ions Affecting the Pulmonary and Cardiovascular Systems
Massimo Corradi and Antonio Mutti
5 Metal Ions Affecting the Gastrointestinal System Including the Liver
Declan P. Naughton, Tamás Nepusz, and Andrea Petroczi
6 Metal Ions Affecting the Kidney
Bruce A. Fowler
xxxii Contents of Volumes in the Metal Ions in Life Sciences Series

7 Metal Ions Affecting the Hematological System


Nickolette Roney, Henry G. Abadin, Bruce Fowler, and Hana R. Pohl
8 Metal Ions Affecting the Immune System
Irina Lehmann, Ulrich Sack, and Jörg Lehmann
9 Metal Ions Affecting the Skin and Eyes
Alan B.G. Lansdown
10 Metal Ions Affecting the Neurological System
Hana R. Pohl, Nickolette Roney, and Henry G. Abadin
11 Metal Ions Affecting Reproduction and Development
Pietro Apostoli and Simona Catalani
12 Are Cadmium and Other Heavy Metal Compounds
Acting as Endocrine Disrupters?
Andreas Kortenkamp
13 Genotoxicity of Metal Ions: Chemical Insights
Woijciech Bal, Anna Maria Protas, and Kazimierz S. Kasprzak
14 Metal Ions in Human Cancer Development
Erik J. Tokar, Lamia Benbrahim-Tallaa, and Michael P. Waalkes

Subject Index

Volume 9 Structural and Catalytic Roles of Metal Ions in RNA

1 Metal Ion Binding to RNA


Pascal Auffinger, Neena Grover, and Eric Westhof
2 Methods to Detect and Characterize Metal Ion Binding
Sites in RNA
Michèle C. Erat and Roland K.O. Sigel,
3 Importance of Diffuse Metal Ion Binding to RNA
Zhi-Jie Tan and Shi-Jie Chen
4 RNA Quadruplexes
Kangkan Halder and Jörg S. Hartig
5 The Roles of Metal Ions in Regulation by Riboswitches
Adrian Ferré-D’Amaré and Wade C. Winkler
6 Metal Ions: Supporting Actors in the Playbook of Small Ribozymes
Alexander E. Johnson-Buck, Sarah E. McDowell, and Nils G. Walter
7 Multiple Roles of Metal Ions in Large Ribozymes
Daniela Donghi and Joachim Schnabl
Contents of Volumes in the Metal Ions in Life Sciences Series xxxiii

8 The Spliceosome and Its Metal Ions


Samuel E. Butcher
9 The Ribosome: A Molecular Machine Powered by RNA
Krista Trappl and Norbert Polacek
10 Metal Ion Requirements in Artificial Ribozymes that Catalyze
Aminoacylations and Redox Reactions
Hiroaki Suga, Kazuki Futai, and Koichiro Jin
11 Metal Ion Binding and Function in Natural and Artificial
Small RNA Enzymes from a Structural Perspective
Joseph E. Wedekind
12 Binding of Kinetically Inert Metal Ions to RNA:
The Case of Platinum(II)
Erich G. Chapman, Alethia A. Hostetter, Maire F. Osborn,
Amanda L. Miller, and Victoria J. DeRose

Subject Index

Volume 10 Interplay between Metal Ions and Nucleic Acids

1 Characterization of Metal Ion-Nucleic Acid Interactions


in Solution
Maria Pechlaner and Roland K.O. Sigel
2 Nucleic Acid-Metal Ion Interactions in the Solid State
Katsuyuki Aoki and Kazutaka Murayama
3 Metal Ion-Promoted Conformational Changes
of Oligonucleotides
Bernhard Spingler
4 G-Quadruplexes and Metal Ions
Nancy H. Campbell and Stephen Neidle
5 Metal Ion-Mediated DNA-Protein Interactions
Barbara Zambelli, Francesco Musiani, and Stefano Ciurli
6 Spectroscopic Investigations of Lanthanide Ion Binding
to Nucleic Acids
Janet R. Morrow and Christopher M. Andolina
7 Oxidative DNA Damage Mediated by Transition Metal
Ions and Their Complexes
Geneviève Pratviel
8 Metal Ion-Dependent DNAzymes and Their Applications
as Biosensors
Tian Lan and Yi Lu
xxxiv Contents of Volumes in the Metal Ions in Life Sciences Series

9 Enantioselective Catalysis at the DNA Scaffold


Almudena García-Fernández and Gerard Roelfes
10 Alternative DNA Base Pairing through Metal Coordination
Guido H. Clever and Mitsuhiko Shionoya
11 Metal-Mediated Base Pairs in Nucleic Acids with
Purine- and Pyrimidine-Derived Nucleosides
Dominik A. Megger, Nicole Megger, and Jens Müller
12 Metal Complex Derivatives of Peptide Nucleic Acids
Roland Krämer and Andrij Mokhir

Subject Index

Volume 11 Cadmium: From Toxicity to Essentiality

1 The Bioinorganic Chemistry of Cadmium


in the Context of Its Toxicity
Wolfgang Maret and Jean-Marc Moulis
2 Biogeochemistry of Cadmium and Its Release
to the Environment
Jay T. Cullen and Maria T. Maldonado
3 Speciation of Cadmium in the Environment
Francesco Crea, Claudia Foti, Demetrio Milea,
and Silvio Sammartano
4 Determination of Cadmium in Biological Samples
Katrin Klotz, Wobbeke Weistenhöfer, and Hans Drexler
5 Imaging and Sensing of Cadmium in Cells
Masayasu Taki
6 Use of 113Cd NMR to Probe the Native Metal Binding
Sites in Metalloproteins: An Overview
Ian M. Armitage, Torbjörn Drakenberg, and Brian Reilly
7 Solid State Structures of Cadmium Complexes
with Relevance for Biological Systems
Rosa Carballo, Alfonso Castiñeiras, Alicia Domínguez-Martín,
Isabel García Santos, and Juan Niclós-Gutierrez
8 Complex Formation of Cadmium(II) with Sugar
Residues, Nucleobases, Phosphates, Nucleotides,
and Nucleic Acids
Roland K.O. Sigel, Miriam Skilandat, Astrid Sigel,
Bert P. Operschall, and Helmut Sigel
9 Cadmium(II) Complexes of Amino Acids and Peptides
Imre Sóvágó and Katalin Várnagy
Contents of Volumes in the Metal Ions in Life Sciences Series xxxv

10 Natural and Artificial Proteins Containing Cadmium


Anna F. Peacock and Vincent L. Pecoraro
11 Cadmium in Metallothioneins
Eva Freisinger and Milan Vašák
12 Cadmium-Accumulating Plants
Hendrik Küpper and Barbara Leitenmaier
13 Cadmium Toxicity in Plants
Elisa Andresen and Hendrik Küpper
14 Toxicology of Cadmium and Its Damage to Mammalian Organs
Frank Thévenod and Wing-Kee Lee
15 Cadmium and Cancer
Andrea Hartwig
16 Cadmium in Marine Phytoplankton
Yan Xu and François M.M. Morel

Subject Index

Volume 12 Metallomics and the Cell


Guest Editor: Lucia Banci

1 Metallomics and the Cell: Some Definitions and General Comments


Lucia Banci and Ivano Bertini
2 Technologies for Detecting Metals in Single Cells
James E. Penner-Hahn
3 Sodium/Potassium Homeostasis in the Cell
Michael J.V. Clausen and Hanna Poulsen
4 Magnesium Homeostasis in Mammalian Cells
Andrea M.P. Romani
5 Intracellular Calcium Homeostasis and Signaling
Marisa Brini, Tito Calì, Denis Ottolini, and Ernesto Carafoli
6 Manganese Homeostasis and Transport
Jerome Roth, Silvia Ponzoni, and Michael Aschner
7 Control of Iron Metabolism in Bacteria
Simon Andrews, Ian Norton, Arvindkumar S. Salunkhe,
Helen Goodluck, Wafaa S.M. Aly, Hanna Mourad-Agha,
and Pierre Cornelis
8 The Iron Metallome in Eukaryotic Organisms
Adrienne C. Dlouhy and Caryn E. Outten
xxxvi Contents of Volumes in the Metal Ions in Life Sciences Series

9 Heme Uptake and Metabolism in Bacteria


David R. Benson and Mario Rivera
10 Cobalt and Corrinoid Transport and Biochemistry
Valentin Cracan and Ruma Banerjee
11 Nickel Metallomics: General Themes Guiding Nickel Homeostasis
Andrew M. Sydor and Deborah B. Zamble
12 The Copper Metallome in Prokaryotic Cells
Christopher Rensing and Sylvia Franke McDevitt
13 The Copper Metallome in Eukaryotic Cells
Katherine E. Vest, Hayaa F. Hashemi, and Paul A. Cobine
14 Zinc and the Zinc Proteome
Wolfgang Maret
15 Metabolism of Molybdenum
Ralf R. Mendel
16 Comparative Genomics Analysis of the Metallomes
Vadim N. Gladyshev and Yan Zhang

Subject Index

Volume 13 Interrelations between Essential Metal Ions


and Human Diseases (this book)

Volume 14 The Metal-Driven Biogeochemistry of Gaseous


Compounds in the Environment (in preparation)
Guest Editors: Peter M.H. Kroneck and Martha E. Sosa-Torres

1 The Early Earth Atmosphere and Early Life Catalysts


Sandra I. Ramírez Jiménez
2 Living on Acetylene, a Primordial Energy Source
(Acetylene Hydratase)
Felix ten Brink
3 Carbon Monoxide, Toxic Gas and Fuel for Anaerobes
and Aerobes: Carbon Monoxide Dehydrogenases
Jae-Hun Jeoung, Jochen Fesseler, Sebastian Götzl, and Holger Dobbek
4 Investigations of the Efficient Electrocatalytic
Interconversions of CO2 and CO by Nickel-Containing
Carbon Monoxide Dehydrogenases
Vincent Wang, Stephen W. Ragsdale, and Fraser A. Armstrong
5 Nature’s Toolbox to Handle Dihydrogen (Hydrogenases)
Alison Parkin
Contents of Volumes in the Metal Ions in Life Sciences Series xxxvii

6 The Making of the Greenhouse Gas Methane (Methanogenesis)


Dariusz A. Sliwa and Stephen W. Ragsdale
7 Light-Dependent Production of Dioxygen (Photosynthesis)
Vittal Yachandra and Junko Yano
8 Production of Dioxygen in the Dark (Dismutases of Oxyanions)
Jennifer DuBois
9 Transition Metal Complexes and Activation of Dioxygen
(Model Compounds, Catalysis)
Gereon M. Yee and William B. Tolman
10 Respiratory Conservation of Energy with Dioxygen
(Respiratory Chain/Cytochrome c Oxidase)
Shinya Yoshikawa
11 Methane Monooxygenase: Breaking up Methane
with Iron and Copper
Matthew H. Sazinsky and Stephen J. Lippard
12 Cleaving the N,N Triple Bond: The Transformation of N2 to NH3
(Nitrogenase)
Chi Chung Lee, Yilin Hu, and Markus W. Ribbe
13 The Production of Ammonia by Multiheme Cytochromes c
Jörg Simon and Peter M.H. Kroneck
14 No Laughing Matter: The Making of the Greenhouse
Gas Dinitrogen Monoxide (N2O Reductase)
Oliver Einsle
15 Hydrogen Sulfide: A Toxic Gas Produced by Dissimilatory
Sulfate Reduction and Consumed by Microbial Oxidation
Larry L. Barton, Marie-Laure Fardeau, and Guy Fauque
16 Anaerobic Oxidation of Methane and Ammonia
Mike S.M. Jetten
17 Transformation of Dimethylsulfoxide
Ulrike Kappler

Subject Index

Comments and suggestions with regard to contents, topics,


and the like for future volumes of the series are welcome.
Chapter 1
Metal Ions and Infectious Diseases.
An Overview from the Clinic

Peggy L. Carver

Contents
ABSTRACT ............................................................................................................................. 2
1 INTRODUCTION ............................................................................................................. 3
1.1 Role of Antioxidants ................................................................................................. 3
1.2 Host Defense Responses to Infection ....................................................................... 3
1.3 Alterations in Serum Levels of Trace Elements ....................................................... 4
1.4 Nutritional Immunity ................................................................................................ 4
1.5 Natural Resistance-Associated Macrophage Protein (Nramp) ................................. 5
1.6 Calprotectin............................................................................................................... 5
2 IRON .................................................................................................................................. 5
2.1 Human Pharmacology and Pharmacokinetics ......................................................... 5
2.2 The Complex Defense-Counter Defense System in the Battle for Iron.................... 6
2.3 Role of Iron in Infectious Diseases .......................................................................... 6
2.3.1 Dialysis Patients ............................................................................................ 7
2.3.2 Malaria ......................................................................................................... 7
2.3.3 Human Immunodeficiency Virus .................................................................. 8
2.3.4 Diabetes ........................................................................................................ 8
2.3.5 Iron Overload ................................................................................................ 8
2.3.6 Role of Iron Chelators in Infection ............................................................... 9
3 ZINC .................................................................................................................................. 10
3.1 Human Pharmacology and Pharmacokinetics .......................................................... 10
3.1.1 Zn-Metallothionein (Zn-MT) ....................................................................... 11
3.1.2 Zn-Metallo β-Lactamases ............................................................................. 11
3.2 Role of Zinc in Infectious Diseases .......................................................................... 11
3.2.1 Cystic Fibrosis .............................................................................................. 11
3.2.2 Prevention of Childhood Diarrhea and Respiratory
Tract Infections ............................................................................................. 12
3.2.3 The Common Cold ....................................................................................... 12

P.L. Carver (*)


University of Michigan College of Pharmacy, Department of Clinical,
Social, and Administrative Sciences, 428 Church St.,
Ann Arbor, MI 48109-1065, USA
e-mail: peg@umich.edu

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 1


Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_1, © Springer Science+Business Media Dordrecht 2013
2 Carver

3.2.4 Prevention or Treatment of Malaria .............................................................. 13


3.2.5 Burn Patients ................................................................................................ 13
3.2.6 Wound Healing ............................................................................................. 13
3.2.7 Critically Ill Patients ..................................................................................... 13
3.2.8 Sickle Cell Disease ....................................................................................... 14
4 SELENIUM ....................................................................................................................... 14
4.1 Human Pharmacology and Pharmacokinetics .......................................................... 14
4.2 Role of Selenium in Infectious Diseases .................................................................. 15
4.2.1 Human Immunodeficiency Virus .................................................................. 15
4.2.2 Intensive Care Unit Sepsis ............................................................................ 16
4.2.3 Role of Selenium in Other Infections ........................................................... 17
5 COPPER ............................................................................................................................ 18
5.1 Human Pharmacology and Pharmacokinetics .......................................................... 18
5.2 Role of Copper in Infectious Diseases ..................................................................... 19
5.2.1 Copper/Zinc Ratio ........................................................................................ 19
6 CHROMIUM ..................................................................................................................... 19
6.1 Human Pharmacology and Pharmacokinetics ......................................................... 19
6.2 Role of Chromium in Infectious Diseases ............................................................... 20
7 MANGANESE .................................................................................................................. 20
7.1 Human Pharmacology and Pharmacokinetics .......................................................... 20
7.2 Role of Manganese in Infectious Diseases .............................................................. 21
7.2.1 Arginase ........................................................................................................ 21
7.2.2 Manganese Superoxide Dismutase ............................................................... 22
8 SUMMARY AND FUTURE DEVELOPMENTS ............................................................ 22
ABBREVIATIONS .................................................................................................................. 22
ACKNOWLEDGMENT .......................................................................................................... 23
REFERENCES ........................................................................................................................ 23

Abstract Trace elements (TEs) are required by both humans and bacterial pathogens.
Although metal ion homeostasis is tightly controlled in humans, growing evidence
suggests that pathogens utilize a variety of means designed to circumvent the
sequestration of TEs. Colonizing pathogenic microorganisms employ a variety of
strategies to sense, acquire, store, and export metal ions in the vertebrate host
which include the biosynthesis and utilization of siderophores, and the expression
of high-affinity metal-ion transporters. For iron, selenium, and zinc, significant
correlations have been shown between TE levels in plasma, serum, or tissues, and
the prevention or treatment of a variety of infectious diseases; fewer such data exist
for copper, chromium, or manganese. TEs are often employed as antioxidants, and
as supplements in patients with TE-deficient states. The role of TE supplementation
in humans as antioxidants remains controversial, but has demonstrated significant
benefit in the role of selenium for patients with sepsis, and of zinc for the prevention
of several infectious diseases.

Keywords burns • chromium • copper • critically ill • Cu/Zn ratio • cystic fibrosis •
diarrhea • human immunodeficiency virus (HIV) • infectious diseases • intensive
care unit • iron • malaria • manganese • mycobacterium • pneumonia • selenium •
sepsis • supplementation • trace elements • zinc

Please cite as: Met. Ions Life Sci. 13 (2013) 1–28


1 Metal Ions and Infectious Diseases. An Overview from the Clinic 3

1 Introduction

Trace elements (TEs) are often defined as minerals that are required by adult humans
in amounts between 1 to 100 mg/day. Nutrition is a two-edged sword when dealing
with the treatment or prevention of infectious diseases, since bacteria, like humans,
have a need for TEs [1]. Since metal ions are both necessary for life, but toxic in
excess, metal homeostasis is tightly controlled by both bacteria and humans. When
infecting humans, bacteria must acquire nutrients required for survival from the
host environment. Colonizing pathogenic microorganisms employ a variety of strat-
egies to sense, acquire, store, and export metal ions, which include the biosynthesis
and utilization of siderophores, and the expression of high-affinity metal-ion trans-
porters [2]. In order to control the availability of metals while restricting access by
bacteria, humans have developed a variety of immune strategies.

1.1 Role of Antioxidants

Much of the research on TEs and infection has evaluated the response of the host to
the onset of infection, particularly in critically ill patients, including those with
trauma or severe burns. Any injured patient will develop an acute-phase response
and a systemic inflammatory response syndrome (SIRS) with the production of
numerous mediators, including cytokines, which modulate the metabolic response.
Oxidative stress is defined as a state in which the level of toxic reactive oxygen
intermediates overcomes the endogenous antioxidant defenses of the host, resulting
in damage to DNA, RNA, proteins, carbohydrates, and unsaturated fatty acids of the
cell membrane. In critically ill patients, hyperinflammation, cellular immune dys-
function, and oxidative stress, combined with pathophysiologic events leading to
mitochondrial dysfunction and SIRS, can result in multiple organ dysfunction and
high rates of mortality. Manzanares et al. [3] recently performed a meta analysis of
the outcomes of 21 randomized clinical trials in patients who received antioxidant
micronutrients versus placebo. The use of antioxidants was associated with a
significant reduction in overall mortality.

1.2 Host Defense Responses to Infection

In the human antioxidant defense armamentarium, a variety of cytosolic, mitochon-


drial, and plasma antioxidants serve to protect tissues from the accumulation of
reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can lead
to target end organ dysfunction and death. In addition to nonenzymatic endogenous
antioxidant defense mechanisms (e.g., uric acid, glutathione, bilirubin, thiols, albumin,
and nutrition factors, including vitamins and phenols), enzymatic defense mechanisms
such as catalase (Cu, Fe), copper-zinc superoxide dismutase (Cu/Zn SOD), manganese
4 Carver

superoxide dismutase (Mn-SOD) and glutathione peroxidase (GPx), are responsible


for neutralizing ROS and RNS [3,4]. ROS activate the nuclear transcription factor
NF kappa beta (NFκB). Activation of NFκB is modulated by Se, Zn, and vitamins
C and E. Cu is also part of ferroxidases such as ceruloplasmin. The SODs initiate
the antioxidant process, transforming the superoxide anion into hydrogen peroxide,
which is further metabolized, first by catalase, then by the different GPxs [5].
Host defense responses to infection include the release of cytokines, including
tumor necrosis factor (TNF), interferon alpha (IFNα), and interleukins (IL) [1,6].
Cytokine-mediated anorexia results in reduced nutrient intake and sequestration of
critical nutrients such as iron, copper, and zinc [1]. During SIRS, low plasma levels
of endogenous TEs are observed, secondary to escape of TEs from the interstitial
compartment due to capillary leakage, hemodilution, and hemodialysis or continu-
ous renal replacement therapies [3,7–9]. Critically ill, burn, and trauma patients are
characterized by an increased free radical production, which is proportional to the
severity of the injury [5]. The most severe depletions of antioxidants occur in the
most critically ill patients.

1.3 Alterations in Serum Levels of Trace Elements

Relationships between TE doses and serum TE concentrations vary for each TE and
in varying underlying clinical conditions. SIRS is characterized by decreased serum
levels of Fe, Se, and Zn, along with increased levels of Cu [5,10,11]. In patients with
major burns, however, Cu deficiency is observed.
A recent study in clinically stable patients undergoing long-term administration
of parenteral nutrition demonstrated a significant dose-response relationship
between weekly TE doses and serum TE concentrations for Zn, Cr, and Mn, but not
for Se, Cu, or Fe [12]. Serum levels of Cu, Zn, Se and Fe in 44 patients with tuber-
culosis (TB) were compared to a control group of healthy individuals, at baseline
and at the end of an intensive phase of anti-TB chemotherapy. Concentrations of Zn,
Se, and Fe were significantly lower (P < 0.05) while that of Cu and the Cu/Zn ratio
significantly higher (P < 0.05) in TB patients versus controls. Further, TB patients
with human immunodeficiency virus (HIV) coinfection had significantly lower
serum Zn and Se concentrations, and significantly higher Cu/Zn ratios compared to
those in TB patients without HIV coinfection (P < 0.05). Serum Cu concentration
and Cu/Zn ratios declined significantly after anti-TB chemotherapy, irrespective of
HIV serostatus (P < 0.05) [13].

1.4 Nutritional Immunity

“Nutritional immunity” is a term used to describe the starvation of pathogens by the


host for the vital metal ions Fe, Zn, and Mn. All bacterial pathogens must have
mechanisms to circumvent nutritional immunity, but complex host defense mechanisms
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 5

to counteract these bacterial counter-defense mechanisms have also evolved [14].


For example, Borrelia burgdorferi, the causative agent of Lyme disease, and the
only pathogen known to be an exception to the obligatory requirement for host Fe,
circumvents host-mediated Fe sequestration by substituting Mn2+ in place of Fe2+
and thus does not require Fe2+ to infect the host. In response, vertebrates encode
additional mechanisms to restrict Mn2+ availability [15].

1.5 Natural Resistance-Associated Macrophage


Protein (Nramp)

The Nramp family constitutes a large class of metal-ion membrane transporters,


localized either at the cell surface or in intracellular vesicles, which translocate
a wide range of divalent metal substrates, including Mn, Fe, Co, Cu, Zn, and Cd.
The first of these to be mechanistically studied was mammalian DMT1 (divalent
metal transporter) [16].

1.6 Calprotectin

Calprotectin (CP) is a metal chelating molecule that acts as a Mn scavenger in the


context of Staphylococcus aureus infection. Although the antibacterial and antifun-
gal properties of calprotectin were first attributed to its ability to sequester Zn, more
recent studies demonstrate that calprotectin-dependent depletion of Mn also occurs
in abscesses caused by S. aureus [2]. CP binds Mn2+ and Zn2+ with high affinity and
essentially “starves” bacteria of these essential nutrients [17].

2 Iron

2.1 Human Pharmacology and Pharmacokinetics

In humans, a complex system of transporters regulates Fe homeostasis, which is


maintained through careful coordination of duodenal absorption and recycling of Fe
stores (see Chapter 8). The role of Fe in infectious diseases has been intensively
studied. Since Fe serves as an important cofactor for enzymes, and is involved in
many basic cellular functions and metabolic pathways of bacteria and fungi, they
have developed sophisticated mechanisms for its acquisition.
6 Carver

2.2 The Complex Defense-Counter Defense System


in the Battle for Iron

Human hosts tightly regulate Fe levels, sequestering this nutrient intracellularly as


a mechanism to prevent bacterial growth. Since Fe3+ is almost insoluble, nearly two-
thirds of the Fe within vertebrates is complexed to the porphyrin heme in hemoglo-
bin. Extracellular Fe is rapidly removed by transferrin and lactoferrin, proteins with
a high affinity for Fe. Inflammation and febrile conditions can increase ferritin and
lactoferrin synthesis. Hemopexin is a heme-scavenging protein found in serum
which binds heme with high affinity. Infection and inflammation alter Fe homeosta-
sis through immune-mediated mechanisms that further restrict the supply of readily
available Fe.
Iron administration alone does not appear to cause bacterial growth; however,
once the transferrin saturation exceeds a critical threshold, free Fe becomes avail-
able for bacterial utilization. In addition, bacteria and fungi have evolved complex
strategies to acquire Fe from vertebrate hosts. Pathogens employ one or more Fe
transport mechanisms, depending on the type of Fe found in the host, while the host
counters by increasing synthesis of Fe binding proteins such as transferrin and lac-
toferrin. The resulting Fe starvation of pathogen limits its growth, allowing the host
time to eradicate the infection via immune-related mechanisms [18].
Two main mechanisms for obtaining host Fe include the development of recep-
tors that can bind transferrin, lactoferrin, or hemoglobin, or the production of sid-
erophores [19]. Siderophores are low-molecular-weight Fe chelators secreted by
bacteria and fungi, that compete with transferrin for available Fe. Siderophores bind
Fe3+ with an affinity stronger than that of transferrin or lactoferrin. Energy-dependent
transport of siderophores across the outer membranes of bacteria is mediated by
TonB-dependent receptors. To counteract siderophores, vertebrates produce neutro-
phil gelatinase-associated lipocalin (NGAL; siderocalin), which binds and seques-
ters siderophores [14].
Staphylococcus aureus uses non-siderophore mechanisms to acquire Fe from
hemoglobin. By secreting hemolytic toxin, S. aureus lyses erythrocytes to release
hemoglobin, which binds to a surface receptor on the bacteria. Fe is transported as
heme into the bacterial cell for use as a nutrient.

2.3 Role of Iron in Infectious Diseases

In vitro evidence and animal studies suggest that increased Fe availability promotes
bacterial growth and virulence. The risk of increased infections with administration
of intravenous (IV) Fe has also been supported in limited animal studies. For exam-
ple, in a murine model of E. coli sepsis, administration of IV Fe sucrose was associ-
ated with a mortality rate of nearly 60% when septic mice were also administered
Fe, as compared to a mortality rate of 0% in mice with sepsis alone, or in those
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 7

administered Fe alone [20]. The relationship between Fe and infection has been
investigated in human patient populations infected with malaria and in those at
high risk for infection.

2.3.1 Dialysis Patients

Renal failure resulting in hemodialysis is an independent risk factor for infection


in hemodialysis patients. Teehan and colleagues [21] evaluated Fe storage levels
in hemodialysis patients receiving IV iron and found that patients with replete
Fe indices were at increased risk for bacteremia compared with patients having
deficient iron stores.

2.3.2 Malaria

Controversy continues over whether the benefit of Fe supplementation in Fe-deficient


individuals outweighs the potential risk of malaria, and whether Fe supplementation
should be restricted to Fe-deficient or anemic patients. In a recent study in 785
Tanzanian children living in an area of intense malaria transmission, the presence of
naturally occurring Fe deficiency significantly decreased the odds of parasitemia,
severe malaria, and malaria-associated mortality [22]. However, international
guidelines support Fe supplementation in children under 2 years of age in areas with
a high prevalence of anemia. Iannotti et al. [23] reviewed 26 randomized controlled
trials of preventive, oral Fe supplementation in young children (<5 years) and found
conflicting data regarding the benefits and adverse effects of Fe supplementation on
malaria. In several smaller studies in Gambia, Nepal, and Tanzania, Fe supplemen-
tation was associated with a significant increase in fever-associated severe malaria,
a 16% greater risk of adverse events due to malaria [24–26]. However, in several
studies [27–30], including two large (832 and 25,490 children) studies in Tanzania
and Nepal [29,30], no significant differences in the rate of parasitemia, parasite
density, or frequency of malaria were observed. They concluded that additional
research is needed in populations affected by HIV and tuberculosis, and that Fe
supplementation in preventive programs may need to be targeted. However, it is also
important to note that nearly all trials of Fe supplementation took place in conjunc-
tion with some form of malaria control (e.g., bed nets, malaria prophylaxis), and
some trials took place in area of lower malaria transmission [31–36].
Pregnant women, like children, are among the populations in greatest need of
Fe supplementation while also being at greatest risk of malaria [31]. As gestation
proceeds, latent malarial protozoa become active, especially in the Fe-rich placenta
[37]. Although some studies have reported that Fe deficiency is associated with a
decreased prevalence and severity of malaria in pregnant women, [38,39], and that
Fe supplementation in pregnant women increases the risk of malaria, these effects
are likely diminished by factors such as host immunity, host Fe status, and effective
malaria surveillance and control. A recent meta analysis of 23 studies conducted in
8 Carver

countries having some risk of malaria concluded that there is no evidence that Fe
supplementation increases placental malaria. However, only 2 of the 23 studies reported
malaria outcomes [40], and the authors qualified their findings by noting that there
was a significantly increased risk of malaria associated with Fe supplementation in
areas without adequate malaria surveillance and treatment programs.
The interaction between Fe level, Fe supplementation and susceptibility to
maternal and childhood malaria remains a concern, in particular in areas without
adequate malaria surveillance and treatment programs [31,35,41]. Several ongoing
studies are currently comparing the risk of malaria in Fe-supplemented versus
non-supplemented pregnant women [31].

2.3.3 Human Immunodeficiency Virus

During chronic diseases, such as acquired immunodeficiency disease, Fe is rapidly


sequestered by macrophages, causing a condition known as anemia of chronic
disease, in which total body stores of Fe range from normal to increased, but are
sequestered and unavailable even to the host, causing an anemia which functions as
a defense against infection [42].

2.3.4 Diabetes

Patients with diabetic ketoacidosis appear uniquely susceptible to infections caused


by Rhizopus species, lending support to the role of Fe uptake in the pathogenesis of
this infection. In the presence of low pH and high glucose, phagocytes are dysfunc-
tional, with impaired chemotaxis and defective intracellular oxidative and non-
oxidative killing [43]. Patients with diabetic ketoacidosis have elevated serum levels
of free Fe, and acidic (7.3–6.88) pHs [44,45]. In vitro, R. oryzae grows profusely at
acidic conditions upon addition of exogenous Fe, but only at pHs ≤7.4.

2.3.5 Iron Overload

In vertebrates, administration of large doses of Fe, or the presence of medical condi-


tions such as thalassemia, solid organ or hematopoietic stem cell transplantation in
which Fe overload is present, are all strong risk factors for the development of bac-
terial and fungal infections.
In patients with thalassemia, the need for frequent transfusions often results in Fe
overload, and infections continue to be among the major (12–46%) causes of mor-
tality. The bacteria isolated most frequently include Staphylococcus aureus,
Klebsiella pneumoniae, Escherichia coli, Streptococcus pneumoniae, Salmonella
typhi, Yersinia enterocolitica and other Gram-negative bacteria [46].
Fe overload is common in patients undergoing hematopoietic stem cell trans-
plantation (HSCT), particularly in patients with myelodysplastic syndromes, in part
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 9

due to frequent administration of red blood cell transfusions prior to HSCT, adding
exogenous Fe loading at a rate of 200–250 mg per unit of red blood cells. In these
patients, hepatic Fe concentrations often approach levels observed in hereditary
hemochromatosis. In HSCT recipients, elevated ferritin, hepatic Fe, hepcidin, and
Fe bone marrow stores have all been shown to correlate with a markedly increased
risk for the development of invasive fungal infections, including those caused by
Aspergillus, Candida, Cryptococcus, Histoplasma, Paracoccidioides, Pneumocystis,
Pythium, Rhizopus, Trichosporon, and Mucor [47–58].
Similarly, in patients undergoing liver transplantation, elevated serum Fe and
hepatic Fe overload is associated with decreased long term survival, regardless of
whether the patient had hereditary hemochromatosis [59,60]. In 153 patients under-
going liver transplantation, 31 invasive fungal infections were observed in 28
patients, of which 21 (68%) were caused by Candida, 7 (23%) by Aspergillus, 2
(6%) by Cryptococcus, and 1 (3%) by Saccharomyces. Stainable Fe in the hepatic
explant was found in 48 patients (31%) and was strongly and independently associ-
ated with the development of post transplantation fungal infections [59].

2.3.6 Role of Iron Chelators in Infection

The predisposition to Mucorales (zygomycoses), Yersinia, and Vibrio vulnificus


infections in patients treated with deferoxamine is now known to be due to the for-
mation of feroxamine, a deferoxamine-Fe chelate which acts as a siderophore,
delivering Fe to the pathogen. Deferoxamine strips ferric Fe from transferrin and
attaches itself on the mold through an inducible receptor, and the Fe is transported
intracellularly by an active reduction of the ferric form into the more soluble ferrous
form [61–65].
In contrast, in a mouse model of diabetic ketoacidosis, mice are protected from
R. oryzae infection by administration of the Fe chelators deferiprone and defera-
sirox, which do not act as siderophores [66–68]. Unfortunately, not all Mucorales
have the same susceptibility to effective Fe chelators. For example, Cunninghamella
bertholletiae and Mucor species display higher deferasirox minimal inhibitory and
fungicidal concentrations than do Rhizopus species.
The possible utility of deferasirox as an adjunctive therapy for mucormycosis
has been evaluated in small studies, with mixed results. In an open label study of
deferasirox in combination with antifungal therapy, seven of eight patients survived
[69]. A recent multicenter, placebo-controlled, double-blinded clinical trial assessed
the potential role of administration of deferasirox, in combination therapy with anti-
fungal agents, in the treatment of 14 patients with mucormycosis. Patients with
mucormycosis treated with deferasirox had a higher mortality rate at 90 days than
in those who received placebo (82% versus 22%, respectively), possibly because
more patients in the deferasirox group had hematologic malignancy, neutropenia,
and/or pulmonary involvement. Further study is necessary to determine the possible
benefits or harms of deferasirox [70,71].
10 Carver

3 Zinc

3.1 Human Pharmacology and Pharmacokinetics

Zinc affects multiple aspects of the immune system, and the response to infection,
and is required for normal development and function of cells mediating innate
immunity, neutrophils and natural killer cells, as well as macrophages. Zinc acts as
a cofactor for more than 3000 metalloenzymes and proteins (Chapter 12), including
Cu-Zn superoxide dismutase and metallothionein, as well as a large family of Zn
proteins involved in gene transcription (such as the Zn finger proteins) which are
important in maintenance of the immune system or in the prevention of infectious
diseases [72–74].
Zn circulates at a concentration of 70 to 120 μg/dL, with 60 percent loosely
bound to albumin and 30 percent tightly bound to macroglobulin. The primary
stores of Zn include the liver and kidney; mostly intracellularly bound to metallo-
proteins. Zn is actively absorbed throughout the small intestine, mainly in the duo-
denum and jejunum via an intricate homeostatic mechanism which is regulated by
metallothionein. Typically, Zn absorption is 20 to 40% bioavailable but metallothio-
nein found in the gut enterocyte binds Cu more avidly than Zn. Zn homeostasis (see
also Chapter 12) is probably maintained by a combination of changes in fractional
absorption and endogenous fecal Zn excretion. Zn is transported bound to albumin,
and taken up by peripheral tissues and by the liver where it may be stored as metallo-
thionein. Zn excretion is primarily via the gastrointestinal tract, although up to 10
percent of the circulating Zn is also excreted through urine; urinary excretion typi-
cally ranges from 0.5 to 0.8 mg/day [72–74].
Because most Zn is bound to albumin, measured Zn levels may be reduced in
patients with hypoalbuminemia; however, the correlation between Zn and albumin
levels is weak, and plasma levels are loosely correlated with Zn stores. Thus, plasma
levels do not reliably identify individuals with Zn deficiency. A recent (2012) meta
analysis of studies correlating dietary Zn intake and serum or plasma levels of Zn in
healthy adults reported that for every doubling of Zn dose, the plasma concentration
changes by 6% [75]. Although plasma levels correlate with doses, and are generally
a good index of Zn status in healthy individuals, these levels are depressed during
inflammatory disease states [12]. In healthy individuals, plasma, urinary, and hair
Zn are reliable biomarkers of Zn status [76].
Zn deficiency is associated with impaired phagocytic function, lymphocyte
depletion, decreased immunoglobulin production, a reduction in the T4+/T8+
ratio, and decreased interleukin-2 production [77–79]. Mild Zn deficiency is com-
mon, especially in developing countries, because the diet is relatively low in Zn
and contains significant amounts of plant or vegetable phytates found in cereal
proteins, which reduce Zn absorption. Zn absorption may also be impaired in
patients with severe liver disease, although levels increase within one week follow-
ing liver transplantation [80,81]. Zn deficiency is also associated with pancreatic
disease or insufficiency, since pancreatic enzymes, while necessary for release of
dietary Zn, also contain Zn-complexing ligands. Rarely, Zn deficiency can occur
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 11

due to malabsorption of Zn in patients with an autosomal recessive disease caused


by mutations in the SLC39A4 gene on chromosome 8q24.3, which encodes
ZIP4, a Zn transporter in the gastrointestinal tract, resulting in acrodermatitis
enteropathica [74,82].
Recent studies suggest that Zn deficiency can result in a significant increase in
the incidence of diarrhea and upper respiratory tract infections, as well as morbidity
and mortality from these infections. Diabetics (both type 1 and type 2) can exhibit
hyperzincuria due to alterations of Zn metabolism, which may have a role in the
immune dysfunction associated with diabetes mellitus [83]. While Zn supplementa-
tion in diabetic patients may improve immune function, it increases the HbA1c
levels and leads to worsening glucose intolerance [84].

3.1.1 Zn-Metallothionein (Zn-MT)

Physiologically, sepsis increases Zn-MT production, likely mediated by the up-


regulation of the metallothionein gene by interleukin-1, leading to Zn sequestration
in the intestinal and liver cells. The physiologic benefit of Zn as a negative acute-
phase reactant may be to minimize Zn availability for bacterial use in its own DNA
replication [72].

3.1.2 Zn-Metallo β-Lactamases

Recent studies in multidrug-resistant Acinetobacter have highlighted the role of


Zn in Zn-metallo β-lactamases. “Starving” Acinetobacter baumannii (with Zn
chelators) restored susceptibility of the organism, highlighting the possibility of
Zn limitation strategies as a possible mechanism to combat carbapenem resistance
in Acinetobacter [85].

3.2 Role of Zinc in Infectious Diseases

In recent years, much interest has been generated by the possibility that subclinical
Zn deficiency may significantly increase the incidence of, and morbidity and mor-
tality from, diarrhea and upper respiratory tract infections. Several studies have
now demonstrated that Zn supplementation of select high risk populations can have
substantial health benefits.

3.2.1 Cystic Fibrosis

Low plasma Zn concentrations have been reported in approximately 30% of young


infants with cystic fibrosis identified by newborn screening. Since fecal Zn losses
correlated with fecal fat excretion, investigators have suggested that cystic fibrosis
interferes with enterohepatic recycling of Zn [86].
12 Carver

The existence of positive correlations between Zn and IL-2, and between Zn or


active thymulin and natural killer (NK) cell activity suggest a close link among Zn
failure, impaired IL-2 activity, low thymulin level, and reduced NK activity in cystic
fibrosis patients with both normal and growth retardation. Although the role of NK
cells is unknown in cystic fibrosis, Zn supplementation has been suggested as a
means to induce a complete saturation of thymulin molecules and correct cellular
immune defects [87].
Abdulhamid et al. [88] performed a double blind placebo controlled study inves-
tigating the effect of oral supplementation of elemental Zn (30 mg daily for 1 year)
on the rate of respiratory tract infections, use of antibiotics and plasma cytokines in
26 children with cystic fibrosis. Zn supplementation reduced the number of days of
oral antibiotics used to treat respiratory tract infections, and was marginally effec-
tive in reducing percentage increase in plasma IL-6 and IL-8 while increasing the
percentage change in ex vivo generation of IL-2 in isolated mononuclear cells; this
effect was greater in patients who exhibited low plasma Zn at baseline than those
who had plasma Zn levels identical to normal subjects. The authors concluded that
a higher daily Zn dose may be needed to decrease respiratory tract infections and
modify immune responses [88]. A later small, retrospective study of cystic fibrosis
patients with normal serum Zn evaluated a higher dosage of Zn (5 mg/kg Zn sulfate
daily to a maximum of 150 mg daily). They reported that Zn supplementation
resulted in a significant decrease in the number of infections [89].

3.2.2 Prevention of Childhood Diarrhea and Respiratory Tract Infections

Several meta analyses [90–92] have concluded that preventive Zn supplementation


(with doses ranging from 15 to 140 mg/week of elemental Zn) for ≥3 months to
Zn-deficient children 2–5 years old in developing countries reduces the frequency
and severity of diarrhea by 13 percent, and the incidence of clinically confirmed
pneumonia by about 20 percent.

3.2.2.1 Treatment of Childhood Diarrhea

A recent meta analysis of 24 randomized trials concluded that in children >6 months
old from populations in which Zn deficiency is common, evidence suggests that oral
Zn supplementation reduces the severity and duration of acute diarrhea [91,93,94].
Zn supplementation is probably helpful for treatment of acute diarrhea even in
populations without Zn deficiency, perhaps because Zn has specific local inhibitory
effects on some enteric pathogens and toxins.

3.2.3 The Common Cold

A large number of studies have evaluated the effect of Zn lozenges on the duration
or severity of common cold symptoms. A recent Cochrane analysis of 13 therapeutic
trials and two preventive trials concluded that 7 days of Zn treatment is associated
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 13

with a significant reduction in the duration and severity of common cold symptoms,
and a decreased rate of developing a cold. However, adverse events including bad
taste and nausea are higher in patients taking Zn [95]. Some investigators feel that
if utilized, Zn lozenges must have a minimal daily dose of elemental Zn of at least
75 mg, and be started within 24h of the onset of the common cold [96].

3.2.4 Prevention or Treatment of Malaria

Trials examining whether Zn supplementation reduces morbidity or mortality from


childhood malaria have had conflicting results. Several small trials from Asia and
Africa showed that supplementation reduced clinic visits or mortality due to malaria
[97], while several others reported non-significant reductions of morbidity or clinic
visits due to malaria [92,98,99].
Zn supplementation does not appear to have a beneficial effect when used as an
adjunct to treatment of the disease. Although Zn levels tend to decrease by ~70%
during the acute phase response in children with Falciparum malaria, a large
placebo-controlled trial of Zn supplementation in children, performed in a malaria-
endemic area of Africa, also failed to show a significant effect of Zn supplementa-
tion on overall mortality or malaria-related mortality [99–102].

3.2.5 Burn Patients

In patients with major burns, IV administration of 2.9 μmol Se, 40.4 μmol Cu, and
406 μmol Zn daily for 3 weeks results in improved wound healing, and a 65%
reduction in the rate of nosocomial pneumonia [103,104]. Current guidelines from
the European Society for Clinical Nutrition and Metabolism recommend enteral
supplementation of Se, Cu, and Zn at a “higher than standard doses” after burn
injury, based upon the above studies [105].

3.2.6 Wound Healing

Application of topical Zn oxide application to post-surgical wounds can signifi-


cantly decrease the occurrence of Staphylococcus aureus, and the need for postop-
erative antibiotics [106]. In burn patients, IV Zn supplementation (in combination
with Se and Cu) was associated with increased skin concentrations of Zn and Se,
improved wound healing, and a reduction in pulmonary infections [5,107–109].

3.2.7 Critically Ill Patients

In 1996, Berger et al. [110] reported that in 11 intensive care unit (ICU) patients,
Zn levels were decreased in the first week of ICU stay. In patients on home total
parenteral nutrition (TPN) with a diagnosis of catheter sepsis or pancreatitis,
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administration of 30 mg IV Zn daily for 3 days resulted in a significantly higher


febrile response, as evidenced by increased temperature in the Zn versus a control
(placebo) group [111].
Heyland and colleagues [112] conducted a systematic review of Zn supplementation
in critically ill patients, including 4 randomized controlled trials, where IV adminis-
tration of Zn showed nonsignificant trends toward reductions in mortality and ICU
length of stay. They concluded that evidence to support the use of IV Zn is lacking, as
the 6 available studies were too small to detect even a moderate effect, and that current
recommendations for high dose Zn supplementation in critically ill patients should
be revised. However, considering the importance of Zn in free radical scavenging,
anabolism, and immunity, large rigorously designed randomized trials are warranted,
to evaluate the effects of Zn supplementation in severe septic patients [113].

3.2.8 Sickle Cell Disease

Gupta and Chaubey conducted a double-blind, placebo-controlled, randomized


controlled trial of 130 sickle cell patients in India. Zn (200 mg orally 3 times daily)
or placebo was administered for 1.5 years. There was a significant reduction of the
mean number of infective episodes in the Zn-supplemented group [114].
Prasad et al. [115] evaluated Zn supplementation in 32 patients with sickle cell
disease who were divided into 3 groups, based upon their level of granulocytic or
lymphocytic Zn levels (mild, moderate deficiency, or no Zn deficiency). Subjects
were administered Zn acetate (50 to 75 mg of elemental Zn orally daily) for 2 or 3
years, or placebo, in the mild, moderate deficiency, or no Zn deficiency groups,
respectively. Prolonged Zn supplementation resulted in increased, lymphocyte and
granulocyte Zn, and IL-2 production, and a decreased number of documented bacte-
riologically positive infections. In a later study by the same investigator, Bao, Prasad
and colleagues [116] conducted a double-blind, randomized, placebo-controlled trial
to further evaluate the role of Zn supplementation (25 mg orally 3 times daily for 3
months) on the incidence of infections, oxidative stress, and biomarkers for chronic
inflammation in patients with sickle cell disease. The Zn-supplemented group
experienced a decreased incidence of infections, and significant decreases in
lipopolysaccharide-induced TNF-α and IL-1 β mRNAs, and TNF-induced NFκB-
DNA binding in mononuclear cells compared with the placebo group.

4 Selenium

4.1 Human Pharmacology and Pharmacokinetics

Serum Se levels vary widely in different parts of the world, as does the ingestion of
dietary Se. As Se has a narrow therapeutic range, the optimal range of dietary intake
of Se is narrow; potentially toxic intakes are closer to recommended dietary intakes
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 15

than for other dietary trace minerals (see also Chapter 16). While Se status can be
assessed by determining the Se concentration of whole blood, plasma, serum, or
erythrocytes, plasma or serum levels are the most commonly used and are reason-
ably accurate biomarkers of Se status, responding to short-term changes in intake
[117–119]. While Se supplementation may be beneficial in individuals with low
levels of Se, it is potentially toxic if administered to those who already have normal
or high levels. People whose serum or plasma Se concentration is ≥122 μg/L should
not supplement with Se [120].
Currently, Se is the most intensively studied TE with respect to the treatment or
prevention of a variety of infections in humans. Se deficiency (serum or plasma Se
≤85 μg/L) has been linked to the incidence, virulence, or disease progression of
viral infections and has correlated with several infectious diseases, including HIV,
sepsis or pneumonia in ICU or burn patients, and prostatitis.
In healthy subjects, Se can be found in plasma associated with selenoprotein-
P (52%), glutathione peroxidase (39%), albumin (9%), and free Se (<1%)
[121,122]. Among the >30 selenoproteins which have been identified, 4 forms of
glutathione peroxidase have been shown to be important in antioxidant defense [120],
while selenoprotein-P appears to play a role in protection versus infection [123].
Cu, Mn, Zn, Fe, and Se are required for the activity of SOD, catalase, and glutathi-
one peroxidase, respectively [3].
Se is found in relatively high amounts in the liver, spleen, and lymph nodes,
which are involved in hematopoietic and immune function potential. Se is incorpo-
rated into at least 25 selenoproteins and thus is a constituent of multiple antioxi-
dant defense systems [124]. In mice, selenoprotein-P appears to provide protection
against the parasitic infection trypanosomiasis [123]. Impaired cell-mediated
immunity has been demonstrated when tissue stores of Se are depleted. Natural
killer cell activity is enhanced when Se is supplemented in the diet of Se-depleted
individuals [125].

4.2 Role of Selenium in Infectious Diseases

4.2.1 Human Immunodeficiency Virus

HIV replication is inhibited by Se [126,127], and a number of studies have shown a


linear relationship between Se deficiency (usually defined as a serum level ≤85
μg/L) and a reduction in CD4 cell counts in HIV-infected patients. In HIV-infected
individuals, several studies [126,128–131] have associated low (not necessarily
deficient) serum Se levels with lower CD4 counts, increased viral load, rapid pro-
gression of HIV, and higher mortality. When serum Se levels are controlled for
serum albumin, or for an acute phase response (defined as an α1-acid glycoprotein
level ≥100 mg/dL or a C-reactive protein ≥1 mg/dL), these associations disappear
[132], which may be attributable to the lowering of blood Se concentration by the
acute-phase response in individuals with more advanced HIV-1 infection.
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4.2.1.1 Selenium Supplementation in HIV

Two randomized controlled trials have shown apparent benefit from Se supple-
mentation in HIV-infected patients [133,134]. Burbano et al. [133] conducted a
randomized, placebo-controlled study evaluating the administration of Se to
HIV-infected individuals in the US who were not Se-deficient (with Se deficiency
defined as a serum Se >85 μg/L). Se supplementation (200 μg orally daily) resulted
in a decreased rate of hospital admissions (P = 0.02) and in the percentage of
hospital admissions due to infection (P = 0.01). Similarly, Hurwitz et al. [134],
conducted a double-blind, randomized, placebo-controlled trial in the US in 174
HIV-infected subjects with Se levels >75 μg/L. Administration of Se 200 μg orally daily
for 18 months significantly increased serum Se concentrations (∆ = 32.2 ± 24.5 versus
0.5 ± 8.8 μg/L; P < 0.001) in adherent subjects. Higher serum Se levels predicted
decreased HIV viral load (P < 0.02) and increased CD4 count (P < 0.04) even after
covariance for demographic factors, antiretroviral therapy regimen and adherence,
HIV-disease stage and duration, and hepatitis-C virus co-infection. Moreover,
Se-treated subjects in whom serum Se levels changed ≤26.1 μg/L displayed
elevations in viral load and, as a result, decreases in CD4 count. However, others
have criticized the method by which the data were analyzed and the relevance of the
differences recorded in CD4+ cell count and viral load [135].
By contrast, Kupka et al. [135] reported that Se supplementation (200 μg of
Se daily, as selenomethionine during the antenatal and post-partum periods) had
no effect on HIV-1 viral load or CD4+ cell count in 913 HIV-infected Tanzanian
pregnant women in whom use of antiretroviral therapy was uncommon, although it
reduced the risk of mortality in children older than 6 weeks. However, the Se status
of these subjects was unknown.
In a case-control study of 259 HIV-infected drug users, 47 (18.1%) patients
whose plasma Se level was < 135 μg/L had a three-fold higher risk of developing
mycobacterial disease (primarily tuberculosis), than did those with higher plasma
Se levels.

4.2.2 Intensive Care Unit Sepsis

As noted above, studies have consistently demonstrated decreased plasma Se con-


centrations in critically ill patients, especially those with septic shock. In ICU
patients with severe septic shock, there is a 40% decrease in plasma Se concentra-
tions [136,137] and in selenoprotein-P, following admission to the ICU. ICU mor-
tality is strongly associated with the minimum Se concentration [137].
Se has emerged as the most important antioxidant micronutrient in the critically
ill, particularly in burn and trauma patients [5]. Intravenous selenite has a biphasic
action: firstly as a prooxidant and, after incorporation into selenoenzymes, as an
antioxidant [138]. Since glutathione peroxidase, the body’s most important antioxidant
enzyme, is directly dependent on Se, strategies focused on replacement of Se have been
evaluated as a means to reduce infections, the length of hospital stay, and mortality.
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 17

The role of supplemental Se (given as sodium selenite) has been evaluated in a


number of studies, with conflicting outcomes. The 2004 and the updated 2008
Cochrane data base reviews [4] of 7 trials ([139–145]) of supplemental IV sodium
selenite concluded that there was limited evidence to recommend supplementation of
critically ill patients with Se, and that additional trials of adequate size and appropriate
methodology were required in order to overcome the defects of previous studies.
Since 2007, several larger clinical trials of Se supplementation [146,147] have
been published, with conflicting outcomes. The studies have varied in patient popu-
lations, method of Se administration (as a bolus followed by continuous infusion
versus continuous infusion), and duration of therapy [4,139,144,146,147]. Few trials
reported on outcomes other than mortality and there were insufficient data to exam-
ine the effect of methodological superiority or dose of Se on the outcomes. However,
two recent meta analyses [3,148] evaluated the results of 21 randomized clinical
trials of antioxidant micronutrients, including Se, in critically ill patients. They con-
cluded that bolus administration of Se followed by transient prooxidant effect of an
IV bolus followed by the antioxidant effect of continuous infusion seems effica-
cious and well tolerated and was associated with a significant reduction in mortality
and in the duration of mechanical ventilation, with a trend towards a reduction in
infections. Reductions in the risk of mortality were highest in those patients with a
higher risk of death. Trials using loading doses, high doses (>500 μg daily) and a
longer duration of Se therapy appear to be associated with lower mortality.

4.2.3 Role of Selenium in Other Infections

In a small study in adult subjects in the United Kingdom, Broome et al. [149] dem-
onstrated a functional outcome of Se supplementation (50 or 100 μg Se orally per
day for 15 weeks as sodium selenite) on the immune system of subjects with fairly
low (<1.2 μmol/L) Se status. When patients were challenged with an oral, live,
attenuated poliovirus, Se-supplemented patients demonstrated an augmented cellu-
lar immune response as evidenced by an increased production of IFN and other
cytokines, an earlier peak T cell proliferation, and an increase in T helper cells.
In addition, they cleared the virus more rapidly than did those given placebo [149].
Most studies in surgical patients report only postoperative or post-ICU admis-
sion Se levels. However, Stoppe et al. [124] recently reported pre- and post-operative
Se, Zn, and Cu levels in patients undergoing elective cardiac surgery. Fifty of 60
patients (83.3%) exhibited Se deficiency prior to surgery, and all patients demon-
strated significant decreases in Se levels postoperatively, as compared to baseline
(pre-operative) levels. The intra-operative decrease in TE concentrations was most
pronounced in patients who developed multi-organ failure post-operatively [124].
As discussed above (under Zinc, Section 3.2.5) administration of Cu, Zn, and Se
supplements for 3 weeks has been found to decrease the incidence of pneumonia
following severe burns [103–105,108]. In cystic fibrosis patients, there is evidence
both for and against antioxidant supplementation with vitamins E and C, β-carotene,
and Se. However, a recent meta analysis concluded that antioxidant supplementation
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in cystic fibrosis is not yet recommended beyond routine care, because while levels
of antioxidants in the blood improved, there was no improvement in lung function,
and quality of life decreased in groups taking supplements [150].

5 Copper

5.1 Human Pharmacology and Pharmacokinetics

Among the biologically important Cu-containing enzymes which have been described,
Cu/Zn SOD (a component of antioxidant defense) and ceruloplasmin play important
roles in the development of infections (see also Chapter 11, in general).
Cu is absorbed in the proximal small intestine and stomach, with absorption
occurring by a saturable active transport process at lower levels of dietary Cu and by
passive diffusion at high levels of dietary Cu [151,152]. The Menkes P-type ATPase
(ATP7A) is responsible for Cu trafficking to the secretory pathway for efflux from
enterocytes and other cells. Absorbed Cu is loosely bound to plasma albumin and
amino acids in the portal blood and taken to the liver, where most of it is taken up
on the first pass and incorporated into ceruloplasmin, a Cu-containing protein which
transports Cu from the liver to peripheral tissues. Ceruloplasmin binds to its
receptors on the cell surface; Cu is then released from its binding protein and enters
the cell [151,152]. Cu homeostasis is largely regulated by excretion of Cu into the
gastrointestinal tract via the bile; ~50% of Cu is excreted in the bile while the
remaining half is excreted through other gastrointestinal secretions. Metallothionein,
synthesized in the liver, may act as a Cu storage protein.
Acquired Cu deficiency is rare, but has been well documented in humans.
Hematologic features of Cu deficiency include anemia (usually microcytic) and
neutropenia, which can be mistaken for Fe deficiency anemia. In this setting, admin-
istration of Fe supplements can worsen Cu deficiency because excess Fe competes
with Cu and decreases net Cu absorption. Similarly, because Cu and Zn are com-
petitively absorbed from the jejunum via metallothionein, high doses of Zn (>150
mg/day) can result in Cu deficiency in normal individuals. Excessive Zn ingestion
can occur due to prolonged use of oral Zn supplements for the treatment of common
colds, administration of parenteral Zn in patients on chronic hemodialysis, or occa-
sionally when trace elements in TPN are withheld in patients with cholestasis.
Patients with major burns are unique for having Cu deficiency, as compared to
trauma patients with the systemic inflammatory response syndrome, in whom serum
levels of Cu are increased, and Fe, Se, and Zn decreased [5].
Ceruloplasmin (like ferritin) is an acute phase reactant, and serum Cu and
ceruloplasmin levels are increased in adult patients with inflammatory processes,
pregnancy, coronary artery disease, cirrhosis, diabetes, malignancies, and renal
failure [153]. Conflicting data have been reported in children; although Teslariu
and Nechifor reported decreased serum levels of Cu and Zn in otherwise healthy
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 19

children with acute urinary tract infections [154], Wang et al. reported no correlation
between Cu levels and severity of illness scores in children admitted to an ICU
[155]. Ceruloplasmin has an independent role in Fe metabolism, in which it serves
as a plasma ferroxidase, converting Fe to a valence that can be bound by plasma
transferrin. Metallothionein, synthesized in the liver, may act as a Cu storage protein.

5.2 Role of Copper in Infectious Diseases

To date, studies examining the relationship between Cu levels and the development
of infections have found no correlation with the development of infectious diseases;
however, in several patient populations, correlations have been found between an
increased Cu/Zn plasma ratio and decreases in immune-related markers or responses
to infection. In addition, as noted above, administration of Cu, Zn, and Se supple-
ments for 3 weeks have been found to decrease pneumonia following severe burns
[103–105,108].

5.2.1 Copper/Zinc Ratio

In patients undergoing peritoneal dialysis, Guo et al. noted the Cu/Zn ratio was
strongly correlated with nutritional abnormalities, oxidative stress, inflammation,
and immune dysfunction, including a negative correlation of the Cu/Zn ratio with the
percentages of B- and T-lymphocyte subsets and the ratio of CD4/CD8 antigens
[156]. As noted earlier, TB patients with HIV coinfection demonstrated significantly
higher Cu/Zn ratios compared to those in TB patients without HIV coinfection
(P < 0.05) [13]. Similarly, children with chronic hepatitis B infection had significantly
lower plasma levels of Mn, Se, Zn (but not Cu), and significantly higher Cu/Zn ratios
prior to interferon therapy (P < 0.001) as compared to a control group [6].

6 Chromium

6.1 Human Pharmacology and Pharmacokinetics

Chromium is absorbed predominantly in the small intestine and is transported in the


circulation bound to albumin and transferrin [157]. The total body Cr concentration
is the main homeostatic control of its gut absorption. Dietary bioavailability of Cr is
very low and almost all of the ingested Cr is excreted via feces [158,159]. Cr absorp-
tion is enhanced in the setting of Zn and Fe deficiency, suggesting that these minerals
compete for intestinal absorption [157]. Patients receiving parenteral nutrition with
usually prescribed doses of Cr can have abnormally elevated serum and urine
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concentrations in part attributable to contamination of amino acid products, especially


in patients with renal dysfunction [160]. Although there appears to be a significant
dose-response relationship between Cr doses and serum Cr concentrations, serum
Cr equilibrates slowly with tissue stores [12]. Cr is excreted mainly through the
urine; however, some Cr is excreted in the feces through bile and small intestinal
losses. Urinary losses increase with metabolic stress, trauma, and ascorbic acid
deficits. Cr deficits induce glucose intolerance, and glucose intolerance can further
drive these urinary losses of Cr (see also Chapter 6, in general).

6.2 Role of Chromium in Infectious Diseases

Chromium is a cofactor for insulin function that enhances insulin effects to improve
glucose metabolism through the glucose tolerance factor [160]. While diabetics,
particularly those with altered glucose levels, are known to have an increased prevalence
of infectious diseases, thus far no studies have evaluated the role of Cr as a risk
factor for infectious diseases.

7 Manganese

7.1 Human Pharmacology and Pharmacokinetics

Manganese is a component of metalloenzymes such as manganese superoxide


dismutase, arginase, glutamate synthetase, and pyruvate carboxylase and is associ-
ated with oxidative phosphorylation and mucopolysaccharide metabolism (see also
Chapter 7). An average adult has 10–12 mg Mn incorporated into the active center
of various metalloenzymes [161]. Particular interest has been paid to Mn-SOD,
which is located primarily in mitochondria, which are important for detoxifying the
superoxide radical to hydrogen peroxide [12]. Mn is excreted mainly from the bile,
and thus can accumulate in patients with cholestasis.
A number of proteins involved in Mn transport have been identified including the
putative uptake proteins divalent metal transporter-1 (DMT1), transferrin receptor
(TfR) and ATP13A2 (also known as PARK9), as well as the efflux protein Fpn.
Previously, the only protein known to be operant in cellular Mn export was the
Fe-regulating transporter, Fpn [162].
Mn absorption, transport, and excretion are tightly regulated because Mn is both
essential at low dose and toxic at higher doses. While Mn is transported by simple
diffusion in the large intestine, Mn is absorbed by active transport in the small intes-
tine [163]. Absorption, efflux, and distribution of Mn appear to be inversely related
to stored Fe, with Fe deficiency facilitating Mn absorption. Only about 5% of dietary
Mn appears to be absorbed; however, absorption is greater in neonates and children
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 21

than in adults, and in females than in males. Fe deficiency increases the absorption,
efflux, and distribution of orally administered Mn into the body, and in delivery to
the brain possibly via Nramp [161,162,164].
Once absorbed, Mn is transported to the liver where ~80% of plasma Mn is
bound to β1-globulin, a small fraction is bound to transferrin, an Fe-binding protein.
Mn in the liver is conjugated with bile and >90% of Mn is excreted by secretion into
the intestine via the hepatobiliary system, where a small fraction is reabsorbed
and the remainder is excreted in the feces. Decreased elimination of Mn in patients
with poor biliary excretion (e.g., neonates and adults with cholestasis) may result
in increased delivery of Mn to the brain and other tissues, increasing the potential
for toxicity [161].
In vivo experiments in mice and rats have defined the range (1–3.5%) of GI
absorption of Mn [161]. While Mn is transported by simple diffusion in the large
intestine, Mn is absorbed by active transport in the small intestine. Mn excretion
into bile is likely active as well because it depends on concentration gradients.
A plethora of plasma proteins or ligands have been implicated as specific Mn carrier
proteins, including transglutaminase, β1-globulin, albumin, and transferrin. In fact,
approximately 80% of plasma Mn is bound to β1-globulin. a small fraction of
plasma Mn is bound to transferrin, while approximately 80% of plasma Mn is
associated with albumin and β1-globulin. Despite the demonstration that Mn
preferentially binds to albumin in the plasma of both rabbits and humans, emerging
evidence has provided evidence for weaker binding of Mn to albumin compared to
Cd and Zn [163].
Because 60–80% of Mn is contained in red blood cells, erythrocyte or whole-
blood Mn concentrations appear to be the most accurate and reproducible parameter
[163]. Several investigators [12,165] have demonstrated a correlation between Mn
supplementation and serum concentrations and in long term (up to 20 years) patients
receiving parenteral nutrition, while Siepler et al. did not [166].

7.2 Role of Manganese in Infectious Diseases

Many organisms can compensate for the loss of antioxidant enzymes by the formation
of catalytic Mn-antioxidants during periods of Mn abundance. It has been proposed
that cells utilize these Mn-antioxidant complexes as a “backup” for Cu/Zn SOD1:
when Mn is abundant, surplus intracellular Mn2+ forms antioxidant complexes and
when Mn is limited, cells rely on the high efficiency of SODs [167].

7.2.1 Arginase

The Mn-containing enzyme arginase down-regulates nitric oxide (NO) production


by competing with nitric oxide synthase (NOS) for arginine. NO is important in host
immune defense, since it is utilized by the immune system to generate peroxynitrite
22 Carver

which kills bacteria; however, septic shock is associated with an overproduction of


NO [168]. In humans, plasma arginase is elevated (and levels of arginine usually
reduced) in a variety of conditions, including sickle cell disease, oxidative stress,
malaria, and cystic fibrosis.
Clinically, the ratio of plasma arginine/(plasma ornithine + citrulline), which has
been termed the ‘global arginine bioavailability ratio’ (GABR) has proven more
useful as a biomarker in some disorders than have plasma concentrations of arginine
alone. For example, a low GABR represent an independent risk factor for morbidity
and mortality in sickle cell patients [169]. In animal studies, Mn-SOD activity in the
heart, and arginase activity in the liver, were lower in piglets fed a low Mn diet, and
the relative arginase activity increased with enhanced dietary Mn and correlated
with Mn concentrations in the liver [170].

7.2.2 Manganese Superoxide Dismutase

Following burns, trauma, and surgery, despite no changes in Mn serum concen-


trations, Mn (but not Cu or Zn) concentrations are increased within burn scars,
emphasizing the importance of Mn-SOD, a mitochondrial antioxidant defense, in
wound healing [5].
Despite the significant and evolving role of Mn in pathogens, to date, no published
studies have correlated Mn plasma or serum levels with the prevention or treatment
of infectious diseases, or addressed the role of supplementation or chelation of
Mn in humans.

8 Summary and Future Developments

The role of trace elements in infectious diseases is complex. Significant correlations


have been demonstrated for Fe, Se, and Zn and infections; fewer data exist for
Cu, Cr, or Mn. However, as the synergistic role of these TEs is further elucidated,
investigators, clinicians, and most importantly, patients, will benefit from a more
complete understanding of this complex biological system and the prevention or
treatment of infectious diseases.

Abbreviations

Cp calprotectin
Cu/Zn SOD copper-zinc superoxide dismutase
DMT1 divalent metal transporter
Fpn ferroportin
GABR global arginine bioavailability ratio
1 Metal Ions and Infectious Diseases. An Overview from the Clinic 23

GI gastrointestinal
GPx glutathione peroxidase
HIV human immunodeficiency virus
HSCT hematopoietic stem cell transplantation
ICU intensive care unit
IFNα interferon α
IL interleukin
IV intravenous
Mn manganese
Mn-SOD manganese superoxide dismutase
NFκB NF kappa beta
NGAL neutrophil gelatinase-associated lipocalin; also known as siderocalin
NK cell natural killer cell
NO nitric oxide
NOS nitric oxide synthase
Nramp natural resistance-associated macrophage protein
RNS reactive nitrogen species
ROS reactive oxygen species
SIRS systemic inflammatory response syndrome
SOD superoxide dismutase
TB tuberculosis
TEs trace elements
TfR transferrin receptor
TNF tumor necrosis factor
TPN total parenteral nutrition
Zn-MT Zn-metallothionein

Acknowledgment I would like to thank Vincent Pecoraro for his invaluable comments, suggestions,
and editing of this manuscript.

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Chapter 2
Sodium and Potassium in Health and Disease

Hana R. Pohl, John S. Wheeler, and H. Edward Murray

Contents
ABSTRACT ............................................................................................................................. 30
1 INTRODUCTION ............................................................................................................. 30
2 PHYSIOLOGY OF SODIUM AND POTASSIUM IN HUMANS................................... 32
2.1 Action of Sodium and Potassium on Membranes..................................................... 32
2.1.1 Nervous System ............................................................................................ 32
2.1.2 Muscular System........................................................................................... 33
2.2 Homeostasis of Sodium and Potassium .................................................................... 33
2.2.1 Absorption and Distribution of Potassium .................................................... 34
2.2.2 Absorption and Distribution of Sodium ........................................................ 34
2.2.3 Potassium Excretion and Secretion in the Kidneys ...................................... 34
2.2.4 Sodium Excretion and Secretion in the Kidneys .......................................... 35
2.3 Mechanism of Other Physiological Systems Influencing Sodium
and Potassium Homeostasis ...................................................................................... 36
2.3.1 Potassium ...................................................................................................... 36
2.3.2 Sodium .......................................................................................................... 37
3 PATHOLOGY ASSOCIATED WITH SODIUM LEVELS .............................................. 38
3.1 Hyponatremia............................................................................................................ 38
3.2 Hypernatremia .......................................................................................................... 40
4 PATHOLOGY ASSOCIATED WITH POTASSIUM LEVELS ........................................ 41
4.1 Hypokalemia ............................................................................................................. 41
4.2 Hyperkalemia ............................................................................................................ 43
5 CONCLUSION.................................................................................................................. 45
ABBREVIATIONS .................................................................................................................. 45
REFERENCES ........................................................................................................................ 46

H.R. Pohl (*) • J.S. Wheeler • H.E. Murray


Agency for Toxic Substances and Disease Registry (ATSDR), US Department
of Health and Human Services, 1600 Clifton Road, Mailstop F-57,
Atlanta, GA 30333, USA
e-mail: hpohl@cdc.gov

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 29


Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_2, © Springer Science+Business Media Dordrecht 2013
30 Pohl, Wheeler, and Murray

Abstract Sodium and potassium are essential for human health. They are important
ions in the body and are associated with many physiologic and pathophysiologic
processes. The chapter summarizes the basic physiologic actions of sodium and
potassium on membranes of the neurologic and muscular systems. It provides
information regarding the kinetics, i.e., absorption, distribution, and excretion of these
ions and their movement between the intracellular and extracellular compartments.
It also explains the physiologic systems that can influence proper homeostasis
between sodium and potassium. Concentrations of sodium in the blood that exceed
or do not reach the normal value range are called hypernatremia or hyponatremia,
respectively. Similarly, the clinicians recognize hyperkalemia and hypokalemia.
Pathologies associated with these states are described and examples of some of the
diseases are presented here.

Keywords homeostasis • hyperkalemia • hypernatremia • hypokalemia • hyponatremia


• potassium • sodium

Please cite as: Met. Ions Life Sci. 13 (2013) 29–47

1 Introduction

This chapter provides an overview of sodium and potassium and their importance in
human physiology and pathology. Sodium and potassium are essential in maintain-
ing cellular homeostasis. Most metabolic processes are dependent on or affected by
these electrolytes. Among the functions of these electrolytes are maintenance of
osmotic pressure and water distribution in various body fluid compartments,
maintenance of proper pH, regulation of the proper function of the heart and other
muscles, involvement in oxidation-reduction (electron transport) reactions, and
participation in catalysis as cofactors for enzymes.
Dietary requirements for sodium and potassium vary widely, but generally, daily
intake should be only in small amounts [1]. Normal plasma levels for sodium in
adults range from 136 to 146 mEq/L, and this balance is normally maintained by an
average dietary intake of 90 to 250 mEq per day. Sodium excretion tends to reflect
sodium intake, and on an average diet, urine sodium excretion will range between
80 and 180 mEq per day.
Potassium is essential for the proper function of all cells, tissues, and organs in
the human body. It is also crucial to heart function and plays a key role in skeletal
and smooth muscle contraction, making it important for normal digestive and mus-
cular function. Normal plasma levels for potassium in adults range from 3.5 to 5.0
mEq/L, and this balance is usually maintained in adults on an average dietary intake
of 80 to 200 mEq per day. It is noted that the normal intake, minimal need, and
maximum tolerance for potassium is almost the same as that for sodium.
2 Sodium and Potassium in Health and Disease 31

Sodium ions are the major cations of extracellular fluid, whereas, potassium ions
are the major cations of the intracellular fluid [2]. To maintain internal fluid and
electrolyte balance, water, sodium, and potassium are in constant movement
between the intracellular and extracellular body compartments. Potassium and
sodium ions are particularly important in the renal regulation of acid-base balance
because hydrogen ions are substituted for sodium and potassium ions in the renal
tubule. Potassium plays a key role in that potassium bicarbonate is the primary
intracellular inorganic buffer. Potassium enters the cell more readily than sodium
and initiates the brief sodium-potassium exchange across the cell membranes.
In the nerve cells, this sodium-potassium flux generates the electrical potential
that aids the conduction of nerve impulses. When potassium leaves the cell, it changes
the membrane potential and allows the nerve impulse to progress. This electrical
potential gradient, created by the “sodium-potassium pump”, helps generate muscle
contractions and regulates the heartbeat. Discovery of the sodium-potassium pump
in the 1950s by a Danish scientist, Jens Christian Skou, marked an important step
forward in our understanding of how ions enter and leave cells. This physiologic
function is of particular significance for excitable cells such as nerve cells, which
depend on this pump for responding to stimuli and transmitting impulses [3].
Cellular uptake of potassium is regulated by the sodium-potassium pump, while
movement of potassium out of the cell is governed by passive forces (cell membrane
permeability and chemical and electrical gradients to the potassium ions). Another
of the pump’s most important functions is preventing the swelling of cells. If sodium
is not pumped out, water accumulates within the cell causing it to swell and
ultimately burst.
Abnormal levels of these electrolytes may result in a variety of pathological
disorders [2]. For example, too high a concentration of sodium, a condition called
hypernatremia, leads to edema (swelling of tissues due to excess fluid retention)
thirst, and lessened urine production. Hyponatremia is a low level of serum sodium
and is usually characterized by headache, confusion, seizures, muscle spasms, nausea,
and vomiting. Too much potassium, called hyperkalemia, characterized by irritabil-
ity, nausea, decreased urine production, and cardiac arrest. Fatigue is the most
common symptom of chronic potassium deficiency. Early symptoms include muscle
weakness, slow reflexes, and dry skin or acne; these initial problems may progress
to nervous disorders, insomnia, slow or irregular heartbeat, and loss of gastrointestinal
tone. A sudden loss of potassium may lead to cardiac arrhythmia. Low potassium
may impair glucose metabolism and lead to elevated blood sugar. In more severe
potassium deficiency, there can be serious muscle weakness, bone fragility, central
nervous system changes, decreased heart rate, and even death.
Potassium is very important in cellular biochemical reactions and energy
metabolism; it participates in the synthesis of proteins from amino acids in the cell.
Potassium also functions in carbohydrate metabolism; it is active in glycogen and
glucose metabolism, converting glucose to glycogen that can be stored in the liver for
future energy. Potassium is important for normal growth and for building muscle.
32 Pohl, Wheeler, and Murray

Though sodium is readily conserved by the body, there is no effective method for
potassium conservation. Even when a potassium shortage exists, the kidneys
continue to excrete it. Since the human body relies on potassium balance for a
regularly contracting heart and a healthy nervous system, it is essential to strive for
this electrolyte’s balance.
The renin-angiotensin-aldosterone system and vasopressin levels play an important
role in regulating the electrolyte levels in the body. Pathological states of the system
can be accompanied by imbalances of potassium and sodium levels.
A complex interplay of physiological control systems maintains fluid, sodium,
and potassium homeostasis. When this interplay of physiological systems is disrupted,
or when homeostatic mechanisms can no longer maintain intracellular, extracellular
or interstitial fluid, an imbalance of sodium and potassium will occur. The following
discussion will address some of the complexities of the physiology and pathology
involved with sodium and potassium interactions.

2 Physiology of Sodium and Potassium in Humans

2.1 Action of Sodium and Potassium on Membranes

2.1.1 Nervous System

One of the major roles of potassium/sodium balance in the body is that of the nerve
impulse. A differential in sodium and potassium concentration forms a polarity
across the nerve membrane that when stimulated (electrical, chemical, mechanical,
or thermal) leads to depolarization and propagation of the nerve impulse along the
cell membrane [4]. In the nerve cell, active sodium-potassium pumps create this
differential by pumping two K+ atoms into the cell for every three Na+ atoms pumped
out of the cell. Active pumping, along with negatively charged ions of other molecules
inside the cell, leads to a voltage potential across the cell membrane. The resulting
voltage is approximately –70mV [5]. Following membrane stimulation the membrane
becomes permeable to Na+ ions, allowing Na+ inside the cell, thus eliminating the
electrical potential across the membrane (depolarization). Depolarization propagates
in all directions from the initial point. For a very brief time, the membrane is unable
to depolarize again and remains unresponsive.
It is the nature of this delicate balance of sodium and potassium across the
neuronal membrane that leads to diseases and physiological imbalances which result
in a number of different neurological problems. Chemicals such as the organochlorine
DDT gain their physiological disrupting power by interfering with the sodium
channel across the axonal membrane, thus leading to variety of toxic effects,
including lethality [6].
2 Sodium and Potassium in Health and Disease 33

2.1.2 Muscular System

Similar in function to the membrane of neurons is the membrane function of the


muscle fiber [2]. The muscle fiber, when stimulated by acetylcholine, depolarizes,
propagating the depolarization into the deeper muscle through the transverse tubules,
leading to the release of calcium ion followed by a contraction of the myofibrils of
the muscle and thus movement of the muscle. Na+ and K+ play a key role in the
depolarization of the muscle cell membrane. Polarization, as with the neuron,
requires an active ion pump and energy in the form of ATP to create an ion gradient
across the cell’s membrane. As with neurons, the muscle cell membrane becomes
impervious to Na+ while Na+ ions are actively pumped out of the cell and K+ ions
into the cell; however, some K+ diffuse back out at a slower rate than Na+ is pumped
out. This ion gradient, along with anions of many organic compounds and proteins
inside the cell, create a voltage across the cell membrane. When the membrane is
stimulated (electrically or mechanically, but usually chemically with acetylcholine),
the membrane becomes permeable to sodium and voltage suddenly drops, thereby
depolarizing the membrane. The depolarization propagates in all directions, moving
into the muscle through transverse tubes, leading to Ca2+ release and the subsequent
contraction of muscle.
Diseases and xenobiotics can interfere with many steps along this complicated
process of muscle cell depolarization and contraction. Interference can occur at
the cell membrane, with Na+/K+ balance, with Ca2+ influx, and with many other
pathways. Many toxins and therapeutic agents work by inhibiting cell depolarization
and repolarization.

2.2 Homeostasis of Sodium and Potassium

Homeostasis of Na+ and K+ is critical to life, especially extracellular K+ levels.


A number of homeostatic mechanisms keep Na+ and K+ regulated. Normal extra-
cellular and intracellular Na+ and K+ are [7]:
intracellular K+ 140 mEq/L
extracellular K+ 5 mEq/L
intracellular Na+ 12 mEq/L
extracellular Na+ 140 mEq/L
Intracellular K+ levels can be affected by insulin, aldosterone, β-adrenergic
stimulation, acid base abnormalities, cell lysis, and strenuous exercise [8]. While short-
term regulation involves cellular redistribution, long-term regulation involves renal
excretion and reabsorption.
34 Pohl, Wheeler, and Murray

2.2.1 Absorption and Distribution of Potassium

The recommended intake of potassium for adolescents and adults is 4700 mg/day
[9]. Following ingestion, K+ is rapidly absorbed by active uptake in the mucosal
lining of the intestine. This rapid uptake could lead to severe K+ imbalance if it was
not for the rapid absorption of K+ into cells (see Section 4.2). Ninetyeight percent of
gastrointestinally absorbed K+ is stored in cells, with 2% being found extracellularly
[8]. Even though cellular storage allows for the rapid regulation of extracellular K+,
long-term regulation is carried out in the kidney.

2.2.2 Absorption and Distribution of Sodium

The average daily intake of sodium for males over 20 in the United States is 4,243
mg/day. For women it is 2,980 mg/day [10]. The Food and Drug Administration
recommends that daily intake not exceed 2,300 mg/day for healthy individuals
and no more than 1,500 mg/day for sensitive individuals (hypertensive, blacks,
middle-aged, and older) [11].
Sodium is rapidly and actively taken up by the mucosal lining of the gastrointestinal
(GI) tract [10]. Unlike K+, however, it is not rapidly sequestered into the cells.
Only around 10% of Na+ body burdens are found in the cells, 40% remains in
extracellular fluid [4]. Na+ is excreted through urine, feces, perspiration, and tears.
It is also secreted back into the intestines at the rate of 25 grams per day. To remain
in homeostasis, the intestines must absorb 25–35 of sodium every day [8]. This
amount plus the amount of Na+ lost from other routes (urine and perspiration) needs
to be reabsorbed every day for Na+ homeostasis to occur. It is easy to see why
diseases such as diarrhea and intestinal influenza can easily upset the Na+ mainte-
nance in the body and quickly lead to life threatening situations.

2.2.3 Potassium Excretion and Secretion in the Kidneys

A small percentage of excess K+ is excreted in the feces, while the bulk of K+


excretion occurs in the urine following filtration, reabsorption, and secretion in the
kidneys (see Figure 1). The kidney filters around 800 mg of K+ per day of which
approximately 65% and 27% is reabsorbed in the proximal tubule and loop of
Henle, respectively [8]. These percentages remain fairly constant from day to day
and do not significantly regulate daily variations from changes in diet and absorp-
tion. The work of regulating daily variations occurs mainly in the secretion of K+ in
the distal tubules and cortical collecting tubules [11].
2 Sodium and Potassium in Health and Disease 35

Figure 1 Nephron.
Image used with permission
of the Regents of the
University of Michigan.
http://www.med.umich.edu/
lrc/secondlook/.

Under normal potassium intake the amount of absorption exceeds what the body
needs, and secretion into the distal tubules and cortical collecting tubules eliminates
the excess through excretion in the urine. Under extreme K+ deficiencies reabsorption
in the distal tubules can actually exceed secretion and thus conserve K+.

2.2.4 Sodium Excretion and Secretion in the Kidneys

Some sodium is lost in feces and sweat, but as was seen with potassium, the majority of
sodium regulation in the body occurs in the kidney (see Figure 1). In the kidney, sodium
ions (approximately 70%) are reabsorbed into the proximal tubules and loop of Henle
after filtration through the glomerulus [4]. However, unlike K+, the driving force of
36 Pohl, Wheeler, and Murray

Na+ homeostasis is the glomerular filtration rate and tubule reabsorption. By the
time the filtrate reaches the distal tubules almost all the Na+ has been reabsorbed.
As the filtrate formed at the glomerulus passes through the proximal tubules, loop
of Henle, and distal tubules, the solution undergoes several transformations in tonicity
that allows (along with active Na+ uptake throughout the loop) for reabsorption of
water and Na+. The ascending limb is impermeable to water yet still actively secretes
Na+ causing the interstitial space around the ascending limb to become hypertonic.
Since the interstitial space around the ascending limb is immediately adjacent to the
descending limb, it creates an osmotic gradient between fluid inside the descending
limb and the interstitial fluid. This gradient drives the removal of water from the
descending limb, thereby increasing the fluid tonicity (forming a hypertonic solution).
As the fluid makes its way out of the descending limb into the ascending limb the
tubule becomes impermeable to water, yet Na+ continues to be actively pumped out.
This results in a hypotonic fluid low in Na+ that leaves the ascending loop of Henle.
Following the reabsorption of Na+ in the ascending loop of Henle, Na+ reabsorption
continues in the distal tubules. It is in this region of the kidney where water retention
occurs. The pituitary gland, in response to decreased water concentration in the blood,
releases stored antidiuretic hormone (ADH) into the circulatory system. ADH causes
the epithelial cells of the distal convoluted tubules to become more permeable to water,
thus concentrating urine and saving water during times of water stress.
Na+ homeostasis is critical to life and thus requires the amount of sodium intake
to equal the amount of Na+ excretion. There are numerous feedback loops and hor-
monal controls in play to regulate Na+ excretion such as blood pressure (pressure
natriuresis and diuresis), blood volume, antidiuretic hormone, angiotensin II, arterial
baroreceptor, low pressure stretch receptors reflexes, aldosterone, and natriuretic
peptide. Regardless of the mechanism (complex or simple), all these feedbacks
work by altering either glomerular filtration rates or by Na+ reabsorption.
Xenobiotics, disease, or even fever can cause any of these mechanisms to alter Na+
balance. It is therefore necessary to have a complex system of redundancy and rapid
response to maintain critical Na+ balance.

2.3 Mechanism of Other Physiological Systems Influencing


Sodium and Potassium Homeostasis

2.3.1 Potassium

Aldosterone: See the discussion of aldosterone’s effects on Na+ below. Aldosterone


increases the Na+/K+ ATPase pump as Na+ is conserved, K+ is secreted into the urine.
β-adrenergic stimulation: Activation of β2-adrenergic receptors by stimulants such
as epinephrine causes K+ to move into cells. Drugs that block β2 receptors can
prevent the uptake of K+ into cells.
Acid-base abnormalities: The activity of the sodium-potassium ATPase pump is
inhibited in the presence of increased hydrogen ion concentration. Therefore
2 Sodium and Potassium in Health and Disease 37

disease or physiological states that affect acid-base balance can affect K+ homeostasis
as well [8].
Cell lysis: Necrosis or major cell death can lead to the release of intracellular K+
causing a disruption in K+ homeostasis.
Strenuous exercise: Muscle cells release K+ during long-duration exercise. Usually
this is not a problem except in individuals that may already be sensitive to K+
disturbances (diabetics, people taking beta blockers).

2.3.2 Sodium

Pressure natriuresis and diuresis: Blood pressure drives both urinary volume and
the amount of Na+ filtered into the proximal tubule. While increases and decreases
in natriuresis pressure can help regulate Na+ homeostasis when such pressure
changes occur as a result of disease (e.g., hypertension) or other causes, the increase
or decrease in pressure can cause imbalances in sodium.
Blood volume: Changes in blood volume quickly lead to changes in cardiac output
and blood pressure. As discussed above, blood pressure changes can lead to changes
in Na+ excretion.
Antidiuretic hormone: As previously discussed (see sodium excretion and secre-
tion in the kidney), the pituitary gland, in response to decreased water concentra-
tion in the blood, releases stored antidiuretic hormone into the circulatory system.
ADH causes the epithelial cells of the distal convoluted tubules to become more
permeable to water, thus concentrating urine and saving water during times of
water stress.
Angiotensin II: Decreased levels of angiotensin II result in decreased reabsorption
of Na+ in the renal tubules. Thus decreases in angiotensin II are seen following
increases in sodium intake. Angiotensin II works by modifying the natriuresis pres-
sure mechanism, decreasing angiotensin II and increasing pressure when sodium
needs to be excreted [12]. It also indirectly stimulates aldosterone secretion and
constricts efferent arterioles. Angiotensin II is decreased by inhibiting renin, an
angiotensin II precursor. In some individuals, this renin-angiotensin system (RAS)
does not operate as efficiently, and greater increases in arteriole pressure are needed
to excrete sodium. This may lead to hypertension in some individuals [8].
Arterial baroreceptor and low pressure stretch receptors reflexes: Sympathetic
activity can constrict renal arterioles, increase tubular reabsorption, and stimulate
renin release, all leading to increased retention of sodium. This type of reflex is
likely to occur from decreased blood volume, as in following a large hemorrhage.
Aldosterone: Na+ absorption in the kidney (the ascending limb of the loop of Henle,
the distal convoluted tubules, and collecting ducts) is greatly influenced by the
amount of aldosterone excreted by the adrenal cortex [4]. When Na+ levels drop, the
adrenal cortex secretes aldosterone, which results in an increase in the active reab-
sorption of Na+.
38 Pohl, Wheeler, and Murray

3 Pathology Associated with Sodium Levels

3.1 Hyponatremia

Hyponatremia represents a decrease in the serum sodium concentration below the


lower end of the normal range (136 mEq/L) [13].
Clinical signs and symptoms associated with hyponatremia include hypotension,
and decreased extracellular fluid osmolarity resulting in intracellular fluid increase
[14]. Hyponatremia is the most common electrolyte disorder. In one study, the prev-
alence of hyponatremia was 28% in acute hospital care patients at the time of admis-
sion and 21% in ambulatory patients [15]. The risk factors for hyponatremia include
use of diuretics, liver failure, heart failure, myocardial infarction, and endocrine
changes which are mostly found in older patients. Hyponatremia is associated with
various conditions that can be grouped into dilutional disorders (characterized by
water intake in excess of output; the condition implies impaired water excretion)
and depletional disorders (caused by sodium depletion in excess of water depletion
or replacement of fluid losses with water alone). See Table 1 for pathologic states
associated with hyponatremia.

Table 1 Pathology associated with hyponatremiaa.


Causes of hyponatremia Associated diseases
Water intake higher than output; always impaired Primary: chronic renal failure, acute renal
water excretion (dilutional disorders) failure (recovery phase), SIADH
Neuroendocrine: adrenal and pituitary
insufficiency
With edema: congestive heart failure, hepatic
cirrhosis, toxemia in pregnancy
Osmotic: severe hyperglycemia
Diuretics: thiazides
Sodium depletion higher than water depletion or Severe diarrhea, vomiting, blood loss,
replacement of fluid losses with water alone excessive sweating
(depletional disorders = extrarenal losses)
a
All tables were modified from Chandrasoma and Taylor [14] and Merck [13].

Dilutional disorders include primary causes such as renal failure and the
syndrome of inappropriate antidiuretic hormone secretion (SIADH). Other causes
of dilutional disorders include neuroendocrine dysfunction (adrenal and pituitary
insufficiency), diseases linked to sodium retention and edema (congestive heart
failure, cirrhosis, nephrotic syndrome), osmotic hyponatremia (severe hyperglycemia
in diabetes), and drug-induced disorders (mercurial diuretics, chlorothiazide diuretics).
Hyponatremia with hypotonicity can also be induced by diets with high water and
low salt intake or by excessive beer drinking.
SIADH is an example of a dilutional disorder. The syndrome was first described
almost 50 years ago [16]. The diagnostic criteria include hyponatremia with
2 Sodium and Potassium in Health and Disease 39

hypotonicity of plasma, high urine osmolarity relative to plasma, increased renal


sodium excretion, absence of edema, and normal renal and adrenal function. SIADH
explains about 60% of all types of chronic hyponatremia and is the most common
type of hyponatremia in hospitalized patients [17]. SIADH is associated with 4
major etiologies: nonmalignant pulmonary diseases, neoplasms with ectopic
hormone production, neurologic disorders, and use of several pharmaceutics [18].
SIADH is linked to euvolemic hyponatremia described as an increase in total water
with normal or near normal sodium levels. It is associated with inappropriate secre-
tion of arginine vasopressin (AVP), the hormone that regulates excretion of water by
kidneys. Excessive release of AVP unrelated to plasma osmolarity occurs in about
40% of patients with SIADH.
In the case of impaired glomerular filtration rate (renal failure) hyponatremia is
caused by inadequate glomerular filtration of water (i.e., the body cannot get rid of
water taken in). However, this usually happens when the filtration rate is substan-
tially reduced to about 20–30% of the normal rate [14].
The common ground of diseases such as congestive heart failure, cirrhosis, and
nephrotic syndrome is the edematous state. Hyponatremic patients with these
diseases have abnormal renal retention of sodium resulting in extracellular fluid
volume overload and edema. They also have retention of water causing hyponatremia
with hypotonicity.
Drugs such as thiazide diuretics are an important cause of hyponatremia espe-
cially in elderly women. The mechanism of action is inhibition of Na+-Cl– symport
(co-transporter) located in the cortical part of the ascending loop of Henle and the
distal convoluted tubules of the kidneys resulting in the failure of these ions to reab-
sorb [19,20]. Thiazides also increase calcium reabsorption in the distal tubule.
Complications of thiazide therapy are hyponatremia, hypokalemia, hypercalcemia,
hyperglycemia, and hyperlipidemia.
Hyponatremia can also be induced by loop diuretics (e.g., furosemide,
bumetanide). These diuretics block the Na+-K+-2Cl– symport which facilitates ion
movement from the tubular lumen into the tubular cells in the ascending part of the
loop of Henle [20]. The mechanism of action of the loop diuretics lays in competing
for the Cl– binding sites of the symport. This may lead to natriuresis and hyponatre-
mia, hypokalemia, hypomagnesemia, and dehydration.
Genetic mutation of the Na+-K+-2Cl– symport encoding gene may lead to
impaired function of the symport; the clinical presentation is severe volume deple-
tion, hypokalemia, and metabolic alkalosis with increased prenatal mortality. The
disease is called type I Bartter’s syndrome [21].
A special case of hyponatremia is with hypertonicity. It was described in patients
with uncontrolled diabetes mellitus with severe hyperglycemia [14]. The increased
glucose concentration causes water to move from the intracellular to the extracellular
compartment resulting in decreased sodium concentration (i.e., dilutional state).
Depletional disorders include severe diarrhea, vomiting, blood loss, and excessive
sweating accompanied by large oral intake of water.
Diarrhea is an example of a condition linked to depletional hyponatremia.
The most common causes of diarrhea are bacterial enterotoxins (e.g., Vibrio cholerae),
bacterial invasion of gastric mucosa (e.g., some Shigella, Salmonella), and enteroviruses.
40 Pohl, Wheeler, and Murray

Diarrhea is also associated with hypokalemia and metabolic acidosis. The condition
may become severe and lead to mortality, especially in susceptible populations such
as the elderly, those debilitated by other diseases, and the very young. In a retro-
spective study in Nepal, 5 children died out of 57 who were admitted to the hospital
with diarrhea [22]. Most patients (70%) were younger than 2 years. Electrolyte
disturbance was reported in 46 (80%) patients, and acid-base disturbance was
reported in all tested. Hyponatremia was present in 56% of patients and was either
isolated (26%) or associated with hypokalemia (26%). Hypokalemia was found in
46% of patients and was isolated in 14%. In a two year prospective study in Nigeria,
191 children under 15 years of age were admitted to the hospital with diarrhea and
protein energy malnutrition [23]. The most often observed disturbance was meta-
bolic acidosis that was reported in 108 (56.3%) of patients. Hypokalemia was found
in 45 (23.4%) and hyponatremia in 25 (13%) of patients. Clinical risk factors
contributing to mortality in children hospitalized for diarrhea were studied in
Turkey [24]. In a cohort of 400 children, 27 (6.75%) died. Significant factors
contributing to fatalities included severe malnutrition, co-existent sepsis, hypogly-
cemia, hypoalbuminemia, Shigella infection, hyponatremia (p = 0.016), hypokalemia
(p = 0.00041) and metabolic acidosis (p = 0.0069).

3.2 Hypernatremia

Hypernatremia represents an elevation in the serum sodium concentration above the


higher end of the normal range (145 mEq/L) [13].
Clinical signs and symptoms associated with hypernatremia include hypertension,
increased extracellular fluid volume, and increased extracellular fluid osmolarity
resulting in intracellular fluid loss [14]. See Table 2 for pathologic states associated
with hypernatremia. Hypernatremia is not as common as hyponatremia. It is associ-
ated with abnormal renal excretion of water with inadequate water intake disorders
such as in pituitary ADH deficiency (central diabetes insipidus) and nephrotic
syndrome (nephrotic diabetes insipidus), in which kidneys are ADH unresponsive, or
with osmotic diuresis such as severe glycosuria and manitol diuresis. Other diseases
and states that may be accompanied by hypernatremia are chronic renal failure, recovery
phase of acute renal failure, hypocalcemia, hypokalemia, and sickle cell anemia.

Table 2 Pathology associated with hypernatremia.


Causes of hypernatremia Associated diseases
Abnormal renal excretion of water with Diabetes insipidus, renal failure,
inadequate intake loop diuretics
Water depletion with normal renal water Excessive sweating;
conservation diarrhea (children)
Excessive intake of sodium with limited Poisoning
water intake
2 Sodium and Potassium in Health and Disease 41

Another mechanism of hypernatremia is water depletion with normal renal


conservation of water but inadequate intake of water; causes include excessive
sweating and diarrhea (pronounced in children). For example, hypernatremia was
reported in 6 children (3.1%) with severe diarrhea in a cohort of 191 (see Section 3.1.)
[23]. However, hyponatremia was far more frequent. i.e., in 13% of the cohort.

4 Pathology Associated with Potassium Levels

4.1 Hypokalemia

Hypokalemia represents the low potassium levels. In adults, potassium blood levels
drop below 3.5 mEq/L, which is the lower range of normal values.
Clinical signs and symptoms associated with hypokalemia include neuromuscu-
lar (weakness, paralysis, fasciculation and tetany), gastrointestinal (ileus, nausea,
vomiting, abdominal distention), and renal effects (polyuria) [14]. Cardiac effects
present themselves as dysrhythmias and conduction defects. ECG manifestations
include decreased amplitude and broadening of the T waves, prominent U waves,
ST segment depression, increased QRS duration, and increase in P wave amplitude
and duration. The changes may lead to atrioventricular block and cardiac arrest
[25–27]. With hypokalemia, cardiac arrest occurs during systole [28]. See Table 3
for pathologic states associated with hypokalemia.

Table 3 Pathology associated with hypokalemia.


Causes of hypokalemia Associated diseases
Increased extrarenal losses Severe diarrhea, laxative abuse, vomiting, excessive sweating,
villous adenoma
Increased renal losses With metabolic acidosis: renal tubular acidosis, diabetic
ketoacidosis
With metabolic alkalosis: diuretics, post hypercapnea,
mineralocorticoid excess syndrome, Bartter’s syndrome
With no specific acid-base disorder: acute renal failure
(recovery phase), post obstructive diuresis, osmotic diuresis,
saline intake
Potassium shifts into cells Alkalemia, β-adrenergic activity, familial hypokalemic
(redistribution) periodic paralysis, theophylline toxicity

Potassium homeostasis depends on external balance (i.e., dietary intake and


absorption versus excretion) and internal balance (i.e., the distribution of potassium
between intracellular and extracellular fluids [14]).
External losses include those through the gastrointestinal tract (e.g., diarrhea,
villous adenoma of recto-sigmoid colon, inadequate intake) or through the skin
42 Pohl, Wheeler, and Murray

(e.g., profuse sweating). Urine potassium is usually <20 mEq/24 hours. In external
losses through the kidneys, urine potassium is usually >20 mEq/24 hours.
Eating disorders and starvation: Anorexia nervosa and bulimia are psychologi-
cal eating disorders. Medical consequences of these eating disorders include heart
damage, failure of the endocrine system, perforation of the stomach or esophagus,
aspiration of vomit, erosions of teeth enamel, and depression [29]. Death by
starvation has been reported in up to 24% of the patients with anorexia. Biochemical
changes are also pronounced [30]. Hypokalemia is the most common electrolyte
disturbance. It is often reflected by changes on the electrocardiograms. Metabolic
alkalosis is found in patients who vomit or abuse diuretics, whereas acidosis is
found in those abusing laxatives. In laxative abuse, potassium is lost directly from
the intestines. In contrast, the loss of potassium in those who vomit is largely due to
metabolic alkalosis, which is secondary to loss of hydrogen ions in the vomitus.
This results in increased availability of bicarbonate from blood and increased
renal excretion of potassium [31]. Hypokalemic nephropathy is also associated with
laxative abuse. Severe chronic hypokalemia in these patients was found to result in
a progressive decrease in renal function and histological changes suggestive of
chronic glomerular damage. Chronic tubulo-interstitial nephropathy has been
also reported [32,33].
Hypokalemia is also associated with starvation related to other causes. For example,
hypokalemia was reported in malnourished children on poor protein-calorie diets all
over the world. In these children, decrease in total body potassium was correlated
with decreased muscle potassium established by analysis of biopsy samples
[34–36]. This result correlated with loss of total muscle mass. In contrast, muscle
water was increased. Wasting is one aspect of the muscle loss; however, a contribut-
ing factor may be a decreased muscle build-up. Several laboratory studies showed
the importance of potassium in protein synthesis. A study in young chicken demon-
strated that there was a significant decrease in the incorporation of injected
L-leucine-1-14C into skeletal muscle of chicken fed a potassium-deficient diet [37].
Similarly, when rats were maintained on a potassium-deficient diet, the animals
stopped growing within a few days and the incorporation of [3H]leucine into
skeletal muscle protein in vivo was reduced by 28–38% [38].
Related to the above topic is the refeeding syndrome. It illustrates the metabolic
and clinical changes in the body that occur in the process of aggressive nutritional
rehabilitation of starved patients. The most important manifestation is hypophos-
phatemia [39]. Hypokalemia, hypomagnesemia, hyperglycemia, fluid overload, and
thiamine deficiency may also be present. During starvation, potassium is depleted in
the cells. During refeeding, increased insulin secretion promotes cellular uptake of
potassium, resulting in hypokalemia. The outcome is an imbalance of electrochemi-
cal potential on membranes leading to cardiac arrhythmias and arrest. Neuromuscular
dysfunction is also observed. The refeeding syndrome was reported in up to 25% of
adults with cancer.
Causes for potassium renal losses are complex [26,27]. Contributing clinical fac-
tors are increased mineralocorticoid-receptor stimulation (primary hyper-reninism
distinguished by increased renin and aldosterone levels that cannot be suppressed
2 Sodium and Potassium in Health and Disease 43

by saline); primary aldosteronism (e.g., Conn syndrome); a primary increase in the


effectiveness and/or amount of non-aldosterone mineralocorticoid-receptor agonist
(e.g., Cushing syndrome, congenital adrenal hyperplasia); and increased distal
sodium delivery and/or non-reabsorbable ions in the distal nephron (e.g., magne-
sium deficit, Bartter syndrome) [27].
Clinical data indicate that renal losses of potassium are often related to adverse
effects to therapy (e.g., penicillin, gentamicin, cisplatin, diuretics). For example,
hypokalemia was reported in 10% to 40% of patients on thiazide diuretics [40].
The mechanism includes increased exchange of Na+ for K+ and increased production
of aldosterone as a response to diuretic hypovolemia [19].
It is well established that acid-base imbalance and electrolyte disorders are
associated with diabetes. Recent reports indicate that low potassium is a possible
risk factor for developing type 2 diabetes [41].
Redistribution losses are the consequence of potassium shifts into cells from
the extracellular fluids. By this mechanism, hypokalemia is present in respiratory
alkalosis, increased β2-adrenergic activity, theophylline toxicity, and in familial
hypokalemic periodic paralysis.
Stimulation of β2-adrenergic receptors redistributes potassium into cells by
increasing the activity of sodium-potassium ATPase. States of increased sympa-
thetic responsiveness can be observed in myocardial infarction, delirium tremens, or
major head trauma. These states are also associated with shifts in potassium levels.
Hypokalemia is common in congestive heart failure due to a defect in sodium-
potassium ATPase activity and intracellular transfer of potassium caused by
oxidative stress and neurohormonal activation [42]. Hypokalemia in the presence of
congestive heart failure may lead to serious outcomes [43]. These include impaired
diuresis because of decreased natriuresis and lack of suppression of renin secretion,
reduced myocardial performance, and elevated risk for ventricular arrhythmia and
sudden death. Recent studies indicated that heart failure itself may stimulate meta-
bolic changes such as insulin resistance [44]. These in turn may worsen the primary
condition. A study in hospitalized patients with heart failure and a depressed left
ventricular ejection fraction reported 30-day and 1-year mortality as 7.1% and
25.5%, respectively [45]. Impaired renal function is a major factor that influences
the prognosis of patients with heart failure [46].

4.2 Hyperkalemia

In adults, hyperkalemia refers to blood values of potassium >5 mEq/L. Clinical


manifestations of hyperkalemia include neuromuscular effects (weakness, ascend-
ing paralysis, and respiratory failure) and ECG changes (peaked T waves, flattened
P waves, widened QRS complex) [14]. The changes in heart conductivity can lead
to sinus arrest, ventricular tachycardia, and fibrillation at >10 mEq/L [25]. With
hyperkalemia, cardiac arrest occurs during diastole [28]. See Table 4 for pathologic
states associated with hyperkalemia.
44 Pohl, Wheeler, and Murray

Table 4 Pathology associated with hyperkalemia.


Causes of hyperkalemia Associated diseases
Decreased excretion Renal failure (acute and chronic), severe oliguria due to severe
dehydration or shock
Endocrine dysfunction Adrenocortical insufficiency,
hyporeninemic-hypoaldosteronism
Potassium shifts out of cells Acidosis, hypertonic states, massive release in burns,
(redistribution) rhabdomyolysis or crush injury, or severe infection

Hyperkalemia is less common than hypokalemia. However, it still affects about


8% of patients in US hospitals [25]. There are two major mechanisms for hyperka-
lemia development. Redistribution hyperkalemia is caused by potassium shifting
from the intracellular space into the extracellular space, thus raising serum potas-
sium concentration. Potassium is forced out of cells in exchange for hydrogen ion
in both metabolic and respiratory acidosis. Similarly, potassium leaks out of cells in
hypertonic states, in burns and injuries, and in massive digitalis overdose.
Hyperkalemia secondary to impaired potassium excretion is the major cause
of this electrolyte disorder. It may be due to aldosterone deficiency (e.g., primary
adrenal failure, Addison’s disease) or tubular unresponsiveness to aldosterone
(e.g., chronic renal diseases, some pharmaceuticals). Hyperaldosteronism is a disease
caused by an excess production of adrenal hormone aldosterone. This hormone is
responsible for sodium and potassium balance, which then directly controls water
balance to maintain appropriate blood pressure and blood volume. With adrenal
insufficiency, there is inappropriate sodium excretion. When adrenal aldosterone
production is increased (as in shock, heart failure, or cirrhosis) sodium excretion is
decreased. People with a deficiency of aldosterone, especially found in association
with cortisol deficiency in Addison’s disease, have low blood volume and therefore
low blood pressure, low sodium and high potassium. Just the opposite is seen in
hyperaldosteronism.
There are several drugs that affect the renin-angiotensin-aldosterone system and
thus may impact potassium levels. A review of studies that administered angiotensin-
converting enzyme inhibitors, angiotensin receptor blockers, aldosterone receptor
antagonists, and direct renin inhibitors alone or in combination to patients with
hypertension, heart failure, or chronic kidney disease revealed that the risk of hyper-
kalemia on monotherapy of hypertension is low (≤2%) but increases to about 5% in
combination therapy [47]. Increased incidence was also observed in patients with
heart failure or chronic kidney disease (5% to 10%).
The syndrome of hyporeninemic hypoaldosteronism (SHH) that also belongs to
this category is associated with several renal diseases. SHH includes low plasma
renin activity, low plasma aldosterone, and hyperkalemia. The syndrome is also
common in patients with diabetes mellitus.
In a study of 210 outpatient diabetics, metabolic alkalosis was the most common
acid-base imbalance [48]. The most common electrolyte disorders were hypernatre-
mia in patients with serum creatinine <1.2 mg/dL, and hyponatremia and hyperka-
lemia in patients with higher creatinine levels (>3.1 mg/dL).
2 Sodium and Potassium in Health and Disease 45

Renal diseases with changes in urine output are another obvious reason for potassium
disbalance. Patients with acute renal failure present with anorexia, nausea, vomiting,
lethargy, and increased blood pressure [28]. The onset of oliguria is sudden; protein-
uria and hematuria are common. There is a progressive increase in serum urea nitro-
gen, creatinine, potassium, phosphate, and sulfate. In contrast, serum sodium,
calcium, and bicarbonate are decreased. The etiology for inducing acute renal failure
is numerous and the disease is classically divided into pre-renal, renal (intrinsic), and
post-renal failure. Multiple animal models have been developed to induce acute renal
failure by different mechanisms [49]. These laboratory studies contribute to a better
understanding of the disease. In chronic kidney disease, the changes develop at a
slower rate. Therefore, the organism has time to compensate for partial loss of func-
tion. For example, uremia and azotemia occur only when renal failure is advanced;
usually when the creatinine clearance decreases to about 30–40% of normal [14].
The inability to concentrate urine, resulting in polyuria, is one of the early signs of
chronic kidney failure. Metabolic acidosis is caused by the failure of hydrogen ion
excretion. Hyperkalemia is one of the later signs of the disease; so is the development
of secondary hyperparathyroidism and renal osteodystrophy. When pre-dialysis mor-
tality was studied in a large cohort of men (N = 1,227), both hypo- and hyperkalemia
were linked to mortality in white patients [50]. Black patients seemed to better toler-
ate hyperkalemia than whites. Hypokalemia was associated with faster chronic kid-
ney disease progression in both races.

5 Conclusion

Sodium and potassium are essential to life. These ions are involved in many
physiological processes, and their imbalance may impair proper function in various
organs and/or entire systems in the body. It is beyond the scope of this chapter to
describe in detail all the diseases. The interested reader is encouraged to find more
information in the medical texts and scientific papers cited here.

Abbreviations

ADH antidiuretic hormone


ATP adenosine 5′-triphosphate
AVP arginine vasopressin
DDT dichlorodiphenyltrichloroethane
ECG electrocardiogram
GI gastrointestinal
RAS renin-angiotensin system
SHH syndrome of hyporeninemic hypoaldosteronism
SIADH syndrome of inappropriate antidiuretic hormone
46 Pohl, Wheeler, and Murray

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Chapter 3
Magnesium in Health and Disease

Andrea M.P. Romani

Contents
ABSTRACT ............................................................................................................................. 50
1 INTRODUCTION ............................................................................................................. 50
1.1 Distribution of Magnesium in the Human Body....................................................... 50
1.2 Intestinal Magnesium Absorption and Release into the Blood................................. 51
1.2.1 Apical Side .................................................................................................... 51
1.2.2 Cellular Transport ......................................................................................... 52
1.2.3 Basolateral Side ............................................................................................ 53
1.3 Renal Magnesium Handling and Reabsorption ........................................................ 53
2 CELLULAR MAGNESIUM HOMEOSTASIS ................................................................ 54
2.1 Cellular Magnesium Transport Mechanisms ............................................................ 55
2.2 Regulation of Magnesium Transport ........................................................................ 55
3 MAGNESIUM IN DISEASE ............................................................................................ 55
3.1 Hypermagnesemia .................................................................................................... 56
3.1.1 Hypermagnesemia in Renal Failure .............................................................. 57
3.2 Hypomagnesemia...................................................................................................... 57
3.2.1 Cardiovascular Pathologies ........................................................................... 59
3.2.2 Hyperaldosteronism ...................................................................................... 62
3.2.3 Diabetes......................................................................................................... 62
3.2.4 Metabolic Syndrome ..................................................................................... 64
3.2.5 Alcoholism .................................................................................................... 65
3.2.6 Inflammation ................................................................................................. 66
3.2.7 Renal Pathologies.......................................................................................... 67
3.2.8 Magnesium and Tumors................................................................................ 69
3.2.9 Magnesium and Prenatal Pathologies ........................................................... 70

A.M.P. Romani (*)


Department of Physiology and Biophysics, School of Medicine,
Case Western Reserve University, 10900 Euclid Avenue, Cleveland,
OH 44106-4970, USA
e-mail: amr5@po.cwru.edu

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 49


Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_3, © Springer Science+Business Media Dordrecht 2013
50 Romani

3.3
Pharmacological Agents Causing Hypomagnesemia ............................................... 71
3.3.1 Proton Pump Inhibitors ................................................................................. 72
3.3.2 Anti-epidermal Growth Factor Receptor Antibodies .................................... 72
4 CONCLUSIONS ............................................................................................................... 73
ABBREVIATIONS .................................................................................................................. 74
ACKNOWLEDGEMENTS ..................................................................................................... 75
REFERENCES ........................................................................................................................ 75

Abstract Mammalian cells tightly regulate cellular Mg2+ content through a variety
of transport and buffering mechanisms under the control of various hormones and
cellular second messengers. The effect of these hormones and agents results in
dynamic changes in the total content of Mg2+ being transported across the cell
membrane and redistributed within cellular compartments. The importance of
maintaining proper cellular Mg2+ content optimal for the activity of various cellular
enzymes and metabolic cycles is underscored by the evidence that several diseases
are characterized by a loss of Mg2+ within specific tissues as a result of defective
transport, hormonal stimulation, or metabolic impairment. This chapter will review
the key mechanisms regulating cellular Mg2+ homeostasis and their impairments
under the most common diseases associated with Mg2+ loss or deficiency.

Keywords alcoholism • cancer • cell cycle • diabetes • homeostasis • hormones


• hypertension • inflammation • insulin • metastases • Mg2+ • Mg2+ transport

Please cite as: Met. Ions Life Sci. 13 (2013) 49–79

1 Introduction

Magnesium is the 4th most abundant element in the human body and the 2nd most
abundant cation within human cells after potassium. The human body contains
about 760 mg of magnesium at birth. This amount increases to 5 g at age 4–5 months
and to 25 g at adulthood [1]. How this increase is regulated or stimulated is
presently unknown.
In the following lines we will provide a general picture of how Mg2+ is absorbed
at the intestinal level, and the role of the kidney in controlling urinary Mg2+ loss.

1.1 Distribution of Magnesium in the Human Body

Approximately 60% of whole body magnesium is found in bones, 30% to 40% in


skeletal muscles and soft tissues, and 1% in the extracellular fluid [2]. In bones, mag-
nesium is mainly distributed along the Havers’s channels, where it contributes to form
hydroxyapatite crystals [3]. In net terms, this magnesium accounts to about 1% of
bone ash [2,3]. At an early stage, most of this magnesium can readily exchange
3 Magnesium in Health and Disease 51

with serum, representing an optimal store to compensate occasional dietary deficiency.


As age progresses the proportion of readily exchangeable magnesium in the bone
decreases significantly. In individuals consuming magnesium-enriched diet a positive
association between bone mineral density and magnesium content within the erythro-
cytes has been reported [4]. Not much is known about the role of magnesium within
skeletal muscles other than that it controls ATP content and utilization for contraction
purposes and reticular Ca2+ uptake and release [5].
In soft tissue, magnesium acts as a cofactor of many enzymes involved in energy
metabolism, protein synthesis, and RNA and DNA synthesis [6]. It also plays a
major role in the maintenance of the electrical potential of nervous tissue and cell
membranes.

1.2 Intestinal Magnesium Absorption and Release


into the Blood

Diet and water are the main sources of magnesium intake. The recommended daily
dose of Mg2+ is ~300 mg for men and 250 mg for women [7] unless pregnant, in
which case an increase to ~350 mg is suggested. These doses correspond to the
amount of Mg2+ eliminated daily through the urinary and digestive systems [7].
Dietary Mg2+ is absorbed at the apical side of intestinal epithelial cells and
transported throughout the cell to be released into the blood at the basolateral side
of the cell [7].

1.2.1 Apical Side

Limited information is available about the modality by which Mg2+ is absorbed


from the intestinal lumen. The operation of specific and saturable Mg2+ accumula-
tion mechanisms has been observed in brush border cells of the ileum [8] but also
duodenum and jejunum [9,10].
More recently, attention has been paid to the distribution and operation of apical
Mg2+ channels, namely TRPM6 [11] and TRPM7 [12]. They are members of the
melastatin subfamily of transient receptor potential (TRP) channels [13]. TRPM6 is
specifically located in the colon and in the distal convolute tubule of the nephron
[11]. TRPM7 is ubiquitously expressed in the majority of mammalian cells [71],
including the various segments of the small intestine [14]. The specific modalities
of operation and regulation of these channels have been amply discussed in several
reviews [15–18], and will not be discussed here. For the purpose of this review, we
will only mention that both TRPM6 and TRPM7 operate as tetramers, and present
a α-kinase domain at their C-terminus, which phosphorylates serine and threonine
residues within an α-helix structure [19–21]. Presently, only annexin I [22], myosin
IIA heavy chain [23,24], and calpain [25] have been clearly identified as phosphory-
lation substrates for the TRPM7 kinase domain while the best known target for
TRPM6 kinase domain is TRPM7 itself [26].
52 Romani

Aside from the specific location, the most striking difference between the two
channels is that TRPM6 but not TRPM7 expression and activity are modulated by
diet and estrogens. Estrogens (17β-estradiol) markedly upregulate TRPM6 mRNA
in both colon and kidney while having no effect on TRPM7 mRNA [27,28]. In the
absence of estrogens, the repressor of estrogen receptor activity (REA) binds to the
6th, 7th and 8th β-sheets of TRPM6 kinase domain in a phosphorylation-dependent
manner and inhibits TRPM6 activity [27]. Short-term estrogen administration dis-
sociates the binding between REA and TRPM6, resulting in increased channel
activity [27]. Dietary Mg2+ restriction increases TRPM6 mRNA expression both in
the kidney and the colon [28,29], whereas Mg2+ enriched diet upregulates TRPM6
mRNA expression only in the colon, increasing intestinal absorption [28]. In con-
trast, neither dietary Mg2+ manipulation affects TRPM7 mRNA expression in the
two organs [28,29]. Thus, evidence is there that genetic factors and variation in
dietary Mg2+ content control TRPM6 expression and activity in the large intestine to
favor Mg2+ absorption, while renal Mg2+ resorption only occurs following dietary
Mg2+ restriction [28,29].
TRPM6 channel activity is also modulated by RACK1 (receptor for activated
protein kinase C), which binds directly to the α-kinase domain of TRPM6, and pos-
sibly TRPM7 due to the high homology (>84%) between the two kinase domains
[30]. RACK1 binds the same β-sheets involved in REA regulation [27], and inhibits
the channel activity of TRPM6 and TRPM7. Activation of protein kinase C (PKC),
the natural ligand for RACK1, completely prevents the inhibitory effect of RACK1
on TRPM6 channel activity [30], suggesting a competition of PKC for TRPM6
towards RACK1.

1.2.2 Cellular Transport

At front of a total cellular Mg2+ concentration of 15 to 20 mM [31–33], cytoplasmic


free Mg2+ concentration accounts for ~0.5 to 1 mM [31–33], suggesting that as Mg2+
enters the cell, it is rapidly buffered by ATP, phosphonucleotides, and proteins.
It is hypothesized that cytoplasmic proteins can bind Mg2+ and contribute to its
buffering within this cellular compartment while transporting it to the basolateral
side for dismissal into the circulation. Aside from calmodulin [34], troponin C [35],
parvalbumin [36], and S100 protein [37], for which a Mg2+ binding consensus
sequence has been reported, the number or nature of Mg2+ binding proteins remains
elusive. Parvalbumin and calbindin-D28k, two proteins abundantly present within
intestinal and renal cells, have been indicated as transcellular transporters of the
Mg2+ accumulated at the apical domain of the cell, accelerating its delivery rate to
the basolateral domain for dismissal. In intestinal cells, these proteins contribute to
Ca2+ binding and transport upon vitamin D stimulation. Whether these proteins
operate in a similar manner for Mg2+ in the intestinal epithelium is presently unde-
fined. The physiological relevance of Mg2+ binding by cytoplasmic proteins and
their ability to transport Mg2+ from the apical to the basolateral side of intestinal and
renal cells is highly questioned since parvalbumin null mice do not exhibit hypo-
magnesemia or significant changes in tissue Mg2+ handling and homeostasis [38].
3 Magnesium in Health and Disease 53

A variable percentage of the Mg2+ present in the diet is not absorbed and is
eliminated through the intestine. This percentage varies based upon the diet com-
position and the complex form in which magnesium is present in the diet and its
solubility. Magnesium sulfate, magnesium hydroxide, magnesium chloride, magne-
sium oxide, magnesium oxalate, magnesium gluconate, and magnesium citrate are
among the most common forms of magnesium salts present in the diet, or available
in multi-vitamin and multi-mineral dietary supplements [39]. Each of these com-
pounds is characterized by different solubility and intestinal absorption rate, varying
from very little solubility for magnesium oxide to good solubility for magnesium
citrate [39].

1.2.3 Basolateral Side

Once delivered to the basolateral domain of the intestinal cell, Mg2+ is extruded into
the bloodstream through a Na+-dependent Mg2+ extrusion mechanism termed Na+/
Mg2+ exchanger.
The first evidence for the operation of such a mechanism was provided by
Gunther, Vormann and Forster in 1984 [40]. In two sequential studies [40,41],
these authors detailed how this Na+/Mg2+ exchanger operates, and its inhibition by
amiloride. Several other groups have confirmed the presence and operation of
this extrusion mechanism in various mammalian cell types (see [42] for a list).
The current consensus is that this Mg2+ extrusion mechanism becomes active upon
phosphorylation by cAMP, and operates as an antiporter, strictly requiring a physi-
ological concentration of extracellular Na+ to be fully operative [43]. Under condi-
tions in which a less than optimal concentration of extracellular Na+ is available,
Mg2+ can be extruded from the cell into the bloodstream through the operation of a
subsidiary, and not fully characterized Na+-independent Mg2+ extrusion mecha-
nism [44], which appears to utilize both anions and cations to promote Mg2+
transport (reviewed in [16]).

1.3 Renal Magnesium Handling and Reabsorption

Upon dismissal into the blood stream, about one third of serum Mg2+ circulates
bound to proteins (mainly albumin), or in a complex with anions [45], whereas the
remaining two thirds (~0.7 mM) is in the free form. This serum Mg2+ concentration
is in equilibrium with the concentration in the extracellular space, and both are just
slightly higher than the free [Mg2+] in the cell cytoplasm. As a result of this distribu-
tion, the majority of mammalian cells is at, or near a zero trans condition in terms
of magnesium concentration across the cell membrane.
Serum magnesium undergoes renal glomerular filtration as other serum cations.
Approximately 25%–30% of filtered Mg2+ is reabsorbed in the proximal tubule, and
~65% is reabsorbed in the thick ascending limb of the Henle’s loop [46]. It is in this
segment of the nephron that various hormones (vasopressin, PTH, etc.) and drugs
54 Romani

(cyclosporine, cisplatin, gentamycin, etc.) operate to increase or decrease Mg2+


reabsorption [46]. The increase in reabsorption occurs through paracellular and trans-
cellular Mg2+ transport mechanisms: passive paracellular transport via claudins
favors bulk Mg2+ absorption while active transcellular transport mechanisms mediate
the fine-tuning of Mg2+ absorption. Claudin 16 (originally known as paracellin-1
[47]) and claudin 19 [48] form the tight junction component through which bulk
Mg2+ absorption occurs. This pathway is controlled by the CaSR (calcium sensing
receptor [49]) via negative feedback of the PKA/cAMP signaling [50], but it is also
influenced by the proper expression of the EGF receptor (EGFR), with defects in
either signaling pathway resulting in increased Mg2+ loss in the urine [51,52]. The
involvement of the CaSR in Mg2+ reabsorption is also supported by the evidence that
this sensor in the basolateral membrane of renal epithelial cells is activated by equiv-
alent concentrations of Ca2+ and Mg2+ [53]. Activation of the CaSR inhibits apical K+
channels and Na+/K+/Cl– co-transporter activity [54], overall reducing lumen positive
potential and paracellular transport of divalent cations. The hormone-mediated
increase in Mg2+ reabsorption and cellular Mg2+ content ultimately results in an
enhanced operation rate of the basolateral, cAMP-modulated Na+/Mg2+ exchanger
previously described. In contrast, exposure to cyclosporine, gentamycin, cisplatin, or
other drugs will decrease the activity of paracellular and transcellular Mg2+ transport
mechanisms, ultimately reducing the operation of the Na+/Mg2+ exchanger.
The residual 5%–10% Mg2+ reabsorption occurs in the distal convolute tubule
[46]. It is in this segment of the nephron that TRPM6 would play a major role in
enhancing Mg2+ accumulation from the lumen into the cells and ultimately into the
bloodstream, and where major changes in TRPM6 expression have been observed
as a result of variations in dietary Mg2+ content [28,29]. Interestingly, EGF also
controls TRPM6 expression through Erk1/2 and AP-1 [54,55].
Because of the key role of the renal apparatus in controlling Mg2+ reabsorption,
it is not surprising that several genetic and iatrogenic diseases primarily impair renal
Mg2+ reabsorption (see below).

2 Cellular Magnesium Homeostasis

Total cellular Mg2+ content ranges between 15 to 18 mM, well below the concentra-
tion predicted by the Nernst equation (~55 mM), whereas cytosolic free Mg2+ con-
centration (0.5–0.8 mM) is slightly below the concentration present in the
extracellular environment [56,57]. Within the cell, Mg2+ is distributed within cyto-
plasm and cellular organelles. In the cytoplasm, more than 95% of Mg2+ located
therein is in the form of a complex with ATP and phosphonucleotides [58,59]. As
for the organelles, Mg2+ is abundantly localized within nucleus, mitochondria, and
endoplasmic reticulum [56], in which it regulates the activity of numerous enzymes,
channels, and genes, directly and indirectly controlling metabolic and bioenergetics
processes [56]. This well defined distribution points to a tightly regulated cellular
Mg2+ homeostasis through a combination of transport and chelating mechanisms.
3 Magnesium in Health and Disease 55

2.1 Cellular Magnesium Transport Mechanisms

Our current understanding of Mg2+ transport across the cell membrane indicates that
Mg2+ exits the cell via an exchange mechanism, tentatively identified as Na+/Mg2+
exchanger [60,61] based upon its strict functional dependence on physiological
extracellular Na+ concentration [56,60], and via an alternative pathway termed Na+-
independent extrusion mechanism, which appears to utilize different cations and
anions in the process (reviewed in [56] and [62]). Entry of Mg2+ into the cell occurs
through channels or electrogenic transporters. Several Mg2+ entry mechanisms have
been identified. Yet, it remains still unclear to which extent these mechanisms coop-
erate in mediating Mg2+ entry, or whether Mg2+ accumulation primarily occurs
through a pre-dominant mechanism, perhaps different in diverse cells.

2.2 Regulation of Magnesium Transport

Several exhaustive review articles have addressed the specific modality of operation
and regulation of the Mg2+ transport mechanisms [56,62–66], and we refer to those
reviews for further information. For the purpose of this chapter, we will only men-
tion that Mg2+ entry and extrusion is under hormonal control. Hormones that
increase cellular cAMP level (e.g., catecholamine, glucagon, PGE2, etc.) all pro-
mote Mg2+ extrusion primarily via the Na+/Mg2+ exchanger [16]. Conversely, hor-
mones (insulin, vasopressin, etc.) that decrease/prevent cAMP production or activate
protein kinase C signaling, all favor Mg2+ accumulation primarily via TRPM6 or
TRPM7 [16]. In the case of Mg2+ entry, the involvement of Erk1/2 and associated
signaling components has been observed or postulated [67,68].
Both Mg2+ extrusion and Mg2+ accumulation are quantitatively and timely limited
processes [69,70], implying the movement of Mg2+ from and to specific cellular
compartments. Cytoplasm is but one of the cellular compartments involved in Mg2+
transport out of the cell or into the cells [71,72], other compartments being mito-
chondria and the endo-sarco-plasmic reticulum [71,72]. The mechanisms involved
in Mg2+ transport in and out of these compartments, however, are not yet fully elu-
cidated. In the case of the cytoplasm, evidence is there that pathological conditions
that decrease cellular ATP content through dysmetabolic processes [73–75] ultimately
cause cellular Mg2+ loss or deficiency.

3 Magnesium in Disease

Both hyper- and hypomagnesemia occur in human patients. Hypermagnesemia is


less common than hypomagnesemia and is mostly iatrogenic (i.e., medically
induced) in nature. Hypomagnesemia, on the other hand, can result from different
56 Romani

causes but because in its initial phase it is associated with vague and non-specific
symptoms, it often goes undetected in the large population until an individual
checks in a hospital or a medical facility for another pathological condition. This
association raises the question as to which extent hypomagnesemia is connected in
a cause-effect relation to the concurrent disease.
The following pages will address the medical concept and the main pathological
causes of hyper- and hypo-magnesemia. In addition, because several of the most
common human pathologies frequently present hypomagnesemia as an associate
condition, efforts will be made to provide a better understanding of the possible
cause-effect relation between hypomagnesemia or magnesium deficiency and the
onset of a specific disease and/or its main complications.

3.1 Hypermagnesemia

Hypermagnesemia is defined as an abnormally elevated Mg2+ level in the blood


[76]. Usually, it is the result of an excess of magnesium in the body. Whole body
Mg2+ homeostasis is the result of equilibrium between absorption (intestine), stor-
age (bones), and excretion (kidneys). Hence, hypermagnesemia is usually the result
of diseases in any of these compartments. Because the kidneys are very effective at
excreting excess Mg2+, hypermagnesemia occurs rarely and is mostly observed
when renal creatinine clearance falls below 30 mL urine per minute. Thus, hyper-
magnesemia develops almost exclusively in patients with kidney failure who are
given magnesium salts or Mg2+-containing drugs such as laxatives or antacids, and
it is usually concurrent with hypocalcemia and/or hyperkalemia [76]. Main symp-
toms are: impaired breathing, hypotension, decreased or absent deep tendon reflexes,
muscle weakness, arrhythmia, and bradycardia as a result of abnormal electrical
conduction at the nervous, muscular, and cardiac level. High serum Mg2+
concentrations are associated with nausea and vomiting, in the attempt to renormal-
ize the electrolyte level. Because some of these symptoms occur following hypocal-
cemia and/or hyperkalemia onset, it is difficult to determine to which extent either
of these two conditions contributes to the appearance of the symptoms. Symptoms
usually worsen based upon the serum Mg2+ concentration: hyporeflexia is present at
serum Mg2+ concentrations ≥4.0 mEq/L, prolonged atrioventricular conduction
occurs at a concentration ≥5.0 mEq/L while heart block and cardiac arrest occur at
concentrations between ≥10 and 13 mEq/L.
One condition in which high levels of serum Mg2+ are usually attained as a result
of therapeutic approach is pre-eclampsia. In this clinical condition, prevention of
pre-eclampic uterine contractions usually requires concentrations between 4.0 and
7.0 mEq/L (≥2.5 and 4.5 mM, respectively) [77]. Serum Mg2+ concentrations at
which maternal toxicity but also neonate depression, hypotonia, and low Apgar
scores are observed are usually in excess of 7.0 mEq/L [77]. Loss of patella reflex
occurs between 7.0 and 10 mEq/L; respiratory depression between 10.0 and 13.0
3 Magnesium in Health and Disease 57

mEq/L: altered atrioventricular conduction and heart block at 15.0 and 25.0 mEq/L,
and cardiac arrest at >25mEq/L.

3.1.1 Hypermagnesemia in Renal Failure

Clinical evaluation of a cohort of patients on hemodialysis indicates that serum


Mg2+ concentration lower than 2.77 mg/dL (1.14 mmol/L) is a significant predictor
for increased all-cause mortality. This mean serum Mg2+ concentration would be
considered indicative of mild hypermagnesemia in the healthy population. Hence, it
appears that a serum Mg2+ concentration higher than normal in hemodialysis patients
will be largely asymptomatic because of better survival of these patients under
hemodialysis conditions [78]. Consistent with this interpretation, serum Mg2+ levels
lower than 1.14 mmol/L appear to be significantly associated with the presence of
vascular calcifications of the hand arteries in the absence of other concauses.
Overall, these results suggest that higher than normal serum Mg2+ concentrations
may play an important protective role in the development of vascular calcification
in hemodialysis patients [78]. Results from a longitudinal study in end-stage renal
disease patients suggest that hypermagnesemia may retard the development of arte-
rial calcifications in this pathological state [79]. Significantly lower values of carotid
intima-media thickness and aortic pulse wave velocity values, two surrogate mark-
ers for vascular calcification, have been observed in patients affected by chronic
kidney disease (CKD) and presenting high serum magnesium levels (0.90–1.32
mmol/L, or 2.18–3.21 mg/dL), indicating a lower arteriosclerotic burden associated
with a lower risk of cardiovascular events and mortality [80]. Consequently, CKD
patients with mildly elevated serum Mg2+ levels could have a survival advantage
over those with lower magnesium levels [80].
Aside from renal insufficiency/failure, conditions predisposing to hypermagne-
semia have been identified with hemolysis, lithium intoxication, adrenal insuffi-
ciency, and hyperparathyroidism. Yet, a full understanding of the mechanisms
whereby these clinical conditions predispose to hypermagnesemia is yet lacking.

3.2 Hypomagnesemia

Hypomagnesemia (or hypomagnesaemia) is an electrolyte disturbance character-


ized by an abnormally low level of magnesium in the blood [81]. Normal serum
Mg2+ levels in humans ranges between 1.5–2.5 mg/dL (or 1.0–1.2mmol/L) [45].
When the serum Mg2+ level is lower than 0.7 mmol/L, we refer to the condition as
hypomagnesemia. This term strictly refers to the Mg2+ level in the serum, and is not
and should not be equated to magnesium deficiency, although the two conditions
can be related in specific patients. Magnesium deficiency refers to an intake of
dietary magnesium below minimal levels, and can result in numerous symptoms
58 Romani

and diseases. The majority of the symptoms and conditions can generally be remedied
by increasing Mg2+ in the diet or via oral supplements. In the most severe cases,
intravenous Mg2+ supplementation is necessary to rapidly restore Mg2+ level within
tissues and serum. Although hypomagnesemia is usually indicative of a systemic
magnesium deficit, hypomagnesemia can be present without Mg2+ deficiency, and
vice versa. Hence, three distinct conditions can be observed:
(a) Hypomagnesemia without magnesium deficiency
(b) Hypomagnesemia with magnesium deficiency
(c) Magnesium deficiency without hypomagnesemia
Hypomagnesemia may result from a number of conditions including inadequate
intake of magnesium, chronic diarrhea, malabsorption, chronic stress, alcoholism,
and (ab)use of medications such as diuretics or antacids of the proton pump inhibitor
family (e.g., omeprazole and similar).
The most common signs and symptoms of hypomagnesemia are: muscle weak-
ness, muscle cramps, cardiac arrhythmia, increased irritability of the nervous sys-
tem, with tremors, athetosis, jerking, nystagmus, and extensor plantar reflex.
Additionally, confusion, disorientation, hallucination, depression, epileptic fits,
hypertension, tachycardia, and tetany may be present in a significant percentage of
cases. Usually, symptoms are bland or not existent when hypomagnesemia is
between 0.5 and 0.7 mmol/L, to become more apparent and severe when magnese-
mia falls below 0.5 mmol/L [82].
Magnesium deficiency is not uncommon in hospitalized patients. Ten to twenty
percent of all hospitalized patients and 60–65% of patient in intensive care units
(ICU) have hypomagnesemia. The condition is usually under-diagnosed because (i)
testing for serum magnesium levels is not routine, and (ii) not always lower cellular
Mg2+ content correlates with low serum Mg2+ level. Low levels of Mg2+ in blood
may be the result of low Mg2+ content in the diet, defective Mg2+ absorption in the
intestines, or increased Mg2+ excretion by the kidneys.
Magnesium deficiency and hypomagnesemia is often observed in acute myocar-
dial infarction, usually within the first 48 hours after a heart attack, or as the result
of drug and medication intake, or gastrointestinal and renal causes.
Drugs: Alcohol intake is one of the primary causes of hypomagnesemia. Hypo-
magnesemia occurs in 30% of patients with alcohol abuse and 85% with delirium
tremens, due to malnutrition, chronic diarrhea, and direct effect of alcohol on liver,
muscle tissues, and neurons. Alcohol also stimulates renal Mg2+ excretion, which is
also increased because of ketoacidosis, hypophosphatemia, and hyperaldosteronism
resulting from liver disease. Hypomagnesemia is also observed in severe cases
of thiamine deficiency because magnesium is required to transform thiamine into
thiamine pyrophosphate.
Medications: Loop and thiazide diuretics; antibiotics that block Mg2+ resorption in
the loop of Henle (i.e., aminoglycoside, gentamicin, tobramycin, amphotericin,
pentamidine, viomycin); proton pump inhibitors (i.e., omeprazole); digitalis; cate-
cholamine and adrenergic agonist; cisplatin and cyclosporin, which both stimulate
renal excretion; and insufficiency in selenium, vitamin D, and vitamin B6.
3 Magnesium in Health and Disease 59

Gastrointestinal causes: The distal portion of the digestive tract secretes high
levels of magnesium. Thus, hypomagnesemia can occur as the result of secretory
diarrhea in Crohn’s disease, ulcerative colitis, Whipple’s disease, and celiac sprue.
Magnesium loss also occur in cases of malabsorption and acute pancreatitis.
Renal causes: Renal magnesium loss is observed in Gitelman/Bartter’s syndrome,
postobstructive diuresis, diuretic phase of acute tubular necrosis, and in kidney
transplant. Massive urinary Mg2+ loss is observed in ~40% of diabetic patients, most
likely as the result of glycosuria and ketoaciduria.
The following pages present an overview of the most common pathologies asso-
ciated with low Mg2+ content within tissues or in the circulation. Where possible, an
indication of the role of hypomagnesemia or low cellular Mg2+ content for the onset
of the main pathology or its complications will be provided.

3.2.1 Cardiovascular Pathologies

Reduced serum Mg2+ content has often been observed in several cardiac and cardio-
vascular pathologies including acute myocardial infarction, specific forms of
arrhythmias, and hypertension. Because the association is usually observed a poste-
riori, at the time the patient seeks medical attention for the concurrent cardiovascu-
lar pathology, it is difficult to determine whether reduced Mg2+ content in the blood,
and perhaps within the affected tissue, is an epiphenomenon or has any causal con-
nection with the onset of the disease or its manifestation.

3.2.1.1 Cardiac Arrhythmias

The effects of low Mg2+ levels on cardiac rhythm have been studied for more than
70 years. Magnesium plays an essential role in maintaining normal cardiac electro-
physiology, mostly by acting as a natural Ca2+ channel blocker or as an antagonist
for Na+, thus limiting the cellular content of this cation. Consequently, it is
hypothesized that inadequate serum and tissue Mg2+ concentrations contribute to the
onset of various cardiac arrhythmias. Among these, we can list ventricular tachycar-
dia (VT), ventricular fibrillation (VF), long QT and torsades de pointes, as well as
atrial and ventricular extra systoles or premature beats, all conditions predisposing
to sudden cardiac death. Magnesium deficit is also observed in the setting of con-
gestive heart failure (CHF), which affects more than 5 million people just in the US,
and in the setting of hypertension, which affects more than 30 million Americans.
In the case of CHF, approximately 600,000–700,000 new patients are diagnosed
with the disease every year. These patients have a very high propensity for ventricu-
lar arrhythmias, which represent one of the prominent causes of death in the group,
and are frequently linked to hypomagnesemia. In these patients, Mg2+ deficiency
may result from elevated circulating levels of catecholamines, aldosterone, and
vasopressin, and from increased urinary Mg2+ excretion consequent to diuretic and
digoxin therapy. With the exception of spironolactone and other diuretics that spare
60 Romani

potassium and magnesium, the treatment with diuretics of the thiazide family
(the most commonly used) increases urinary Mg2+ excretion by a minimum of 25%
to as much as 400% above basal level. This increased loss of Mg2+ affects the
response to digitalis therapy in CHF patients, who may eventually necessitate a dose
that is twice the amount administered to CHF patients with normal serum Mg2+ level
to control cardiac performance and rhythm. The concomitant administration of
magnesium instead can reduce the dose of digitalis required to control the disease,
therefore decreasing the risk of toxicity.
Several forms of arrhythmias including ventricular tachyarrhythmias and tors-
ades de pointes are attenuated or sedated with Mg2+ replacement or Mg2+ boluses
[83]. Examination of the effects of pharmacological i.v. doses of Mg2+ on heart rate
and rhythm suggests an inverse relationship between sudden death from arrhyth-
mias and serum Mg2+ levels, prompting the idea that patients with low Mg2+ levels
may require Mg2+ administration either orally or parenterally. Candidates for i.v.
Mg2+ treatment include patients that respond less than optimally to conventional
antiarrhythmic therapy. The notion that arrhythmias precipitated by digitalis can be
effectively reversed by injections of magnesium dates back to 1930 [84], and has
been confirmed by several other studies thereafter (reviewed in [85]). Similarly,
several studies have found that the use of oral Mg2+ may decrease the risk of arrhyth-
mias associated with long QT syndrome [86], coronary artery disease [87], and
mitralic valve replacement [88]. Interestingly, patients that received an oral combi-
nation of magnesium and potassium supplementation presented significant increase
in the serum concentration of both cations. Hence, it would appear that due to the
simplicity, cost-effectiveness, and safety of magnesium salts, such a supplementa-
tion could be a first-line option for treating patients with frequent but not life-
threatening ventricular tachyarrhythmias. Several trials have attempted to delineate
the usefulness of Mg2+ supplementation in other cardiovascular diseases including
myocardial infarction and coronary diseases [87]. The results of the studies, how-
ever, have been inconsistent and inconclusive [89], not fully supporting the imple-
mentation of Mg2+ treatment for these diseases. Whether this lack of results depends
on the severity of the condition, the bioavailability of Mg2+, or the possibility that
for certain diseases Mg2+ supplementation is more preventive than curative, still
remains to be elucidated.

3.2.1.2 Hypertension

Several epidemiological studies have highlighted an inverse relationship between


serum Mg2+ level and hypertension, with higher blood pressure values being observed
in the presence of lower Mg2+ levels [90]. Because serum Mg2+ level represents <1%
of total Mg2+ content, from this relationship it has been inferred that Mg2+ is low not
only in the circulation but also within tissues. In good agreement with this hypothe-
sis, several authors have observed that hypertensive patients have reduced Mg2+ con-
tent within erythrocytes [91,92], platelets [93], and other cell types. In the majority
of these studies, the decrease in cellular Mg2+ content was associated with an increase
in cellular Na+ and/or Ca2+. In keeping with these results, using an in vitro system
3 Magnesium in Health and Disease 61

Altura and coworkers demonstrated that low Mg2+ causes vasoconstriction of blood
vessels while increasing external Mg2+ content dilates the vessels and blocks vaso-
constriction induced by epinephrine or other agents [94]. Further, Resnick and his
group observed intracellular Mg2+ deficit in hypertensive patients [95], and noted that
blood pressure was inversely related to basal fasting cellular free Mg2+ level irrespec-
tive of whether the patients were hypertensive or normotensive. These results paral-
leled a report from a Swiss group showing that blood pressure is directly proportional
to cellular free Ca2+ [96], along the lines that Ca2+ and Mg2+ have opposite effects
within tissues and that their tissue contents are inversely related. Despite the consis-
tency of these reports, evidence for a direct and clear effect of Mg2+ supplementation
on blood pressure has remained largely inconclusive.
Recently, Kass et al. [97] undertook an extensive reviewing of this topic by
screening 141 relevant articles in the literature. By applying several stringent crite-
ria, the authors narrowed down the pertinent articles to 22, which were used for their
meta-analysis. These articles provided information from 12 different countries
around the world, and from studies with both parallel (13) and cross-over (10)
design. Although not all individual trials showed significant reduction in blood pres-
sure, the combination of all trials indicated a significant decrease in both systolic
(3–4 mmHg) and diastolic (2–3 mmHg) blood pressure, the effect becoming more
evident in trials of cross-over design and with an intake >370 mg/day [97]. In addi-
tion, Mg2+ appeared to have a more pronounced blood pressure lowering effect
when it was administered with normal to high potassium intake and with low sodium
intake [97]. A similar beneficial effect has been observed when Mg2+ has been
administered with taurine, and attributed to the ability of these two agents to reduce
intracellular Ca2+ and Na+ levels [98]. Whatever the mechanism, patients with higher
24 h urinary levels of Mg2+/creatinine and taurine/creatinine presented significantly
lower incidence of cardiovascular risks including cerebrovascular accidents, coro-
nary heart disease, congestive heart failure, and myocardial infarction [98].
Presently, not a single comprehensive hypothesis on how Mg2+ exerts its
antihypertensive effect is available. An in-depth reviewing of the possible modali-
ties of Mg2+ action in the field suggests that Mg2+ can exert its effect through differ-
ent mechanisms. In addition to the mentioned ability of Mg2+ to operate as a natural
Ca2+-channel blocker, which explains the observed antithesis of free Ca2+ and Mg2+
levels within cells and their direct and inverse relationship, respectively, with hyper-
tension, several other possibilities are at hand. Cellular Mg2+ level has been reported
to inversely relate to IP3-mediated mobilization of reticular Ca2+ and Ca2+-ATPase
activity [99], and to reactive oxygen species formation [100], the enhancement of
both processes being implicated with increased vascular tone and hypertrophic vas-
cular remodeling. Additional effects observed at the vasculature level include
increased production of nitric oxide [101] and prostacyclins [102], which promote
endothelium-dependent and endothelium-independent vascular relaxation. Other
possible mechanisms of action for Mg2+ include antiinflammatory and antioxidative
effects, modulation of cell growth [103], and reduction of circulating LDL levels
and cholesterol delivery to endothelial cells [104].
All these mechanisms can have direct implications for atherosclerosis onset
and progression and for maintenance of proper vascular structure and function.
62 Romani

Yet, comparison of serum Mg2+, vascular dysfunction, hypertension and atherosclerosis


has failed to support a direct association among these parameters. Thus, low serum
Mg2+ level is not currently considered a risk factor for the development of these
conditions [105]. In fact, several studies have reported no differences in serum Mg2+
level in hypertensive patients (reviewed in [106]). Thus, evidence is there that not all
hypertensive patients are hypomagnesemic, and not all Mg2+-deficient patients are
hypertensive. Some hypertensive subgroups, however, consistently present altered
Mg2+ homeostasis. These subgroups include African-Americans or individuals of
African descent, elderly patients, or patients with malignant hypertension, meta-
bolic syndrome, or obesity [107]. While emphasis has been placed in identifying
genetic causes of hypertension, limited information is available as to whether
genetic alterations of Mg2+ homeostasis and transport play a significant role in
hypertension onset and progression in the indicated subgroups. In this respect, the
group of Touyz has proposed that dysregulation or alteration of the Mg2+ entry chan-
nel TRPM7 may play an important role in abnormal cellular Mg2+ homeostasis in
hypertension [107]. This group, in fact, observed an altered magnesium influx in
vascular smooth muscle cells in SHR rats, associated with down-regulation of
vascular TRPM7 [107] as well as cardiovascular and renal remodeling, fibrosis, and
inflammation associated with down-regulation of renal TRPM7 following infusion
of aldosterone in mice [108]. Noteworthy, several of these responses were amelio-
rated by dietary Mg2+ supplementation [109]. The studies of Touyz’s group have
predominantly been carried out in animals. Yet, the obtained results forebode the
likelihood that similar changes and alterations also occur in the human setting.

3.2.2 Hyperaldosteronism

The results discussed at the end of the previous paragraph shed some light on the
clinical observation that hyperaldosteronism is one of the main endocrinopathies
associated with hypomagnesemia [10]. The disease is characterized by urinary Mg2+
loss and low level of circulating Mg2+, but the mechanisms behind these events are
not fully elucidated. One possibility is that the Mg2+ loss is due to the elevated Na+
retention resulting from aldosterone hypersecretion, which exchanges with cellular
Mg2+ perhaps through the Na+/Mg2+ exchanger, triggering a significant loss of
cellular Mg2+ in various tissues. At the same time, it is conceivable that urinary Mg2+
loss depends on a defect in expression or activity of the ubiquitous Mg2+ entry chan-
nels TRPM7 but also TRPM6, which is deputed to specifically reabsorb Mg2+ in the
distal convolute tubule of the nephron. This possibility is supported by the data from
Touyz’s group [107] mentioned above.

3.2.3 Diabetes

Diabetes is one of the best known diseases that induces Mg2+ loss in both animals
and humans. Despite the large body of evidence in the medical literature, the majority
of the reports are correlative at best.
3 Magnesium in Health and Disease 63

Both type-1 (T1DM) and type-2 diabetes (T2DM) are characterized by hypo-
magnesemia, hypermagnesuria, and lower Mg2+ level within tissues. Because
T2DM presents the majority of all the diabetic cases diagnosed every year in the
human population, more attention has been paid to this condition, in the attempt to
determine whether the Mg2+ loss observed in the disease is a predisposing condition
or an epiphenomenon.
Insulin has long been recognized as one of the hormones playing a major role in
regulating cellular Mg2+ homeostasis. Experimental and clinical evidence indicates
that insulin increases cellular Mg2+ content although the mechanism of action is not
completely clear. One possibility evidenced by Romero and collaborators in eryth-
rocytes is that insulin can directly modulate the Na+/Mg2+ exchanger [110].
Alternatively, insulin could increase cellular Mg2+ indirectly by enhancing cellular
K+ content while decreasing cellular Na+ content [111]. It is currently unclear
whether insulin has a direct effect on the expression and activity of TRPM6 and
TRPM7. Recent epidemiological studies, however, indicate the occurrence of defec-
tive mutations in the intestinal expression and activity of TRPM6 involved in dietary
Mg2+ absorption [112] in a cohort of diabetic women.
Magnesium accumulation directly correlates with the rates of glucose accumu-
lation within tissues following insulin administration. This has been observed in
liver cells [113], cardiac myocytes [114], and β-cells [115]. Moreover, experi-
ments conducted in our laboratory have consistently indicated that the decrease in
tissue Mg2+ content observed in T1DM animals correlates directly with the level
of K+, but inversely with the level of Na+ and Ca2+ within liver, skeletal muscle,
and heart [75], i.e., tissues directly involved in controlling glycemia. In addition,
the extrusion rate of Mg2+ from diabetic hepatocytes [75] and cardiac myocytes
[116] is dramatically enhanced both in intact cells and in purified plasma mem-
brane [117], and is renormalized by the exogenous insulin administration [116],
or artificial increase of glucose and glycogen within plasma membrane vesicles
[117]. Diabetic animals also exhibit a marked increase in urinary Mg2+ loss [75],
mimicking what has been reported to occur in diabetic patients. Whether this
effect depends on the absence of an insulin stimulatory effect on renal Mg2+
reabsorption is presently undefined.
The interplay between insulin and Mg2+ is reciprocal in that Mg2+-deficient
animals present reduced levels of insulin receptor phosphorylation [118], at least
in skeletal muscles, with consequent reduction in muscle glucose accumulation.
Determination of cellular [Mg2+]i under similar experimental conditions indicates
the reduction of cytosolic Mg2+ from physiological ≥0.7 mM [45] to half those
values [119], thus affecting several Mg2+-dependent enzymes requiring phosphor-
ylation. In addition, these conditions can set the basis for reduced insulin-stimu-
lated cellular metabolism, and predispose to insulin resistance. Conversely,
Mg2+ addition can restore several of these dysmetabolic conditions, if not all. In
particular, Mg2+ intake appears to be directly and significantly associated with
insulin sensitivity in a threshold fashion [120]. Overall, these results strongly
suggest that cellular Mg2+ deficiency can actually be the cause rather than the
result of insulin resistance.
64 Romani

In keeping with the dependence of Mg2+ accumulation on an effective glucose


uptake, hypomagnesemia appears to be prevalent in individuals with poor glycemic
control [120], and actually relate inversely to the effectiveness of the metabolic
control and glycated hemoglobin (HbA1C) levels [120]. On the other hand, several
treatments for T2DM appear to increase cellular Mg2+ levels. Oral antidiabetic drugs
such as metformin or pioglitazone increase Mg2+ levels within hepatocytes [121] or
adipocytes [122] as well as circulating Mg2+ levels.

3.2.3.1 Diabetes Complications

Mg2+ deficiency has also been implicated as a predisposing factor to the onset and
development of diabetic complications, along the lines of what has already been
indicated for hypertension. Inflammation, atherosclerosis, oxidative stress, i.e., the
main functional and metabolic changes observed in hypertensive patients, also play
an essential role in the progression of diabetic cardiomyopathy, nephropathy,
neuropathy, and retinopathy. All these complications as well as diabetic hyperalgesia
are attenuated to a varying extent by magnesium supplementation [123].

3.2.4 Metabolic Syndrome

About 20 years ago, Resnick formulated the ‘ionic hypothesis’ for hypertension and
other metabolic disorders. Based on his hypothesis, hypertension, insulin resistance,
and type 2 diabetes are associated with an increase in intracellular Ca2+ and a
decrease in intracellular Mg2+ [124]. Yet, the exact mechanisms behind the onset of
the metabolic syndrome are not completely defined. Most of the patients are of
middle age, sedentary, and with varying degrees of obesity, mostly central obesity,
and insulin resistance. Stress is considered a contributing factor. The most important
factors implicated in the disease onset are weight gain, genetics, endocrine disor-
ders (e.g., polycystic ovary syndrome in women of reproductive age), aging, and
sedentary lifestyle, (i.e., low physical activity and excess caloric intake). The exact
sequence of events is also not clear. Is it obesity or insulin resistance that causes the
metabolic syndrome? Or, does the metabolic syndrome cause obesity and insulin
resistance? Or, are these three conditions an expression of a more far-reaching meta-
bolic and hormonal derangement? To complicate the issue, several inflammatory
markers including C-reactive protein, interleukin 6 (IL-6), and tumor necrosis factor
α (TNFα) are usually increased in these patients [125].
Irrespective of whether the metabolic syndrome (aka syndrome X) is cause or
consequence of insulin resistance and obesity, all three conditions are associated
with a deranged cellular and serum Mg2+ homeostasis. Due to the limited number
of studies on the topic, it is unclear whether Mg2+ deficiency predisposes to the
disease or it is the results of the incurrent dysmetabolism and/or insulin resistance
(see previous paragraph).
3 Magnesium in Health and Disease 65

3.2.5 Alcoholism

Alcoholism is another of the most common human pathologies associates with


Mg2+ deficiency. Experimental evidence indicates that in the case of alcoholism, it
is the alcohol consumption that induces Mg2+ loss from tissues, and ultimately with
the urine. This pattern has been observed in both human patients and in animal
models. At the liver level, ethanol administration induces a marked decrease in
cytoplasmic ATP content through the change in pyridine nucleotides associated
with ethanol metabolism by the alcohol dehydrogenase [126]. This effect occurs in
a time- and dose-dependent manner [126]. The observed decrease in ATP content
removes an essential complexing agent from the cell cytoplasm that ultimately
results in an increase in cellular free [Mg2+] and a detectable Mg2+ extrusion from
the hepatocyte via the Na+/Mg2+ exchanger [126]. Inhibiting the exchanger or the
alcohol dehydrogenase prevents Mg2+ loss [126]. Similar effects have been
observed following acute [126] and chronic [127] ethanol administration, as well
as addition of repeated doses of alcohol with a small interval in between [128].
More importantly, acute and chronic ethanol administrations exert an inhibitory
effect on the protein kinase C signaling involved in favoring Mg2+ accumulation
[129]. As a result of this inhibition, Mg2+ cannot be effectively accumulated within
the cell until ethanol has been removed from the system and protein kinase C can
properly translocate to the cell membrane [129]. This inhibitory effect lasts for
more than 1 hour following an acute ethanol administration [129], and for almost
two weeks in a chronic ethanol model [129]. Under both acute and chronic condi-
tions, Mg2+ is lost from the cytoplasm as well as mitochondria and endoplasmic
reticulum [130].
Magnesium losses qualitatively similar to those observed in the liver have been
reported to occur in other tissues including skeletal muscles [131], vascular smooth
muscle cells [132], and neurons [133], and they have been associated with the high
incidence of vasospasm and stroke plaguing chronic alcoholics, and possibly delir-
ium tremens insurgence [134]. In the case of the skeletal muscles, alcohol acceler-
ates protein catabolism and muscle atrophy but it is presently unclear whether Mg2+
loss plays any role in the process (reviewed in [135]).
Magnesium deficit appears to play a significant role in modulating the inflamma-
tory response induced by EtOH. Physiological Mg2+ levels inhibit the release of
proinflammatory cytokines while promoting production and release of antiinflam-
matory cytokines [136]. Because inflammation plays a key role in the onset of ste-
atohepatitis and its progression towards alcohol liver disease (ALD) [137], it can be
easily hypothesized that cellular and systemic Mg2+ deficit modulates the immune
response of liver resident macrophages (Kupffer), and circulating monocytes, and
lymphocytes, with major consequences for liver function and cyto-architectonics.
Lastly, physiological Mg2+ levels have been related to proper cell cycle progression
[103]. Hence, it is possible that lower than normal cellular Mg2+ levels affect hepa-
tocyte regeneration following alcohol-induced liver apoptosis [137].
While alcohol administration promotes Mg2+ wasting, Mg2+ supplementation
ameliorates several neuronal, muscular, and hepatic biochemical functions. Because
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of the variety of symptoms and functions ameliorated by Mg2+ supplementation, it


would appear that Mg2+ acts as a coenzyme in biochemical reactions but also as a
regulator of cellular signaling pathways such as adenylyl cyclase [138], protein
kinase C [139], and Erk1/2 MAPKs [140]. In the case of the heart, for example,
dietary Mg2+ supplementation ameliorates the myocardial dysfunction induced by
acute or chronic ethanol administration, and renormalizes heart size as well as iso-
metric force and isotonic shortening [141]. The mechanism(s) behind these effects
are not elucidated. Magnesium is considered to act as a natural Ca2+-channel blocker.
Thus, it is possible that the changes in force development and cell shortening depend
on the restoration of a normal cellular Ca2+ level that directly impacts the contractile
myofilaments. Less clear is whether the renormalization of heart size depends on
the restoration of normal cellular Ca2+ levels or, alternatively, on direct effects of
Mg2+ on protein synthesis and mRNA translation.

3.2.6 Inflammation

As indicated in the previous paragraph, seminal work by different laboratories


supports the notion that an increase in systemic inflammation is associated with
Mg2+ deficit. This response is characterized by increased serum levels of TNFα and
inflammatory cytokines [136] while the production and release of antiinflammatory
cytokines is reduced [136]. Currently, two main mechanisms are invoked to explain
the increase in inflammatory cytokines in the case of Mg2+ deficiency: (i) the Ca2+-
channel blocking effect of Mg2+ is attenuated, resulting in increased Ca2+ entry
within the immune-competent cells, with enhanced cell priming towards an inflam-
matory phenotype/response, and/or (ii) the reactive oxygen species production,
which is increased under Mg2+ deficiency conditions through non fully-elucidated
mechanism(s), promotes membrane oxidation and activation of NFκB [142].
Irrespective of whether these two mechanisms cooperate or act independently to
increase the inflammatory response, the net result in an increased production of
inflammatory cytokines that can be detected in the circulation. In addition, Mg2+-
deficient animals are more susceptible to septic shock [143]. Administration of lipo-
polysaccharide (LPS) results in a mortality rate in excess of 70% within 3 hours in
these animals as compared to no lethal effect in control animals [143]. Magnesium
supplementation prior to LPS administration significantly increases the survival
rate of the animals.
In agreement with the above observation, Altura and collaborators reported that
Mg2+ deficiency results in an increased production of specific cytokines [144] via de
novo synthesis of ceramide in vascular smooth muscle cells [145], and that inhibi-
tion of ceramide synthesis attenuates NFκB activation and cytokines production
[145]. Recently, in collaboration with Dr. Bernstein’s laboratory, we have provided
evidence that monocytes from women undergoing preterm labor synthesize elevated
levels of pro-inflammatory cytokines, and that pharmacological doses of MgSO4,
commonly used as a tocolytic agent to stop preterm labor, completely block
cytokines production [146,147].
3 Magnesium in Health and Disease 67

All together, these results indicate that optimal levels of Mg2+ are key to modu-
late systemic and local inflammation by regulating inflammatory cytokine produc-
tion and release.

3.2.7 Renal Pathologies

The kidney plays an important role in controlling human body Mg2+ content through
reabsorption in the Henle’s loop and the distal convolute tubule. Thus, it is not sur-
prising that several renal diseases impact the ability of the organ to reabsorb Mg2+
to a significant extent, causing Mg2+ wasting and Mg2+ deficit. In the following
paragraphs, the most common renal Mg2+ handling diseases and the predominant
location within the nephron will be commented. We refer to several recent reviews
for a more in-depth description of the causes and complications [148,149].

3.2.7.1 Bartter’s Syndrome

Bartter’s syndrome is characterized by Na+ and Cl– wasting, hypokalemia, meta-


bolic alkalosis, and increased production of renin and aldosterone [150]. The dis-
ease affects the thick ascending limb (TAL) of the Henle’s loop, and is the result of
autosomal recessive mutations of various genes involved in Na+ and Cl– transport
including that encoding for the Na+/K+/Cl– co-transporter (NKCC2 or SLC12A1).
The disease is associated with hypermagnesuria and hypomagnesemia in approxi-
mately 50% of the cases. The precise explanation for this variability is not clear,
especially if we consider that all the various forms of Bartter’s syndrome are
characterized by inhibition of ion transport in the TAL and by a varying level of
dissipation of the electrochemical gradient that drives the reabsorption of divalent
cations such as Ca2+ and Mg2+. Consequently, it is difficult to determine the electro-
chemical gradient responsible for Mg2+ but also Ca2+ reabsorption in the TAL since
the associated polyuria will determine volume depletion and changes in tubular and
systemic ionic concentrations. Moreover, patients presenting specific mutations of
the chloride channel (CLC-Kb) located in the basolateral domain of the distal
convolute tubule (DCT) present a combined phenotype of Bartter’s syndrome plus
Gitelman’s syndrome, and more persistent hypomagnesemia [151]. The reason as to
why mutations in the CLC-Kb channel in this segment distal to the TAL are associ-
ated with such a phenotype is not completely understood.

3.2.7.2 Gitelman’s Syndrome

Gitelman’s syndrome is a salt wasting condition characterized by metabolic and


ionic conditions reminiscent of the Bartter’s syndrome. Also in this condition the
production of renin and aldosterone are increased but to a lower extent than in the
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Bartter’s syndrome. The disease affects specifically SLC12A3 isoform, located in


the DCT. The syndrome can be mimicked by the chronic administration of thiazide
diuretics, which specifically block the SLC12A3 encoded transporter NCC [152].
The patients affected by this syndrome present consistently hypomagnesemia, and
a decreased expression of the Mg2+ selective channel TRPM6 in the DCT has been
indicated as the most likely reason for the Mg2+ deficit. However, the mechanism(s)
responsible for the reduced TRPM6 expression remain(s) speculative at the moment.
Aldosterone has been indicated as a possible culprit of the reduced channel expres-
sion, as the secretion of this hormone is increased in Gitelman’s syndrome, and the
treatment with spironolactone, which antagonizes aldosterone, ameliorates urinary
Mg2+ loss, and increases serum Mg2+ level.

3.2.7.3 Defects in Claudin Expression

Defects in paracellin-1 (a.k.a. claudin 16, encoded by CLDN16) result in an autoso-


mal recessive disorder termed familial hypomagnesemia with hypercalciuria and
nephrocalcinosis, or FHHNC [47]. These defects consist in single amino acid muta-
tion of this protein, which forms tight junction in the TAL and the DCT. Based upon
the single amino acid mutation, a more or less severe phenotype is observed, with a
variable degree of urinary Mg2+ and Ca2+ loss. These mutations affect the regulation
of the tight junction by cAMP and consequently the reabsoprtion of Mg2+ and Ca2+
by cAMP-mediated hormones like parathyroid hormone.
Claudin 19 contributes to form tight junction in the TAL and DCT by forming a
heterotetramer with claudin 16 [48]. Consequently, single amino acid mutations in
claudin 19 sequence have also been associated with Mg2+ wasting and hypomagne-
semia [48].

3.2.7.4 Defects in TRPM6 Expression

As indicated in Sections 2 and 3, TRPM6 constitutes the Mg2+ entry mechanism of


choice in the DCT and in the distal portion of the intestine in which it promotes
Mg2+ reabsorption and absorption, respectively.
Autosomal recessive mutations in this gene product are cause of hypomagnese-
mia with secondary hypocalcemia (HSH), with consequent increase in neuromuscu-
lar excitability, muscle spasm, tetany and convulsions [11]. Interestingly,
supplementation with high doses of Mg2+ is sufficient to renormalize calcemia while
serum Mg2+ levels remain suboptimal and indicate a defective intestinal absorption
of Mg2+ via TRPM6. It has to be noted that urinary Mg2+ wasting may not be notice-
able in most patients under day-to-day conditions, becoming more detectable fol-
lowing the supplementation with high doses of Mg2+ especially if administered
intravenously [51].
3 Magnesium in Health and Disease 69

3.2.7.5 Defects in Epidermal Growth Factor Signaling

Hypomagnesemia associated with psychomotor and mental retardation, and epileptic


seizures has been observed in patients with mutation in the EGF (epidermal growth
factor) gene [52]. Because of these mutations, the patients present a defective secre-
tion of EGF in the interstitium and a limited or absent activation of the EGF receptor
in the DCT [52]. Subsequent studies have indicated that EGF-receptor activation
promote Mg2+ entry through TRPM6 [54] by enhancing the channel expression in
the cell membrane from the endosomal compartment. As described more in details
in the following Section 3.3.2, this EGF/EGFR/TRPM6 pathway becomes inacti-
vated by specific monoclonal antibodies used therapeutically to treat metastatic
forms of colon cancer.

3.2.8 Magnesium and Tumors

Magnesium plays an essential role in numerous cell functions including progression


through the cell cycle [103]. Since the 1970s, the group of Rubin has advocated an
essential role of Mg2+ as a regulator of cell proliferation and protein synthesis irre-
spective of the cell type [153,154]. Results from this laboratory and from the groups
of Touyz, Maier, and Wolf have provided significant lines of evidence that low lev-
els of cellular Mg2+ impact the ability of the cells to properly synthesize proteins and
to progress through the various mitotic steps [103,142,154,155]. Conversely, an
increase in cellular Mg2+ correlated well with DNA and protein synthesis, and with
tissue growth, while quiescent tissues present lower levels of Mg2+.
Because of this role of Mg2+ in cell growth, the attention of several researchers
has obviously focused on the possible involvement of Mg2+ in tumor development
and metastatization. The results obtained so far support an intriguing scenario, with
very clear and interesting distinction.
At variance of normal cells, which depend on Mg2+ content and availability for
proper growth, tumor cells are essentially independent on magnesium availability
and stop growing only when extra-cellular Mg2+ is reduced to a very low level (e.g.,
0.2 mM or less [155]). Under these conditions, p21, p27, and p53 cell cycle regula-
tory proteins become up-regulated [155] or activated [156] while the cell cycle pro-
moting proteins cyclin D and cyclin E, and several cyclins-dependent kinases
become down-regulated [155]. These effects are associated to changes in the level
of several MAP kinases including Erk1/2 and p38 [155]. Associated cDNA studies
have indicated that more than 30 genes are affected by up-and-down changes in
Mg2+ content [157]. Many of these genes control cell proliferation while other con-
trol cell-matrix interaction (e.g., integrin), or antioxidant defenses (e.g., glutathione
S-transferase). The latter gene is of particular relevance because it not only contrib-
utes to the antioxidant defenses of the cell but also regulates cell proliferation and
differentiation. Consistent with this widespread inhibitory role of a low Mg2+ level
on cell growth, mice exposed to a Mg2+-deficient diet and grafted for various solid
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tumors exhibited a reduced tumor growth compared to mice maintained on a normal


Mg2+ diet [158]. This observation strikes an interesting correlation with the clinical
evidence that colon cancer patients treated with monoclonal antibodies anti-EGF
receptor presents a reduction in the primary tumor size and in metastases while
presenting hypomagnesemia and increased urinary Mg2+ wasting [159] (see also
Section 3.3.2).
Because of the role of Mg2+ as chemo-attractant for endothelial cells [160], low
Mg2+ levels negatively affect endothelial cell proliferation [161]. In line with this
observation, Mg2+-deficient mice presented less developed and vascularized tumors
[158]. The mechanism responsible for the reduced vascularization and angiogenesis
is not clear. In part, it can be attributed to the reduced ability of endothelial cells to
proliferate and migrate. In part, however, it may depend on the accelerated senes-
cence associated with growth arrest which the endothelial cells and the tumor cells
experience [161]. At the same time, Mg2+ deficiency is associated with a higher level
of basal inflammation, and the higher level of proinflammatory cytokines may cer-
tainly play a role in limiting angiogenesis and accelerating the deterioration of
endothelial cells.
The protective or at least limiting effect of low Mg2+ levels for the growth of
primary tumors and the associated angiogenesis process does not apply to the meta-
statization process. Based upon the reduction in cell growth, a lower number of
metastases would be expected to be present in Mg2+-deficient mice carrying solid
tumors, and many of these metastases would be of smaller size. In fact, quite the
opposite, as a larger number of lung metastases have been observed in Mg2+-
deficient mice [158]. This could be explained by the observed overexpression of
metalloproteinases and other proteinases and the increased expression of vascular
cell adhesion molecules (VCAMs) within the primary tumor cells [162]. On the
other hand, Mg2+ is essential for the proper activity of NM23-H1, an 8 member
genes family with very well established antimetastatic activity [163]. It is therefore
possible that in a low Mg2+ environment this gene complex does not operate effec-
tively in controlling and suppressing the diffusion of metastasis.
Taken together, the effect of Mg2+ deficit or deficiency appears to span to both
sides of the aisle. On one side, Mg2+ deficit limits tumor growth and proliferation
and tumor-related angiogenesis. On the other side, it promotes tissue invasion and
metastatization. In the middle, we have multiple effects of up- and down-regulation
of a variety of enzymes, molecules, and inflammation agents that contribute to the
final outcome in this complex scenario.

3.2.9 Magnesium and Prenatal Pathologies

Magnesium has been widely used for more than 60 years in the US in maternal/
perinatal settings [164]. Its use has been mainly in two areas: (i) preterm labor, and
(ii) preeclampsia.
The rationale behind the use of Mg2+ (mostly MgSO4) in preterm labor is that it
decreases muscle contractility by limiting Ca2+ accumulation within the muscle cell.
The utilization as a tocolytic has been widespread although the mechanism of action
3 Magnesium in Health and Disease 71

is not completely understood and some studies have indicated a limited or nihil
beneficial effect as tocolytic [165].
As for preeclampsia, MgSO4 is commonly used for seizure prophylaxis. In this
case, MgSO4 has to be administered at doses that rapidly increase serum Mg2+ level
to 2.5 mM or higher [166]. Its anticonvulsant effect can be attributed to slowing
down neuromuscular conduction, depression of vasomotor center, and blockade of
peripheral neuromuscular transmission [166]. One of the main complications of
pre-term labor is the occurrence of cerebral palsy in premature infants born as early
as 27 weeks of gestations or earlier [167]. The disease is ~80-fold higher in preterm
delivered babies than in babies delivered at term, and is characterized by permanent
abnormal gross and fine motor functioning. The main cause has been attributed to
disturbances in the developing fetal or infant brain [167]. Several clinical observa-
tions in the 1990s indicated that newborns of very low birth weight exposed to
MgSO4 while in utero, mostly as a tocolytic for preterm labor or as prevention for
eclamptic seizures, presented a much lower incidence of cerebral palsy than new-
borns of similar birth weight not exposed to the agent [168]. The suggestion that
MgSO4 could act as a neuroprotective agent for at risk newborn was the object of
several other studies many of which although not all confirmed the notion. Because
of this inconsistency, it was not until 2009 that the role of MgSO4 as a neuroprotec-
tive agent for at risk newborn was ultimately confirmed [169]. This led the American
College of Obstetrics and Gynecology (ACOG) to issue a committee opinion on the
use of MgSO4 for neonatal neuroprotection, and to establish clear guidelines for the
dosage and modality of administration [170].
While the beneficial effect of MgSO4 treatment in at-risk perinatal conditions
appears to be finally accepted, the mechanism(s) behind this effect is not completely
elucidated. Recent studies by Bernstein’s and our laboratories [146,147] provide
compelling evidence that Mg2+ may act as an antiinflammatory agent on maternal
and perhaps neonatal monocytes, reducing the synthesis and production of circulat-
ing proinflammatory cytokines including TNFα and IL-1 among others by modulating
NFκB signaling. These results are well in line with the reports by the Altura group
discussed earlier [144], coincidentally and independently published at the same time
as ours [146]. Interestingly, high levels of inflammatory cytokines have been reported
to be present in the cerebral fluid of newborns with cerebral palsy [167], thus provid-
ing a far reaching relevance to our reports and to those by the Altura group.
Because of obvious ethical restrictions, however, it is currently undefined
whether the inflammatory cytokines present in the fluids of the newborns are mater-
nal in origin or they are generated endogenously by the newborn’s immune system
in response to proinflammatory stimuli released by the mother.

3.3 Pharmacological Agents Causing Hypomagnesemia

Increasing evidence in the literature indicates that proton pump inhibitors and anti-
EGFR antibodies have become the two fast rising groups of pharmacological agents
inducing hypomagnesemia and magnesium waste in patients.
72 Romani

3.3.1 Proton Pump Inhibitors

The first observation of hypomagnesemia associated with the use of a proton


pump inhibitor (PPI) dates back to 2006. Since then, the incidence of episodes has
progressively increased due to the wide utilization of prescription PPI products
(in excess of 70 million in 2009 [171]) and availability of several PPI products over
the counter. The increasing trend of episodes prompted the FDA to issue a warning
in 2011 to the public indicating that prescription PPI may cause severe hypomagne-
semia when taken for prolonged periods of time (1 year or longer) [172]. Recent
reviewing of the data in the literature indicates that hypomagnesemia: (i) can be
severe (as low as 0.35 mM [173], as compared to the normal lower limit of 0.76 mM
[45]); (ii) it is often (>60%) associated with hypokalemia [173] and hypocalcemia,
the latter springing from altered parathyroid hormone release; (iii) it regresses rap-
idly upon discontinuation of PPIs [173]; but (iv) it reoccurs just as rapidly following
PPIs re-introduction in the therapy [173].
Presently, the mechanism responsible for the onset of hypomagnesemia is not
fully elucidated. Modeling of intestinal Mg2+ absorption under conditions resem-
bling the decrease in intestinal pH elicited by the proton pump inhibitor esomepra-
zole suggests a reduced intestinal absorption of Mg2+ in the distal portion of the
intestine [171]. This inhibition is likely to be the result of a proton neutralizing
effect on carboxyl side chains of glutamic acid and aspartic acid residues deemed
essential for Mg2+ binding and conduction in the pore-forming region of TRPM7
and TRPM6 [171]. Frequent but small doses of Mg2+ supplementation appear to be
beneficial in preserving proper circulating Mg2+ levels following PPI-induced hypo-
magnesemia [174]. Because of the association between low Mg2+ levels and diseases
like diabetes, osteoporosis, hypertension, arrhythmias, and congestive heart failure,
patients taking PPI for extended periods of time should be advised to consistently
monitor their serum Mg2+ level and perhaps discontinue at time the use of the proton
pump inhibitor.

3.3.2 Anti-epidermal Growth Factor Receptor Antibodies

Anti-EGF receptor antibodies represent the second class of pharmaceutical agents


consistently associated with an increased incidence of hypomagnesemia and mag-
nesium waste. These agents are commonly used for the treatment of various forms
of cancer, including cancer of the colon, ovary, lung, prostate, and kidney among
others. Persistent and/or abnormal activation of the EGF receptor has been observed
in all these neoplastic conditions. Following engagement by its ligand, the EGF
receptor becomes activated and recruits several signaling pathways including MEK,
ERK, PI-3-K, STATS, and PLC-γ [175], which are potent oncogenic regulators of
tumor cell growth, invasion, angiogenesis, and metastasis. On the other hand, acti-
vation of the EGF receptor results in the upregulation of TRPM6 via Erk1/2 [54],
thus promoting physiological intestinal absorption and renal reabsorption of Mg2+.
Administration of anti-EGFR antibodies will then inhibit the activation of the
3 Magnesium in Health and Disease 73

oncogenic regulators mentioned above but also Mg2+ absorption and reabsorption,
inducing hypomagnesemia.
As the use of these antineoplastic agents has increased, so has the occurrence of
hypomagnesemia increased [159]. As in the case of the proton pump inhibitors,
hypomagnesemia is associated with hypocalcemia and hypokalemia [159].
Comparison among the different forms of anti-EGFR antibodies indicates that pni-
tumumab, a fully human monoclonal antibody used for metastatic colon cancer,
presents the highest incidence of hypomagnesemia, often as severe as 0.9 mg/dL, or
half the physiological level [176]. The occurring hypomagnesemia does not appear
to lead to major complications other than neuroexcitability and neuromuscular
spasms [176]. Nevertheless, an aggressive treatment in patients with severe hypo-
magnesemia is recommended, with very high doses of magnesium (up to10 g) being
needed to achieve a clinically significant reversal of the symptoms. Weekly Mg2+
administration is usually inadequate as serum Mg2+ return to low baseline level
within 3–4 days. In several patients daily to twice-a-week doses of intravenous Mg2+
as high as 6–10 g/dose have been required, and some of these cases have registered
a continuous or worsening hypomagnesemia despite the treatment [177].
The most likely explanation for such a negative outcome is that as long as the EGF
receptor in the kidney and intestine is inhibited, renal Mg2+ wasting and ineffective
intestinal Mg2+ absorption will persist or worsen. Moreover, because the inhibition of
the EGF receptor is rapid and long-lasting during anti-EGFR therapy, the effectiveness
of intravenous Mg2+ supplementation is largely diminished. Also, patients treated
with these antineoplastic agents show a marked hypoalbuminemia [176]. The causes
for this effect are unknown. Nevertheless, a reduced level of circulating albumin has
a two-fold effect in promoting hypomagnesemia and other ionic alterations: (i) it
exacerbates the loss of Ca2+ and Mg2+ as lower amounts of these cations are protein-
complexed, and (ii) it increases indirectly the doses of anti-EGFR antibodies present
in the circulation, thus promoting more pronounced and long lasting effects of the
antineoplastic agent on the EGFR, and consequently on Mg2+ homeostasis.

4 Conclusions

Due to space constrains, and the complexity of the field, we have only tapped on the
main pathologies and iatrogenic conditions associated with hypomagnesemia and
altered cellular Mg2+ homeostasis, and attempted to provide the reader with a frame-
work to appreciate the perhaps incomplete intricacies of Mg2+ homeostasis and its
regulation, as well as its physio-pathological implications.
Each pathological condition mentioned here has been the topic of several ad hoc
reviews in recent years, which are cited in the preceding sections; we refer the inter-
ested reader to them for a more in-depth evaluation. As our understanding of the
regulation of Mg2+ homeostasis progresses, we are confident that new tools will
become available to properly address the key physiological role Mg2+ plays inside
the cell and in the whole human body.
74 Romani

Abbreviations

ACOG American College of Obstetrics and Gynecology


ALD alcohol liver disease
AP-1 activator protein-1
ATP adenosine 5′-triphosphate
cAMP 3′,5′-cyclic adenosine monophosphate
CaSR calcium sensing receptor
CHF chronic heart failure
CHF congestive heart failure
CKD chronic kidney disease
CVD cardiovascular disease
DCT distal convolute tubule
EGF epidermal growth factor
EGFR epidermal growth factor receptor
Erk extracellular signal regulated kinases
FDA Food and Drug Administration
FHHNC familial hypomagnesemia with hypercalciuria and nephrocalcinosis
HbA1c glycated hemoglobin A1c
HSH hypomagnesemia with secondary hypocalcemia
i.v. intravenous
ICU intensive care unit
IL interleukin
IP3 inositol triphosphate
LPS lipopolysaccharide
MAPKs mitogen activated protein kinases
NFκB nuclear factor kappa-light-chain-enhancer of activated B cells
NKCC 2 Na+/K+/Cl– cotransporter
PKA protein kinase A
PKC protein kinase C
PPI proton pump inhibitor
PTH parathyroid hormone
QT interval between Q and T wave in electro cardiogram.
RACK1 receptor for activated protein kinase 1
REA repressor of estrogen receptor activity
SLC12A1 solute transporter class 12 isoform A1
SLC12A3 solute transporter class 12 isoform A3
T1DM type 1 diabetes mellitus
T2DM type 2 diabetes mellitus
TAL thick ascending limb
TNFα tumor necrosis factor α
TRMP6 transient receptor potential melastatin subfamily isoform 6
TRP transient receptor potential
TRPM7 transient receptor potential melastatin subfamily isoform 7
3 Magnesium in Health and Disease 75

VCAM vascular cell adhesion molecule


VF ventricular fibrillation
VT ventricular tachycardia

Acknowledgements This work was supported by NIAAA-11593 and in part by NIH-HL090969.

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Chapter 4
Calcium in Health and Disease

Marisa Brini, Denis Ottolini, Tito Calì, and Ernesto Carafoli

Contents
ABSTRACT ............................................................................................................................. 82
1 INTRODUCTION ............................................................................................................. 83
1.1 Calcium in Nature and in Living Organisms ............................................................ 83
1.2 Regulation of Calcium in Biological Fluids ............................................................. 84
1.3 Calcium in the Mineralized Compartment of the Organisms ................................... 85
2 GENERAL PROPERTIES OF CALCIUM AS A SIGNALING AGENT ........................ 88
3 INTRACELLULAR CALCIUM HANDLING ................................................................. 93
3.1 Transport of Calcium Across Membrane Boundaries .............................................. 93
3.2 Spatiotemporal Dynamics of the Calcium Signal ..................................................... 94
3.3 Regulation of the Calcium Signal by the Cell Organelles ........................................ 97
4 CALCIUM AS A REGULATOR OF BIOLOGICAL PROCESSES ................................ 100
4.1 Gene Transcription.................................................................................................... 100
4.2 Intracellular Proteolysis ............................................................................................ 101
4.3 Protein Phosphorylation and Dephosphorylation ..................................................... 103
4.4 Calcium and Bioenergetics ....................................................................................... 106
4.5 Muscle Contraction ................................................................................................... 108
4.6 Secretion ................................................................................................................... 110
4.7 Calcium in the Beginning of Cell Life...................................................................... 112
4.8 Apoptotic Cell Death and Autophagy....................................................................... 113

M. Brini (*) • D. Ottolini • T. Calì


Department of Biology, University of Padova, Via U. Bassi 58/B, I-35131 Padova, Italy
e-mail: marisa.brini@unipd.it
E. Carafoli (*)
Venetian Institute of Molecular Medicine (VIMM), Via G. Orus 2, I-35129 Padova, Italy
e-mail: ernesto.carafoli@unipd.it

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 81


Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_4, © Springer Science+Business Media Dordrecht 2013
82 Brini, Ottolini, Calì, and Carafoli

5 THE AMBIVALENCE OF THE CALCIUM SIGNAL: DEFECTS


OF CALCIUM REGULATION AND DISEASE ............................................................. 116
5.1 Neuronal Diseases..................................................................................................... 116
5.1.1 Ataxia ............................................................................................................ 116
5.1.2 Migraine ........................................................................................................ 118
5.2 Neurodegenerative Diseases ..................................................................................... 118
5.2.1 Parkinson’s Disease ...................................................................................... 119
5.2.2 Alzheimer’s Disease...................................................................................... 120
5.2.3 Huntington’s Disease .................................................................................... 120
5.2.4 Amyotrophic Lateral Sclerosis ..................................................................... 121
5.3 Genetic Hearing Loss................................................................................................ 122
5.4 Cardiac Diseases (Cardiomyopathies) ...................................................................... 123
5.5 Skeletal Muscle Diseases .......................................................................................... 124
5.5.1 Malignant Hyperthermia ............................................................................... 124
5.5.2 Central Core Disease..................................................................................... 125
5.5.3 Brody’s Disease ............................................................................................ 125
5.5.4 Duchenne Muscular Dystrophy .................................................................... 125
6 CONCLUSIONS ............................................................................................................... 126
ABBREVIATIONS .................................................................................................................. 127
ACKNOWLEDGMENTS........................................................................................................ 129
REFERENCES ........................................................................................................................ 130

Abstract Evolution has exploited the chemical properties of Ca2+, which facilitate
its reversible binding to the sites of irregular geometry offered by biological macro-
molecules, to select it as a carrier of cellular signals. A number of proteins bind Ca2+
to specific sites: those intrinsic to membranes play the most important role in the
spatial and temporal regulation of the concentration and movements of Ca2+ inside cells.
Those which are soluble, or organized in non-membranous structures, also decode
the Ca2+ message to be then transmitted to the targets of its regulation. Since Ca2+
controls the most important processes in the life of cells, it must be very carefully
controlled within the cytoplasm, where most of the targets of its signaling function
reside. Membrane channels (in the plasma membrane and in the organelles) mediate
the entrance of Ca2+ into the cytoplasm, ATPases, exchangers, and the mitochondrial
Ca2+ uptake system remove Ca2+ from it. The concentration of Ca2+ in the external
spaces, which is controlled essentially by its dynamic exchanges in the bone system,
is much higher than inside cells, and can, under conditions of pathology, generate a
situation of dangerous internal Ca2+ overload. When massive and persistent, the Ca2+
overload culminates in the death of the cell. Subtle conditions of cellular Ca2+
dyshomeostasis that affect individual systems that control Ca2+, generate cell disease
phenotypes that are particularly severe in tissues in which the signaling function of
Ca2+ has special importance, e.g., the nervous system.

Keywords bones • calcium binding proteins • calcium regulated functions • calcium


signaling • calcium transporters • cardiomyopathies • muscle diseases • neurode-
generative diseases • teeth

Please cite as: Met. Ions Life Sci. 13 (2013) 81–137


4 Calcium in Health and Disease 83

1 Introduction

1.1 Calcium in Nature and in Living Organisms

Calcium is the third most abundant metal in nature: it follows aluminium, which is
by far the most abundant, and sodium, and it is followed by magnesium. Na, Ca,
Mg, and Fe are found in nature in comparable abundance, each of them making up
about 3% of the earth’s crust [1,2]. However, in the earth’s mantle, i.e., the layer
immediately underneath the crust, Mg2+ is instead much more abundant than Ca2+.
Ca2+ is found in rocks, soil, and waters: in the sea its concentration is about 10 mM
(however, in sea water Mg2+ is about five fold more abundant than Ca2+).
In nature, Ca2+ is present in salts of various compositions. In living organisms these
salts have long been known to be essential in the formation of skeletal structures: in
higher organisms, Ca2+ phosphate is the major salt of bones and teeth, whereas in
lower organisms other salts, e.g., Ca2+ carbonates and sulfates, are the major contribu-
tors to skeletal or other structural components. In plants, Ca2+ oxalate precipitates are
found, and Ca2+ picolinate is abundant in spore-forming microorganisms [3,4].
In animal organisms there is a large difference between the concentration of Ca2+
in the body fluids and extracellular spaces and that within cells: this difference is the
basis for the signaling role of Ca2+ that will be discussed below. In man, the concen-
tration of Ca2+ in plasma is between 2.1 and 2.6 mM [5] and is in the same mM
range in most extracellular spaces, including the lymph, which is considered equiv-
alent to the extracellular spaces. However, there are significant exceptions, a promi-
nent one being for instance the endolymph of the inner ear, where the concentration
of extracellular Ca2+ is in the low μM range. An important problem is the relation-
ship between total and free (ionized) Ca2+, which may vary from fluid to fluid and,
in any case, is not easy to determine.
Ca2+ exists in at least three basic forms: ionized, complexed to organic compounds,
and bound (precipitated) in the inorganic salts mentioned above. An equilibrium
exists between these forms, which is regulated by hormones (see below) and diet,
and of course by the rules of chemistry. For instance, in blood plasma (where most
Ca2+ of the blood is found) Ca2+ is divided roughly equally between the ionized and
complexed forms. By contrast, in milk, which contains about 30 mM total Ca2+,
about 2 mM is free Ca2+, about 20 mM is associated with casein micelles, and about
8 mM is Ca2+ bound to phosphate (Ca2+-hydrogen phosphate) and citrate. The
cerebro-spinal fluid is also worth mentioning, because of its unusually large
percentage of ionized Ca2+: 1.1 mM ionized versus 1.4 mM total (i.e., about 80% is
ionized). Especially large differences between free and total Ca2+ are found in the
intracellular ambient, but the matter of the intracellular space, where not only Ca2+
binding ligands but also organellar transport and storage are active in determining
the ratio between free (ionized) and bound Ca2+, has special complexities. It will be
discussed in more detail later on.
84 Brini, Ottolini, Calì, and Carafoli

1.2 Regulation of Calcium in Biological Fluids

The concentration of Ca2+ in the blood of mammals (including humans), on which


the concentration of Ca2+ in the extracellular spaces and, eventually, inside cells
depends, is regulated by three hormones: parathyroid hormone, calcitonin, and the
active form of vitamin D (1,25-OH vitamin D3, calcitriol). The major function of
parathyroid hormone is to increase Ca2+ in the blood: in the absence of parathyroid
hormone plasma Ca2+ may decrease by up to 50%, whereas an excess of parathy-
roid hormone results in hypercalcemia. The hormone maintains the blood plasma
Ca2+ concentration by acting on the bones, the kidney, and the intestine. In bones,
it regulates the dynamic equilibrium between Ca2+ adsorbed to the surface of bones
[in the form of Ca10(PO4)6(OH)2] and Ca2+ in plasma by promoting its outflow from
the bone. In the absence of the hormone, the reverse process is favored, leading to
a lowering of blood Ca2+. In the kidneys, parathyroid hormone decreases the excre-
tion of Ca2+ essentially by promoting its reabsorption from the glomerular filtrate,
but also, indirectly, by promoting the production of calcitriol through the action of
renal 25-hydroxyvitamin D1 α-hydroxylase. The resulting calcitriol then increases the
intestinal absorption of Ca2+ (see below). The stimulation of the renal hydroxylase
is the indirect mechanism by which parathyroid hormone promotes the absorption
of Ca2+.
Calcitonin lowers plasma Ca2+ by inhibiting osteoclasts’ motility and spreading.
Osteoclasts are the target cells of the hormone: when incubated with calcitonin, they
rapidly loose the ruffled borders typical of resorbing bone, decreasing the flow of
Ca2+ from bones to blood (osteoblasts and osteoclasts will be discussed in detail in
Section 1.3). Calcitonin decreases the intestinal absorption of Ca2+, and inhibits its
reabsorption by kidney tubules. The level of calcitonin in blood increases when
plasma Ca2+ rises, and drops when it decreases and its secretion by the producing
cells (the C cells of the thyroid) has been proposed to be stimulated by the gastroin-
testinal hormones.
The active forms of the vitamin D endocrine system are the derivatives 1,25(OH)2D,
calcitriol, which is the major biologically active form, and 24,24(OH)2D3: the latter is
produced in the kidney by the activation of the 24-hydroxylase by calcitriol, and has
a role in bone development and parathyroid function. Calcitriol increases bone miner-
alization by increasing the absorption of Ca2+ in the intestine, i.e., it makes more of it
available to osteoblasts. It also stimulates the proliferation of the osteoblasts. The
increased absorption of Ca2+ in the intestine is due to the promotion of the expression
of the Ca2+-binding protein calbindin, an EF-hand protein of which two forms exist:
calbindin D-28k, which has 6 EF-hand Ca2+-binding motifs (of which, however, only
four are operationally active), and calbindin D-9k, which has two EF-hand motifs.
Calbindin D-9k is closely related to the S-100 Ca2+-binding proteins, and is considered
to be a member of their family. Calbindin D-28k, instead, has only minimal sequence
homology to calbindin D-9k and to the S-100 proteins, and is closely related to cal-
retinin, which also has 6 EF-hand motifs. Calcitriol stimulates the synthesis of calbin-
dins by acting on a specific nuclear receptor (the VDR receptor) that recognizes a
4 Calcium in Health and Disease 85

specific response element in the promoter of the gene. Calbindin D-9k is very abundant in
the intestinal mucosa, and present in smaller amounts in other tissues. Its concentration in
the intestine parallels the rate of Ca2+ absorption. Calbindin D-28k is present in brain and
numerous other tissues, including avian intestine. However, it is not expressed in
mammalian intestine, and its expression in the brain is not vitamin D-dependent.
The absorption of Ca2+ in the intestine occurs by a saturable active transport
process dependent on vitamin D, and by a vitamin D-independent passive paracel-
lular process (it has been claimed that also the passive paracellular absorption is
stimulated by vitamin D). The saturable transport process consists of the penetration
of Ca2+ into the mucosal cell through luminal Ca2+ channels, of its buffering by cal-
bindin D-9k (calbindin D-28k as well in birds) which increases the rate of its trans-
cytosolic diffusion to the basolateral membrane, and by its ejection to the
extracellular fluid of the lamina propria by the basolateral plasma membrane Ca2+
ATPase. Calcitriol stimulates the expression of the Ca2+ entry channels, of calbindin
D-9k, and of the Ca2+-exporting plasma membrane pumps.

1.3 Calcium in the Mineralized Compartment


of the Organisms

About 1.5 billion years ago, a large transfer of geologic minerals (including CaCO3)
occurred into the oceans due to the violent moves of tectonic plates. The natural selec-
tion forced the living organisms of the sea to develop more protective body parts (such
as shells or scales) to cope with the new mineral-rich environment. The evolution of
exoskeletons increased enormously the pace of animal evolution but limitations such as
small body size, lack of surface sensory organs and reduced movement/locomotion
inspired a new evolutionary step culminating with the dislocation of mineralized skel-
eton from the outside to the inside of animal bodies, as well as with the replacement of
the Ca2+ carbonate, used to build marine exoskeletons, with the chemically more stable
Ca2+ phosphate Ca3(PO4)2 in the form of Ca2+ hydroxyapatite Ca5(PO4)3(OH) (usually
written Ca10(PO4)6OH2) [6,7]. The development of endoskeletons (bones and teeth)
gave vertebrates improved mobility and mechanical competence. It also provided them
with a ready source of key inorganic ions like Ca2+, Mg2+, and phosphate.
The earliest mineralized structures in the vertebrate lineage were tooth-like
structures in the mouth or in the skin arranged to form a protective shield, while
the first endoskeleton appeared as cartilagineous and gradually evolved through
the process of endochondral ossification [8]. Mineralized tissues are composite
structures consisting of an inorganic mineral phase, an organic phase, and cells.
The crystals in bone have a length of ~20–50 nm and a width of 12–20 nm, depend-
ing on age and species; in dentin they are of similar size, but enamel crystals are ~10
times larger [9,10]. Bone apatite nanocrystals exhibit a variety of substitutions and
vacancies that make the Ca/P molar ratio distinct from the stoichiometric hydroxy-
apatite ratio of 1.67 [11].
86 Brini, Ottolini, Calì, and Carafoli

The cell-produced organic matrices speed up biomineralization and, as crystals


grow, the association with proteins, such as osteocalcin, also regulates bone remod-
eling, maintaining the hydroxyapatite bone mineral in a dynamic state. Dentin min-
eral is remodeled to a much lesser extent, although the roots are remodeled as a
response to disease, and the remodeling is under cellular control. Enamel mineral is
not remodeled, but the enamel matrix is degraded as mineralization takes place.
With time, depending on tissue site and animal diet, bone and dentin mineral pro-
gresses from a poorly crystalline apatite with high HPO42 − content and a low level of
crystallinity to a mineral with somewhat higher crystallinity, lower acid phosphate
content, and a more organized structure, albeit with more carbonate substitutions
[12,13]. Bone and dentin consist of apatite crystals deposited in an oriented fashion
on a collagen scaffold. Type I collagen is predominant and is associated with bone,
dentin, cementum, skin, ligaments, and tendons. Collagen is an insoluble fibrous
protein consisting of three polypeptide chains wound into a repeating triple-helical
fibril [14]. The fibrils line up head-to-tail to form repeating arrays on which the
mineral particles align with their long axes parallel to the fibril axis. The apatite
crystals deposit first within the holes and then spread throughout the matrix [15],
the initial mineralization occurring at the cellular level.
Osteoblasts and odontoblasts control the production and mineralization of the
extracellular collagen protein matrix in bone and teeth, osteoclasts instead remove
bone mineral and bone matrix. Thus, bone cells regulate the formation and resorp-
tion of bone, which is a key step in regulating body Ca2+ (body Mg2+ and phosphate
as well). In the bone formation phase, clusters of osteoblasts on the bone surface
produce the bone matrix constituents by rapidly depositing collagen on which, after
maturation of osteoid matrix, mineralization occurs. At the end of the matrix-secret-
ing period, osteoblasts entrapped in the new bone matrix differentiate into osteo-
cytes, characterized by long cell processes rich in microfilaments that form a
network permeating the entire bone matrix during its formation and before its calci-
fication. The exact function of osteocytes is still unclear but they possibly respond
to bone tissue strain and enhance bone remodeling activity by recruiting osteoclasts
to sites where bone remodeling is required [16]. As mentioned above, osteoclasts
are responsible for bone resorption. They lower the pH within the bone-resorbing
compartment to as low as 4.5 thanks to the action of their plasma membrane H+
pump, which helps mobilize bone mineral [17] as they secrete lysosomal enzymes
like acid phosphatase, cathepsin K, matrix metalloproteinase 9, and gelatinase [18]
that digest the organic matrix. During resorption, mature osteoclasts absorb vast
amounts of Ca2+ and their survival is ensured via the Na+/Ca2+ exchangers, which
extrude Ca2+ into the extracellular space preventing the development of deleterious
Ca2+ overload in their cytoplasm [19,20].
Interestingly, recent evidence has shown that isoforms 1 and 4 of the plasma
membrane Ca2+ ATPases also mediate Ca2+ extrusion from mature osteoclasts
contributing to their differentiation and survival [21]. The function of osteoblasts
and osteoclasts is regulated locally by cytokines and by systemic hormones (see
Section 1.2) ([22,23] and references therein). Vitamin D maintains general Ca2+
homeostasis (see above) by acting on kidneys and small intestine but also directly
4 Calcium in Health and Disease 87

on bones, where it increases osteoclastic resorption [24]. The parathyroid (PTH)


hormone also stimulates bone resorption (but also stimulates bone formation when
administered intermittently). The secretion of PTH is controlled by a negative
feedback of Ca2+ on the parathyroid gland cells through the plasma membrane Ca2+
sensor [25]. The end result is a constant Ca2+ concentration in the extracellular fluid
due to the maintenance of the appropriate concentration of PTH in body fluids [25].
Biomineralization is thus a dynamic process characterized by the constant equi-
librium between crystal deposition and removal. The chemistry of bone hydroxy-
apatite requires that bone formation includes a supply of Ca2+ and H2PO4− and some
way to dispose of 1.4 H+ per each Ca2+ equivalent deposited. The source of Ca2+ is
obviously the extracellular fluid, but the metal must be taken up by osteoblasts and
the mechanism of this process is still poorly understood. Calbindin [26] has been
suggested to have a role but calbindin-negative osteoblasts still transport Ca2+ at a
normal rate [27]. As for phosphate, osteoblasts express high levels of alkaline phos-
phatase (ALP), that cleaves the pyrophosphate produced within the osteoclasts
[28,29] and is likely to have a role. An additional transport mechanism that removes
H+ is required for the rapid deposition of hydroxyapatite. High concentrations of
phosphate and Ca2+ at neutral pH will form an initial precipitate, but mineral forma-
tion becomes limited as the pH falls below 5.6 [30]. A sodium-hydrogen exchange
appears to be the system that holds the pH at slightly elevated levels to permit
mineral deposition [31].
The biggest obstacle in the understanding of the molecular process of bone for-
mation is the mechanism by which osteocytes cause the precipitation of Ca2+ phos-
phate. The solubility limit of Ca2+ phosphate in human bone is approximately 7 ×
10–5 M at physiological pH and temperature. The concentration of free Ca2+ in the
plasma and extracellular fluids (ECF) is higher than 10–3 M, that of PO43 − ion, mostly
in the form of the HPO42 −, is very low in plasma. Under these conditions, when
plasma or ECF come in contact with bone surfaces, plasma Ca2+ is supersaturated
with respect to bone. Nevertheless, the free Ca2+ level in the ECF and plasma is still
maintained at approximately 10–3 M, without continuous deposition of hydroxyapa-
tite crystals. Two main theories have been proposed: the first relies on an organic
or inorganic precursor seeding that directs the formation of apatite from soluble inor-
ganic ions by the action of noncollagenous proteins, such as the SIBLING family of
proteins [32], that would act at the surface of bones and teeth to modify the solubility
of hydroxyapatite, thus promoting or inhibiting mineralization [33,34]. The second
theory is based on the formation of matrix vesicles (MV) as the initial site of primary
nucleation in the mineralization of calcified cartilage, bone, and dentin. MVs are
extracellular vesicles of about 20–200 nm in diameter derived from the plasma mem-
brane of mineral forming cells (chondrocytes, osteoblasts, and odontoblasts) [35] by
a budding process. They are enriched in numerous proteins, among them annexins
(A2, A5, and A6) [36–38] and in phosphatidylserine, which facilitate Ca2+-dependent
annexin binding and enable annexins to form Ca2+ channels (see above) [39,40]. The
MVs would initiate intravesicular mineral formation either by regulating the ratio of
Pi to pyrophosphate (PPi) and by serving as nucleation sites for apatite deposition.
Thus, mineralization would begin with the formation of hydroxyapatite crystals
88 Brini, Ottolini, Calì, and Carafoli

within the MVs and proceed with the propagation of hydroxyapatite through the
membrane into the extracellular matrix [32]. The hydroxyapatite would then be prop-
agated in clusters around MVs and fill the space between collagen fibrils. PPi, which
inhibits the formation of hydroxyapatite [41] formed by nucleotide pyrophosphatase/
phosphodiesterase 1 (NPP1) from nucleotide triphosphates, but also by ankylosis pro-
gressive homolog (ANKH, a homolog of the mouse progressive ankylosis (ank) gene
product), would be hydrolyzed by ALP [42].
Other sites of intracellular mineral deposition are the mitochondria. Their inter-
play with Ca2+ will be discussed in Section 3. For the discussion of their possible
role in the biomineralization process it will be sufficient to mention that in addition
to Ca2+ they accumulate inorganic phosphate [43], precipitating hydroxyapatite in
the alkaline environment of their matrix. The precipitates are frequently seen as
electron-dense granules within mitochondria that have accumulated massive
amounts of Ca2+ and phosphate [44]. They are also observed within the mitochon-
dria of cells that experience conditions of pathological cytosolic Ca2+ overload and
within the mitochondrial profiles of cells normally exposed to high Ca2+ traffic in
the cytosol, e.g., those of mineralized tissues [45]. The granules have been isolated
and found to contain, in addition to hydroxyapatite, a number of organic compo-
nents. Surprisingly, however, even under conditions of high saturation of Ca2+ and
phosphate found in the mitochondrial matrix, they remain amorphous [46]. These
amorphous granules have been suggested to be involved in the process of biological
mineralization (it may be significant that osteoclasts are rich in mitochondria) [45]:
they could in other words be a means to store high concentrations of Ca2+ and phos-
phate in a non-crystalline and more readily available form. A hypothesis for their
possible role has been proposed by Lehninger [46] who postulated that the amor-
phous granules would be somehow stabilized as micropackets by biological factor(s)
and transported to mineralization sites where they would form apatitic bone min-
eral. Phosphocitrate (PC), which has been identified in mammalian mitochondria,
may be one factor involved in the process of granule stabilization [47] and in the
prevention of Ca2+ phosphate precipitation in cells, or cellular compartments, that
maintain a high concentration of Ca2+ and phosphate (e.g., the mitochondria).

2 General Properties of Calcium as a Signaling Agent

Na+ and Ca2+ are the major cationic components of extracellular spaces, whereas K+,
Mg2+ (and Zn2+) are the major intracellular metals. Within cells, nearly all of the K+
and about 75% of the Na+ is free, whereas a much higher proportion of the other three
metals is present in bound forms. This is particularly true for Ca2+, the ionized con-
centration of which within cells is a negligible fraction of its total concentration (see
above). However, the issue of total and ionized Ca2+ inside cells is complex. The
usual measurements of total cell Ca2+ yield values in the 1 to 10 mM range. These
values cover ionized Ca2+, and Ca2+ bound to the usual inorganic ligands and small
molecular weight organic molecules, and to specific binding proteins. They cover
4 Calcium in Health and Disease 89

also, and especially, Ca2+ sequestered within organelles like the endo(sarco)plasmic
reticulum, the Golgi system and, under special conditions (see below), the mito-
chondria. The ionized Ca2+ in the cytosol, which is the cell compartment where most
of the targets of its signaling function reside, is on the order of 100 nM. Clearly, this
concentration is much lower than in all other districts of the organism, where the
ionized/free ratio is solely determined by the action of “inert” Ca2+ ligands.
Within cells the control of Ca2+ concentration is a dynamic operation: inert
ligands like small molecular weight components and binding proteins do have a role
in it, as they have in all other parts of the organism. But they could not possibly
lower free Ca2+ in the ambient to values in the nM range. These extremely low con-
centrations are only achieved thanks to the concerted operation of membrane trans-
porting systems (pumps, exchangers, and channels): their activity maintains
(cytosolic) Ca2+ at the nM level, which is demanded by its signaling function.
Transporters that exchange Ca2+ across the membrane barriers separating the
intracellular organelles from the cytosol generate Ca2+ stores in the former that
ensure the availability of the adequate supply of Ca2+ to the cytosol. As mentioned,
the main Ca2+-storing organelles are the endo(sarco)plasmic reticulum, the Golgi
system and, under special conditions, the mitochondria. Very large amounts of Ca2+
are contained in the reticulum, in which the ratio of ionized versus bound Ca2+ can
be considered similar to that of the extracellular spaces, yielding free Ca2+ concen-
trations in the mM range. Very likely, a similar situation also prevails in the Golgi
system. Under conditions of normal cell life, mitochondria are not a quantitatively
significant Ca2+ store. Their matrix could be considered similar to the cytosol, with
free Ca2+ concentrations in the nM range. Mitochondria, however, can accumulate
very large amounts of Ca2+ under the conditions of cytosolic Ca2+ overload fre-
quently occurring in pathology. They do so because they take up inorganic phos-
phate together with Ca2+ (see above), precipitating amorphous hydroxyapatite in the
matrix. Under these conditions, mitochondria become very large Ca2+ stores, but
nearly all the Ca2+ they contain is unavailable for rapid exchanges with the cytosol.
This massive accumulation of Ca2+ by mitochondria is an important defense device:
it enables cells to clear out excess Ca2+ from the cytosol, giving them the time to
survive cytosolic Ca2+ storms.
Irrespective of the difference in the various parts of the organism, the difference
between free and bound Ca2+ in cells is determined by the unusual propensity of the
metal to be ligated, which in turn reflects its peculiar coordination chemistry.
According to the rules of coordination chemistry the interaction of metal ions with
coordinating ligands is determined by valency, which determines the charge of the
metal, the ionic radius, the polarizability, i.e., the ease with which the electron cloud
of the metal is distorted by external electrical forces, the hydration energy, which
expresses the ease with which the attached water molecules are stripped off the
metal, and the radius of the hydrated ion, which determines the charge density. The
combination of these properties explains why Ca2+ is so easily complexed, and why
it can fit optimally in binding (coordination) sites of irregular geometry, such as
those offered by biological molecules like proteins. One can for instance compare
Ca2+ to the other important Group 2 metal, Mg2+ (Table 1).
90 Brini, Ottolini, Calì, and Carafoli

Table 1 Some properties of un-hydrated and hydrated Ca2+ and Mg2+.


Ionic radius Polarizability Hydration energy Hydrated ions
Å α0 × 1024 cm3 kcal/g ion Å
Ca2+ 0.99 0.531 410 4.5
Mg2+ 0.65 0.012 495 5.9

The smaller size of divalent Mg2+ and its very low polarizability value do not
permit much flexibility in the geometry of the coordinating site (the ligands, as in
the case of Ca2+, are usually oxygens) which tends to be a more or less perfect octa-
hedron: perfect octahedral cavities, naturally, do not easily come about in biological
macromolecules. The properties of Ca2+, by contrast, are compatible with coordinat-
ing sites of irregular geometry, as one expects to find in biological macromolecules.
Synthetic low-molecular-weight compounds offer a visual representation of the dif-
ferences in the coordinating demands of Ca2+ and Mg2+ (Figure 1) [48]. The distance
between the metal and the ligating oxygen atoms may vary by as much as 0.52 Å in
the case of Ca2+, but by only 0.12 Å in the case of Mg2+.

Figure 1 Hypothetic comparison of the binding of Ca2+ and Mg2+ to an EF-hand protein motif.
The chemical properties of the two ions described in Table 1 determine the higher ability of Ca2+
to fit into binding sites of irregular geometry.

In tissues and fluids of animal organisms Ca2+ is ligated by a number of inorganic


and organic low-molecular-weight molecules. Normally, this type of binding occurs
with low affinity. High affinity (and specific) Ca2+ binding, such as necessary for the
4 Calcium in Health and Disease 91

regulation of its signaling function demands coordinating sites of higher complexity,


such as those offered by proteins. Evolution has developed several such motifs,
some of them are present in hundreds of proteins: the regulation of the signaling
function of Ca2+ relies most frequently on three of them: the EF hand helix-loop-
helix motif, the C2 motif, and the annexin Ca2+-binding fold. The EF hand motif is
found in a large number of protein families (about 66 sub-families are known [49]).
The typical EF hand structure (Figure 2) consists of two α-helical domains inter-
rupted by a loop usually containing 12 residues with the pattern X●Y●Z●-Y●-
X●●-Z; X,Y,Z,-X,-Y,-Z are the residues that coordinate Ca2+, and the dots (●) are
residues not involved in the coordination: the residue that follows Z is a central
invariant Gly. At positions X and Y the side chains of Asp or Asn contribute coordi-
nating oxygens, whereas Asp, Asn, or Ser are found at position Z. A peptide carbonyl
oxygen coordinates Ca2+ at position -Y, and a water oxygen usually coordinates Ca2+
at position -X. An Asp or a Glu are conserved residues at position -Z. EF hand
motifs usually occur in pairs, but proteins have also been found that contain only
one EF hand motif or an odd number of EF hand motifs: for instance, the C-terminal
domain of the large subunit of calpain (see below) contains 5. However, this penta-
EF hand domain forms a heterodimer with the small subunit of calpain that also
contains 5 EF hand motifs.

Figure 2 Top: 3D structure of the calmodulin (CaM) (PDB file 3CLN) EF-hand domain (top left),
of the synaptotagmin I (PDB file 1TJX) C2b motif (top middle), and of the full-length annexin A1
(PDB file 1MCX) (top right, repeats 1 to 4 are shown in red, yellow, purple, and green, respectively).
The calcium ions are depicted as orange spheres and the residues involved in its coordination are
shown as sticks. Bottom from left to right: typical EF-hand of CaM, synaptotagmin I C2 motif,
and annexin I AB, AB’, and DE calcium-binding sites are shown with the coordinating residues
(sticks). Calcium ions are green and water molecules cyan, distances in Å are shown as yellow
dotted lines.
92 Brini, Ottolini, Calì, and Carafoli

The C2 domain, initially identified in a Ca2+-dependent protein kinase, protein


kinase C, is a 130 residues domain now found in numerous proteins involved in cell
signaling and other important intracellular processes, e.g., membrane trafficking [50].
The domain forms an eight-stranded antiparallel β-sandwich consisting of a pair of
four-stranded β-sheets. Ca2+ is coordinated, in a depression formed at the edge of the
β-sandwich by loops connecting β-sheets 2–3 and 6–7 (Figure 1), by carbonyl oxygens,
mono- and bidentate Asp side chain oxygens, and a water oxygen.
Annexins are a broad family of Ca2+-dependent phospholipid-binding proteins
that are widely distributed in eukaryotic cells [51]. They are intracellular, but some
(e.g., annexins A1, A2, A5) are found outside of cells. They are involved in diverse
biological processes, among them the inhibition of phospholipase, trafficking of
membrane vesicles (endo-exocytosis), Ca2+ channel formation, anchoring of other
proteins to the cell membrane. Outside cells, annexins play roles in the mechanism
of blood coagulation and in fibrinolysis, and are important actors in the antiinflam-
matory responses. All annexins are composed of a divergent N-terminal domain
(the “head” region) and a conserved C-terminal domain (the “core” domain) [52].
The core domain contains 4 (8 in annexin A6) repeats with 5 α-helices A–E. Helices
A, B, D, and E form a coiled-coil structure with the shape of a curved disk with
loops connecting helices A and B, and D and E on the convex side of the disk. The
loops harbor the Ca2+-binding sites, whereas the N-terminal domain of the molecule
is on the concave side of the disk, from which it is expelled upon binding of Ca2+.
Annexins contain 3 types of Ca2+ binding sites: type II (AB loop), type III (DE
loop), and the AB’ site (Figure 2). In site II 3 backbone carbonyl oxygens coordinate
Ca2+ in a conserved sequence (M,L)-K-G-(A,L)-G-T. The side chain of an acidic
residue 39 amino acids downstream the conserved sequence, coordinates Ca2+ in a
bidentate fashion, and two more coordinating oxygens are contributed by water
molecules. In site III the coordination sphere comprises 2 backbone carbonyl oxygens
from the DE loop which is not part of the conserved sequence. A bidentate acidic
residue close in sequence and 3 water molecules complete the coordination sphere.
The coordination sphere of the AB’ site comprises a backbone carbonyl oxygen down-
stream of the conserved sequence for the AB loop, the bidentate side chain of an acidic
residue close by, and 5 water oxygens: the coordination in site AB’ is thus 8, rather
than 7 as in sites II and III. Numerous X-ray structures of annexins are now available.
Many contain empty or partially occupied Ca2+-binding sites: annexin A1 has 6 to 8
Ca2+ ions bound, annexin A2 and A3 have 5, annexin A5 up to 10. The affinity of
annexins for Ca2+ is rather low, compared, for instance, to that of EF hands: this could
be due to the large number of water oxygens involved in the coordination of Ca2+.
The ease with which Ca2+ can be ligated in sites of great geometric variability
explains its evolutionary choice as a cellular signaling agent. As is self-evident,
signaling agents must be bound reversibly by diverse molecules that control their
concentration in the vicinity of the targets of their messenger function. The coordi-
nation chemistry properties discussed above permit the control to occur optimally
for the case of Ca2+: they would, by contrast, not permit it for that of Mg2+. There is
one additional dividend to the evolutionary choice of Ca2+ as a signaling agent. As
is self-evident, to prevent prohibitive energetic costs to modulate their concentration,
signaling agents must be maintained within cells at very low free concentrations:
4 Calcium in Health and Disease 93

this is easily achieved in the case of Ca2+, thanks to the ease with which it can be
complexed. The resulting sub-μM concentration of cytosolic free Ca2+ prevents the
precipitation of Ca2+-phosphate salts, which would otherwise inevitably occur if
both Ca2+ and phosphate were in the mM range. This has made possible the use of
phosphate as a universal energy currency.

3 Intracellular Calcium Handling

3.1 Transport of Calcium Across Membrane Boundaries

The proteins containing the Ca2+-binding motifs described in Section 2 are soluble,
or associated to non-membranous structures. Their role in the regulation of cell Ca2+
is certainly important, but is quantitatively limited, as cell physiology may demand
the (temporary) ligation of amounts of Ca2+ that could overcome their total Ca2+-
binding capacity. Cells, however, also contain a wealth of proteinaceous Ca2+-
binding systems that are intrinsic to membranes: they play the major role in the
control of cell Ca2+, as they do not only bind Ca2+, but also move it back and forth
across the plasma membrane and the membranes of the organelles. They can “buf-
fer” it even if present in the membranes in minute amounts. The systems that medi-
ate the traffic of Ca2+ across membranes are channels, ATPases (routinely termed
pumps), exchangers (normally Na+/Ca2+-exchangers), and a specific mitochondrial
electrophoretic transport system (the Ca2+ uniporter). These systems have been
described in numerous detailed reviews, including one we have published in the
preceding issue of the present series [53]. They will thus only be described very
briefly to facilitate the understanding of the discussions in the following Sections.
The systems that mediate the entry of Ca2+ into the cytosol from the external
spaces, or the lumen of the organelles, are homo- or hetero-polymeric protein com-
plexes that can be gated, (i) by voltage across the plasma membrane, (ii) by the
binding of specific external ligands to their extracellular portions (e.g., neurotrans-
mitters in neurons), or, (iii) by the binding of ligands generated by stimulatory ago-
nists in the cytosol. These ligands (second messengers) act on channels in the
membrane of the endo(sarco)plasmic reticulum (ER, SR) and the Golgi system
(e.g., InsP3) and possibly on that of the acidic organelles. A fourth type of channels,
which have been defined molecularly and functionally only recently, are the so
called store-operated plasma membrane channels, that are activated by the empty-
ing of the Ca2+ store in the ER [54]. They will have to be discussed in some more
detail later on, since they are especially relevant to the content of this contribution.
The Ca2+ ATPases are located in the plasma membrane (the PMCA pump) and
in the membrane of the ER, SR, and the Golgi system [55]. Their mechanism of
transport is now understood in atomic detail thanks to the solution of the crystal
structure of the sarcoplasmic reticulum pump (the SERCA pump). They are high
Ca2+ affinity transporters, i.e., they interact efficiently with Ca2+ even in the
sub-μM concentrations of the cytosol, and are regulated by a number of
mechanisms, from phosphorylation processes that could be direct or mediated by
94 Brini, Ottolini, Calì, and Carafoli

accessory proteins, to the interaction with regulatory proteins, e.g., CaM in the
case of the PMCA pump.
The Na+/Ca2+-exchangers are present in the plasma membrane and in the inner
membrane of mitochondria. They are lower Ca2+ affinity systems, which transport
bulk amounts of Ca2+, e.g., across the plasma membrane whenever the physiological
need arises to rapidly extrude large amounts of Ca2+, e.g, in heart myocytes at the
end of the contraction phase. Mitochondria take up Ca2+ by an electrophoretic
system that responds to the membrane potential which is negative inside and
maintained across the inner membrane by the operation of the respiratory chain.
Its molecular identity has been clarified only recently [56,57]: the system has
very low affinity for Ca2+, yet, it works efficiently in the intracellular environment.
This apparent paradox will be discussed in more detail later on, as it is central to the
subject matter of this contribution.
The acidic compartment of the cell has also recently been claimed to contain
Ca2+ uptake and release systems (see for instance [53] for a recent review in which
this aspect is mentioned). The Ca2+ release system from its organelles has been
claimed to by a special channel type, the two-pore channel (TPC). The matter of the
acidic compartment in the control of cell Ca2+ is a controversial issue, and will be
discussed in more detail in Section 3.2.

3.2 Spatiotemporal Dynamics of the Calcium Signal

Changes in the intracellular Ca2+ concentration regulate numerous important


biological processes, ranging from cell origin to cell death. The versatility of Ca2+
as an intracellular messenger depends on the tight control of its spatial and temporal
distribution. The spatio-temporal pattern of Ca2+ signals is shaped by a sophisticated
machinery that regulates precisely its amplitude and duration in a site-specific
manner. The components of this machinery which include membrane transport
systems and binding proteins have been succinctly described in Sections 2 and 3.1.
Their concerted operation generates cellular Ca2+ microdomains which have a
distinct physical localization and specific functional significance. The participation
of intracellular organelles in the regulation of Ca2+ signaling has been introduced in
Section 3.1, and will be discussed in more detail later on.
Excitable cells, i.e., neurons and muscle cells, are particularly responsive to the
spatio-temporal regulation of the Ca2+ signal, as they are essential in the tuning of
special processes such as contraction, secretion, synaptic transmission, etc. Not sur-
prisingly, their dysregulation generates major human pathologies, e.g., cardiac and
neurodegenerative diseases.
Temporally, Ca2+ signals occur in two main forms: waves and oscillations, each
of them generated by the specific activation of groups of membrane channels that
are differently distributed throughout the cell. Propagating Ca2+ waves originate mainly
from the release of Ca2+ from internal stores through the opening of intracellular
channels, the most important being those in the InsP3R and RyR. Polycistin-2 and
TPC have also recently been claimed to release Ca2+ from the ER and the acidic Ca2+
4 Calcium in Health and Disease 95

stores (lysosomes and/or endosomes), respectively, thus activating neighboring


InsP3R and RyR channels which are sensitive to Ca2+ itself (see below). On the TPC
channels a controversy has recently arisen, as it has been claimed that they are Na+-
selective, rather than Ca2+-selective, and that the main cation they release from the
endosomes/lysosomes is Na+ and not Ca2+ [58].
According to general consensus a transient increase of cytosolic Ca2+ may propagate
as a wave when the initial localized increase triggers a regenerative process, known
as Ca2+-induced Ca2+ release (CICR), originally described by Ford and Podolski
and by Endo and coworkers in 1970 [59,60]. The process, first described in skeletal
muscles, is now known to take place also in other cell types, e.g., cardiac myocytes
and neurons.
The opening of specific membrane channels generates two main types of intra-
cellular waves. The most common ones are those mediated by the opening of
InsP3Rs. They were first reported in the eggs of a medaka fish during fertilization
[61], and were then found in different egg species, including those of frogs and
rodents. Recently, it has emerged that they may serve specific functions also in other
cell types, e.g., neurons. The main factors determining the generation of the wave
format are the site and magnitude of InsP3 generation. Localized events of InsP3R
opening, known as “puffs”, are responsible for a local Ca2+ release that may act as
trigger to generate a global Ca2+ wave. The rate of intracellular Ca2+ wave propaga-
tion is a complex function of InsP3 and Ca2+ diffusion, sequential CICR via sensi-
tized InsP3Rs (and RyRs), and the distribution and composition of the ion channel/
receptor clusters.
The other Ca2+ wave type, which is mediated by the regenerative activation of
RyRs is rare. It has been observed in cardiac myocytes, and it is not clear whether it
occurs under normal physiological conditions. Spontaneous localized Ca2+ release
events, often called ‘sparks’, result from the opening of clusters of RyRs in the sar-
coplasmic reticulum by local CICR, and have been suggested to cause the large
regenerative Ca2+ release that controls contraction. The generation of this type of
wave depends on the sensitivity and the distribution of the RyR channels, and Ca2+
entry through voltage-gated plasma membrane Ca2+ channels modulate their fre-
quency in myocytes, thus attributing a role to extracellular Ca2+ as well [62].
An important question is the propagation of Ca2+ waves among cells, which has
a role in the physiology and pathology of different tissues [63]. Emerging evidence
has shown that cell communication through gap junctions, and the diffusion of sec-
ond messengers like InsP3, are basic to the propagation of the intercellular waves by
paracrine signaling between adjacent cells. ATP appears to be the most common
paracrine messenger, but the mechanism of its release is still obscure. The signaling
cascade starts from the release of ATP in the extracellular ambient and its diffusion
to the plasma membrane purinergic receptors. Two type of purinergic receptors are
activated by the extracellularly released ATP: the ionotropic receptors that are
ligand-gated ion channels, (i.e., those of the P2X family) and the metabotropic
receptors (i.e., those of the P2Y family) that are coupled to the generation of InsP3.
The generation of the intercellular Ca2+ waves appears to depend on the stimulation
of metabotropic receptors and to the release of Ca2+ from the ER by InsP3. External
ATP is the major extracellular messenger utilized by many cell types, but other
96 Brini, Ottolini, Calì, and Carafoli

molecules, such as glutamate in astrocytes and neurons [64,65] and Ca2+ itself by
acting on the membrane Ca2+ sensor receptor [66] may act as paracrine messengers.
Thus, the waves do not only transfer information from one side of the cell to the
other, they also propagate the Ca2+ signal among adjacent cells. The transfer of informa-
tion through Ca2+ waves is not exclusively determined by its diffusion, as the cytoplasm
contains an elevated concentration of Ca2+ binding proteins and other molecules that
hinder its diffusion. In addition, free Ca2+ is captured by the Ca2+ transport systems that
either eject it or sequester it in the organelles. They also hinder the propagation of its
message, thus making the waves a sophisticated tunable way to transmit the message.
The generation of Ca2+ hot spots at the mouth of the Ca2+ release/influx channels
activates sensors with different affinities for Ca2+. This is the concept of Ca2+ micro-
domains that has forcefully emerged in the last 15–20 years. Among these domains,
those generated by the juxtaposition of ER mitochondrial membranes (the so-called
mitochondria-associated ER membranes, MAMs), and those generated by the close
contacts between the plasma membrane (PM) and the ER [where the ORAI1/STIM1
complexes are formed (see Section 3.3)] are the most important. Both will be dis-
cussed in more detail in the next section.
Whereas waves shape the spatial regulation of the Ca2+ signal, its temporal regu-
lation is determined by oscillations. The rhythmic changes of the plasma membrane
potential in the heart, or the burst of action potential in neurons have long been
known to produce fluctuations in cytosolic Ca2+. The seminal observations by
Cobbold and coworkers in the mid-1980s [67] have then shown that Ca2+ oscilla-
tions may also occur in non-excitable cells, e.g., during fertilization of oocytes and
in hormone-stimulated hepatocytes.
By general assumption, the oscillatory behavior has a physiological advantage
over the sustained elevation of Ca2+ as the latter could be deleterious to the cell.
Ca2+-dependent processes that require activation by high Ca2+ are satisfied by the
oscillatory regime that prevents persistent Ca2+ overload. Oscillations also avoid
long-lasting receptor desensitization. In the oscillatory regime, the concentration of
Ca2+ would be permitted to periodically exceed the threshold for enzyme activation,
but its sustained global level would remain below the threshold. Usually, Ca2+ oscil-
lations are characterized by a constant amplitude and a variable frequency, which
ranges from 5 to 60 seconds depending to the cell type, and on the nature and the
strength of the stimulus that initiates them.
In excitable cells such as neurons, heart, and neuroendocrine cells, the transient
[Ca2+] elevation is due to Ca2+ entry through voltage-operated (VOCCs) or receptor
operated Ca2+ channels (ROCCs) activated by neurotransmitters. In non-excitable
cells, instead, the main mechanism of the oscillatory Ca2+ elevation is through the
activation of plasma membrane receptors coupled to G proteins and the generation
of InsP3. Two possible mechanisms have been proposed for the generation of the
InsP3 generated oscillatory signals: either an oscillatory production of InsP3 or the
oscillatory inactivation of InsP3 receptors. Both mechanisms appear to operate in
different cell types, the common denominator being the positive and negative
feedback by Ca2+ on the release system. For example, in hormone-stimulated
hepatocytes and in pancreatic acinar cells oscillations are driven by the cycling of
the InsP3 channels between a fully open and a largely closed state, rather than by
4 Calcium in Health and Disease 97

oscillations in InsP3 levels. But in kidney epithelial cells spatiotemporal changes in


the concentration of InsP3 appear instead to be synchronous with Ca2+ oscillations.
Other molecules, i.e., cyclic ADP-ribose (cADPR) and nicotinic acid adenine
dinucleotide phosphate (NAADP), have also been shown to mediate the mobiliza-
tion of Ca2+ from internal stores.
An intriguing aspect that has recently emerged is that the influx of Ca2+ across
the plasma membrane also plays a critical role in delivering the oscillatory signal to
the correct cellular locus. As ER Ca2+ stores empty, Ca2+ enters through store-
operated channels (SOCCs). As will be discussed in the next section, these channels
are controlled by STIM proteins, which are sensors of ER luminal Ca2+ levels. The
extra-reticular portion of the STIM1 protein has been shown to move cyclically in
and out of the ER plasma membrane junctions during each Ca2+ oscillatory spike,
thus reversibly activating ORAI1 channels and Ca2+ entry.

3.3 Regulation of the Calcium Signal by the Cell Organelles

Intracellular organelles have a dual role in Ca2+ signaling: they are both the target of
regulation and its effectors. Specific organellar function are Ca2+ regulated pro-
cesses, but at the same time organelles play an essential role in the definition of the
spatiotemporal characteristics of the Ca2+ signal. On this, mitochondria absolve the
main role. They have received increasing attention in the last few years since their
coupling with ER and the plasma membrane is the essential feature in the process
of local regulation of Ca2+ signaling. The mitochondrial inner membrane contains a
specific Ca2+ transport machinery composed by an uptake uniporter (MCU [68])
which is composed by a tetrameric pore forming subunit plus two regulatory com-
ponents, MICU1, MICU2, and MCUR1 [69–71], and by a Na+/Ca2+ extrusion sys-
tem [72], which operates in most cell types (in some cells mitochondria operate
instead of a H+/Ca2+ exchanger). The uptake of Ca2+ uses as driving force the elec-
trochemical gradient generated across the inner mitochondrial membrane (IMM) by
the chemiosmotic operation of the respiratory chain. It also depends critically on
microdomains of high Ca2+ concentration generated by the opening of Ca2+ channels
in the neighboring ER that satisfy the low Ca2+ affinity of the uniporter, thus permit-
ting the accumulation of Ca2+ into the matrix. The outer mitochondrial membrane
(OMM) has been traditionally considered freely permeable to Ca2+, thus excluding
it from a specific regulatory role in the handling of Ca2+. However, more recent
evidence has shown that its VDAC channels favor the Ca2+ transfer from the ER to
mitochondria thanks to their coupling between the InsP3R and MCU [73]. Thus,
OMM proteins would also have a role in the handling of Ca2+ by mitochondria.
As mentioned, the concept of microdomains and of signal compartmentalization
has recently received wide attention, and general consensus now supports the notion
that, in many cases, these microdomains have a specific physical organization and
biochemical properties. This is the case of the previously mentioned MAMs, which
are specialized regions where ER and mitochondria become tethered by specific
proteins that maintain their distance in the range of 10–30 nm [74–76]. MAMs have
98 Brini, Ottolini, Calì, and Carafoli

been identified in the 1990s [77,78], but only recently a signaling role has been
attributed to them. They are involved in several important cellular functions, rang-
ing from Ca2+ signaling, lipid biosynthesis, mitochondrial division, dynamics regu-
lation of ER and mitochondria membranes [79]. The physical link between ER and
mitochondria depend on mitofusin 2, which is partitioned between ER and mito-
chondria [80] and which is crucial for the transfer of Ca2+ from the former to the
latter. This transfer is guaranteed by the chaperone Grp75-mediated interaction
between the mitochondrial outer membrane voltage-dependent anion-channel pro-
tein 1 (VDAC1) and the InsP3R [73]. The transfer of Ca2+ is not only important as a
response to cell stimulation, it is constitutively critical to proper cell bioenergetics,
as documented by bioenergetics defects, and by increase in autophagy observed in
InsP3 silenced cells [81].
Another important example of the compartmentalization of the Ca2+ signal is the
2+
Ca influx from the extracellular ambient in response to the depletion of intracel-
lular stores. The plasma membrane store-operated Ca2+ entry (SOCE) is a wide-
spread and conserved Ca2+ influx pathway, that mediates Ca2+ influx following the
loss of Ca2+ from the ER. Its gating is regulated by mitochondria: by buffering the
Ca2+ released from the ER and that entering through store-operated Ca2+ channels
(SOCCs), they reduce the Ca2+-dependent inactivation of the latter, increasing the
extent of store depletion and the activation of SOCCs. The mechanism by which the
decrease of Ca2+ concentration in the ER initiates the SOCE process has now been
clarified. When the ER luminal Ca2+ decreases, Ca2+ is released by the N-terminal
low-affinity EF hand of the single pass STIM protein in the ER lumen, causing the
association of the C-terminal portion of STIM molecules to form clusters that make
contacts with the plasma membrane, originating the so-called “puncta” structures.
The targets of the STIM1 clusters are ORAI1 proteins, that are the pore-forming
subunits of the SOCCs. The STIM1 proteins recruit ORAI1 channels and gate their
pore opening.
Other organelles such as the Golgi apparatus, the acidic compartment of the cell,
and the nucleus are also involved in the dynamical shaping of the Ca2+ signal.
The Golgi apparatus is equipped with its own Ca2+ transporters and Ca2+ bind-
ing proteins, thus making it an ER-like Ca2+ store. InsP3R and, especially in neu-
rons and cardiac myocytes, RyR channels mediate the Ca2+ release from the Golgi
vesicles. The resident Ca2+ ATPase SPCA, and a SERCA pump are responsible for
Ca2+ reuptake in their lumen. The relative contribution of these different transport-
ers varies with the cell type: interestingly, it has recently emerged that their dif-
ferential distribution on the Golgi membranes generates heterogeneity in the
Golgi intraluminal Ca2+ concentration. At variance with the other Ca2+-ATPases,
the SPCA pump also mediates Mn2+ transport. The transport of Mn2+ from the
cytosol to the lumen of Golgi has an important detoxifying role, but is also essential
to the function of the resident Golgi enzymes involved in the process of protein
glycosylation [82,83].
Whereas the Golgi apparatus is generally recognized as a releasable Ca2+ res-
ervoir, the role of the acidic organelles as Ca2+ stores is instead still controversial.
4 Calcium in Health and Disease 99

Two pore Ca2+ channels have been identified in the membranes of endosomes and
lysosomes and have been proposed to be gated by the second messenger NAADP
(see above), triggering Ca2+ release from them [84]. The primary structure of TPC
contains two six-transmembrane domain repeats, unlike all other Na+ and Ca2+
channels that contain four. They have been first identified in sea urchin eggs [85]
and then also in mammalian cells [86] and have received considerable attention as
they have been proposed to regulate global Ca2+ through the crosstalk with both
intracellular Ca2+ channels in other membrane systems, i.e., the InsP3R and the RyR
channels, and the plasma membrane Ca2+ channels. Ca2+ released from acidic stores
has been proposed to be promoted by NAADP, to trigger further Ca2+ release via a
CICR mechanism and to modify plasma membrane excitability by modulating the
Ca2+ release from endosomes or lysosomes specifically positioned beneath the
plasma membrane. However, some aspects of the process by which lysosomes,
endosomes, and acidic organelles handle Ca2+ are still unclear, beginning with the
mechanism by which they accumulate Ca2+ in their lumen. The driving force has
been claimed to be a proton gradient generated by the vacuolar H+-ATPase [87], but
the precise details of the Ca2+ uptake mechanism are still elusive. Very recently, the
Ca2+ releasing function of the TPCs in the acidic organelles has been questioned, as
the direct recording of TPC currents in endolysosomes has shown that it is carried
by Na+ rather than Ca2+, and is activated by PI(3,5)P2, an endolysosome-specific
phosphoinositide, and not by NAADP. Na+ would thus be the principal cation in the
lysosome, casting doubts on this compartment as a Ca2+ store [58].
As for the nucleus, the matter of Ca2+ permeability of the nuclear envelope is still
an open issue. Alternative proposals suggest that the pores of the nuclear envelope
exist in freely permeable or gated states depending on physiological conditions and
demands. Numerous experiments with fluorescent Ca2+ dyes but also with selec-
tively targeted recombinant probes have shown that the kinetics of cytosolic and
nuclear Ca2+ increases induced by cell stimulation were temporally nearly indistin-
guishable, suggesting that the envelope does not represent a barrier to the free diffu-
sion of Ca2+. Other experiments, however, have found that the Ca2+ signals evoked
by the stimulation of cells were temporally delayed in the nucleus. A conciliatory
view could propose that nuclear pores may be either passively permeable to Ca2+, or
restrict its passage depending on different cell types or metabolic condition. Earlier
work had shown that the nuclear envelope contains InsP3Rs and RyRs and a Ca2+
pump identical to that of the ER. Most enzymes of the phosphoinositide cycle
have also been found in the nuclear envelope, suggesting an independent Ca2+ regu-
lation in the nucleus. Clearly, the problem is to understand how plasma membrane
agonists that activate the phosphatidylinositol cycle would be coupled to the process
occurring at the nuclear envelope. The finding that the nuclear envelope folds inside
the nucleoplasm forming invaginations suggests that this structural arrangement
may facilitate the agonist-induced delivery of Ca2+ to selective sub-compartments of
the nucleoplasm.
Irrespective of the mechanism that governs the nuclear envelope permeability to
Ca2+, a specific nuclear function, gene transcription, is selectively regulated by Ca2+.
100 Brini, Ottolini, Calì, and Carafoli

4 Calcium as a Regulator of Biological Processes

4.1 Gene Transcription

The ability of Ca2+ to influence the expression of genes only became known at the
end of the last century, but rapidly developed into one of the most important Ca2+
signaling areas. Since the Ca2+ signals, including those in the nucleus, generally
occur in the form of rapid transients, most of the work on their effects has focused
on immediate early genes, i.e., on genes, which generally code for short lived
transcription factors without the intermediation of de novo protein synthesis. The
expression of late response genes occurs with much slower kinetics, and is instead
dependent on de novo protein synthesis. Late response genes are frequently acti-
vated by transcription factors that are the products of immediate early genes, thus,
they may also be under the control of (nuclear) Ca2+.
The first clear evidence that nuclear Ca2+ had a role in gene regulation was per-
haps the demonstration [88] that a non-diffusible Ca2+ chelator, microinjected into
the nucleus of AtT20 cells, attenuated the nucleoplasmic Ca2+ transients induced by
the activation of voltage-gated plasma membrane Ca2+ channels, and simultaneously
blocked gene expression mediated by the transcription factor cAMP responsive
element-binding protein (CREB). The cytosolic Ca2+ transients and the transcriptional
activation mediated by the serum response DNA regulatory element (SRE), which is
known to be a target of the Ca2+ sensitive extracellular signal-regulated kinases-
microtubule-associated protein (ERK-MAP) kinase, which is activated by Ca2+ in the
cytoplasm, were instead unaffected. In addition to targeting CREB [89], nuclear Ca2+
also stimulates the CREB co-activator CBP [90] (CREB binding protein), which,
however, also binds to other DNA binding proteins, transmitting the Ca2+ message to
a number of other transcription factors. CREB-CBP-driven transcription is driven by
the phosphorylation of CREB by nuclear CaMK IV [91], activated in turn by the
increase of nuclear Ca2+ (other kinases may also phosphorylate CREB). The kinase
phosphorylates CREB on Ser133, promoting its interaction with CBP, however, for
transcription to start, CBP itself must also be phosphorylated by CaMK IV. CaMK
IV has also recently been found to regulate the process of alternative splicing of the
primary transcripts of numerous genes (see Section 4.3).
Gene transcription, however, can also be regulated by the Ca2+ binding EF hand
protein downstream regulatory element antagonist modulator (DREAM) [92,93], a
multifunctional protein that also has roles outside the nucleus. At low levels of
nuclear Ca2+ DREAM interacts with downstream responsive element (DRE) sites
present in the promoter of a many genes, repressing transcription. As nuclear Ca2+
increases, DREAM binds it, leaving the DRE sites and allowing transcription to
initiate. DREAM was initially found to regulate the transcription of the dynorphin
gene, but is now known to regulate a number of other genes, including some that
code for Ca2+-regulating proteins, e.g., one of the Na+/Ca2+ -exchangers [94], and
one subunit of the L-type Ca2+ channels [95].
Another mechanism by which Ca2+ can influence the transcription of genes involves
the translocation of transcription factors from the cytoplasm to the nucleus. One well
4 Calcium in Health and Disease 101

understood case is that of nuclear factor of activated T cells (NFAT), a transcription


factor that is transported into the nucleus by calcineurin after the latter has dephos-
phorylated it in response to the increase of cytoplasmic Ca2+ [96] (discussed in more
detail in Section 4.3). Another case is that of the nucleo-cytoplasmic shuttling of the
Forkhead transcription factor FoxO3a, which has a role in cell death processes. FoxO3a
is translocated to the nucleus in response to several cell-death promoting stimuli, caus-
ing transcriptional activation of cell death-inducing genes. In hippocampal neurons the
activation of extrasynaptic NMDA receptors [97] causes the translocation of FoxO3a
to the nucleus. The activation of synaptic NMDA receptors, instead, inhibits the trans-
location, protecting the neurons from death-inducing signals. The protection process
involves CaMK IV, stimulated by nuclear Ca2+ transients induced by the activation of
synaptic NMDA receptors. The protection process is likely to be related to a gene tran-
scription process, which would modulate the translocation of the transcription factor;
i.e., its release from the DNA and its export from the nucleus.
Ca2+ can regulate gene transcription by still another mechanism, which involves
a dual function of the L-type Ca2+ entry channels. As just mentioned, it regulates
gene transcription by promoting nuclear Ca2+ transients. However, a C-terminal
fragment of the pore-forming subunit of the channel which is located in the nucleus
is produced in neurons [98] of developing and adult brains. Its production and
nuclear localization are developmentally regulated, and the influx of Ca2+ through
the L-type channel themselves, or through NMDA receptors, causes its export from
the nucleus. Within the nucleus, the fragment affects the transcription of a number
of genes, including some that are involved in the regulation of Ca2+.

4.2 Intracellular Proteolysis

The Ca2+-dependent intracellular cysteine proteases now known as calpains were


discovered in 1964 [99]. They are regulatory, rather than strictly degradative,
enzymes, i.e., they catalyze the limited proteolysis of proteins involved in numerous
cell functions, irreversibly modulating them [100]. They are considered to be cyto-
plasmic enzymes, but recent research has shown that they also exist in mitochon-
dria, where they are claimed to cleave a number of substrates, including
apoptosis-inducing factors like the AIF. Calpains do not have a strict substrate rec-
ognition sequence. The tertiary structure, rather than primary structure elements, of
the attacked proteins appears to determine their substrate preference. In small pep-
tides, calpains have specificity for sites in which a small hydrophobic amino acid is
in position P2, and a large hydrophobic amino acid in position P1 [101]. The calpain
family now comprises numerous isoforms (15 in the human genome), some of
which have multiple spliced variants. Some isoforms are ubiquitous, i.e., calpain 1
(u-calpain), calpain 2 (m-M calpain), and calpain 10, others are tissue-specific, e.g.,
calpain 3 (also termed p94) for muscle, calpain 8 (also termed nCl-2) for stomach,
calpain 9 (also termed nCl-4) for the digestive tract. Ubiquitous calpains play
roles in all cells: their physiological functions are still incompletely understood, but
their involvement in the regulation of processes like cell differentiation, cell cycle
102 Brini, Ottolini, Calì, and Carafoli

regulation, cell death, embryonic development, and a number of nervous tissue


functions is fairly well documented.
Defects of ubiquitous calpains may be lethal, e.g., the knocking out of either the
calpain 1 or calpain 2 genes, but may also be compatible with life; positional clon-
ing has identified variants of the calpain 10 gene that are associated with increased
susceptibility to type 2 diabetes in American and European populations [102]. By
contrast, ubiquitous calpains tend to become hyperactivated in disease conditions,
in which a dysfunction of Ca2+ homeostasis creates a condition of cellular Ca2+
overload, e.g., cardiomyopathies, muscular dystrophies, neuronal excitotoxicity,
Alzheimer disease, cataract formation; permanent (i.e., not regulated) calpain acti-
vation in these conditions promotes the unregulated cleavage of target, but also
non-target, protein substrates, leading to irreversible cell damage. Defects of tissue-
specific calpains may generate tissue-specific disease phenotypes: limb-girdle mus-
cular dystrophy type 2A is caused by mutations in the calpain 3 gene [103], and the
digestive tract-specific calpain 9 is down-regulated in gastric cancer cell lines. Its
depletion in fibroblasts using antisense RNA was found to be tumorigenic, suggest-
ing that in these systems calpain 9 acts as a tumor suppressor.
The calpain superfamily is divided into the typical and atypical families depend-
ing on the domain structure. Typical calpains are heterodimers of a large (about 80
kDa) catalytic subunit, and a small (28 kDa) regulatory subunit. The most exten-
sively investigated typical calpains are calpain 1 and calpain 2. They differ in Ca2+
sensitivity, calpain 1 being optimally stimulated by μM Ca2+, and calpain 2 by mM
Ca2+. The catalytic subunit can be divided into four domains. The N-terminal domain
I becomes autocatalytically hydrolyzed, leading to the activation of the enzyme and
to the detachment of the regulatory subunit from the heterodimer. Domain II
(divided in subdomains IIa and IIb) is the catalytic core of the enzyme. It contains
the catalytic triad (Cys-His-Asn) typical of cysteine proteases. Domain III contains
a C2 Ca2+-binding domain typical of other Ca2+-binding proteins. It is the linker
between the catalytic core of the protein and its Ca2+-binding domain. The C-terminal
domain IV has homology to CaM, and contains 5 EF hand Ca2+ binding motifs. The
fifth one is not operational, but interacts with the analogous EF hand domain of the
regulatory subunit, forming the heterodimer. The small regulatory subunit is com-
posed of domain V and domain VI. Domain V has a high Gly content: its hydropho-
bicity suggests that it may be involved in the binding of calpain to the plasma
membrane. A proline-rich stretch separates it from domain VI, which is homolo-
gous to domain IV of the catalytic subunit. It thus has 5 EF hand Ca2+ binding
motifs, of which the fifth does not bind Ca2+, but mediates the interaction with
domain IV of the large subunit.
Several partial and complete crystal structures of calpains are now available: they
have confirmed the predictions from sequence studies. They have shown that subdo-
main IIa, which contains the catalytic Cys, and subdomain IIb, which contains the
catalytic His and Asn, are held apart in the absence of Ca2+, disrupting the catalytic
triad and keeping the enzyme inactive. The binding of Ca2+ to domain IV, but possibly
to domain III as well, induces a conformational change that moves sub-domain IIa
closer to sub-domain IIb, reconstituting the catalytic triad, and activating protease
4 Calcium in Health and Disease 103

activity. Two non-EF hand Ca2+ binding domains that have recently been identified
[104] in sub-domains IIa and IIb are also involved in the conformational change.
The reason for the difference in Ca2+ sensitivity between calpain 1 and calpain 2 has
never been satisfactorily explained, and has implications for the physiological role
of calpain 2 (but for that of calpain 1 as well, since the concentration of Ca2+ neces-
sary to activate it is 10- to 100-fold higher than that known to prevail in normal
cytoplasms). A number of factors that could somehow lower the concentration of
Ca2+ necessary to activate these calpains have been proposed, but none has been
convincingly validated. The general idea has thus gained consensus that calpains
would only become activated when the concentration of Ca2+ in the cytosol increases
abnormally, as frequently occurring in several disease conditions (see above). The
idea would also make sense if one considers that the effect of calpains on target
proteins, be it an activation or an inhibition, is in any case irreversible, i.e., hardly
compatible with a physiological regulatory role. Related to this point is the matter
of autoproteolysis as a mechanism for calpain activation. Work on calpain 1 and 2
has shown that their incubation with Ca2+ induces the rapid autoproteolysis of both
the large and the small subunits, reducing substantially the Ca2+ requirement for
their activation. This has led to the proposal that calpains would be pro-enzymes
activated by autoproteolysis. However, other studies have shown that both the intact
and the cleaved forms of the enzyme are capable of cleaving substrates, and the
crystal structure of calpain 2 has indeed confirmed that the autoproteolysis removes
an N-terminal fragment from the large subunit that does not block the catalytic site.
Atypical calpains do not contain the small regulatory subunit, and some do not
even contain the penta EF hand domain 4. Their Ca2+ sensitivity is thus an open
problem, although they may contain the Ca2+-binding sites outside domain IV. For
instance, in ubiquitous calpain 10 domain IV is replaced by a domain structurally
related to domain III (which is also found in two other atypical calpains, calpain 5
and 6). In addition to the absence of domain IV, calpain 10 does not contain the
Ca2+-binding motifs in domain II (which are instead present in atypical calpain 5),
and its Ca2+-dependent activation mechanism is thus unclear.
Calpastatin [105] is a natural protein inhibitor of calpains. It has 4 repeated,
poorly homologous inhibitory domains of about 140 amino acids (domains I, II, III,
and IV) and an N-terminal domain L that has no inhibitory activity. Three sub-
domains have been identified in each inhibitory domain: sub-domain A binds to
domain IV of calpain, sub-domain B, which has little inhibitory activity by itself, is
essential for calpastatin activity, and sub-domain C binds to domain VI in the small
regulatory subunit of calpain.

4.3 Protein Phosphorylation and Dephosphorylation

Ca2+ has an important role in the phosphorylation and dephosphorylation of pro-


teins, which is a major mechanism for the regulation of metabolism. A large num-
ber of enzymes functioning in diverse metabolic pathways are phosphorylated on
104 Brini, Ottolini, Calì, and Carafoli

serine or threonine residues by Ca2+-dependent kinases, and are dephosphorylated by


calcineurin, the only Ca2+-dependent protein phosphatase so far known. Most of
the Ca2+-dependent protein kinases are CaM-stimulated enzymes, but other
kinases, the most prominent example being protein kinase C, decode the Ca2+
signal without the intermediation of CaM. The protein kinase C family contains
several members, some of which are not Ca2+-regulated. Five or six of them are
binding Ca2+ to a C2 domain normally located in the N-terminal moiety of the
protein. Binding of Ca2+ to the C2 domain helps targeting the kinase to the plasma
membrane, where it can effectively search for its membrane embedded activatory
ligand, diacylglycerol.
The CaM-dependent kinases (CaMKs) phosphorylate serine or threonine resi-
dues in specific consensus sequences of the substrates, although their substrate
specificities can overlap. The catalytic domain of most of them contains acidic
residues that interact with the basic residues in the consensus sequences. CaMKs
can be divided into narrow-specificity and broad-specificity CaMKs [106–108].
The former comprise myosin light chain kinase (MLCK), phosphorylase kinase
(PhK), and eukaryotic elongation factor 2 kinase (eEF-2K, also called CaMK III).
MLCK has only the regulatory light chain of myosin as substrate. Apart from the
CaM-binding domain, it contains an AID domain (atypical interacting domain; a
domain of unknown function found in many proteins, which is part of a broader
consensus sequence termed octicosapeptide). MLCK is normally activated by
Ca2+ release from the sarcoplasmic or endoplasmic reticulum, and the phosphory-
lation of myosin initiates contraction of smooth muscle and potentiates that of
skeletal muscle. PhK phosphorylates and activates glycogen phosphorylase,
accelerating glycogen degradation: it is present in many tissues, but is particularly
abundant in liver and muscle. It is a holoenzyme of 4 catalytic γ-subunits, com-
plexed to 4 each regulatory α-, β-, and δ-subunits. The δ-subunit, actually, is CaM,
which remains unusually associated to the holoenzyme even in the absence of
Ca2+. PhK is activated by the binding of Ca2+ to the δ-subunits, and the activation
is potentiated by the phosphorylation of the α- and β-subunits by protein kinase A.
eEF-2K phosphorylates elongation factor 2, promoting ribosomal translocation
along mRNA during translation: the phosphorylation inactivated eEF-2. The cata-
lytic center of eEF-2k bears no homology to those of the other CaMKs. Its regula-
tion is complex, and entails binding of CaM, autophosphorylation, and
phosphorylation by a variety of kinases.
The broad specificity CaMKs comprise CaMK I, CaMK II, CaMK IV, and CaM-
dependent kinase kinase (CaMKK). CaMK I, a protein of about 40kDa, is abundant
in brain, liver, and intestine. The binding of Ca2+/CaM activates the enzyme by dis-
rupting the inhibitory interaction of an AID with the ATP-binding pocket. CaMKK
phosphorylates CaMK I, activating it up to 20-fold. CaMK I phosphorylates a num-
ber of proteins, including brain proteins, but its physiological role remains elusive.
CaMK II is a ubiquitous kinase that has been involved in the regulation of a large
number of processes [109]. It is the product of 4 separate genes, the isoforms having
propensity to form a holoenzyme structure thanks to the presence of a C-terminal
association domain. The holoenzyme forms a double hexameric ring-shaped struc-
4 Calcium in Health and Disease 105

ture that facilitates regulatory properties. When subunits in the holoenzyme bind
Ca2+/CaM, they become activated, and trans-phosphorylate adjacent subunits at
Thr-286 increasing their affinity for CaM and making them Ca2+-independent. Since
the number of subunits in the holoenzyme that become active and autophosphory-
lated at Thr-286 depends on the Ca2+ concentration, CaMK II is able to “decode” the
frequency and the amplitude of the Ca2+ oscillations. This ability to “prolong” the
signaling of Ca2+ after its transient has abated has been exploited to involve CaMK
II in processes like learning and memory. CaMK II is present in the cytosol and is
also associated with organelles, in line with the large number of proteins it phos-
phorylates. One variant of CaMK II contains a nuclear localization sequence and
has been shown to regulate gene transcription, at least in cardiac myocytes. CaMK
II plays an important role in the regulation of synaptic transmission: indeed, several
neuronal proteins are phosphorylated by CaMK II, among them the NMDA and
AMPA glutamate receptors.
CaMK IV has a more restricted tissue distribution: it is expressed abundantly in
neuronal cells, in T-cells, and in the testis. It is activated by the binding of CaM, and
further activated by phosphorylation by CaMKK: unusually, the phosphorylation by
CaMKK renders CaMK IV Ca2+-independent. CaMK IV has a nuclear localization
sequence and has a prominent role in the nucleus, where it phosphorylates numer-
ous transcription factors. It also phosphorylates the heterogeneous nuclear ribonu-
cleoprotein (hnRNP) L which is the transactive factor that then interacts with the
CA (cysteine-adenosine) repeat in the CAMK IV-responsive RNA elements
(CaRRE) of numerous genes to regulate the splicing process of their primary tran-
scripts [110,111]. Interestingly, one of these genes encodes a plasma membrane
Ca2+ pump. CaMKK is similar in the organization of domains and function to the
other CaMKs, however, it also has distinctive features: it does not contain the acidic
residue that is used by other CaMKs to recognize basic residues next to the phos-
phorylated Ser or Thr, but contains instead an Arg- and Pro-rich insert that is impor-
tant in the phosphorylation of CaMK I and IV. Unusually, both CaMKK and its
substrates (CaMK I and CaMK IV) must bind CaM for phosphorylation to occur.
The only Ca2+-dependent protein phosphatase so far known is calcineurin (Cn),
a dimer of a 58–64 kDa catalytic subunit (CnA), and a tightly bound 19 kDa, Ca2+-
binding regulatory subunit (CnB), which is a canonical EF hand protein with 4 Ca2+-
binding motifs [112]. It is the product of three human genes, and is expressed in
most tissues. However, it is particularly abundant in the brain (hence, its name),
where isoform α predominates: it represents about 1% of the total brain protein. In
brain, Cn triggers a phosphatase cascade that opposes the stimulatory effects of
PKA and CaMKs: it has been implicated in a large series of brain processes, from
the expression and activity of ion channels, to the release of neurotransmitters, to
the recycling of synaptic vesicles.
The catalytic domain of the phosphatase is located in the N-terminal moiety of
CnA, and is followed by a domain that binds CnB and by two further domains, one
that binds CaM and one that acts as an autoinhibitory sequence. Importantly, in the
absence of CaM calcineurin is inactive, and is thus peculiarly under dual Ca2+ regu-
lation, by CaM and by its own “calmodulin”, i.e., the CaM-like 19 kDa subunit.
106 Brini, Ottolini, Calì, and Carafoli

This multiple Ca2+ regulation mechanism is reminiscent of that of calpain (see


above), which, however, instead of exogenous CaM, has, its own “calmodulin”
incorporated in its sequence (the penta EF hand domain IV). Cn is a phosphatase
with a binuclear iron-zinc binuclear active center, which has a dual substrate speci-
ficity: it dephosphorylates both phosphoseryl/threonyl and tyrosyl substrates. It
prefers substrates in which a basic residue lies at the N-terminal side of the phos-
phorylated amino acid, and no acidic residue lies at its C-terminal side. CnB is
tightly bound to CnA even in the absence of Ca2+, and is required for phosphatase
activity. The binding of CaM to CnA displaces the autoinhibitory domain, expos-
ing the catalytic center and activating phosphatase activity: however, prolonged
exposure of the active site facilitates the oxidation of catalytic Fe2+, inactivating the
enzyme. Paradoxically, then, CaM can be either activating or inhibiting depending
on the duration of the Ca2+ signal. A 240 kDa protein termed Cain/Cabin 1 has been
described as an endogenous inhibitor of calcineurin. Cn is also inhibited by fungal
immunosuppressive compounds (FK-506, cyclosporin), which bind to their respec-
tive immunophilins and then bind to calcineurin. The immunosuppressive drugs
are used to prevent organ rejection after transplant operations and in the treatment
of autoimmune diseases.
Cn plays an important role in the regulation of gene expression: this has been
established mostly through studies of T cell activation: the liberation of Ca2+ in the
cytosol by the activation of InsP3-linked receptors activates calcineurin to dephos-
phorylate the transcription factor NFAT, which exposes its nuclear localization
sequence and translocates it to the nucleus together with calcineurin. NFAT dephos-
phorylation also increases its DNA binding and transcriptional activity. The export
of NFAT from the nucleus depends on its rephosphorylation by GSK 3, and on
calcineurin inactivation upon Ca2+ removal/decrease [96].

4.4 Calcium and Bioenergetics

Shortly after initial findings on the transport of Ca2+ in mitochondria, it was discov-
ered [113,114] that three enzymes of the citric acid cycle of the mitochondrial
matrix (the pyruvate, the α-ketoglutarate, and the NADH-dependent isocitrate dehy-
drogenases) are activated by Ca2+ in the micromolar range (Km 1 to 50 μM) [115].
Thus, it became evident that the mitochondrial Ca2+ transport process had an essen-
tial role in the regulation of ATP production and in maintaining the proper bioener-
getics balance of the cells: a problem, in those early days, was the low affinity of the
mitochondrial Ca2+ uptake by uniporter, which in principle would not have permit-
ted mitochondria to efficiently take up Ca2+ in the physiological ambient of the
cytosol. As discussed above, the problem was solved decades later by the demon-
stration that the release of Ca2+ from the ER exposed neighboring mitochondria to a
Ca2+ concentration high enough to overcome the low affinity of the uniporter.
The machinery for energy production by mitochondria is the electron transport
chain (ETC) of the inner mitochondrial membrane, which is composed of five
4 Calcium in Health and Disease 107

multiprotein complexes. Three of them (I, III, IV) pump protons (H+) across the
inner membrane, ejecting them from mitochondrial matrix, thus establishing
the electrochemical gradient which is used by complex V (the ATP synthase) to
produce ATP. Reducing equivalents from the citric acid cycle are transported by the
respiratory chain from NADH and FADH2, to oxygen which is converted to H2O.
The negative-inside electrochemical gradient generated by their travel down to O2 is
not only used to synthetize ATP. It is also used to drive the transfer of Ca2+ across
the inner membrane into the mitochondrial matrix. Since the entry of Ca2+ dissipates
the membrane potential, it temporarily abolishes the synthesis of ATP. However, the
entry of Ca2+ into the matrix stimulates the activity of the citric acid cycle, thus
enhancing the delivery of reducing equivalents to the respiratory chain.
The mechanisms of the regulation of the three citric acid cycle enzymes by transient
Ca2+ increases in the matrix are different [115]. The pyruvate dehydrogenase
complex represents “the point of no return” in carbohydrate metabolism. The complex
is therefore subject to stringent regulation and its activity is directly inhibited by the
end product acetylCoA/CoA and NADH/NAD+ ratios, but also, more importantly,
by reversible phosphorylation by highly specific kinases and phosphatases in the
mitochondrial matrix. The phosphorylated pyruvate dehydrogenase is activated by
the Ca2+-dependent dephosphorylation by the pyruvate dehydrogenase phosphatase.
Two phosphatase isoforms are present in mammalian mitochondria, PDP1 and
PDP2, each containing a Mg2+-dependent catalytic subunit, designated as PDP1c
and PDP2c, of which only the first is activated by Ca2+. Ca2+ regulation of pyruvate
dehydrogenase may thus vary according to the distribution of the two isoforms in
different tissues, or physiological situations, e.g., the nutrition status [116,117].
Mammalian NAD-isocitrate dehydrogenase consists of three subunits associated
to form an octamer. It has complex regulatory properties: it is inhibited by increas-
ing ATP/ADP and NADH/NAD+ ratios (a property shared with the pyruvate dehy-
drogenase system and oxoglutarate dehydrogenase). Ca2+ causes a marked decrease
in the Km of the dehydrogenase, the Ca2+ sensitivity of which is influenced by the
ATP/ADP ratio (it becomes more sensitive to Ca2+ at lower ratios). Two Ca2+ ions
are bound per dehydrogenase octamer, to motifs different from the canonical Ca2+-
binding motifs discussed above.
Oxoglutarate dehydrogenase is a multienzyme complex that has similarities to
pyruvate dehydrogenase. It is also end-product inhibited by increases in the succi-
nyl CoA/CoA and NADH/NAD+ ratios. However, unlike pyruvate dehydrogenase,
it is not regulated by reversible phosphorylation. Ca2+ acts directly on the enzyme
markedly decreasing its Km. The Km is also decreased by the decrease in the ATP/
ADP ratio. As in the case of the NAD-isocitrate dehydrogenase, decreases in this
ratio also markedly increase the sensitivity of the enzyme to Ca2+. Between 2.5 and
5 Ca2+ are bound to each oxoglutarate dehydrogenase complex. As in the case of the
NAD-isocytrate dehydrogenase, canonical Ca2+-binding sites have been found in
the subunits of the complex.
The possibility to directly monitor mitochondrial Ca2+ transients generated by
cell stimulation has permitted to analyze in detail the activation of the three
matrix enzymes by Ca2+. Thus, it has been shown that the Ca2+ transients monitored
108 Brini, Ottolini, Calì, and Carafoli

directly within the mitochondria paralleled the increase of NADH [118] and of ATP
production [119]. The entity of the increase was proportional to the amplitude of
the matrix Ca2+ transients. Interestingly, imaging studies on single cells have shown
that oscillations of cytosolic Ca2+ were transmitted to mitochondria resulting in the
sustained activation of the matrix enzymes, thus extending the NADH increase for
times longer than those of the Ca2+ transient [120].
In addition to the three citric acid cycle enzymes, a number of other potential
mitochondrial targets of Ca2+ regulation have also been proposed that may directly or
indirectly influence respiration and hence, ATP synthesis. For instance, the mito-
chondrial F1F0 ATPase itself may be activated by μM concentrations of Ca2+ ions
by a mechanism involving the release of a small inhibitory protein [121].
Ca2+ can also improve energy metabolism by favoring the transport of the NADH
equivalents produced in the cytosol during the glycolysis to the mitochondrial
matrix. The transport is mediated by two shuttle mechanisms: the glycerol phos-
phate shuttle and the malate-aspartate shuttle, both of which can be activated by
extramitochondrial Ca2+. The FAD-glycerol phosphate dehydrogenase is located on
the cytoplasmic surface of the inner membrane: together with the cytoplasmic
NAD-glycerol phosphate dehydrogenase it forms the glycerol phosphate shuttle.
The aspartate/glutamate carrier (AGC1, or aralar), is a component of the malate-
aspartate shuttle [122,123]. The proteins of both shuttle pathways contain EF hand
Ca2+-binding sites that face the intermembrane space and are sensitive to Ca2+
increases occurring in the proximity of mitochondria.

4.5 Muscle Contraction

The history of Ca2+ as intracellular messenger actually initiated with studies of heart
muscle contraction. It is traced back to 1883, when S. Ringer discovered that Ca2+
was essential for cardiac contractility [124]. It took a long time to realize that Ca2+
acts as a messenger not only in the contraction of heart, but also in that of skeletal
muscles. The concept of “excitation-contraction coupling” (ECC) was eventually
established, i.e., the concept of a mechanism that links electrical phenomena occur-
ring at the plasma membrane with the activation of contractile proteins [125].
The mechanism by which muscle contraction is regulated by Ca2+ is now well
understood, and will thus only be described very succinctly, to focus on its different
molecular details in skeletal, cardiac, and smooth muscles. Contractile proteins
include myosin, actin, tropomyosin, and troponin, which are organized into func-
tional units (the sarcomere). Myosin thick filaments are surrounded by actin poly-
mers thin filaments organized in a hexagonal array together with tropomyosin and
troponin. Troponin is distributed along the entire length of the thin filament at inter-
vals of about 40 nm. The periodicity is determined by the arrangement of tropomyo-
sin molecules which fit in the grooves of the double stranded actin filaments.
The myosin and actin filaments slide along each other utilizing energy from
ATP hydrolysis, thus shortening the sarcomere unit in the contraction process.
4 Calcium in Health and Disease 109

Tropomyosin and troponin confer Ca2+ sensitivity to it, troponin being the Ca2+
sensor that allows contraction to occur. It is a complex of troponin C, I and
T. Troponin I is the subunit that inhibits the ATPase activity of the actin-myosin
complex, troponin T promotes the binding with myosin and regulates the interaction
between the troponin components, and troponin C binds Ca2+ to four EF hand
motifs. The mechanism of the regulation by Ca2+ is similar in skeletal and cardiac
muscles, but differs in smooth muscle, where, instead of the troponin-tropomyosin
complex, a Ca2+ CaM-dependent myosin light chain kinase operates.
In skeletal muscles, ECC occurs by mechanical coupling involving the interac-
tion between L-type channels in specialized structures of the plasma membrane, the
T tubules. They are formed by PM invaginations that establish physical contact with
specialized portions of the SR (the terminal cisternae) permitting the coupling
between the voltage gated L-type Ca2+ channels with the RyR channels in the SR.
The plasma membrane depolarization is sensed by the L-type channels in the
T-tubules and directly transmitted to the RyR. Several proteins participate in the
junction between PM and SR [126,127], among them the transmembrane proteins
triadin and junctin that mediate the contact between RyRs and the SR protein calse-
questrin, which senses the luminal Ca2+ concentration in the SR, and transmits the
information to RyR via triadin. The contraction of skeletal muscles depends on the
Ca2+ release from the SR store by RyRs, the relaxation phase is instead mediated by
Ca2+ reuptake in the SR by isoform 1 of the SERCA pump.
In the cardiac muscle, instead, even if the release from SR is the triggering event
for contraction, Ca2+ entry from L-type channels is required to induce the CICR
mechanism and thus the opening of RyRs. The depolarizing action potential
originating from the sino-atrial node induces contraction starting from the right
atrium forcing blood into the ventricles. When the action potential travels across the
heart the membrane of cardiac myocytes becomes depolarized causing the opening
of L-type voltage-gated channels and the influx of Ca2+ into a restricted region
between the plasma membrane and the membrane of SR (the junctional zone or
dyadic cleft). This Ca2+ influx is not sufficient per se to activate contraction, but it
induces the opening of a clusters of RyRs located in the SR membrane opposed to
the PM and thus activates the CICR mechanism and the consequent mobilization of
Ca2+ from SR. The diffusion of Ca2+ from the junctional zone then generates a global
Ca2+ increase that activates the contractile machinery [128]. As in skeletal muscle,
after Ca2+ has activated the contractile proteins it is rapidly extruded from the cyto-
sol to permit the next action potential to trigger a new contraction. In cardiac myo-
cytes the main systems that remove Ca2+ from the cytosol are the plasma membrane
Na+/Ca2+ exchanger and isoform 2 of the SERCA pump of the SR. The relative
contribution of these systems differs according to the species. The PMCA pumps of
the plasma membrane do not have a quantitatively significant role in the extrusion
of Ca2+ from the cardiomyocyte but can regulate contraction in a subtler way, linked
to the modulation of the NO synthase [129].
The ECC in smooth muscles differs from that of skeletal and cardiac muscles
[130]. Smooth muscle cells form a layer that wraps up hollow organs such as
blood vessels, intestine, bladder, airways, uterus etc. Their contractile properties are
110 Brini, Ottolini, Calì, and Carafoli

functionally important in these organs, as they permit dynamic changes in their


luminal volume that may regulate the movements of the content of the organs, as in
the case of peristalsis or urine expulsion. Uterine smooth muscle exerts a dual
action: it relaxes during gestation to accommodate fetal growth, and contracts
during parturition. Similarly, the contractility of smooth muscle in the vessel wall
controls blood pressure and flow to the body.
In most cases the changes in plasma membrane depolarization driven by action
potentials are sufficient to promote Ca2+ influx into the myocytes through the
voltage-gated channels that directly activate contraction. In arterial smooth muscle
cells, where the membrane potential across the plasma membrane is about −50/–40
mV, oscillations of approximately 10 mV above or below these values trigger
changes in global Ca2+ that engage the contractile apparatus and cause maximal
dilatation or constriction.
Contraction of smooth muscle cells occurs even in the absence of extracellular
Ca2+, when driven by agonists that trigger the opening of the intracellular InsP3R
and RyR channels and the activation of a CICR mechanism. This is the case of the
urinary bladder, the gastrointestinal trait, and the airways, but also of arteries where
Ca2+ sparks have been detected. It is also the case of smooth muscle cells from the
colon or the portal vein, where the InsP3-mediated Ca2+ release originates a Ca2+
wave [131].

4.6 Secretion

Ca2+ is especially important in the fusion of the secretory vesicles with the plasma
membrane [132], but it also has a role in the process of vesicle maturation [133].
Conceptually, the release of the vesicle content can be divided into four steps: vesi-
cle docking, vesicle priming, Ca2+ triggering, and the vesicle fusion reaction itself.
Two secretion processes are especially well characterized: the synaptic trans-
mission in neurons and the insulin secretion in pancreatic β cells. Synaptic and
endocrine exocytosis use the same Ca2+-triggering mechanisms, but differ in the
mechanism by which the vesicles are docked and prepared for fusion (i.e.,
primed). Vesicle exocytosis is managed by the SNARE (soluble N-ethylmaleimide-
sensitive factor attachment protein receptor) fusion machinery [134,135]. The
complex includes a number of proteins having different localization and roles:
synaptobrevin-2 (syb2) on the vesicle, syntaxin-1 (syx1), and SNAP-25 on the
plasma membrane interact with each other to form a very stable bundle of four
coiled α-helices. Accessory factors, including complexins, Munc13, Munc18, and
synaptotagmins also participate in the assembling of the complex (for reviews
see, e.g., [136–138]).
Synaptotagmins are single pass transmembrane proteins that bind Ca2+ with rela-
tively low affinity (Kd > 10 μM) at two C2 domains in their C-terminal portion
(these domains are not functional in all synaptotagmin isoforms). Interestingly,
membrane phospholipids also participate in Ca2+ binding to C2 domains, which,
due to their different affinity for Ca2+, could operate cooperatively to regulate both
4 Calcium in Health and Disease 111

constitutive secretion, triggered by spontaneous fluctuation in cytosolic Ca2+ and


thus occurring in resting condition, and evoke exocytosis triggered by massive Ca2+
entry promoted by the action potential.
Synaptotagmin I (Syt1) is the Ca2+ sensor in many Ca2+-sensitive processes of
neurotransmission; it binds both syntaxin and SNAP-25 and transduces the Ca2+
signal into a nanomechanical activation of the membrane fusion machinery, thus
causing vesicle fusion. Complexin, which is another Ca2+-binding protein, activates
SNARE-complexes before synaptotagmin action, and clamps fusion by preventing
complete SNARE-complex assembly until Ca2+ binds to synaptotagmin [139].
The molecular details of the fusion process have been investigated in depth, and
the data obtained by studying secretion events in reconstituted membranes have
shown that Ca2+ entry through the VOCCs is necessary to recruit vesicles at the
plasma membrane rather than to trigger their fusion. According to this model
docked vesicles are tethered to the membrane through a non-primed or primed
excitosome complex. Synaptotagmin 1 is a member of the complex that can be in
either a Ca2+-bound or a Ca2+-unbound form, differentiating releasable pools. Only
docked vesicles primed by Ca2+ binding to synaptotagmin would be released fol-
lowing the depolarization signal. Ca2+ binding to the sensor in the transmembrane
sector of the voltage-gated channel induces conformational changes to the channel
transmitted through the interaction between the intracellular loop connecting trans-
membrane domain II and III of the VOCCs and the TM domain of synaptotagmin.
Thus, according to this view, synaptotagmin acts as a priming protein rather than
as a fusion inducing protein, and the sensors for fusion are instead the VOCCs
channels themselves.
This model, named excitosome model, adequately explains secretion events
occurring in the sub-millisecond timescale, which especially characterizes synaptic
transmission, satisfying the time requirements of the of excitation-secretion cou-
pling process [140].
As for endocrine secretion, the role of Ca2+ in regulating insulin secretion is of
special interest as it has been proposed to be linked to the generation of local Ca2+
microdomains beneath the plasma membrane would differentially control the
release of different pools of vesicles. Nutrient-induced increases in intracellular free
Ca2+ concentrations are the key trigger for insulin release from pancreatic islet
β-cells [141]. In healthy β-cells, the uptake of glucose by a facilitated glucose trans-
porter and glycolytic and mitochondrial metabolism increases the ATP concentra-
tion. The increased ATP/ADP ratio promotes the closure of the ATP-sensitive K+
(KATP) channels, plasma membrane depolarization, and Ca2+ influx through voltage-
gated channels: ultimately, it promotes the fusion of insulin containing large dense
core vesicles (LDCVs) with the plasma membrane. The subsequent opening of
voltage-gated K+ channels repolarizes the cell to terminate exocytosis.
Convincing evidence shows that in β-cells L-type (voltage-gated) Ca2+ channels
are most active close to sites where LDCVs are clustered [142] and it has also been
proposed that these channels may physically interact with them [143]. In addition,
it has been suggested that Ca2+ release from the ER or even from the LDCVs by
CICR or NAADP-gated channels, may locally contribute to the increase of the Ca2+
levels close to the vesicle surface.
112 Brini, Ottolini, Calì, and Carafoli

4.7 Calcium in the Beginning of Cell Life

The initiation of embryo development depends on the intracellular Ca2+ increase in


the egg during fertilization. In most species, including mammals, Ca2+ is also
responsible for inducing changes in oocytes that make them mature for fertilization.
In particular, the resumption and progression of the meiosis, which is a step neces-
sary to activate oocytes to initiate embryo development, is directly associated with
an activating Ca2+ signal generated during sperm-oocyte fusion [144,145]. This gen-
eral mechanism, however, differs among species with respect to the spatiotemporal
configuration of the Ca2+ signal.
In some species, e.g., in sea urchins, starfish, frogs, and fish, a single Ca2+ tran-
sient in the oocyte is sufficient for the activation, in others, e.g., ascidia and mam-
mals, an oscillatory pathway is instead required. The molecular machinery of the
signaling cascade also differs among species, being essentially dependent on the
activation of the phosphoinositide pathway in sea urchins, starfish, and frogs, and on
the release of a protein moiety containing a molecule named sperm factor (SF) in
mammals. This factor has been molecularly identified only in mammals and found
to be a sperm-specific isoform of phospholipase, PLCζ [146]. Thus, if in one case
the generation of the diffusible second messenger InsP3 is driven by the activation
of a Src-family kinase and PLCγ, in mammals the sperms directly delivers PLCζ,
which hydrolyzes membrane PIP2 to InsP3 and diacylglycerol (DAG). The genera-
tion of InsP3 induces the release of Ca2+ from the intracellular stores (cADPR/ryano-
dine receptor channels may contribute to the Ca2+ mobilization). The role of InsP3 in
the globalization of the Ca2+ signal is now generally accepted. However, recent
work has demonstrated that the initial liberation of Ca2+ is promoted in the cortex of
the egg by NAADP acting on special stores, that would then promote the liberation
of Ca2+ from the InsP3 stores via a CICR process [147]. The NAADP sensitive stores
which initiate the liberation of Ca2+ have been proposed to be the acidic organelles
[84] which, as has been mentioned above, have been recently questioned as Ca2+
stores [58]. It has also been proposed that the initial increase of Ca2+ in the cortex of
starfish eggs could be promoted by NAADP acting on a novel plasma membrane
channel [148].
Irrespective of the mechanism by which the elevation of intracellular Ca2+ at
fertilization is produced and shaped, its immediate consequence is the exocytosis of
cortical granules (CG) which modifies the component of zona pellucida to prevent
polyspermy and ensures the formation of the diploid zygote. The actin cytoskeleton
is prominently involved in the reorganization of the cortical domain of the cell that
promotes these processes [149]. The release from meiosis arrest is mediated by a
Ca2+-CaM dependent protein kinase II degradation of cyclin B [150]. Interestingly,
it has been shown that each sperm-induced Ca2+ increase is accompanied by a parallel
increase in CaMKII activity [151].
Considering that the elevation of Ca2+ occurs in a relatively rapid temporal
sequence, e.g., minutes, oscillations may extend the effect of Ca2+ for hours after
sperm fusion, and may thus be advantageous for other processes in mammals.
4 Calcium in Health and Disease 113

Interestingly, it has been shown that the recruitment of maternal mRNA for new
protein synthesis occurs during the oscillation, and is proportional to the magnitude
of Ca2+ stimulation. Pulsatile activation of CaMKII appears to underline enhanced
gene expression [152], however, it has also been shown that cell cycle progression
is required to recruit mRNA, implying that the process is not under exclusive Ca2+
control [153].

4.8 Apoptotic Cell Death and Autophagy

As has now become clear Ca2+ does not only regulate biological processes necessary
to cell survival and wellness: it also participates in processes that may culminate in
cell death, e.g., apoptosis and autophagy. However, it must be understood that these
processes are not just ways for the cells to die: they also represent a sophisticated
mechanism for cell quality control and rescue, which are necessary to the harmoni-
ous development of the organism. Apoptosis is necessary to normal cell homeosta-
sis as it eliminates cells that are damaged or unnecessary. Autophagy is the general
term used to define a cellular process responsible for the delivery of proteins or
organelles to lysosomes.
Apoptosis occurs through two conventional pathways: (i) the extrinsic pathway
which is typically initiated by death receptors acting on the plasma membrane and
the activation of the death-inducing caspase cascade [154], and (ii) the intrinsic
pathway, which acts through the permeabilization of the mitochondrial outer mem-
brane that releases cytochrome c and induces caspase activation [155]. The second
pathway is regulated by Ca2+, and will thus be discussed here. Ca2+-mediated apop-
tosis can be triggered by physiological signals, but multiple cytotoxic agents also
lead to it by disrupting Ca2+ homeostasis: the inhibitor of SERCA pumps thapsigar-
gin and the alkaloid staurosporine are the best known. They have been used to dis-
sect the molecular details of the pathway and have established that the Bcl-2 family
of proteins is important to it. Bcl-2 is overexpressed in a number of cancer cells as
it promotes their survival [156]. That Ca2+ was involved in the Bcl-2-linked apopto-
sis process was indicated by the finding that the protein controlled the Ca2+ signal,
and by work showing that Bcl-2, which is not only localized in the cytoplasm and in
the nuclear envelope, but also associates with the ER and mitochondrial membranes,
regulates the InsP3-mediated Ca2+ release [157]. The first evidence that Bcl-2 over-
expression directly affected Ca2+ homeostasis came from work on hematopoietic
cells, where its overexpression prevented the reduction of the cytosolic free Ca2+
induced by the withdrawal of interleukin-3, at the same time protecting the cells
from apoptosis [158]. It was later shown that the overexpression of Bcl-2 reduced
the Ca2+ content of the ER and Golgi lumina, and reduced the Ca2+ release following
InsP3 stimulation [159,160]. As a result, it also prevented the mitochondrial Ca2+
overload and the cell death it would induce.
Different mechanisms have been proposed for the control of ER luminal Ca2+ by
Bcl-2, and it has been concluded that it could be linked to differences in Bcl-2
114 Brini, Ottolini, Calì, and Carafoli

expression and localization in various cells, or to the isoform of InsP3 receptor


expressed in them. Initial studies showed that Bcl-2 increased the ER Ca2+ leak by
increasing membrane permeability [159,160], but other studies showed instead that
Bcl-2 depleted Ca2+ pools by interacting with the SERCA pump [161], and still
other studies proposed that Bcl-2 interacted directly with the InsP3 receptor, either
stimulating or inhibiting it [162,163]. A more recent study even suggested that
Bcl-2 inhibited Ca2+ entry by downregulating L-type channels, thus eventually pre-
venting mitochondrial Ca2+ uptake [164].
In addition to Bcl-2 (and Bcl-xL, another antiapoptotic member of the family),
also the proapoptotic members of the Bcl-2 family Bax and Bak regulate Ca2+
homeostasis. Their knockdown reduces the Ca2+ content of the ER, whereas Bax
overexpression increases it [165,166]. Accordingly, Bax/Bak MEF double knockout
cells were found to be resistant to apoptotic stimuli [167].
As for autophagy, it is classified in three main classes: microautophagy,
chaperone-mediated autophagy (CMA) and macroautophagy. They differ in the
way by which cellular organelles and proteins are phagocytosed and delivered to
lysosomes, so that their constituents are recycled to satisfy the energy demands dur-
ing metabolic stress. Microautophagy implies a direct delivery of a small portion of
cytoplasm by invagination and fission of the lysosomal membrane. CMA is a very
selective process that relies on the help of chaperones that recognize specific
sequence motifs in proteins, e.g., the KFERQ pentapeptide, and of the lysosome-
associated membrane protein type 2A (LAMP-2A) to target unfolded or aggregated
proteins to the lysosomes. Macroautophagy is characterized by the formation of
double membranous vesicles called autophagosomes. Under non-stress conditions,
low levels of autophagy guarantee normal cellular homeostasis by removing dys-
functional proteins or even organelles: an intensively studied autophagy specializa-
tion is the removal of dysfunctional mitochondria, a process named mitophagy
[168,169]. Autophagy must thus be considered a positive process: not surprisingly,
therefore, its impairment or alteration are often involved in pathologies like cancer
and neurodegenerative disorders [170].
More than 30 atg genes have been identified that regulate macroautophagy, and
in mammals the serine/threonine kinase mTOR has a central role in the process.
mTOR controls cell growth and metabolism in response to nutrients, growth factors,
ATP, and stress. Emerging evidence indicates that, as in the case of apoptosis, intra-
cellular Ca2+ regulates autophagy, even if the exact role of Ca2+ remains still ambig-
uous. Numerous reports suggest an inhibitory role, others a stimulatory role
[171,172]. The inhibitory role has been linked to InsP3R activity since the silencing
of the receptor by siRNA or its inhibition by xestospongin or Li+ induced autophagy
[81,173]. An intriguing explanation for the control of autophagy by the InsP3R pro-
poses that it could act as a scaffold and, by simultaneously binding beclin 1 (a pro-
tein required for autophagy) and Bcl-2, could participate in the formation of
antiautophagic Bcl-2/beclin 1 complexes, which would sequester beclin 1 and pre-
vent the activation of autophagy [174].
By converse, the activating role of Ca2+ has been associated to agents that induce
its increase in cytosolic concentration, e.g., the SERCA pump inhibitor thapsigargin
4 Calcium in Health and Disease 115

Figure 3 The cartoon illustrates the most important players in the control of cellular Ca2+ homeostasis
and in the systems that decode its function. The free cytosolic Ca2+ concentration is maintained in
the nM range by buffering proteins and by the action of pumps and other transporters on the plasma
membrane or in the membrane of the organelles (ER and Golgi). When the cell is stimulated,
channels in the plasma membrane and in the organelles are opened, generating the immediate
increase of cytosolic Ca2+ which is then returned to the basal concentration by the system above. The
precise control of Ca2+ homeostasis is fundamental for important activities of the cell, e.g., gene
transcription in the nucleus, energy transformation in mitochondria, exocytosis (secretion)
mechanisms, muscle contraction (T-tubules). The values of Ca2+ concentration indicated in the
cartoon are only representative and may vary depending on the cell type. The insets surrounded by
black lines represent specific compartments or organelles. The filling color indicates the concentra-
tion of Ca2+ in the compartment/organelle (e.g., dark blue: high Ca2+ concentration, light blue: low
Ca2+ concentration). All symbols and acronyms in the cartoon are explained and described in the
text. Some clarifications: in the lysosomes the putative system that accumulates Ca2+ in the lumen
of the organelle is indicated with a question mark, as it has not been characterized. In the ER/SR
numerous Ca2+-buffer proteins are present, however only calsequestrin (Calseq) is indicated. Inside
the vesicles , the little green shapes with spikes represent the secreted molecules. The arrows indi-
cate the direction of Ca2+ ion fluxes. CaR, IP3R, STIM, Mfn2, and NCX refer to Ca2+ sensors,
InsP3R, STIM1, mitofusin 2, and Na+/Ca2+ exchanger, respectively, in the text.

or the Ca2+ ionophore ionomycin. Increased cytosolic Ca2+ activates autophagy via
a signaling pathway involving the CaMKK, the ΑMP-activated protein kinase (AMPK),
and mTOR. An independent pathway in which the elevation in cytosolic Ca2+ activates
autophagy in AMPK-knock out fibroblasts has also been proposed [175]. While the
unambiguous correlation between cytosolic Ca2+ levels and autophagy activity is
116 Brini, Ottolini, Calì, and Carafoli

difficult to demonstrate, a clear correlation has been established with the state of
filling of the ER Ca2+ store. This underlines the close relationship between the
autophagic and apoptotic pathways, since the latter is also strictly related to the
concentration of luminal ER Ca2+ and to the amount that can be released by the ER.
Briefly, Bcl-2 acts a suppressor of both Ca2+-dependent apoptotic and autophagic
processes. In the autophagy pathway, the proposal that Bcl-2 acts by binding beclin
1, and also by lowering ER Ca2+ concentration has been convincingly documented
[176–178].
It has also been shown that treatments which enhance autophagy, e.g., that with
the mTOR inhibitor rapamycin, or the deprivation of nutrients, also remodeled the
intracellular Ca2+ signaling machinery [179,180].
The comprehensive cartoon of Figure 3 summarizes visually the information
provided up to this point on the systems for the control of cell Ca2+, and on the
processes regulated by its signal.

5 The Ambivalence of the Calcium Signal:


Defects of Calcium Regulation and Disease

As has been made clear in the preceding section, the Ca2+ message is vital to the
correct functioning of most cell processes. It has also been made clear that Ca2+
within cells must be controlled with utmost precision, even when the aim of the Ca2+
signal is to terminate cells in the apoptotic process. Conditions may arise, however,
in which the control of cell Ca2+ fails, in which cases cells predictably develop vari-
ous forms of pathological dysfunctions. Conditions in which the lack of control of
cell Ca2+ is massive and global terminate rapidly cell life: these are the cases of toxic
cell death induced by massive conditions of Ca2+ overload. However, subtler Ca2+
defects that affect single systems for the control of Ca2+ permit cell life to continue,
albeit with various degrees of discomfort. A number of these conditions are genetic,
and their study has even contributed to the understanding of the mechanisms for the
control of Ca2+ and the decoding of its message. A brief description of these condi-
tions will be offered in the next sections, which will cover only the most important
(and interesting) among them.

5.1 Neuronal Diseases

5.1.1 Ataxia

Ataxias are neurological disorders characterized by lack of coordination in voluntary


movements. There are 3 types of ataxia: cerebellar, sensory, and vestibular. The last
two types involve problems in the dorsal spinal cord (due to impaired proprioception)
and in the vestibular system, respectively: they represent a minor percentage of all
4 Calcium in Health and Disease 117

ataxias. The cerebellar ataxias, and more precisely the spinocerebellar type (SCA),
are the most intensively studied: Ca2+ frequently has been involved in their pathogenesis.
They are caused by defects in more than 30 genes [181]. Their correlation to the
impairment of Ca2+ homeostasis is obvious in patients which present mutations in
proteins involved in Ca2+ regulation. Mutations in CACNA1A, a subunit of CAV2.1
voltage-dependent P/Q-type Ca2+ channels, which are expressed abundantly in cerebellar
Purkinje cells indeed induce SCA6 [182]: the most frequent mutation is the expansion
of CAG repeats, that specify glutamines (more than 19Q in SCA6) at the C-terminal
of the subunit. SCA6 is thus one of the poly-glutamine (poly-Q) neurological diseases.
The N-terminal portion of the protein containing the long poly-Q chain specified by
the CAG repeats is processed by proteases, producing a (presumably toxic) fragment
that can aggregate or translocate to other cell compartments. The Ca2+ channel that
generates the poly-Q fragment seems not to be damaged [183], and its mutant subunit
can aggregate even if not cleaved by the protease [184]. Other point mutations in the
CACNA1A gene induce different ataxic phenotypes, e.g., episodic ataxia type 2 and
early-onset cerebellar atrophy [185]. Partial deletions in the gene of InsP3R1, instead,
cause SCA15, SCA16, and SCA29 [186,187] and a marked downregulation of the
InsP3R has been found in several SCA15 patients [181].
Alterations of Ca2+ influx into neurons due to glutamate excitotoxicity can be
involved in the pathogenesis of SCA5, a condition caused by a mutation in the
SPTBN2 gene (β-III spectrin): the mutant protein becomes unable to stabilize the
glutamate transporter EEAT4 in the plasma membrane of Purkinje cells, predispos-
ing them to excitotoxicity [188]. Also SCA1, SCA2, and SCA3 are poly-glutamine
diseases: in these cases the mutation affects ataxin-1, -2, and -3, respectively [181].
The major function of ataxin-1 is the regulation of the transcription of several genes
[189]. Ataxin-2 is instead probably involved in the control of mRNA regulation
[190] and ataxin-3 is a potent transcriptional repressor with deubiquitinating activ-
ity [191]. Mouse ataxin-1 mutants have altered levels of several proteins involved in
Ca2+ homeostasis, most of them with Ca2+ buffering function and located in the ER
[192]. Evidence has been provided that the InsP3R interacts with mutant forms of
ataxin-1, ataxin-2, and ataxin-3 [191,192]: accordingly, mice with InsP3R deletions
display an ataxic phenotype [193]. Mitochondrial Ca2+ handling has also been stud-
ied in a SCA28 disease model: the ablation of the AFG3L2 gene, which encodes a
mitochondrial protease mutated in this form of ataxia, causes impaired mitochon-
drial Ca2+ uptake and respiratory chain dysfunction. The impaired mitochondrial
Ca2+ uptake is due to the increased organelle fragmentation and to the loss of
ER-mitochondria connections [194].
Ca2+ pumps have also been involved in ataxias. A defect of one of the PMCA
pump isoforms, PMCA3, has recently been discovered in a case of X-linked human
cerebellar ataxia. The defect impairs the ability of the pump to properly eject Ca2+
from cells overexpressing it [195]. Genetic defects of a special PMCA2 isoform
which cause deafness (see Section 5.3) also causes equilibrium defects. The ataxic
“Wriggle Sagami” mouse model, in which a genetically defective PMCA2 has been
detected, displays impaired development of Purkinje cells dendrites and synaptic
connections [196].
118 Brini, Ottolini, Calì, and Carafoli

As repeatedly mentioned above, the role of the release of Ca2+ from lysosomes
through a two-pore channel (TPC) is controversial. Emerging evidence has never-
theless shown that in Niemann-Pick disease type C, a metabolic neurodegenerative
disease causing ataxia with selective loss of Purkinje neurons, there is an alteration
of lysosome-related Ca2+ signaling [197].

5.1.2 Migraine

Migraine is a disorder characterized by headache in combination with nausea,


photophobia, phonophobia, and vomiting. The headache, which is unilateral and
pulsating, is generally preceded by an “aura”, a neurological symptom characterized
by a visual (or other sensory) disturbance [198]. Migraine has generally a polygenic
and multifactorial inheritance, but some monogenic types also exist. Among them
the best studied is the rare autosomal dominant familial hemiplegic migraine (FHM)
[199]. Three genes have been linked to the disease (see [200] for a brief review):
CACNA1A, that encodes the pore-forming subunit of the voltage-gated CAV2.1
P/Q-type Ca2+ channel [201], ATP1A2, that encodes the α-2 subunit of the Na+/K+
pump, and SCN1A, that encodes the neuronal voltage-gated sodium channel.
A novel mutation has recently also been found in the SLC4A4 gene, encoding
the Na+/HCO3 transporter. CAV2.1 channels are involved in the initiation of
action potential-evoked neurotransmitter release and are expressed in the presyn-
aptic terminals and the somato-dendritic membrane of spinal cord and brain neurons
[202] (their mutations have also been linked to SCA6 and episodic ataxia type 2,
see above). Two mice models that recapitulate FHM have contributed signifi-
cantly to the study of the human disease. A number of reports have described a
gain-of-function of mutant CAV2.1 channels with increased open probability
and activation at lower voltage [203]. Other reports have described alterations
of the inactivation of CAV2.1 due to the increasing dissociation of regulatory
G proteins [203].

5.2 Neurodegenerative Diseases

The evidence that connects Ca2+ dyshomeostasis with neurodegenerative diseases


like Parkinson’s (PD), Alzheimer’s (AD), Huntington’s diseases (HD), and amyo-
trophic lateral sclerosis (ALS) is now abundant [204]. All these diseases are charac-
terized by the loss of specific neurons somehow linked to the deposition of abnormal
proteinaceous aggregates. Ca2+ dysregulation and alteration of Ca2+ signaling have
also been detected in animal and cellular model of all these diseases, suggesting that
they could have a role in the apoptotic death of neurons [205]. Multiple mitochon-
drial defects are also present. Mitochondria move along axons and supply the energy
necessary to the pumps for the extrusion of Ca2+ (and other) ions. Neurons are
uniquely exposed to glutamate excitotoxicity, which can lead to massive Ca2+
4 Calcium in Health and Disease 119

influx and to the activation of detrimental enzymes like phospholipases, endonucle-


ases, and proteases (e.g., calpains). As discussed in Section 3.2, mitochondria can
temporarily counteract the effects of cytosolic Ca2+ overload by buffering the exces-
sive level of Ca2+ in the cytosol.

5.2.1 Parkinson’s Disease

The progressive death of dopaminergic neurons (DN) of the substantia nigra pars
compacta containing proteinaceous aggregates of α-syn called Lewy bodies is the
hallmark of Parkinson’s disease. Only about 5% of PD cases are genetic, with muta-
tions in several proteins, among them α-synuclein (α-syn), parkin, DJ-1, PINK1, and
LRRK2. All these proteins are somehow involved in the functions of mitochondria
[205], suggesting the possible involvement of the organelles in the etiology of PD.
The first indication came from the discovery that the ingestion of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP), a poison of complex I of the respiratory chain,
or the exposure to pesticides like rotenone and paraquat (which also inhibit complex I),
induced a phenotype that recapitulated almost all the aspects of idiopathic PD. In
addition to the toxicity of α-syn aggregates and mitochondria damage, oxidative
stress and Ca2+ homeostasis impairment have also come into focus as possible fac-
tors in the molecular etiology of PD [206].
The role of Ca2+ dyshomeostasis is supported by a number of observations [207]:
DNs are peculiarly susceptible to “Ca2+ insult” since they use L-type Ca2+ channels for
their normal pacemaking activity, instead of the Na+ channels used by other types of
neurons. They are constitutively exposed to the risk of Ca2+ overload, and it has indeed
been shown that those DNs that express high levels of Ca2+-buffering proteins (e.g.,
calbindin D28K, calretinin, and parvalbumin) are protected from degeneration [208].
Another interesting characteristic of DNs is the lower total mitochondrial mass with
respect to other cells, which decreases their Ca2+-buffering power [208]. As for the
oxidative damage, it could be linked to the dyshomeostasis of Ca2+, as the Ca2+ defect
could be responsible for the excessive production of ROS. A protein called DJ-1, which
is involved in the defense of cells against oxidative stress [209], counteracts the ROS
produced during the partial uncoupling of mitochondria in the course of pacemaking
activity, which is a mechanism used by DNs to limit the uptake of Ca2+ [210]. Thus, the
DJ-1 KO-DNs have enhanced vulnerability to Ca2+-induced ROS production.
Evidence for the participation of DJ-1 in Ca2+ homeostasis is limited, but our
laboratory has recently shown that the protein increases the ER-mitochondria
connection, and thus the correct Ca2+ transfer between the two organelles [211].
Interestingly, this effect is shared with α-syn, the other protein that is frequently
mutated in PD, and with parkin [212–214]. Abundant information is also available
on the toxic effect of α-syn aggregates (or mutants) on Ca2+ homeostasis [204,206].
As for PINK1, it has been claimed to control the mitochondrial Na+/Ca2+
exchanger, its deletion predisposing cells to mitochondrial Ca2+ overload [215]. It
has also been claimed that PINK1 deletion directly impairs mitochondrial Ca2+
accumulation [216].
120 Brini, Ottolini, Calì, and Carafoli

5.2.2 Alzheimer’s Disease

Alzheimer’s disease, which is the most common form of dementia, represents one
of the most important pathologies in developed countries [217]. Although ageing
is acknowledged as a primary risk factor, the cause of the disease is still obscure.
The extracellular space of cerebral cortex in AD patients presents aggregates, called
“plaques”, of β-amyloid (Abeta), a 29–43 amino acid peptide, originated by the
cleavage of a large transmembrane protein (the amyloid precursor protein (APP)),
by the β and γ secretases. Proteinaceous aggregates of the phosphorylated tau
protein, called “neurofibrillary tangles”, are also present within neurons. 95% of
AD cases are sporadic [208], but familial forms of the disease occur in patients with
mutations in APP, and in the ER proteins presenilin 1 and 2 (PS1 and PS2, respec-
tively), which are the catalytic core of γ-secretase.
Ca2+ dyshomeostasis is increasingly recognized as an important factor in the
etiology of AD. Elevated levels of intracellular Ca2+ have been observed in the areas
of brain affected by AD pathology, with stimulation of several Ca2+ activated enzymes,
phosphorylation of tau, and processing of APP to Abeta [208]. The latter peptide
may then initiate a vicious circle in which its oligomers would form pores in the cell
membranes that potentiate Ca2+ entry and increase cytosolic Ca2+. However, Abeta
has also been claimed to impair glutamatergic signaling, by somehow reducing the
number of NMDA receptors and thus the influx of Ca2+ into the neurons.
Interestingly, the intracellular portion of APP, that is released after secretase
cleavage, modulates Ca2+ efflux from ER, thus also altering intracellular Ca2+
homeostasis [208]. Mutant forms of PS1 and PS2 also modify InsP3R and RyR
activity, thus altering ER Ca2+ release. PS1 has been claimed to form Ca2+-conducting
pores in the ER membrane. Its mutation reduced ER Ca2+ leak and thus enhanced
the ER Ca2+ levels. Mutant PS were also shown to increase the expression and the
sensitvity of ER Ca2+ release channels, thus promoting exaggerated Ca2+ release
after stimulation. However, the pore-forming ability of PS is controversial, as other
reports have not confirmed it and failed to measure enhanced ER Ca2+ levels in cells
overexpressing mutant PSs [218]. PSs also regulate the activity of other Ca2+-related
proteins as sorcin, calmyrin, and calsenilin/DREAM [219], and modify the activa-
tion of store-operated Ca2+ channels, as they alter the expression of the STIM pro-
tein [218]. Very recently, some works describe a direct role of PSs in the regulation
of MAM activities, e.g., Ca2+ transfer from ER to mitochondria [213].

5.2.3 Huntington’s Disease

Huntington’s disease is a purely genetic disease, caused by a mutation in the first


exon of the gene that encodes the ubiquitous protein huntingtin (Htt). The muta-
tion increases the length of the poly-glutamine tract normally present in the
N-terminus portion of the protein and eventually causes the death of neurons of
brain regions involved in motor circuit (neostriatum). HD is thus one of the poly-
glutamine diseases. The number of CAG repeats determines the age of onset of
4 Calcium in Health and Disease 121

HD and its severity (35Qs being the upper limit for a normal life). Htt is a 348
kDa protein of unclear function, however, it has been proposed to be involved in
important processes like gene transcription, apoptosis, and organelle regulation
[204]. Its abnormal poly-Q tract is cleaved by proteases (caspase 6), and the frag-
ment has the propensity to aggregate to form fibrils or oligomers, which have been
variously proposed to be toxic or even protective to cells [204,220]. The idea is
now gaining momentum that the toxic species is the Htt monomer, which could
explain why the larger aggregates, which may “sequester” the monomer, could be
protective. Ample evidence suggests an action of Htt on Ca2+ signaling [221], and
a disruption of mitochondrial Ca2+ homeostasis in HD cells has indeed been docu-
mented by different groups (it has even been proposed that Htt can interact directly
with mitochondria [220]). The Htt fragment can migrate to the cell nucleus, where
it would be involved in the regulation of the expression of gene, including that of
the InsP3R [220]. However, Htt has also been shown to directly interact with
InsP3R and to regulate ER Ca2+ release [204]. Augmented Ca2+ leak from RyR,
and subsequent cell death, has also been observed in neurons expressing mutant
Htt [222].
Work in our laboratory has shown that the Ca2+ dyshomeostasis condition is
associated to a severe damage in mitochondrial dynamics [223]. HD neurons dis-
play elevated expression of metabotropic glutamate receptors, which can activate
InsP3 signaling. The NMDA glutamate receptor appears to be hypersensitized by
mutant Htt with subsequent increased Ca2+ influx in the neurons [208]. A recent
report has linked extracellular Ca2+ and glutamate toxicity, by showing that a novel
compound protects mutant Htt expressing neuronal cells from apoptosis by TRPC1-
mediated SOCE inhibition [224].

5.2.4 Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis is caused by the loss of motor neurons in the motor
cortex and the spinal cord [225]. The molecular/cellular phenotype is characterized
by oxidative stress, organelle dysfunctions, and Ca2+ imbalance [226]. About 5–10%
of ALS cases are familiar, and 20% of them present a mutation in the gene that
encodes superoxide dismutase 1 (SOD1) [227]. The remaining genetic cases are
caused by mutations of numerous other genes, e.g., TDP-43 (TAR DNA binding
protein 43), VAPB (vesicle-associated membrane protein-associated protein B/C)
and FUS (fused in sarcoma). As in PD, HD, and AD, ALS neurons also present
proteinaceous inclusions in the soma and in the axon which are composed by ubiq-
uitin and the proteins cited above. Most ALS research is now concentrated on the
mutations of SOD1: since the protein is critical in the defense against oxidative
stress, the notion that oxidative stress is at the basis of ALS cellular phenotype has
traditionally occupied the central stage.
However, a number of aspects of the cellular phenotype of ALS are not solely
explained by oxidative stress: they could instead be explained by Ca2+ signaling
dysfunctions. Motor neurons are normally exposed to numerous and rapid Ca2+
122 Brini, Ottolini, Calì, and Carafoli

transients, which are necessary for their physiological rhythmic activity. As a result,
they have lower Ca2+ buffering capacity than other neurons [208]. This exposes
them to the risk of Ca2+ overload, which is exacerbated by their higher number of
AMPARs [204]. Interestingly, the Ca2+-permeability of AMPARs is augmented by
the mutations of SOD1, and the Arg-Gln substitution in the GluR2 subunit, which
blocks Ca2+ influx, is defective in ALS patients. Surprisingly, however, even if
glutamate excitotoxicity explains several aspects of the ALS phenotype, the
deletion of the glutamate transporter in astrocytes (that induces an increased
concentration of the neurotransmitter around the neurons) has no deleterious effects
on motor neurons [204].
Ca2+-buffering defects have been found in the mitochondria of synapses of
mutant SOD1 mice, suggesting the possibility that impairment of mitochondrial
Ca2+ handling is important in the pathogenesis of ALS. Interestingly, recent studies
have indeed shown that VAPB (one of the proteins mutated in genetic forms of ALS)
has a role in mitochondrial dynamics and in the ER-mitochondria Ca2+ transfer
(as other proteins do in AD and PD (see above)) [204,213,228].

5.3 Genetic Hearing Loss

Hearing depends on the conversion of the sound waves transmitted through the
endolymph of the inner ear into signals that are transduced by the hair cells of the
Corti organ through the mechanoelectric transduction (MET) process. The sound
waves deflect the stereociliar bundle that protrude from the hair cells, inducing the
opening of the MET channels that mediate the penetration of K+ and Ca2+ into the
stereociliar cytoplasm: only about 0.2% of the total MET current is carried by Ca2+,
that must be exported back to the endolymph by a special variant of isoform 2 of the
plasma membrane Ca2+ pump: a splicing insert in the first cytosolic loop directs the
variant to the apical portion of the hair cell [229], and a second insert in its C-terminal
cytosolic CaM-binding domain truncates it about 50 residues short of the normal
C-terminus (PMCA2wa). PMCA2 is unique among the isoforms of the PMCA
pump because of its ability to function very effectively even in the absence of the
natural activator CaM, and the doubly spliced variant wa has lower Ca2+-pumping
activity than the full length, unspliced isoform. These special characteristics of the
PMCA pump have evidently been evolutionarily adjusted to satisfy the require-
ments of the Ca2+ balance between the stereociliar cytoplasm and the endolymph: an
extracellular fluid in which the uniquely low Ca2+ concentration (see above) must be
constantly maintained at its very low μM level. This demands a PMCA variant that
is able to pump Ca2+ with limited efficiency, protected by the oscillations in the
natural activator CaM that would instead greatly influence the activity of all other
PMCA pump isoforms.
In the endolymph, Ca2+ binds reversibly to a single pass stereociliar EF hand
2+
Ca -binding protein, cadherin 23, which, together with protocadherin 15, forms the
tips links that organize the stereociliar bundle to promote its deflection. The balance
4 Calcium in Health and Disease 123

of Ca2+ between the stereocilia and the endolymph is vital to the correct operation
of the stereocilia bundle, and must be maintained with the exquisite precision that is
necessary for the correct functioning of the MET process: it is thus not surprising
that mutations of the stereociliar PMCA pump (and/or of the Ca2+ binding ability of
cadherin 23) should have been found to generate a deafness phenotype. Such phe-
notypes have been described in mice and also in humans [230–232]: the defects of
the PMCA pump are invariably characterized by an efficiency of Ca2+ pumping that
is lower than that of the wt PMCA2 wa pump (which, as mentioned, is lower than
that of the full length unspliced PMCA2 pump): the pump defect has been analyzed
molecularly in both mice and humans, and found to predominantly affect the long
term, unstimulated basal ability of the pump to export Ca2+ to the endolymph
rather that the burst of pump activity in response to the arrival of a large Ca2+
load. Depending on the functional severity of the pump mutations, the deafness
phenotype may or may not demand a concomitant loss of function mutation of
cadherin 23.

5.4 Cardiac Diseases (Cardiomyopathies)

Ca2+ links the electrical signals at the heart sarcolemma with the contraction of
the myocytes [233]. The concerted operation of the proteins/systems involved in the
myocyte contraction process allows heart to function normally. Its disruption leads
to diverse disease phenotypes, which are generally classified into four categories:
hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), restrictive
cardiomyopathy (RCM) and arrhythmogenic right ventricular dysplasia (ARVD)
(reviewed in [234]). A large number of mutations in the genes of Ca2+-dependent
contractile proteins have been identified in HCM, DCM, and RCM (reviewed in
[235]), but none of the two disease-causing gene defects identified in familial forms
of ARVD are related to them. HCM- and RCM-causing mutations increase the Ca2+
sensitivity of cardiac muscle contraction because they impair the interaction of TnT
with TnI, whereas DCM-causing mutations decrease it due to an increased affinity
of TnT for tropomyosin. HCM-causing mutations in myosin and myosin-binding
protein C have also been described that lead to increased Ca2+ sensitivity of cardiac
myofilaments.
The diastolic and systolic dysfunction observed in HCM and DCM, respectively,
fits well with the increase and decrease in the myofilament Ca2+ sensitivity and might
lead to increase and decrease of the ventricular wall stress. Genetic defects of cardiac
ryanodine receptor (RyR2) have instead been identified in ARVD [236]. The cardiac
RyR associates with four molecules of FKBP12.6 (the immunophilin mentioned
above that binds the immunosuppressive agent FK506). Four mutations map in the
cytosolic portion of the receptor, two of them clustering in the central FKBP12.6-
interacting domain. The mutations cause hypersensitivity of the receptor to activate
levels of Ca2+, and lead to abnormal excitation-contraction coupling and arrhythmias
(they eventually also trigger apoptosis and/or necrosis of the cardiomyocytes).
124 Brini, Ottolini, Calì, and Carafoli

Impairments in Ca2+ cycling are also considered early signs for the adaptive hyper-
trophic response upon heart damage or increased volume load [237]. During this adap-
tation, a number of changes associated with the process of Ca2+ handling have been
observed: the activity of the SERCA pump increases, thus resulting in augmented SR
store loading. With the progression of the disease, failing hearts exhibit increased sen-
sitivity of RyRs to activation by luminal Ca2+. The potentiated spontaneous Ca2+ release
[238] would contribute to the decreased SR Ca2+ content. The expression level of
another important Ca2+ homeostasis actor, i.e., the InsP3R, is also critical for the main-
tenance of rhythmic heart Ca2+ signals. InsP3Rs are typically 50-fold less abundant in
healthy cardiomyocytes than RyRs [239], but their expression increases significantly
during hypertrophy and heart failure [240]. The location of InsP3R next to the RyRs
might be important in the process of CICR and thus in EC-coupling.

5.5 Skeletal Muscle Diseases

Malignant hyperthermia, central core disease, and Brody’s disease are three Ca2+-
related pathologies that affect skeletal muscles. Another muscular disease (Duchenne
muscular dystrophy) also involves Ca2+ control deficiencies, but the central genetic
defect of the disease affects a protein that is not directly Ca2+-related.

5.5.1 Malignant Hyperthermia

Malignant hyperthermia (MH) is a disorder in which the exposure to volatile anes-


thetics and muscle relaxants (e.g., succinylcholine) causes a dysregulation (increase)
of myoplasmic Ca2+ that produces hypercontraction of muscles in susceptible
patients. The condition occurs with a frequency of 1:3000 cases, with a mortality
rate which originally was 70–80%, but is now less than 5% [241] thanks to the dis-
covery of the positive therapeutic effects of dantrolene, a drug that inhibits the
release of Ca2+ through the RyR [242].
During the MH episode the release of Ca2+ from the SR, probably activated by
the anesthetic drugs, becomes uncontrolled, overwhelming the Ca2+ homeostatic
capacity of myocytes, and resulting in a sustained muscle contracture that con-
sumes completely the cellular ATP and dramatically increases heat production.
The death of myocytes is eventually caused by the loss of membrane integrity with
leakage of muscle cell contents [243]. The principal cause of the susceptibility are
mutations in the RyR1 gene [244], but mutations in the gene that encodes the α1
subunit of the L-type voltage Ca2+ channel (CACNA1S [245]) have also been
detected. The current view on the molecular etiology of the disease claims that the
mutations predispose the RyR1 to become more readily opened. An alternative
proposal involves instead the participation of ECCE (excitation-coupled Ca2+
entry) [246] and does not consider the increased leakage of Ca2+ from the SR [247].
4 Calcium in Health and Disease 125

5.5.2 Central Core Disease

Central core disease (CCD), a congenital myopathy that induces weakness of


the muscles of lower extremities, is also caused by mutations of the RyR1 [248].
The disease is characterized by a particular amorphous area (called “core”), frequently
located in the center of the fibers of muscle, in which no mitochondria are found
[249]. Lack of glycogen granules and myofibrillar disorganization has also been
described [250]. The mechanisms by which the mutations cause the CCD molecular
phenotype, and why different mutations in the same protein can generate two
pathologies as different as MH and CCD is still obscure.
However, four types of channel defects have been recognized: (i) mutations
that cause hyper-sensitization to electrical and pharmacological stimuli (as in MH);
(ii) mutations that result in leaky channels and in depletion of Ca2+ from SR; (iii)
mutations that cause excitation–contraction uncoupling (“E-C hypothesis”), with
lack of ability of the Cav1.1 channel to activate the initial release of Ca2+ from
the SR; (iv) mutations that cause a decrease of RyR1 channels expression in SR
membranes [251]. The trigger event of the myopathy seems to be the inability of
myocytes to achieve a cytosolic Ca2+ concentration sufficient to induce the con-
traction of muscle fibers [244].

5.5.3 Brody’s Disease

Brody’s disease (BD) is a rare early-onset myopathy caused by mutations in the


ATP2A1 gene that encodes isoform 1 of the SERCA pump. The disease is character-
ized by muscle stiffness: the contraction phase is normal, but the time of relaxation,
after vigorous exercise or rapid movement, increases significantly and slight atrophy
in type 2 fibers is usually present [252]. The pathophysiology of BD is now clarified:
defective SERCA activity causes a slow and difficult relaxation, since the reuptake
of the Ca2+ released from the SR is impaired. Some patients show decreased activity
of the pump, without mutations in its gene: these cases are classified as “Brody syn-
dromes” [253].
Two drugs are available for the therapy of the disease: dantrolene, which inhibits
the release of Ca2+ from the SR (see above), and verapamil, a blocker of the L-type
channels, and thus of the membrane depolarization that triggers the initial SR-Ca2+
release. However, they only partially cure the symptoms [254].

5.5.4 Duchenne Muscular Dystrophy

Mutations in dystrophin, a protein encoded by a very large and complex gene, cause
Duchenne muscular dystrophy (DMD), an invariably fatal X-linked disease that
causes progressive muscle weakness with subsequent muscular degeneration.
Dystrophin is part of a large complex of at least 10 proteins [255] and forms a
transmembrane bridge between intracellular actin and the extracellular matrix.
126 Brini, Ottolini, Calì, and Carafoli

The dystrophin gene is susceptible to deletions, splicing errors and frame shifts
which decrease or abolish its expression and disrupt the complex causing plasma
membrane instability [256]. In parallel with these dystrophin defects, the Ca2+
content of DMD myocytes has been found to be extremely elevated, suggesting the
involvement of Ca2+ in the pathophysiology of the disease [257,258]. Normal muscles
that undergo intensive and stressing exercises can experience pain, swelling, and
inflammation, all effects that appear to be correlated to excessive Ca2+ entry into the
myocytes [259]. In DMD muscles that are predisposed to be weak and fragile, the
situation is more dramatic [260]. Plasma membrane ruptures conclusively allow
the entering of excessive Ca2+ in the myocyte cytosol, but it has also been proposed
that stretch-activated channels that normally allow the entering of Ca2+ are more
active in DMD muscles [257]. The condition of Ca2+ overload, which is likely to be
exacerbated by the defective Ca2+-buffering capacity documented in DMD [261],
would activate detrimental proteases, e.g., calpains, that induce myonecrosis [262].
Another important effect of the Ca2+ imbalance in the myocytes is the increased
production of ROS. Recent evidence has linked the mechanical stress-mediated
entering of Ca2+ into DMD myocytes to the potentiation of ROS production [263].
DMD, however, also affects the heart: abundant data in literature have discussed the
relationship between Ca2+ imbalance and heart function in DMD hearts [264]. DMD
patients generally display dilated cardiomyopathy and heart failure, which could
probably be due to the altered handling of Ca2+ by SR [265]. However, the proposal
is debated: no differences in heart SR Ca2+ content have been found in some reports
[266], whereas other reports have instead shown increased SR Ca2+ level [267].

6 Conclusions

The Ca2+ signal controls the most important processes which shape cell life, from its
origin at fertilization to its end in the process of programmed death. Ca2+ must thus
be very precisely controlled in the cell ambient: to this aim evolution has developed
numerous means from specific binding proteins to systems that transport Ca2+ across
membrane boundaries. They maintain cell Ca2+ at a basal (very low) set point, that
is selectively and transiently increased according to the demands of the targets of its
message.
Cells are sealed to external Ca2+ by the plasma membrane barrier, that only
admits the passage of Ca2+ in a carefully controlled way from the virtually unlimited
source in the external spaces. The fact that the concentration of Ca2+ is much higher
in the external spaces than inside cells is a dynamically favorable situation, as it
ensures that even slight increases of the permeability of the plasma membrane, such
as those produced by the opening of specific channels, promptly generate signifi-
cant swings of Ca2+ within the cytosol.
The main reservoir of Ca2+ in the organism is the bone compartment, in which
dynamic exchanges reversibly regulate Ca2+ in the circulating fluids and in the
extracellular spaces of the tissues. The dynamics of Ca2+ exchanges in bones is
4 Calcium in Health and Disease 127

controlled by hormones, as is the absorption of Ca2+ in the intestine and its excretion
and resorption in the kidneys.
One distinctive feature of the Ca2+ signal is its ambivalence: the correct functioning
of cell life demands its absolute spatial and temporal control within the cell ambient.
Should this control become defective, various degrees of cell dyscomfort will ensue,
that in extreme cases culminate in cell death.

Abbreviations

AD Alzheimer’s disease
AGC aspartate/glutamate carrier
AID atypical interacting domain
AIF apoptosis-inducing factor
ALP alkaline phosphatase
ALS amyotrophic lateral sclerosis
AMP adenosine 5′-monophosphate
AMPA 2-amino-3-hydroxyl-5-ethyl-4-isoxazolepropionic acid
AMPAR 2-amino-3-hydroxyl-5-ethyl-4-isoxazolepropionic acid receptor
AMPK AMP-activated protein kinase
ANKH ankylosis progressive homolog
APP amyloid precursor protein
ARVD arrhythmogenic right ventricular dysplasia
ATP adenosine 5′-triphosphate
BD Brody’s disease
cADPR cyclic adenosine diphosphate ribose
CaM calmodulin
CaMK calmodulin-dependent kinase
CaMKK calmodulin-dependent kinase kinase
cAMP cyclic adenosine 5′-monophosphate
CaRRE CaMKIV-responsive RNA elements
CBP CREB-binding protein
CCD central core disease
CG cortical granules
CICR Ca2+-induced Ca2+ release
CMA chaperone-mediated autophagy
Cn calcineurin
CoA coenzyme A
CREB cAMP responsive element-binding protein
DAG diacylglycerol
DCM dilated cardiomyopathy
DMD Duchenne muscular dystrophy
DN dopaminergic neuron
DRE downstream responsive element
DREAM downstream regulatory element antagonist modulator
128 Brini, Ottolini, Calì, and Carafoli

ECC excitation-contraction coupling


ECCE excitation-coupled Ca2+ entry
ECF extracellular fluid
eEF-2K eukaryotic elongation factor 2 kinase = CaMKIII
ER endoplasmic reticulum
ERK-MAP extracellular signal-regulated kinases-microtubule-associated protein
ETC electron transport chain
FADH2 flavin adenine dinucleotide (reduced)
FHM familial hemiplegic migraine
FKBP FK506 binding protein
FUS fused in sarcoma
GSK3 glycogen synthase kinase 3
HCM hypertrophic cardiomyopathy
HD Huntington’s disease
Htt huntingtin
hnRNP heterogeneous nuclear ribonucleoprotein
IMM inner mitochondrial membrane
InsP3 inositol 1,4,5-trisphosphate
InsP3R inositol 1,4,5-trisphosphate receptor
KO-DNs knock out dopaminergic neurons
LAMP-2A lysosome-associated membrane protein type 2A
LDCV large dense core vesicle
MAM mitochondria-associated endoplasmic reticulum membrane
MCU mitochondrial Ca2+ uniporter
MET mechanoelectric transduction
MH malignant hyperthermia
MICU1 mitochondrial calcium uptake 1
MLCK myosin light chain kinase
MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
mTOR mammalian target of rapamycin
MV matrix vesicle
NAADP nicotinic acid adenine dinucleotide phosphate
NAD nicotinamide adenine dinucleotide
NADH nicotinamide adenine dinucleotide (reduced)
NFAT nuclear factor of activated T cells
NMDA N-methyl D-aspartate
NPP1 nucleotide pyrophosphatase/phosphodiesterase 1
OMM outer mitochondrial membrane
ORAI1 calcium release-activated calcium channel protein 1
P2X, P2Y purinergic receptors type X and type Y
PC phosphocitrate
PD Parkinson’s disease
PDP pyruvate dehydrogenase phosphatase
PhK phosphorylase kinase
Pi inorganic phosphate
4 Calcium in Health and Disease 129

PI(3,5)P2 phosphatidylinositol 3,5-bisphosphate


PIP2 phosphatidylinositol 4,5-bisphosphate
PKA protein kinase A
PL phospholipase
PM plasma membrane
PMCA plasma membrane Ca2+-ATPase
poly-Q poly-glutamine
PPi inorganic diphosphate = pyrophosphate
PS presenilin
PTH parathyroid hormone
RCC restrictive cardiomyopathy
ROCC receptor-operated Ca2+ channels
ROS reactive oxygen species
RyR ryanodine receptor
SCA spinocerebellar ataxia
SERCA sarco/endoplasmic reticulum Ca2+-ATPase
SF sperm factor
siRNA small interference ribonucleic acid
SNARE soluble N-ethylmaleimide sensitive factor attachment protein receptor
SOCCs store-operated Ca2+ channels
SOCE store-operated Ca2+ entry
SOD superoxide dismutase
SPCA secretory pathway Ca2+ ATPase
SR sarcoplasmic reticulum
SRE serum response DNA regulatory element
STIM sensors stromal interaction molecule
Syt1 synaptotagmin I
TAR transactivation response element
TDP-43 TAR DNA-binding protein 43
TM transmembrane
Tn troponin
TPC two-pore channel
TRP transient receptor potential channel
VAPB vesicle-associated membrane-associated protein B/C
VDAC1 voltage-dependent anion channel 1
VDR voltage-dependent receptor
VOCCs voltage-operated Ca2+ channels

Acknowledgments The original work by the authors has been supported over the years by
grants from the Italian Ministry of University and Research (FIRB2001 to E.C., PRIN 2003,
2005 and 2008 to M.B), the Telethon Foundation (Project GGP04169 to M.B.), the FP6 program
of the European Union (FP6 Network of Excellence NeuroNe, LSH-2003-2.1.3-3 to E.C. and
Integrated Project Eurohear to E.C.), the Human Frontier Science Program Organization to E.C.,
to ERANet-Neuron (nEUROsyn), and CARIPARO Foundation to E.C, the Italian National
Research Council (CNR) and by a grant from the University of Padova (Progetto di Ateneo 2008
CPDA082825) to M.B.
130 Brini, Ottolini, Calì, and Carafoli

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Chapter 5
Vanadium. Its Role for Humans

Dieter Rehder

Contents
aBSTRACT.............................................................................................................................. 139
1 Introduction.............................................................................................................. 140
2 Distribution and Cycling of Vanadium....................................................... 142
2.1 Vanadium in Nature................................................................................................... 142
2.2 Pharmacokinetics and Pharmacodynamics................................................................ 144
3 The Aqueous Chemistry of Vanadium
and the Vanadate-­Phosphate Antagonism.................................................. 147
4 The Medicinal Potential of Vanadium.......................................................... 152
4.1 Diabetes Mellitus....................................................................................................... 152
4.2 Activity in Health Hazards Other than Diabetes........................................................ 156
4.2.1 Treatment of Cancer....................................................................................... 156
4.2.2 Cardiovascular Effects; Bacterial and Viral Diseases.................................... 159
4.2.3 Diseases Caused by Parasites......................................................................... 162
5 Concluding Remarks and Prospects............................................................. 164
Abbreviations................................................................................................................... 166
References......................................................................................................................... 167

Abstract  Vanadium is the 21st most abundant element in the Earth’s crust and the
2nd-to-most abundant transition metal in sea water. The element is ubiquitous also
in freshwater and nutrients. The average body load of a human individual amounts to
1 mg. The omnipresence of vanadium hampers checks directed towards its essentiality.
However, since vanadate can be considered a close blueprint of phosphate with
respect to its built-up, vanadate likely takes over a regulatory function in metabolic
processes depending on phosphate. At common concentrations, vanadium is non-toxic.
The main source for potentially toxic effects caused by vanadium is exposure to high
loads of vanadium oxides in the breathing air of vanadium processing industrial

D. Rehder (*)
Chemistry Department, University of Hamburg, D-20146 Hamburg, Germany
e-mail: rehder@chemie.uni-hamburg.de

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 139
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_5, © Springer Science+Business Media Dordrecht 2013
140 Rehder

enterprises. Vanadium can enter the body via the lungs or, more commonly, the
stomach. Most of the dietary vanadium is excreted. The amount of vanadium
resorbed in the gastrointestinal tract is a function of its oxidation state (VV or VIV)
and the coordination environment. Vanadium compounds that enter the blood stream
are subjected to speciation. The predominant vanadium species in blood are vana-
date and vanadyl bound to transferrin. From the blood stream, vanadium becomes
distributed to the body tissues and bones. Bones act as storage pool for vanadate.
The aqueous chemistry of vanadium(V) at concentration <10 μM is dominated by
vanadate. At higher concentrations, oligovanadates come in, decavanadate in par-
ticular, which is thermodynamically stable in the pH range 2.3–6.3, and can further
be stabilized at higher pH by interaction with proteins.
The similarity between vanadate and phosphate accounts for the interplay
between vanadate and phosphate-dependent enzymes: phosphatases can be inhib-
ited, kinases activated. As far as medicinal applications of vanadium compounds
are concerned, vanadium’s mode of action appears to be related to the phosphate-­
vanadate antagonism, to the direct interaction of vanadium compounds or frag-
ments thereof with DNA, and to vanadium’s contribution to a balanced tissue level
of reactive oxygen species. So far vanadium compounds have not yet found
approval for medicinal applications. The antidiabetic (insulin-enhancing) effect,
however, of a singular vanadium complex, bis(ethylmaltolato)oxidovanadium(IV)
(BEOV), has revealed encouraging results in phase IIa clinical tests. In addition,
in vitro studies with cell cultures and parasites, as well as in vivo studies with
animals, have revealed a broad potential spectrum for the application of vanadium
coordination compounds in the treatment of cardiac and neuronal disorders, malig-
nant tumors, viral and bacterial infections (such as influenza, HIV, and tuberculo-
sis), and tropical diseases caused by parasites, e.g., Chagas’ disease, leishmaniasis,
and amoebiasis.

Keywords  antiparasitic vanadium compounds • antiviral potential • cardiovascular


effects • essentiality of vanadium • insulin-enhancing action • vanadate-phosphate
antagonism

Please cite as: Met. Ions Life Sci. 13 (2013) 139–169

1  Introduction

Vanadium is a versatile and omnipresent element that can attain the oxidation states
–III to +V. Low-valent vanadium is stabilized by strongly π-accepting ligands, car-
bon monoxide in particular, high valent vanadium by σ and π donors represented by
hard, oxygen and nitrogen functional ligands. Soft ligands such as thio-functional
ones are predominantly found in vanadium compounds with vanadium in interme-
diate oxidation states. Vanadium nitrogenase is an example for a naturally occurring
vanadium compound where vanadium switches in-between the oxidation states +II
5  Vanadium. Its Role for Humans 141

and +IV: In vanadium nitrogenase from nitrogen fixing bacteria such as Azotobacter,
vanadium – an integral constituent of the Fe7VS9 M-cluster – is coordinated to three
sulfides, a histidine-N, and two oxygen functions of homocitrate. Vanadium(III)
coordinated to water molecules is present in the vanadocytes of sea squirts. The +IV
and +V oxidation states, which are the by far predominating ones in physiologically
relevant vanadium systems, typically contain the VIVO2+, VVO3+ or VVO2+ ‘core’,
although there are exceptions. An example for a ‘bare’ vanadium(IV) complex is
the naturally occurring amavadin, where V4+ is coordinated to two tetradentate
N-oxyimino-2,2′-dipropionate ligands. Amavadin is found in mushrooms belonging
to the genus Amanita, such as the fly agaric. The oxidovanadium(V) core is present in
vanadate-dependent haloperoxidases from, inter alia, marine algae, with vanadate
H2VO4− coordinatively linked to an active center histidine-N.
To date, vanadate-dependent haloperoxidases and vanadium nitrogenases have
remained the only identified naturally occurring vanadium-based enzymes. Whether
or not vanadium is an essential element for evolutionary younger organisms, includ-
ing vertebrates, remains to be verified. A functional role of simple vanadium
­compounds (vanadate in particular) in vertebrates, and hence also in humans, is
likely, an assumption which is based on the similarity between vanadate and phos-
phate. In this context, the vanadate-dependent haloperoxidases are of particular
interest since they mimic, or model, enzymes involved in phosphate metabolism,
where the protein binding domain for phosphate is blocked by vanadate.
The competitive behavior of vanadate with respect to phosphate is likely also the
clue for the insulin-mimetic/insulin-enhancing potential of vanadium compounds,
and hence the surge in the design of antidiabetic vanadium complexes during the
past two decades. These auspicious developments have also initiated research
towards the design of biologically active vanadium complexes in the search of phar-
macological control of cancer, cardiovascular imbalances, and diseases caused by
viruses, bacteria, amoebae, and flagellate protozoan parasites. In several cases,
ligands have been employed that relate to original pharmacologically applied drugs,
with the aim to increase the efficacy of the drug, and to widen the spectrum of thera-
peutic use by exploiting the cooperative effect of the metal and the ligand. The
research into these medicinal applications includes functional alternatives to the
phosphate-vanadate antagonism, e.g., the direct interaction of the vanadium com-
pound with the DNA of a tumor cell or the pathogen.
The broad medicinal potential of vanadium will be extemporized in Section 4 of
this chapter. Section 2.1 is dedicated to vanadium’s distribution and speciation in
nature, and hence, its availability, potential inalienability, and occasional toxicity
for humans. Section 2.2 addresses resorption, speciation in the blood stream and in
the cytosol, tissue distribution, and excretion of vanadium within the human body,
and hence, vanadium’s gateways commonly referred to as pharmacokinetics and
pharmacodynamics. Given the importance of the similarities (and, to some extent,
also the dissimilarities) between vanadate and phosphate for the potentiality of
vanadium in toxic as well as in beneficial issues (such as regulatory function and
medicinal applications), an extra section, Section 3, is devoted to the vanadate-­
phosphate antagonism.
142 Rehder

2  Distribution and Cycling of Vanadium

2.1  Vanadium in Nature

The Earth’s crust, including the aqua- and atmosphere, contains ≈130 ppm (by
mass) vanadium, making vanadium the 21st most abundant element in the outer-
most sphere of our home planet. The abundance of vanadium in the Earth’s crust
thus exceeds its occurrence in the Universe (≈1 ppm) and in the Sun (≈ 0.4 ppm) by
about two orders of magnitude [1]. Volcanic areas with basaltic layers are particu-
larly rich in vanadium, as are hard coal (up to 0.34%) and some oil shales and crude
oil. Venezuelan crude oil can contain up to 0.12% V, mostly in the form of
oxidovanadium(IV) porphyrins (Figure 1a). VO2+ ions are strongly complexed by
porphyrins and related chelators, and the enrichment of crude oil with vanadium is
due to its extraction from vanadium-bearing rock by porphyrins present in oil that
had passed through rocky layers. Natural release of vanadium mainly goes back to
weathering of vanadium-containing rock and the erosion of soil.
Vanadium concentrations in seawater and freshwater are around 30 nM. At the
prevailing oxic conditions and about neutral to slightly alkaline pH, soluble
vanadate(V), H2VO4−, is the dominant species. The high Na+ concentration in seawa-
ter implies that ion pairs Na+[H2VO4−] are formed. Under non-oxic conditions, spar-
ingly soluble oxidovanadium(IV) hydroxide ‘VO(OH)2’ is generated, transported in
water in the form of colloids and absorbed to floating particulate sediment, or solu-
bilized by complexing ligands.
Vanadium is the second-to-most abundant transition metal in seawater, out-
classed only by molybdenum (MoO42 −, c = 100 nM). The nanomolar concentration
excludes the formation of vanadates of higher nuclearity otherwise typical for
vanadates (see below). Vanadate concentrations in potable water are around 10 nM.
In volcanic areas with basalt layers, the concentration of vanadium in underground
water, and consequently also in drinking water, can rise to 2.5 μM [2]. Where drinking

a b
C2H5 CH3 CH3
H3C 2-
H3C C2H5
N O
N O N O O
V O O
V
N N O O
H3C CH3
O O
O N
(H2C)2
CH3
CO2H H3C

Figure 1 (a) The porphinogenic vanadium compound oxidovanadium(IV)-deoxiphylloerythrin


from crude oil. (b) Amavadin, present in the fly agaric (Amanita muscaria), is a non-oxido vanadium
compound containing the ligand (S,S)-N-oxyimino-2,2′-diisopropionate(3–).
5  Vanadium. Its Role for Humans 143

water is supplied through lead water pipe systems, vanadate is removed through the
formation of deposits of sparingly soluble vanadinite, PbCl2⋅3Pb3[VO4]2. A decrease
of pH and an increase of the phosphate concentration (e.g., by addition of phosphate-
based corrosion inhibitors to drinking water) can re-mobilize vanadate (eqns 1
and 2) and thus eventually increase vanadate concentrations beyond a tolerable
level [3].
PbCl2 ⋅ 3Pb3 [ VO 4 ]2 + 12H +  10Pb 2 + + 6H 2 VO 4− + 2Cl− (1)

PbCl2⋅ 3Pb3 [ VO 4 ]2 + 3HPO24 − + H + → PbCl2⋅ 3Pb3 [ PO 4 ]2 ↓ + 2H 2 VO 4− (2)

Vanadinite is a common mineral containing vanadium in the oxidation state +V.
Vanadium’s first discovery by the Spanish mineralogist Manuel del Rio y Fernández
in1801 goes back to this mineral. Vanadium-based minerals are otherwise compara-
tively rare, i.e., most of the vanadium in the Earth’s crust is dissipated in other
minerals, rocks, and sediments. Examples for defined minerals with vanadium in
oxidation states other than +V are minasragrite, VOSO4⋅5H2O, and patronite,
V(S2)2, with vanadium(IV), and karelianite, V2O3, with vanadium(III). Note that,
under oxic conditions, only the oxidation states +V and, to some extent, also +IV
are stable. Vanadium(II) has been found in forsterite (Mg2SiO4) and enstatite
(MgSiO3) in chondrules of meteorites such as the Vigarano meteorite [4]. Here, V2+
can partially occupy Mg2+ and Ca2+ sites.
High contents of vanadium are found in various sea squirts (ascidians), in fan
worms, and in Amanita mushrooms such as the fly agaric [5]. In ascidians, vana-
dium concentrations in specialized blood cells, where the predominant form of
vanadium is [VIII(H2O)5HSO4]2+, can go up to 0.3 M. The concentration of vanadium
in the fly agaric exceeds that in other plants by a factor of 102. In the fly agaric, also
known as toad stool, vanadium is present as amavadin, a low molecular mass non-
oxido VIV complex (Figure 1b).
Vanadium contents in food average 30 μg kg–1, the daily intake via food and
beverages is 10 μg to 2 mg, only a minute proportion of which becomes resorbed.
The body pool of an average human being (70 kg body mass) amounts to ca. 1 mg
V, the average blood plasma concentration to 45 nM. Oral intake of vanadium is
somewhat increased for sportsmen and bodybuilders resorting to preparations con-
taining VOSO4. This ‘vanadyl fuel’ allegedly helps increasing the muscle mass.
Since almost all of the vanadium is excreted in the form of insoluble VO(OH)2 prior
to resorption, potential harm due to vanadium overload is not likely to occur.
Still another – and more critical – source of vanadium intake is breathable air
in urban and industrialized areas. Vanadium, in the form of vanadium(IV) and
vanadium(V) oxides, VOx, is present in air in particulate form or absorbed to tiny
dust particles and aerosols, and thus enters the lungs and the pulmonary system, from
where it becomes distributed in the body after solubilization. The main natural
sources for vanadium loads in the atmosphere are continental dust, marine aerosols,
and volcanic emissions [6]. In rural areas, the concentration of vanadium oxides is in
the range of 50 ng m–3; pollution can go up to >103 ng m–3 in urban settings, and in
industrial areas in particular [7], where combustion of petroleum and oil are the main
144 Rehder

contributors to aerial VOx. Potential toxic effects of vanadium overloads [8], in


particular irritations of the respiratory tract in workers exposed to high loads of
vanadium oxides at their working place, will briefly be addressed in the next section.

2.2  Pharmacokinetics and Pharmacodynamics

As noted, critical exposure to vanadium compounds, vanadium oxides in particular,


is confined to inhalation in the frame of occupational exposure, including mining
and milling of vanadium ores, metallurgical processing involving ferrovanadin, pro-
duction of catalysts, batteries and glass melt additives based on vanadium oxides,
and cleaning of oil-fired boilers. Inhaled vanadium oxides cause rhinitis, irritations
of the respiratory tract and – commonly transient – pulmonary malfunctions such as
bronchitis, pneumonia, and asthma. Whether or not vanadium oxides can promote
lung cancer has yet to be shown. In any case, the maximum allowable concentration
of V2O5 at the working place, the MAC value, has been set by the World Health
Organisation to 0.05 mg m–3 (40-h week, 8-h time-weighted average).
Vanadium oxides are readily absorbed in the lungs and enter the blood stream
after solubilization in the form of vanadate, H2VO4−. Skin does not appear to allow
for an appreciable import of vanadium. The main ‘natural’ source for the body’s
vanadium supply thus is dietary uptake, a comparatively ineffective process because
up to 99% of the dietary vanadium is usually excreted with the feces. The main
routes of vanadium uptake and distribution in the body are sketched in Figure 2.
Dietary forms of vanadium are either vanadate, H2VO4−, present mainly in drinking
water, and oxidovanadium(IV) compounds {VOL}, where {VOL} represents any
ligand-stabilized VO2+. Free VO2+ is, as noted, essentially unavailable, since it forms
sparingly soluble oxidovanadium hydroxide, allowing for nanomolar concentration
of ‘free’ VO2+ (actually [VO(OH)3]–) at the best. H2VO4− is more easily taken up in
the gastrointestinal tract. However, vanadate(V) is partially reduced in the stomach
and precipitated in the form of VO(OH)2 in the slightly alkaline medium of the
intestines. Vanadium can also enter the blood stream by injection or infusion, either
intentionally when injected intravenously (or intraperitoneally), or accidentally
when present as a ‘contaminant’ in infusion solutions [9].
Vanadium compounds ending up in the blood stream either after resorption in the
gastrointestinal tract, or via the lungs, or by infusion/injection, are subjected to
redox interconversion between VV and VIV, depending on the oxygen tension and the
presence of redox-active agents. The main transporter for both anionic vanadate(V),
cationic VO2+, and neutral or charged {LVIVO} is transferrin [10]. Transferrin (Tf)
forms binary complexes {VO2+-Tf} and ternary complexes {VOL-Tf} and
{VOL′-Tf}, where L is a ligand originally coordinated to VO2+, and L′ a low molec-
ular mass (lmm) ligand provided by blood serum, such as lactate [11], the serum
lmm compound with the highest concentration, i.e., 1.5 mM. The VO2+ ion binds
into the same protein pocket as Fe3+, and hence to two tyrosinates, an aspartate, and
the Nε of a histidine, plus a synergistic carbonate (Figure 3a). With increasing blood
5  Vanadium. Its Role for Humans 145

Respiratory tract
Lungs
V2O5, VO2, V2O3 Bone

Dietary vanadium
Blood plasma Heart, Kidney,
VO2+ H2VO4
H 2VO 4 Liver, Spleen
L e L VO 2+ -Tf
{LVO} {L/L'VO}-Tf
Brain, Muscle,
Adipose tissue
Feces Infusion; Injection
VO(OH)2 H2VO4 , {LVO} Urinary excretion
H2VO4
Urinary excretion
H2VO4

Figure 2 Uptake, distribution and excretion of vanadium compounds. Uptake routes are indicated
by broad arrows, excretion routes by broken arrows, and distribution routes by standard arrows
and equilibrium arrows, respectively. Main vanadium compounds are indicated. Abbreviations:
Tf = transferrin, L is any ligand provided by the nutritional matrix or in a medicinally applied
vanadium compound, L′ is a low molecular mass ligand present in blood serum, and {L/L′VO} is
the abbreviation for a VO2+ complex with L and/or L′.

a
Arg NH
NH2+
H2N Carbonate
O H
O O
C b N
Asp O O
O O N His
V O
O V
N O Tyr O
O
HN O
His HO

O
Tyr

Figure 3  Likely binding modes of VO2+ in (a) the ternary VO2+-transferrin complex [12b], and
(b) in the ternary complexes LVO2+-albumin or LVO2+-immunoglobulin (L = ethylmaltol), coordinating
through a histidine [12a]. In (a), the Tyr trans to the oxido ligands binds just weakly.

serum concentrations of vanadium, high-molecular mass transporters other than Tf


come in, namely serum albumin (Ab) and immunoglobulin G (Ig) [12]. These pro-
teins preferentially form ternary complexes, {VOL-Ab} and {VOL-Ig}. As shown
in Figure 3b, the protein binds to the VOL moiety via a histidine residue.
Plasma vanadium contents decline in three phases: The first phase is a rapid
decline with a half-life t1/2 of 1 hour, followed by a second intermediate decline
146 Rehder

(t1/2 ≈ 26 hours) and a third slow decline with t1/2 ≈ 10 days. Vanadium contents in
blood are thus reduced to about 30% within the first 24 hours [9]. Clearing occurs
directly via urinary excretion, and after distribution over tissues of the inner com-
partment (heart, liver, kidney, spleen), the outer compartment (brain, muscle, adi-
pose tissue), and bones. About 50% of the vanadium is recovered in urine after
12 days. The ­residence time of vanadium in bones, where it replaces phosphorus
in hydroxyapatite, Ca5(PO4)3OH, is ca. 1 month [13], corresponding to a half-life
of 4–5 days.
There are several alternative routes by which vanadium compounds can be trans-
ported from the blood plasma into blood and tissue cells. Vanadate is essentially
present, at pH ≈ 7, in the form of dihydrogenvanadate, H2VO4− (the pKa is 8.2), and
may use phosphate and sulfate channels: Vanadate and phosphate HPO42 −/
H2PO4− (pKa = 7.2) are structurally very similar (see also the next section). Vanadate
and oxidovanadium(IV) bound to transferrin can enter the intracellular space by
endocytosis – analogously to Fe3+, the main target ion for transferrin. An additional
conceivable path – for a stable vanadium coordination compound with a sufficiently
lipophilic coordination sphere – is diffusion across the cell membrane. The feasibil-
ity of this latter route of entry has been demonstrated for the uptake of the complex
[VO(pyridinone)2H2O] by erythrocytes [14].
The low absorption rate of dietary vanadium and the rather efficient desorption
of excess vanadium that has entered the blood and body tissues diminish toxic
effects that contemporarily can emerge, such as irritations of the conjunctivae and
the respiratory system on exposure to vanadium oxides in the breathing air (see
above), or (mild) gastrointestinal and renal problems in the course of medicinal
applications of vanadium compounds. The no-effect level has been set to a daily
intake of 10 mg V per kg body mass. The respective limit values for intravenous
application is 7 mg kg–1, for breathing air 35 mg m–3. Acute poisoning in animals
fed an about tenfold excess of vanadium compounds causes paralysis, convulsion,
and eventually death [5,15]. Vanadium compounds are considered potentially
genotoxic and thus mutagenic, teratogenic, and ‘suspected carcinogenic’.
Classification as a carcinogen is based on the fact that vanadium induces the for-
mation of tumor-­associated antigens, and that it can directly and indirectly damage
DNA and affect DNA repair [1b,16]. ‘Indirectly’ here refers to the potentiality of
VO2+ to effect the formation of reactive oxygen species (ROS) such as the OH radi-
cal in a Fenton-like reaction (eqn. 3a), and superoxide when directly interacting
with O2 (eqn. 3b).
VO2 + + H 2 O2 + H + → VO3+ + H 2 O + • OH (3a)

VO2 + + O2 + 3H 2 O → H 2 VO −4 + • O2− + 4H + (3b)

Superoxide in turn can cause the release of iron from the iron storage protein ferritin
[17] and thus contributes to the disruption of iron homeostasis. In rat models, vana-
dium provokes neuro-toxicological effects in the brain, such as demyelination, i.e.,
damage of the myelin sheet of neurons. Myelin is a lipid-rich membrane of the
nerves, and vanadium apparently promotes its peroxidative destruction [8].
5  Vanadium. Its Role for Humans 147

While carcinogenic properties of vanadium compounds via ROS formation and


interference with phosphokinases [18] are well in the realm of possibility, it should
also be noted that vanadium compounds damage DNA in tumor cells more effec-
tively than in healthy cells. As elaborated in Section 4.2.1, many vanadium
­compounds have an antitumor activity. Vanadium species can also annihilate reac-
tive oxygen species. This is demonstrated by the sequence in equation (4) for the
oxidation of peroxide to superoxide and further to O2 [19].

( )
2+ +
 V IV O  + O22 − →  V V O O22 −  + e –

O ( O )  ( )
+ 2+
VV 2−
→  V V O • O2−  + e−
 2

O( )
2+ 2+
VV •
O2− (4) →  V IV O  + O2

In reference to Paracelsus, who noted that “All substances are poisons; it is solely
the dose that differentiates between a poison and a remedy” (“Alle Ding’ sind Gift, und
nichts ohn’ Gift; allein die Dosis macht, daß ein Ding kein Gift ist”), there is so far no
solid basis for categorizing vanadium compounds as harmful when administered in
sensible amounts. Rather, as discussed in more detail in the oncoming sections, vanadium
is likely an essential element in as far as vanadate can interfere with phosphatases,
phosphorylases, and kinases and, more generally, is involved in regulating the
phosphate metabolism and phosphate-dependent energetic processes. In addition, the
participation of VIV and VV in levelling ROS suggests that vanadium can be beneficial
in the treatment of several diseases and malfunctions related to ROS imbalances.
Generally, vanadium(V), in particular when present as vanadate, is more toxic
than vanadium(IV). As noted, VO2+ either forms a sparingly soluble hydroxide or is
‘masked’, through coordination, by a variety of physiologically available ligand sys-
tems. Biological detoxification of vanadate occurs via integration into the hydroxy-
apatite structure of the bones (vide supra) and by reduction to vanadium(IV) [20].
Glutathione, ascorbate, NADH, and NADPH are examples for agents that can reduce
vanadate. In ascidians, reduction equivalents for the reduction of H2VO4− to VO2+ are
supposedly delivered by NADPH (generated in the pentose phosphate pathway) via
the redox couple 2GSH ⇌ GSSG + 2H+ + 2e–, where GSH and GSSG are the reduced
and oxidized forms of glutathione, respectively [21]. Vanadium(III) plays, if any, a
minor role only, since vanadium(IV) is not easily reduced to vanadium(III) at physi-
ological conditions – and if so, rapidly re-oxidized to vanadium(IV).

3  T
 he Aqueous Chemistry of Vanadium
and the Vanadate-­Phosphate Antagonism

At strongly acidic conditions (pH <2), cationic octahedral aqua complexes of VIII,
VIVO2+, and VVO2+ can be present in aqueous media, i.e., [V(H2O)6]3+, [VO(H2O)5]2+,
and [VO2(H2O)4]+. While [V(H2O)6]3+ has Oh symmetry, the structures of the aqua
148 Rehder

[VIII(H2O)6]3+ [VIVO(H2O)5]2+ [VVO2(H2O)4]+

OH2
O O
H2O OH2
V H2O V OH2 H2O V O
H2O OH2
H2O OH2 H2O
OH2 OH2
OH2 OH2

d(V-OH2): 1.99 Å d(V=O): 1.6; d(V-OH2cis): 2.0; d(V-OH2trans): 2.2 Å

Figure 4  Cationic aqua complexes of vanadium(III, IV, and V) that can exist in strongly acidic
aqueous media. For structure data see ref. [22].

complexes of VO2+ and VO2+ are somewhat distorted due to the trans influence
exerted by the doubly bonded oxido ligand(s), giving rise to comparatively weakly
bonded water trans to the oxo group(s). In addition, there are distortions in the octa-
hedral arrangement as shown in Figure 4, reducing the local symmetry to C4v in
[VO(H2O)5]2+ and C2v in [VO2(H2O)4]+ [22] (for the respective distances d(V-O) see
Figure  4). Strongly acidic conditions are provided in the stomach, but otherwise
physiologically irrelevant – with the exception of the vanadium sequestering blood
cells of ascidians, where V3+ mainly exists in the form of [V(H2O)5HSO4]2+. In the
presence of a ligand L, partial or complete replacement of H2O provides stability of
the resulting complex(es) also in the less acidic, neutral, and slightly alkaline
regimes. If L is a bidentate, singly negatively charged ligand, such as lactate,
the composition of the resulting mono-ligand complexes is [VIII(H2O)4L]2+,
[VIVO(H2O)3L]+, and [VVO2(H2O)2L]. Depending on the pH, mixed aqua-hydroxido
complexes can form, such as [VIII(OH)(H2O)3L]+, [VIVO(OH)(H2O)2L], and
[VVO2(OH)(H2O)L]–. Further, monooxidovanadium(V) complexes come in, for
instance [VVO(H2O)3L]2+ and [VVO(OH)(H2O)2L]+. As the denticity and charge of
the ligands increases – an example is citrate [23] – the diversity of potential vana-
dium species present around pH 7 rises substantially.
Vanadium(IV) and vanadium(V) are easily interconverted by physiologically rel-
evant redox agents such as NAD+/NADH, NADP+/NADPH, FAD2+/FADH2, gluta-
thione, and ascorbate. Further, VIV and VV redox-interact with reactive oxygen
species. The redox potential for the couple H2VO4−/VO2+ (eqn. 5) at pH 7 is −0.34 V,
which compares to −0.32 V for the couple NAD+/NADH.

[H 2 VO4 ]

+ 4H + + e –  VO2 + + 3H 2 O (5)

The most prominent inorganic vanadium species present at micromolar concentra-
tions and pH 7 is dihydrogenvanadate, H2VO4−: At the physiological ionic strength
of I ≈ 0.15 M, the pKa for the protonation/deprotonation equilibrium (eqn. 6), is
8.2 [24].

[H 2 VO4 ]  [ HVO 4 ] + H +
– 2–
(6)

5  Vanadium. Its Role for Humans 149

O O
O 2 O 3
HO Va Va O
V O
O H O O
HO O
H H H O O O Vb
OH Vb Vc
O O O
H 2VO 4 2- O Vb Vc Vb
H 2V2O 7 O O O
O
O O O
O Va O Va OH
4 5 H
O O
3-
H 3V10O 28
4- 5-
V4O12 V5O 15

Figure 5  Vanadate(V) species that can be present in aqueous media, depending on concentration,
pH, and stabilization by electrostatic interaction with, e.g., proteins. The protonation grade of
decavanadate shown here corresponds to that at pH ≈ 2.7.

With increasing concentration, condensed vanadate species form. In the slightly


acidic to alkaline regime (ca. pH 5–9), these are predominantly divanadate
[H2V2O7]2–, cyclic tetravanadate [V4O12]4–, and cyclic pentavanadate [V5O15]5–. In
the pH range 2.3–6.3, decavanadate [HnV10O28](6–n)– (n = 3–0) comes in. For the
structural formulae of these oligovanadates see Figure 5. Commonly, physiological
vanadate concentrations at subtoxic levels are too low to allow for the formation of
oligovanadates. Locally, however, concentration enhancement or template-directed
nucleation [25] may occur, and the oligovanadate(s) then formed can interact with
proteins and DNA. An example is the incorporation of tetravanadate into Cu,Zn
superoxide dismutase [26a], and the promotion of the hydrolytic cleavage of the
ester bond in a phosphodiester RNA model by tetravanadate [26b]. Decavanadate is
thermodynamically unstable at pH values exceeding 6.3; its decay into vanadates of
lower nuclearity (including monovanadate) is, however, kinetically hampered,
allowing for half-lives at pH 7, and even somewhat above pH 7, of several hours.
Additional stabilization takes place by close interaction with proteins such as tubu-
lins [27] and myosin [28] as well as with mitochondria [29]. Tubulins are proteins
that assemble to structural elements (termed microtubules) of cytoskeletons. Myosin
is involved in muscle contraction. Myosin binds decavanadate with high affinity,
forming a myosin-MgATP-decavanadate complex that blocks the contractile cycle.
Decavanadate that targets mitochondrial proteins inhibits mitochondrial oxygen
consumption. Stabilization of decavanadate can also be achieved by its incorpora-
tion into tiny water pools of intracellular compartments – as suggested by model
experiments with reverse micelles with nanoscopically confined water in the
micelles’ cavities [30].
Vanadate and phosphate are structural analogues, and vanadate is only slightly
larger than phosphate: The geometrical diameters are 6.2 Å for vanadate and 5.8 Å
for phosphate, the volumes of the circumscribing spheres amount to 125 Å 3
for vanadate and 102 Å3 for phosphate. These values are based on the following
150 Rehder

Asp
O Glu
Arg NH O- O HN Arg
2 Gly, Ile
H HN Arg O
N + HN
+
H2N NH
H NH2 O +H NH2 H
O- 2N O O
HN HO V +H N NH2 O
N O O- 2
NH HO V O
N H2N O HO V
His HN O OH
NH HO Ser S
His Arg Ser
Cys

Asp O
O
O HO O HO
HO R O R
H R H OH OH
R O O O
O O
O P OH O H
H H O O P
O P O OH
OH O P
O OH H
N OH
N N
HN N
HN HN
His HN

Figure 6  Top: The active sites of vanadate-substituted (and thus inhibited) rat acid phosphatase
(left) [32a], the Cys215Ser mutant of protein tyrosine phosphatase 1B (center) [32b], and bovine
phosphotyrosyl phosphatase (right) [32c]. Bottom: The mechanism of phosphate ester hydrolysis
as catalyzed by a phosphatase. The transition state {} is ‘fixed’ as vanadate becomes coordinated
into the active center.

structure parameters: r(O2–) = 1.36 Å, d(V-O) = 1.72 Å, d(P-O) = 1.54 Å; V-O and
P-O distances for tetrahedral coordination geometry, where r(VV) = 0.36 and r(PV)
= 0.17 Å. From a geometrical point of view, the two anions are thus essentially
indistinguishable, making vanadate an efficient competitor for phosphate in binding
sites commonly targeted by phosphate. There are, however, also substantial differ-
ences: At pH ≈ 7 and physiological ionic strength, vanadate is almost exclusively
present in the form of dihydrogenvanadate, H2VO4−, while phosphate exists in
approximately equal amounts of mono- and dihydrogenphosphate, HPO42 − and
H2PO4−. The higher average charge enables phosphate to interact more efficiently
than vanadate with dipoles (such as water) and anions (e.g., anionic amino acid resi-
dues in protein matrices). On the other hand, phosphorus can attain the coordination
number 5 in transitional states only, while the d-block element vanadium easily
extends its coordination number by forming stable penta- and hexa-coordinate
complexes. Hence, once incorporated in lieu of phosphate into the active site of a
phosphate-­dependent enzyme, this enzyme is commonly deactivated with respect
to its original function.
Naturally occurring enzymes relying on vanadate in a penta-coordinate trigonal-­
bipyramidal environment are the haloperoxidases in fungi, lichen, marine algae
[5,31a], and Streptomyces [31b]; an example for a vanadate-inhibited phosphate-­
dependent enzyme is the vanadate variant of rat acid phosphatase [32] (Figure 6,
top left), with the same first coordination sphere environment for vanadium as in
5  Vanadium. Its Role for Humans 151

O Base
O

3' O O
O O O
O O O O P O Base
O P O Base V V
V V O HO O
HO O O OH O O O O
5'

O O
H2O

O
O O HO P O Base
O +
V V O
HO O OH O
O
O

Figure 7  Proposed mechanism for the pyrovanadolysis of the DNA primer [36]: Divanadate
attacks the primer at the 3′ position, a process which affords mediation by Mn2+. The transiently
formed 3′-divanadophospho-nucleotide is hydrolytically split into divanadate and the phosphonu-
cleotide to which DNA polymerase falls back.

vanadate-­dependent haloperoxidases. Interestingly, vanadate-inhibited phosphatases


do have some haloperoxidase activity [33], while vanadate-dependent haloperoxi-
dases can exhibit phosphatase activity [34]. In the lower part of Figure 6, the mecha-
nistic sequence of the phosphoester hydrolysis as catalyzed by phosphatases is
illustrated. Apart from vanadate, many complexes of VIII, VIV, and VV with organic
ligands that are related to or mimic physiologically available ligands, effectively
inhibit phosphatases [35], likely after off-dissociation of all or part of the ligands
and thus formation of vanadate or {VOxL} intermediates with an ‘open’ coordina-
tion site capable of interacting with, and thus blocking, the active site of the enzyme.
Vanadates can also activate biochemical processes. An example for the activa-
tion of an otherwise phosphate-dependent process is the linkage of divanadate
(‘pyrovanadate’, H2V2O72 −) to the 3′-end of the primer nucleotide sequence of DNA
[DNA primers serve as a starting point for DNA replication (and hence DNA syn-
thesis) through DNA polymerase]. This activates degradation (‘pyrovanadolysis’)
of the primer, making available nucleotides for DNA replication by DNA poly-
merase [36]. The proposed mechanism for pyrovanadolysis is shown in Figure 7.
Activation of kinases will be addressed in the context of the antidiabetic action of
vanadium compounds in Section 4.1.
In solutions containing vanadate and phosphate, phosphovanadates, [HVPO7]3–
and [H2VPO7]2–, are present at pH 7 (the pKa is 7.2 at physiological ionic strength)
[37] (eqn. 7). This mixed anhydride is labile; the formation constant at pH 7 is
about 20 M–1, as compared to the formation constant of 350 M–1 for divanadate.
Phosphovanadate hence is less stable against hydrolysis than divanadate by an order
of magnitude, but more stable than diphosphate, [H2P2O7]2–, by six orders of magni-
tude. Phosphate can also form mixed species with VO2+ [38]: The dominant species
152 Rehder

O 2 O O 3 O
HO V O O V O P OH HO V R
O O
O O O O O
HVO3(O2)2 HVPO6(O2)3 HVO3(OR)

Figure 8 Examples for physiologically potentially relevant peroxidovanadates, peroxidovanado-


phosphates, and vanadate esters (from left to right).

in the slightly acidic regime is ‘VO(HPO4)’, where hydrogenphosphate is a ligand


for VO2+ rather than a counter-ion. Stabilization against hydrolysis in the neutral to
slightly alkaline range is again achieved by nutritionally or physiologically avail-
able organic ligands.

[H 2 VO4 ] + [ HPO4 ] + H +  [ H 2 VPO7 ] + H 2 O


– 2– 2–
(7)

In view of the omnipresence of hydrogen peroxide which, at low concentration
levels, is an important signalling and regulatory molecule in many biological pro-
cesses, the potential formation of peroxidovanadates [39] and peroxidovanadophos-
phates [40] such as HVO3(O2)2– and HVPO6(O2)3– is also to be considered, as is the
generation of vanadate esters according to equation (8). The monoesters of vanadate
and divanadate – analogues of the physiologically important phosphate and diphos-
phate esters – are, however, hydrolytically labile. Formation constants in the case of
aliphatic residues R are in the order of 0.1 M–1, for aromatic residues (phenyl esters)
around 1 M–1 [41]. Figure 8 contains peroxidovanadates and vanadate esters of
potential importance.

[H 2 VO4 ] + ROH →  HVO3 ( OR )  + H 2 O


– –
(8)

4  The Medicinal Potential of Vanadium

4.1  Diabetes Mellitus

Worldwide, about 10% of the population are suffering from diabetes mellitus –
knowingly and unknowingly. Approximately 90% of all diabetic cases are ascribed
to type 2 diabetes, the remaining 10% to type 1 diabetes [42a]. Type 1 diabetes
(“juvenile diabetes”) goes along with absent or only residual insulin supply by the
β cells in the Langerhans islets of the pancreas, commonly caused by degeneration
of the β cells in the frame of autoimmune reactions, or by accidental dysfunction or
loss of the pancreas. Type 2 diabetes (“adult onset diabetes”) is related, in its initial
stage, to insufficient response of the cellular insulin receptors to insulin. In its
5  Vanadium. Its Role for Humans 153

advanced stage, a feed-back mechanism comes in, provoking β cell failure caused
by de-differentiation of the β cells [43]. Type 2 diabetes typically concerns people
beyond the age of 50; its onset can be kept in check by physical exercise and reason-
able nutritional behavior. However, diabetes type 2 is nowadays increasingly diag-
nosed also with young people and even children; this juvenile onset type 2 diabetes
appears to be correlated to obesity [42b].
Intact β cells produce proinsulin, a peptide hormone consisting of an A-chain
with 21 amino acids (aa), a B-chain (30 aa), and a C-chain (31 aa) (for the role of
the C peptide see [86]), connecting the A- and B-chains. The C-chain is detached
in the final step of insulin synthesis; in genuine insulin, the A- and B-chains are
linked through two cystines. Insulin is stored as a C3-symmetric hexamer, with the
monomers linked through histidines via zinc ions [44]. The discharge of the active,
monomeric form of insulin is initiated by elevated blood glucose levels. Insulin
targets the cellular insulin receptor, triggering a complex mechanism by which
glucose becomes internalized into the cytosol, followed by glucose metabolism.
Further, insulin is involved in the inhibition of gluconeogenesis (the synthesis of
glucose from smaller building blocks, for example amino acids), and in glycogen-
esis (the synthesis of glycogen). Insulin is thus strongly involved in glucose
homeostasis. In addition, insulin stimulates lipogenesis and inhibits lipolysis, and
thus prevents ketoacidosis caused by the accumulation of ketonic bodies such as
acetyl acetic acid in the blood. Ketonic bodies are causative for the severe disease
patterns accompanied with progressive states of diabetes, such as retinopathy and
dying off of limbs.
Many inorganic (vanadate, vanadyl sulfate, peroxidovanadates) and organic
vanadium compounds have been tested positive with respect to effectuating cellular
glucose uptake and controlling free fatty acid levels. A selection of vanadium com-
plexes of the general composition {VOL}, where L represents an organic ligand
(system) in the coordination sphere, is provided in Figure 9; test conditions and
results are summarized in Table 1 [45–52]. The compounds have been successfully
tested in vitro (i.e., with cell cultures) and/or in vivo with diabetic rats or mice and,
in the case of the maltolato complex 1b, with human individuals. Complex 1b
(bis(ethylmaltolato)oxidovanadium(IV), BEOV), has passed clinical trials phase I
and IIa with type 2 diabetic volunteers, essentially with encouraging response [13].
The ligand L in {VOL} largely influences the efficacy of a vanadium compound
by steering resorption, transport, and stability of the complex, and thus the avail-
ability of the actual antidiabetic species, i.e., vanadate, at the locus operandi.
Commonly, vanadium complexes are clearly more effective than inorganic vana-
dium compounds, underlining the advantageous bioavailability and pharmaceutical
efficacy of organic vanadium compounds [13]. Where inorganic vanadium com-
pounds, vanadate (H2VO4−) in particular, induce hypoglycemic effects, such as tea/
vanadate decoctions (last entry in Table 1) [52], this effect is likely due to the inter-
mittent formation of a coordination compound with tea ingredients.
Normal insulin supply and insulin ‘sensing’ provided, insulin docks to the car-
boxyterminal segment of the extracellular α-subunits, IRα, of the trans-membrane
insulin receptor (a tyrosine kinase), de-repressing the tyrosine kinase activity of the
154 Rehder

R1 O R3 O
O O O O
V R2 V
O O
O O O N O
R2 R3 R1 X = H: 2a;
X O X = OH: 2b
R1 = CH3; R2, R3 = H: Maltol; 1a
R1 = C2H5; R2, R3 = H: Ethyl maltol; 1b
R1 = C5H11; R2 = CH3; R3 = OCH3: Allixin; 1c
O
H3C O O CH3
O V
O O OH2 O O O
CH3 O H3C OH2 CH3
V H2O O 5
N N O V
O O
H3C O N
O O 3 O 4 O
H2O O
V
O
O O
O O 8
O S Poly- O OH2 N
N V V CH3
glutamate O
S O N OH2 (CH2)3
6 7 [Co] [Vitamin B12]
O

Figure 9  A selection of vanadium coordination compounds with antidiabetic in vitro and/or in


vivo potential. Compounds 2 and 8 contain VV, the other complexes VIV. See Table 1 and text for
details and references.

intracellular β subunit, IRβ. In such a way, phosphorylation of the tyrosine residues


of IRβ is promoted and the cytosolic signal cascade initiated, ending up in the
­activation of the glucose transporter (GLUT4). Once activated, GLUT 4 becomes
translocated to the cell membrane where it picks up glucose for delivery into, and
metabolism within, the cytosol. The basic signal transduction is illustrated in
Figure 10. Among the various interposed and branching steps [45], insulin receptor
substrates (IRS), phosphatidylinositol 3-kinase (PI3K, also known as Akt), and pro-
tein kinase B (PKB), have been considered.
IRS are cytosolic proteins with tyrosine residues, and PKBs are kinases which
have available serine and/or tyrosine residues for phosphorylation. PKB directly
targets the glucose transporter. In the case of missing insulin supply (type 1 diabe-
tes), or insufficient receptivity of the insulin receptor for insulin (type 2 diabetes),
IRβ is dephosphorylated through the action of a protein tyrosine phosphatase
(PTPase, PTP-1B), annulling signalling for glucose intake by GLUT4 and thus pro-
voking hyperglycemia. A possible mechanism of action of antidiabetic vanadium
compounds in stimulating cellular glucose uptake in the case of hyperglycemia is
included in Figure 10. Accordingly, any vanadium compound {VOL} will be bro-
ken down in the extra- and/or intracellular space to generate vanadate. As noted in
Table 1  A selection of vanadium coordination compounds with antidiabetic potential.
Mode of Complex no.
Target application (see Figure 9) Antidiabetic effect (for abbreviations cf. text and Figure 10) Ref.
Human individuals per os 1b Reduction in blood glucose and glycosylated hemoglobin [13]
STZa mice per os 1c Lowering of blood glucose, phosphorylation of Akt and [45]
glycogen synthase kinase, improvement of gene expression
5  Vanadium. Its Role for Humans

levels
STZa rats intraperitoneal 2a Lowering of blood glucose [46]
STZa rats per os 2a, 2b Alleviation of hyperglycemia and hyperlipidemia [46]
SVb transformed mice fibroblasts in vitro 3 Stimulation of glucose uptake and metabolism [47]
Rat adipocytes in vitro 3 Inhibition of lipolysis [47]
Rat adipocytes in vitro 4 Phosphorylation of protein kinase B [48]
Differentiated 3T3-L1 mouse adipocytes in vitro 5 + serum Phosphorylation of IRβ and IRS; glycogen accumulation [49]
albumin, 6
KK-Ay micec per os 7 Alleviation of hyperglycemia, hypercholesterolemia, [50]
and hyperleptinemia
STZa rats tail vein injection 8 Lowering of blood glucose [51]
STZa rats per os vanadate/black Lowering of blood glucose [52]
tea decoction
a
STZ stands for streptozotocin, a naturally occurring glucosamine derivative that destroys the β cells in the Langerhans islets.
b
SV (for Simian virus) 3T3 fibroblasts are pseudo-adipocytes.
c
KK-Ay mice derive from cross-breading of female glucose-intolerant KK (Kyoji Kando)-mice with male obese Ay mice.
155
156 Rehder

{VOL}
H2O

H 2VO 4 O
IR Glucose
α α extra
β β PKB intra
PI3K O
{VOL} OPO3H
OPO3H
H2O OPO3H
GLUT4
H 2VO 4 IRS
OPO3H
PTP Pyruvate Glycogen

OVO3H

Figure 10  Signal cascade for the internalization of glucose by the glucose transporter GLUT4, as
triggered by the phosphorylated insulin receptor (IR). In the absence of insulin (diabetes 1) or
insufficient insulin response (diabetes 2), protein tyrosine phosphatase 1B (PTP) dephosphorylates
the IR, and the glucose intake is annulled. Vanadate can block PTP and thus restore the signalling
path. Several of the steps of the signal cascade are shown: IRS = insulin receptor substrate,
PI3K = phosphatidylinositol 3-kinase (which activates protein kinase B, also known as Akt),
PKB = protein kinase B.

Section 3, vanadate is an efficient phosphatase inhibitor. Consequently, inhibition


of PTP-1B by vanadate [53] prevents dephosphorylation of IRβ and thus restores
the signalling path.

4.2  Activity in Health Hazards Other than Diabetes

4.2.1  Treatment of Cancer

Several animal and in vitro studies have revealed the virtue of inorganic and, more
pronounced, organic vanadium compounds in reducing or preventing neoplasia (the
malignant proliferation of cells), and thus tumors, including cancer and its meta-
static potential, in various target tissues (see [54] and [55] for comprehensive
reviews on cancer prevention and treatment with vanadium compounds). Selected
examples are collated in Figure 11, and specific test results are provided in Table 2
[56–59].
The vanadocene derivative 10 is a recent advancement of the more basic vana-
docene (η5-C5H5)2VCl2 (= Cp2VCl2; Cp = cyclopentadienyl), introduced a quarter
of a century ago by Köpf and Köpf-Maier, who demonstrated that the compound
effectively degenerates and kills Ehrlich ascites tumor cells [60]. Ehrlich ascites are
derived from breast tumors of female mice. The o-phenanthroline complex 11,
dubbed “metvan”, stands for another group of closely related ‘classical’ vanadium
coordination compounds that turned out to be particularly operant against various
5  Vanadium. Its Role for Humans 157

C2H5 O O
O O
O O OCH3
N O HO
V V
O O O Methoxybenzyl-
H3C N O Cl
H O O V cyclopentadienyl
NH
Ethylcarboxy-4- NH Cl
pyrimidinone O N CH3 O N
C2H5 CH3
O OCH3
9a C2H5 9b 10

H3C C2H5 Ethylidenehydrazine-


O O
N OSO3 O N carbothioamide
V Salicylidene- V NH
N tryptophan N S
N O N CH3
N HN
H3C CH3
O N
o-Phenanthroline S
11 CH3 12 Thiazole

Figure 11  Vanadium(V) and vanadium(IV) coordination compounds with anticancer potential.
Simplistic names of the ligands are provided. 9b is a hydrolysis product of 9a. For details of the
mode of action see Table 2 and text.

Table 2  Selected vanadium coordination compounds with antitumor activity.


Complex
Target; no. (see
mode of application Figure 11) Effect Remarks Ref.
HeLaa tumor cells 9a Sufficiently more Species present [56]
toxic (IC50 = 44 μM) at pH 7: 9a
towards tumor than (= [VO2L2]–), 9b
towards non-tumor (= [VO2(L)OH]–),
cells vanadates
Human kidney cancer 10 IC50 = 0.55 μM [57]
cells (CAKI-1)
CAKI-1 mice; 10 Loss of body weight, Maximum tolerable [57]
intraperitoneal deadly toxic in some dose: 20 mg kg–1
cases; reduced tumor d–1
growth
Leukemia and myeloma 11 Effective at nM and Effective [58]
cells, cells derived low μM conc. Cell also against
from breast and apoptosis associated cisplatin-­resistant
testicular cancer with the generation ovarian cancer
of ROS and depletion
of GSH
Colon cancer cells 12 Decrease of cell viability; More efficient than [59]
IC50 = 48 μM cisplatin
a
HeLa tumor cells derive from cervical cancer (the cervix is the lower part of the uterus, connecting
to the vagina).
158 Rehder

cancer cell lines, including cisplatin-resistant ovarian and testicular cancer [58].
The oxidovanadium(V) pyrimidinone complex 9 and the oxidovanadium(IV)
complex 12 are examples for more recent developments in the search of vanadium-­
based anticancer compounds.
The operating mode of anticancer vanadium compounds is still elusive. Modes
of action that have been proposed include
( i) Inhibition of protein tyrosine phosphatases and activation of protein kinases;
(ii) Activation of tyrosine phosphorylases, with concomitant activation of signal
transduction pathways, followed by apoptosis and/or activation of tumor
suppressor genes;
(iii) Cleavage of, or intercalation into, DNA, resulting in cell cycle arrest;
(iv) Enhanced formation of reactive oxygen species (ROS);
(v) Down-regulation of ferritin expression and disruption of ferritin with concomi-
tant, iron-induced mediation of ROS.
As in the case of antidiabetic vanadium species, the ligand system mediates
the resorption and pharmacokinetics of the anticancer compound. In many cases,
the active species again is likely vanadate, formed by (partial) degradation of the
original drug: Speciation studies of the anionic pyrimidinone (L) complex,
[VO2L2]– (9a), have demonstrated that at pH 7 the complex 9a coexists with the
hydrolysis products, [VO2(OH)L]– (9b), and mono-, di-, and tetravanadate [56].
Intervention of vanadate with phosphatases, phosphorylases, and kinases will
alter and eventually disrupt or enforce signalling paths involved in the regulation
of the proliferation of malignant cells. Inorganic vanadate generated under physi-
ological conditions from, for example, [VO(maltol)2] (1a in Figure 9) has been
shown to discriminate between hepatocytes and hepatoma cells in as far as vana-
date significantly increases the generation of ROS (superoxide and hydrogen per-
oxide), and concomitantly causes cell cycle arrest in hepatoma but not in normal
liver cells (hepatocytes) [61]. Similarly, the formation of ROS by Fe2+ – after
vanadium-induced disintegration of ferritin – may be responsible for the vulner-
ability of astrocytoma cells, while astrocytes (cells of the brain and spinal chord)
remain unaffected [62].
Vanadocenes, Cp′2VCl2 (Cp′ stands for substituted Cp), such as complex 10 in
Figure 11 will become partially hydrolyzed under physiological conditions to form
[Cp′2V(H2O)x(OH)2–x]x+ (x = 0, 1, 2), and hence in a fashion comparable to the
hydrolysis of cisplatin, cis-[(NH3)2PtCl2]. The {Pt(NH3)2 +
2 } moiety of cisplatin can
directly interact with DNA by coordinating to the N-bases of DNA. The harder
(with respect to Pt2+) V4+ in the {Cp′2V2+} moiety supposedly prefers coordination
to oxo functionalities of the phosphoester linkages (Figure 12a). Alternatively,
Cp′2V(OH)2 can interact with the phosphates via hydrogen bonds. In any case, the
resulting ‘kink’ in the DNA will counteract DNA’s replication. Compounds such as
metvan (11 in Figure 11), having an aromatic system strongly coordinated to vana-
dium, may interact with DNA, and thus deactivate DNA and cell proliferation, via
intercalation (π−π interaction) (Figure 12b).
5  Vanadium. Its Role for Humans 159

NH2
a b
HN N
O O
N H2N Guanosine
Adenine N O O P
N O
HN O
N O
O O O N
P R O
O O O X P O
O N V
V O O
H2N N X
O
N O O
NH P
Cytosine O O R
N O
O
N Thymidine
H O

Figure 12  Possible interactions of stable (fragments of) vanadium compounds with DNA.
(a) The Cp2V2+ moiety of vanadocene (e.g., compound 10) binds to two adjacent phosphates.
(b) The o-phenanthroline unit of compound 11 intercalates in-between two nucleobases. X can be
OH or H2O. The π stacking is indicated by broken lines.

4.2.2  Cardiovascular Effects; Bacterial and Viral Diseases

Interference of vanadium with protein tyrosine phosphatase (PTPase), in particular


the inhibition of PTPase discussed in the context of the insulin-enhancing properties
of vanadium compounds in the preceding section, appears to be a major mode of
action also in beneficial effects for the vascular system in general, and vanadium’s
cardio-protective effects in particular. The inhibition of PTPase by coordination of
vanadate to the active site cysteine, as well as oxidative inhibition (cysteine → cys-
tine) by vanadium-induced production of peroxide, result in an up-regulation of
protein kinase B (Akt). Akt plays an important role in the regulation of cardiac
hypertrophy and angiogenesis (the formation of new blood vessels from preformed
ones), and thus in the prevention of, and recovery after, myocardial infarction.
The up-regulation of Akt in turn enhances the expression of epithelial nitrogen
oxide synthase (NO synthase) and thus the release of the vasodilator NO [63].
The maltolato complex 1a in Figure 9 [64], the pyridinethiolato complex 13, and
the picolinato-­bis(peroxido)vanadium(V) complex 14 in Figure 13 [65] perform
significant cardio-protection in test animals (Table 3) [63–71].
Several studies on vanadium’s potentiality in deactivating viral and bacterial
infections have appeared during the last decade, and selected compounds and results
are provided in Figure 13 and Table 3. The vanadium-substituted p­ olyoxidotungstate
[(VIVO)2(VVO)(SbW9O33)2]11– exhibits antiviral activity against viruses causing
influenza and Dengue fever, and is also active against HIV-1 (human immune
deficiency virus) and SARS (severe acute respiratory syndrome) in vitro [67a]. The cluster
contains a linear {O=VO4}3 core (with the vanadium centers in a tetragonal-­pyramidal
160 Rehder

O
O S O
N V N O
S O N VO
13 O O O O 14

R
N(C2H5)2 NH O HN
N O N O
V
R V S NH
NH Cl N
N N O Cl
N HN
V
R R
15 16 NH O HN

H
O
OH O
O V N O
O V
N H3C N S

(His496) NH 17 18 N CH3

Figure 13  Vanadium complexes with cardio-protective effects (13 and 14) or operant against
viral (15, 16, and 17) and bacterial infections (17 and 18). The cut-out 17 is the active center of
vanadate-­dependent haloperoxidase, e.g., from Corallina inaequalis (see text and Table 3).

environment), sandwiched by two {SbW9O33} halves in which the tungsten ions


are octahedrally coordinated [67b]. At physiological conditions, this so-called
‘Keggin sandwich’ likely decomposes into the genuinely active species vanadate(V)
and tungstate(VI). Anti-HIV activity has also been reported for the physiologically
stable, water-soluble oxidovanadium(IV)-porphyrin 15 [68]. Water solubility of
15 is provided by the aminosulfonyl substituent at the porphyrin skeleton. The
complex inhibits HIV-1 replication in virus infected Hut/CCR5 cells, possibly
by targeting and deactivating the viral reverse transcriptase [Hut/CCR5 cells are
derived from Hut78 cells, a human T cell line. Hut/CCR5 cells express the chemo-
kine receptor CCR5, a protein on the surface of T lymphocytes (a group of white
blood cells)].
The oxidovanadium(IV) xylyl-bicyclam complex 16 is a further example for an
agent that is highly effective against strains of HIV-1 and HIV-2 [69], with IC50
values of 0.1–0.3 μM (IC50 values denote the concentration of a drug or prodrug at
which the life function of 50% of the viable cells is inhibited). A possible mode of
action is through binding of the complex to the chemokine receptor type 4 (CXCR-­4),
a receptor protein that HIV can use to infect T lymphocytes. Docking of compound
16 to CXCR-4 can occur via direct coordination of the metal center to aspartate and
glutamate residues of the protein, or via weak interaction of the oxidovanadium
moiety with tryptophan residues, and/or by hydrophobic interaction between
tryptophan residues and the bicyclam rings.
Antiviral and antibacterial activity has also been noted for vanadate-dependent
haloperoxidase isolated from the marine alga Corallina inaequalis, and its alkalophilic
mutant Pro395Asp/Leu241Val/Thr343Ala in particular [70]. The enzyme contains
5  Vanadium. Its Role for Humans 161

Table 3  Vanadium coordination compounds in the treatment of cardiovascular diseases, viral, and
bacterial infections.
Complex
Target; no. (see
mode of application Figure 13) Effect Mode of action Ref.
Female rats 13 Amelioration Activation of Akt [63]
of myocardial signalling →
injuries expression of NO
synthase
Rats; intravenously 1a Limitation of Inhibition of tyrosine [64]
reperfusion phosphatase
injury
Rats; orally 14 Neuro-protection Enhancement of [65]
downstream Akt
Rat cardiac myocites 14 Amelioration of Increase of levels of [66]
cardio-­ NO synthase
dysfunction
Influenza virus, Dengue see text Antiviral activity (not reported) [67]
fever virus, HIV-1,
SARS; in vitro
HIV-1 15 Inhibition of HIV-1 Inhibitory activity [68]
replication in towards HIV-1
host cells reverse
transcriptase
HIV-1 and HIV-2 strains 16 Anti-HIV activity Binding to the [69]
receptor CXCR-4b
Herpes simplex, 17a Antiviral and Formation of HOBr [70]
Coxsackievirus B4, antimicrobial and 1O2
Staphylococcus aureus, activity
Pseudomonas aeruginosa;
pH 8, H2O2 + Br–
Mycobacterium tuberculosis 18 Growth inhibition (not reported) [71]
a
The active center of the Pro395Asp/Leu241Val/Thr343Ala mutant of the vanadate-dependent
chloroperoxidase from the alga Corallina inaequalis.
b
See text for details.

vanadate (H2VO4−) coordinated to the Nε of a histidine residue in the active center


(17 in Figure 13). The native peroxidase catalyzes the oxidation of halides, for
example, bromide to hypobromous acid, HOBr (eq. 9a), and – in a side-reaction –
generates singlet oxygen 1O2 (eq. 9b), commonly at slightly acidic conditions.
The mutant is active at pH ~ 8, allowing for the disinfection, through HOBr and 1O2,
of medicinal equipment under particularly mild and thus moderate conditions. Strong
microbial abatement was observed for both enveloped (Herpes simplex) and non-­
enveloped viruses (Coxsackievirus B4), as well as for Gram-positive (Staphylococcus
aureus) and Gram-negative bacteria (Pseudomonas aeruginosa).
Br – + H 2 O2 + H + → HOBr + H 2 O (9a)

HOBr + H 2 O2 → 1 O2 + Br – + H 2 O + H + (9b)

162 Rehder

Antibacterial action has also been reported for complex 18 in Figure 13. The VV
thiosemicarbazone (tsc) complex [VO2(tsc)] and its VIV precursor [VO(acac)tsc]
(acac = acetylacetonate(1–)) inhibit the pathogen of tuberculosis, Mycobacterium
tuberculosis [71]. Minimal inhibitory concentrations (MIC values) of these vana-
dium complexes are lower than for the free tsc ligand, supporting a role of the vana-
dium center in these antituberculosis drugs.

4.2.3  Diseases Caused by Parasites

Vanadium coordination compounds have also been shown to exhibit potential in the
abatement of epidemic diseases caused by parasites (amoebae and flagellates)
­predominantly in tropical and subtropical countries. Examples are amoebiasis,
Chagas’ disease (American trypanosomiasis), and leishmaniasis. As in the case of
antiviral and antibacterial vanadium compounds, none of these compounds has so
far achieved the status of clinical tests, and studies have thus been restricted to in
vitro tests with cultures of the parasites. In many cases, these studies reveal antipara-
sitic properties of the vanadium species, which are about comparable to or even
more effective than established medications. In this section, selected examples of
antiparasitic vanadium complexes are briefly described. For a recent overview see
also the review by D. Gambino [72]. Respective vanadium compounds are illus-
trated in Figure 14; selected results are summarized in Table 4 [73–77].

O O NH2
2
O Salicyl-
V N
O semicarbazone O
N N O
N 5-Methyl-
V
O O N
salicyl H3CO
2-Furoyl- N
2 o-Phenan-
hydrazide OH
throline
19 20

HO
Galactose
OH VO 2+ (LH -1)2 O
HO HL =
O +H N
Galactomannan 3 NH2
O O
Glutaminate
HO OH O O
V (Gln)
O O
O O O
O O
Mannose O
OH (Gln)
21 22

Figure 14  Complexes and formulations that have been shown to exert antiparasitic potential
against parasites causing amoebiasis (19), Chagas’ disease (20), and leishmaniasis (20, 21, 22). For details
see text and Table 4.
5  Vanadium. Its Role for Humans 163

Table 4  Vanadium compounds that kill parasites causing (tropical) diseases.


Complex
no. (see
Target Figure 13) Effect Remarks Ref.
Tropozoites of 19 Amoebocidal More effective than [73]
Entamoeba Metronidazole; slightly
histolytica toxic against HeLa cells
(in vitro)
Epimastigotes of 20 Trypanocidal Causes conformational [75]
Trypanosomas changes in supercoiled
cruzi (in vitro) plasmid DNA
Promastigotes and 20 Leishmanicidal Cytotoxic against leukemia [74]
amastigotes HL-60 cells
of Leishmania
(in vitro)
Promastigotes and 21 Leishmanicidal on Diminishes also superoxide [76]
amastigotes amastigotes; growth production by macrophages
of Leishmania inhibition of
(in vitro) promastigotes
Leishmania-infected 22 Reduction in parasite Similar effects are brought [77]
mice (in vivo) burden; expansion about by [HVO2(O2)2)]2–
of antileishmanial
T-cells

According to the World Health Organisation, about 50 million people are affected
worldwide by amoebiasis, with infections clearly cumulating in tropical countries.
The etiologic agent of the disease is the amoeba Entamoeba histolytica, present in
contaminated food and water in particular in areas of low sanitary and hygiene stan-
dards. Transmission occurs mainly by the fecal-oral route and through direct con-
tact. 90% of the infected people are asymptomatic. For the remaining 10% the
symptoms include diarrhea and, in more serious cases, dysentery (an inflammatory
disorder mainly of the colon) with mucus and blood, the latter stemming from
amoebae that succeed to overcome the epithelium of the intestines and thus travel to
other organs, the liver in particular, where they cause deadly abscesses. The death
toll amounts to ca. 70,000 per year. The hydrazone complex 19 in Figure 14 is an
example for an efficient amoebocidal (pro)drug [73]. With an IC50 = 0.36 μM, the
compound is more efficient than the standard drug Metronidazole (IC50 = 1.89 μM),
and somewhat more toxic than Metronidazole against human cervical HeLa cancer
cell lines.
Chagas’ disease is caused by the flagellate protozoan parasite Trypanosomas
cruzi, transmitted by the feces of “kissing bugs”, blood-sucking bugs belonging to
the subfamily Triatominae. About 10 million people are infected, with ca. 10,000
deaths per year. The disease is mainly distributed in Latin America, but also increas-
ingly spreading into North America and Europe. Primary symptoms are skin lesions
and swelling of the eye lids; secondary symptoms include digestive and neurologi-
cal alteration, and cardiac disorder up to heart failure. The oxidovanadium complex
164 Rehder

20 [74] contains a salicylidene semicarbazone ligand and hence a ligand that has
also been shown to convey anticancer (compound 12) and antiamoebiasis activity
(19). The additional ligand in 20 is phenanthroline, capable of intercalating DNA
(Figure  12b). The metabolic pathways of parasites such as Trypanosoma and
Leishmania are similar to those in tumor cells, suggesting a similar mode of action
of anti-parasitic and anti-tumor drugs. Complexes such as compound 20, which are
about as trypanocidal against epimastigotes (a developmental state of the parasite in
the bug) of T. cruzi as the reference drug Nifurtimox, are in fact cytotoxic against
leukemia cells [74] and cause conformational changes in plasmid DNA [75].
Complex 20 is also active on the promastigote and amastigote forms (the flagel-
late and non-flagellate stages, respectively) of Leishmania parasites, responsible for
the tropical and subtropical disease leishmaniasis. The vectors for this disease,
which is associated with malnutrition and weakness of the immune system, are
sandflies of the subfamily Phlebotomina. About 12 million people are affected
worldwide. The most serious form of this disease is visceral leishmaniasis, which
goes along with high fever, weight loss, swelling of the spleen and liver, and anemia.
Visceral leishmaniasis is mainly distributed in Brazil, India, Bangladesh, and Sudan,
and accounts for 60,000 deaths each year.
Oxidovanadium complexes of galactomannan (21 in Figure 14) have been
shown to be leishmanicidal on amastigotes of L. amazonensis, and to inhibit the
growth of the promastigotes of this parasite [76]. Galactomannan is a polysaccha-
ride with a mannose backbone and galactose side groups, isolated from the lichen
Ramalina celastri, which is abundant in South Brazil. Antileishmanial effects have
further been noted, with infected mice, for bis(peroxido)vanadate, [HVO2(O2)2]2–,
and combinations of glutaminate and [HVO2(O2)2]2– (see 22 in Figure 14 for a
tentative formulation of a formula unit) [77]. Finally, decavanadate deters the
growth of the leishmania parasite, likely by interaction of decavanadate’s hydrolysis
product [H2VO4]– with phosphatases [78], hence a mechanistic aspect which is
again reminiscent of the antidiabetic, insulin-enhancing action of vanadate and
(hydrolytically labile) vanadium complexes. For the molecular built-up of deca-
vanadate see Figure 5.

5  Concluding Remarks and Prospects

The element vanadium was discovered in 1801 by Andrés Manuel del Rio y
Fernández in vanadinite from the district Zimapán in Mexico [5]. It was rediscovered
by Nils Gabriel Sefström in iron ore from the Taberg in Småland (Sweden) in 1830
by treating a “black powder obtained from the manufacturing of bar iron”, and
in 1869/70 Sir Henry Enfield Roscoe in England was the first one who succeeded
to isolate (an impure form of) metallic vanadium by reduction of VCl2 with H2
[79,87], nowadays widely employed in particularly tough and hard chromium-
vanadium-­iron alloys. Along with metallic vanadium, vanadium oxides (“VOx”) are
5  Vanadium. Its Role for Humans 165

in use as catalysts in oxidation reactions (an example is the production of sulfuric


acid from SO2), as an UV-absorbing additive in glass ware, and in the form of lithium
and silver vanadates in lithium batteries [80]. The first indication for the presence
of vanadium in a living organism, i.e., the sugar beet, goes back to 1888 [81]; the
essentiality of vanadium for some life forms became established in 1982/83 by the
discovery of vanadate-dependent bromoperoxidase in the marine macro-alga
Ascophyllum nodosum [82].
Toxic effects of vanadium were first reported by John Priestley [15], who applied
large doses of sodium vanadate to animals, tediously describing their ailment and
final death. Exploratory medicinal applications of vanadium, again in the form of
vanadate, were carried out in Lyon (France) back in 1897–98 [83] on probands with
health problems as diverse as anemia, tuberculosis, rheumatism, neurasthenia, and
diabetes where, in the latter case, some decrease of blood glucose was observed.
The prospective use of vanadium compounds in the treatment of diabetes thus has a
long-standing tradition. The presently most advanced study of vanadium’s potential
in the treatment of human diabetics are clinical tests phase IIa, carried out in 2007/8
with bis(ethylmaltolato)oxidovanadium(IV), BEOV [13], a compound developed in
the group of Chris Orvig in Vancouver, Canada.
The successful application of vanadium compounds in low doses with diabetic
animals (streptozotozin-induced rats in particular) and with type 2 diabetic humans
has initiated research into medicinally active vanadium compounds for other
medicinal applications, among these tropical and subtropical diseases caused by
infectious parasites responsible for leishmaniasis, Chagas’ disease, and amoebiasis,
just as diseases caused by viruses (HIV, influenza), and bacteria (tuberculosis). In all
cases, in vitro tests with cell cultures or cultured parasites, as well as sporadic in
vivo tests with infected test animals, have provided results that should encourage
further investigations into the field of vanadium-based medications. Given that
classical pharmaceuticals tend to have, sometimes severe, side effects, and parasites
can develop resistance, the development of potential (pro-)drugs based on the
essentially non-toxic element vanadium certainly is a future challenge.
The possibly most important aspect in choosing vanadium as the central metal in
these coordination compounds is their (partial) rebuilt to vanadate(V) and
oxidovanadium(IV) in the physiological broth. Vanadate is an antagonist of phos-
phate – as demonstrated for the first time by Cantley in 1977 for the inhibition of the
Na,K-­ATPase, the sodium-potassium pump [84]. Based on the interference of vana-
date with metabolic processes depending on phosphate in general, and on phos-
phate-regulated enzymatic activity in particular, vanadate has also been introduced
into the amelioration of cardiac dysfunctions, and in fighting malignant tumors. The
latter application, first investigated systematically by Köpf and Köpf-Maier [60], is
based, at least in part, on the direct interaction of stable vanadium-ligand fragments
with the DNA of cancer cells – and is thus reminiscent of the action of the well
established anticancer drug cisplatin. Stable fragments in vanadium compounds are
represented by the cyclopentadienide ligands in vanadocenes, and by N-functional,
oligodentate aromatic ligands such as ortho-phenanthroline and derivatives thereof.
166 Rehder

Last but not least, yet another functional aspect of vanadium compounds is the
ability of VO2+ to interfere – in a Fenton-­like reaction – with the tissue concentrations
of reactive oxygen species [85].

Abbreviations

Ab albumin
acac acetonylacetonate(1–)
Akt protein kinase
ATPase adenosine triphosphate cleaving enzyme
BEOV bis(ethylmaltolato)oxidovanadium
CAKI human renal carcinoma cell line
CCR5 chemokine receptor of T lymphocytes
Cp cyclopentadienide
CXCR-4 chemokine receptor type 4
FAD flavin adenine dinucleotide (oxidized form)
FADH2 flavin adenine dinucleotide (reduced form)
GLUT glucose transporter
GSH glutathione
GSSG oxidized form of glutathione
HeLa cells cervical cancer cell line derived from Henrietta Lacks
HIV human immune deficiency virus
IC50 50% inhibitory concentration; the values denote the concentration of
a drug or prodrug at which the life function of 50% of the viable cells
are inhibited
Ig immunoglobulin
IR insulin receptor
IRS insulin receptor substrate
lmm low molecular mass (ligand)
MAC maximum allowable concentration at the working place
MIC minimal inhibitory concentration
NAD nicotinamide adenine dinucleotide (oxidized form)
NADH nicotinamide adenine dinucleotide (reduced form)
NADP nicotinamide adenine dinucleotide phosphate (oxidized form)
NADPH nicotinamide adenine dinucleotide phosphate (reduced form)
PIK phosphatidyl-inositol kinase
PKB protein kinase B
PTPase protein tyrosine phosphatase
ROS reactive oxygen species
SARS severe acute respiratory syndrome
STZ streptozotocin
SV Simian virus
Tf transferrin
tsc thiosemicarbazone
5  Vanadium. Its Role for Humans 167

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Chapter 6
Chromium: Is It Essential, Pharmacologically
Relevant, or Toxic?

John B. Vincent

Contents
ABSTRACT ............................................................................................................................. 172
1 INTRODUCTION ............................................................................................................. 172
2 IS CHROMIUM ESSENTIAL?......................................................................................... 173
2.1 Current Opinions ....................................................................................................... 173
2.2 Evidence .................................................................................................................... 173
2.2.1 “Low Chromium” Rodent Diets .................................................................... 173
2.2.2 Absorption and Transport.............................................................................. 176
2.2.3 Total Parenteral Nutrition .............................................................................. 179
3 IS CHROMIUM PHARMACOLOGICALLY RELEVANT? ........................................... 180
3.1 Rodent Disease Model Studies.................................................................................. 180
3.2 Clinical Studies ......................................................................................................... 182
3.3 Proposed Mechanisms of Action .............................................................................. 186
3.3.1 Insulin Signaling ........................................................................................... 186
3.3.2 Cholesterol and Fatty Acid Metabolism........................................................ 189
3.3.3 Inflammation and Oxidative Stress ............................................................... 190
4 IS CHROMIUM TOXIC? .................................................................................................. 191
4.1 Chromate ................................................................................................................... 191
4.2 Chromium Picolinate and Other Cr(III) Complexes ................................................. 191
5 CONCLUDING REMARKS AND FUTURE DIRECTION ............................................ 192
ABBREVIATIONS AND DEFINITIONS .............................................................................. 193
ACKNOWLEDGMENT .......................................................................................................... 194
REFERENCES ........................................................................................................................ 194

J.B. Vincent (*)


Department of Chemistry, University of Alabama, Tuscaloosa, AL 35487-0336, USA
e-mail: jvincent@bama.ua.edu

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 171
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_6, © Springer Science+Business Media Dordrecht 2013
172 Vincent

Abstract Over fifty years ago, the element chromium (as the trivalent ion) was
proposed to be an essential element for mammals with a role in maintaining proper
carbohydrate and lipid metabolism. Evidence for an essential role came from dietary
studies with rodents, studies on the effects of chromium on subjects on total
parenteral nutrition, and studies of the absorption and transport of chromium. Over
the next several decades, chromium-containing nutritional supplements became so
popular for weight loss and muscle development that sales were second only to
calcium among mineral supplements. However, the failure to identify the responsible
biomolecules(s) that bind chromium(III) and their mode of action, particularly a
postulated species named glucose tolerance factor or GTF, resulted in the status of
chromium being questioned in recent years, such that the question of its being
essential needs to be formally readdressed. At the same time as chromium(III)’s
popularity as a nutritional supplement was growing, concerns over its safety
appeared. While chromium has been conclusively shown not to have beneficial
effects on body mass or composition and should be removed from the list of essential
trace elements, chromium(III) compounds are generally nontoxic and have beneficial
pharmacological effects in rodents models of insulin insensitivity, although human
studies have not conclusively shown any beneficial effects. Mechanisms have been
proposed for these pharmacological effects, but all suffer from a lack of consistent
supporting evidence.

Keywords chromium • insulin sensitivity • insulin signaling • rats • type 2 diabetes

Please cite as: Met Ions Life Sci. 13 (2013) 171–198

1 Introduction

Recently, a paradigm shift has occurred in terms of the status of chromium. While first
proposed to be an essential element in the late 1950s and accepted as a trace element
in the 1980s, scientific studies have continued to fail to produce convincing evidence
of this status. In the 1990s, statements to justify the status of chromium despite the
results of studies such as “Chromium is a nutrient and not a drug, and it will therefore
benefit only those who are deficient or marginally deficient in Cr” [1] were common
in review articles [1–3]. Recent studies have led to a reinterpretation of the status of
chromium. The status of chromium as an essential element is no longer supported by
experimental data. In fact, chromium is now best understood as a therapeutic agent.
However, the potential benefits of the use of chromium as a therapeutic agent are
uncertain, and its mechanism of action in increasing insulin sensitivity and possibly
influencing lipid metabolism at a molecular level is poorly understood.
This review will examine the data on which chromium was proposed to be an
essential element and describe the problems with this interpretation, discuss the
evidence for a therapeutic role for chromium in animal models of diabetes and
insulin resistance, and evaluate the potential toxicity as chromium(III) complexes
when used at pharmacologically relevant doses.
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 173

2 Is Chromium Essential?

2.1 Current Opinions

Chromium reduces body fat, causes weight loss, causes weight loss without exercise,
causes long-term or permanent weight loss, increases lean body mass or builds
muscle, increases human metabolism, and controls appetite or craving for sugar,
while 90% of US adults do not consume diets with sufficient chromium to support
normal insulin function, resulting in increased risk of obesity, heart disease, elevated
blood fat, high blood pressure, diabetes, or some other adverse effect on health.
Any or all of the above representations may come to mind when thinking about
chromium and its relationship to human nutrition. Most people think of chromium
in terms of weight loss and lean muscle mass development as a result of nutraceutical
product marketing. However, the Federal Trade Commission (FTC) of the United
States ordered entities associated with the nutritional supplement chromium
picolinate to stop making each of the above representations in 1997 because of the
lack of “competent and reliable scientific evidence” [4].
Overwhelming scientific evidence currently indicates that chromium does not
affect body mass and body composition of healthy individuals and that chromium
nutritional deficiency is rare (if it exists at all) [5]. Yet, although the ruling by the
FTC is over 15 years old, such representations can still be found in the popular
media. In the United States, the National Research Council of the National
Academies of Science recognized chromium as an essential trace element in 1980
and reviewed this position in 1989 and 2002 [6–8]. However, in 1980 and 1988,
chromium was determined to have an estimated safe and adequate daily dietary
intake (ESADDI) of 50–200 μg, while in 2002 this was changed to an adequate
intake (AI) of 30 μg. The Panel on Additives and Products or Substances Used
in Animal Feed (FEEDAP) [9] in 2009 determined that chromium deficiency in
farm animals had never conclusively been observed such that ‘no evidence of the
essentiality of Cr(III) as a trace element in animal nutrition’ exists. As discussed in
Section 2.2, the status of chromium is at best uncertain currently, and the element
should probably be removed from the list of essential trace elements.

2.2 Evidence

2.2.1 “Low Chromium” Rodent Diets

Over fifty years ago, Cr was suggested to be an essential trace element in the
mammalian diet. In this work reported by Mertz and Schwarz [10], previously
considered the pioneering work in the field, rats were fed a torula yeast-based
diet, which compromised the health of the rats. The rats developed necrotic liver
degeneration and apparently impaired glucose tolerance in response to an intravenous
174 Vincent

glucose load [10]. Selenium was discovered to reverse the liver disorder but not the
glucose intolerance; as a result, the authors proposed a new dietary requirement,
coined glucose tolerance factor (GTF) was absent from the torula yeast-based diet
and responsible for the glucose intolerance [11]. In an effort to identify the missing
dietary component, a variety of chemicals and some foods were added to the diet.
Most notably, inorganic compounds containing over 40 different elements (200–
500 mg element/kg body mass) could not restore glucose tolerance, while several
inorganic Cr(III) complexes (200 mg Cr/kg body mass) restored glucose tolerance
[12]. Brewer’s yeast and acid-hydrolyzed porcine kidney powder were identified as
natural sources of the missing dietary component and were found to contain appre-
ciable quantities of Cr [12]. When given by stomach tube (500–1000 mg/kg body
mass), brewer’s yeast, porcine kidney powder, and concentrates made from them
restored proper glucose metabolism in rats on the torula yeast-based diet [12].
Consequently, Mertz and Schwarz proposed the active ingredient of GTF was Cr3+,
making Cr an essential trace element for the mammalian diet [12].
As this became the primary evidence for an essential role for Cr, one must ask
what exactly this work established? The Cr content of the regular laboratory rat
diet and of the torula yeast-based diet have not been determined; thus, the rats were
not shown to actually receive a diet lacking or deficient in Cr; the studies only
indicated that adding Cr to the diet could lead to potential effects on apparent glu-
cose intolerance. Subsequently, the Cr content of torula yeast has been determined,
but the Cr content has been found to range significantly in value [13,14]; the con-
tent probably varies based on the growth conditions. As a result, the content of the
original diet simply cannot be established. In addition, the rats were housed in wire
mesh cages, possibly with stainless steel components (as the metal composition of
the wire was not reported), allowing the rats to obtain Cr by chewing on these
components. Thus, the actual Cr intake of the rats in these studies is impossible to
gauge. As subsequent studies have shown that rat in metal free cages on purified
diets fail to develop Cr deficiency, these studies fail to establish that the animals
developed a Cr deficiency.
The results do raise another possible explanation, one that was not originally
considered – the Cr added to the torula yeast-based diet was having a pharmacologi-
cal or therapeutic effect and not correcting a nutritional deficiency. The magnitude
of the doses of Cr utilized in these studies need to be put into perspective. An
American consuming a nutritionist-designed diet [15] or self-selected diet [16] con-
sumes about 30 μg of Cr daily. This value, 30 μg Cr/day, is the value set as the
adequate intake (AI) by the Food and Nutrition Board of the Institute of Medicine
of the National Academy of Sciences (USA) [8]; as defined, the AI indicates that
>98% of the population receiving this quantity of an item display no health prob-
lems from deficiency. Given the average body mass of a human, 65 kg, gives an
adequate Cr intake of less than 0.5 μg Cr/kg per day. Rats on the torula yeast-based
diet that was supplemented with Cr compounds received at least 400 times this
quantity, a supra-nutritional dose. These comparisons, of course, make the assump-
tion that the biochemistry of Cr is similar in rodents and primates.
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 175

Attempts have been made to establish the nutritional status of Cr using nutritionally
compromised diets supplemented with Cr, most notably in the 1990s [17–19]; the
rationale behind these diets was that stresses that increase urinary Cr loss could
potentially lead over time to chromium deficiency. However, these studies suffer from
some of the same flaws in assumptions as the initial studies. Rats were provided a
high-sugar or high-fat diet (supposedly a “low-Cr” diet with ca. 30 μg Cr/kg diet) with
additional mineral stresses for 24 weeks, resulting in compromised lipid and carbohy-
drate metabolism in the rats. The addition of 5 ppm Cr to the drinking water of rats on
the stressed diets led to plasma insulin levels tending to be higher in intravenous
glucose tolerance tests after 24 weeks on the diet [17]. Unfortunately, the Cr intake
compared to the Cr loss in the rats was not determined so that whether the rats were
maintaining a Cr balance cannot be established. However, as described in Section 2.2.2,
the amount of urinary Cr loss is directly dependent on the amount of Cr intake so that
the rats should not have developed a Cr deficiency. Consequently, the results should
be interpreted in terms of supplemental Cr having a beneficial effect on diet-induced
insulin resistance, a pharmacological rather than nutritional effect.
An analysis of the actual Cr content of the diet is in order. A male Wistar rat (as
used in Refs [17–19]) on average in a subchromic study consumes 20 g of food a
day and has an average body mass of 217 g [20]. Twenty grams of food containing
33 μg Cr/kg food provides 0.66 μg Cr. Thus, 0.66 μg Cr/d for a 217 g rat is 3.0 μg
Cr/kg body mass per day, six times what a human intakes. Thus, the “low-Cr” diet
was not deficient, unless rats require more than six times the Cr dose that humans
do. In contrast, a male Wistar rat on average drinks 147 mL of water [20]. This vol-
ume of water supplemented with 5 ppm Cr provides 735 μg Cr daily or 3.39 mg/kg
body mass. This is approximately 100 times the adequate intake of an American
male (35 μg Cr/day) [8]. Again, indicating the lowering of plasma-insulin levels by
addition of Cr can only be considered a pharmacological effect.
Finally, a most recent study appears to have unambiguously demonstrated that Cr
has a pharmacological rather than a nutritional effect in mammals [21]. Whether Cr
is an essential element was examined for the first time in carefully controlled metal-
free conditions using a series of purified diets containing various Cr contents. Male
lean Zucker rats were housed in specially designed metal-free cages for six months
and fed the AIN-93G diet with no added Cr in the mineral mix component of the diet
(containing 16 μg Cr/kg diet), the standard AIN-93G diet (containing added 1,000
μg Cr/kg), the standard AIN-93G diet supplemented with 200 μg Cr/kg, or the stan-
dard AIN-93G diet supplemented with 1,000 μg Cr/kg. The Cr content of the diet
had no effect on the body mass or food intake. Similarly, the Cr content of the diet
had no effect on the glucose levels in glucose tolerance or insulin tolerance tests.
However, a distinct and statistically significant trend toward lower insulin levels
under the curve after a glucose challenge was observed with increasing Cr content
in the diet; rats on the supplemented AIN-93G diets had significantly lower areas
(P <0.05) than rats on the low-Cr diet. The study revealed that a diet with as little Cr
as reasonably possible had no effect on body composition, glucose metabolism, or
insulin sensitivity compared with a “Cr-sufficient” diet; however, pharmacological
176 Vincent

quantities of Cr had a concentration-dependent effect on lowering insulin levels


in glucose tolerance tests, indicating that Cr may have a pharmacological effect
increasing insulin sensitivity in healthy rats [21].
In summary, a complete paradigm shift has occurred in the field of Cr nutrition,
where for four decades, Cr had been considered to have only a nutritional, not a
pharmacological effect. Now Cr is realized to have a pharmacological effect rather
than a nutritional one. Nutritional studies cannot be used to determine whether Cr is
an essential element. Studies using as little Cr as possible in the diet have failed to
establish any signs of Cr deficiency. Without conclusive positive evidence, Cr can-
not be considered an essential trace element. A demonstration that Cr could poten-
tially be an essential element will probably require the isolation of a biomolecule
that is essential to some critical biological process and requires Cr to perform its
essential function. As described below (Section 3.3), this has not occurred.

2.2.2 Absorption and Transport

Cr is absorbed by passive diffusion when intaken orally. This has been convincingly
demonstrated by a double perfusion technique using segments of the small intestine
of rats; these studies revealed that over a 100-fold range of [Cr3O(propionate)6
(H2O)3]+ (Cr3) concentrations (10–1000 ppb), chromium absorption was a nonsatu-
rable process [22]. Additionally, studies following the fate of orally administered
51
Cr have not observed a change in % Cr absorption over a range of intakes [23,24].
Most recently, rats gavaged with a dose of CrCl3 absorbed approximately 0.2% of
the Cr over a 2000-fold range of doses (0.01–20 mg Cr) [24]. Another interesting
conclusion that can be drawn from the intestinal perfusate studies is that Cr appears
to be actively transported out of the intestinal cells, as approximately 94% of the Cr
entering the cells was cleared from the cells (leaving only approximately 6% behind
to be stored). However, no transporter is known for Cr3+. This suggests the possibil-
ity that Cr3+ may be bound to some chelating ligand and actively transported in this
form; this is an area requiring further research. Changes in diet could affect the
amount of Cr absorption and potentially affect the mechanism, although changes in
mechanism have not been demonstrated. For example, the presence of added amino
acids, phytate (high levels), ascorbic acid, and oxalate, but not low levels of phytate,
in the diet reportedly altered the extent of Cr uptake (reviewed in [25]), although the
changes (while statistically significant in some cases) were relatively small in a
small percentage of absorption.
Once in the bloodstream, Cr3+ binds almost exclusively to the Fe-transport pro-
tein transferrin. The association of transferrin and Cr has been reviewed previ-
ously [26]. Cr-loaded transferrin has been demonstrated to transport Cr in vivo
[27,28]. Injection of 51Cr-transferrin into rats resulted in incorporation of 51Cr into
tissues. The transport of Fe into tissue by endocytosis of transferrin has been
found to be insulin sensitive, as the transport of Cr; injection of labeled transferrin
and insulin resulted in a several fold increase in urinary Cr [28]. Thus, transferrin,
in an insulin-dependent fashion, can transfer Cr to tissues from which it is excreted
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 177

in the urine. The binding of Cr to transferrin is quite tight, although the apparent
binding constants for the two metal binding sites differ by approximately 105 [29];
the in vitro binding of Cr3+ from inorganic salts has been shown to be quite slow
[29], although these studies were performed in the presence of ambient bicarbon-
ate concentrations. This also suggests that Cr may be carried to transferrin as a
chelate complex. However, recent studies in the author’s laboratory reveal that at
the bicarbonate concentration of human blood (~20 mM) the binding of Cr3+ is
quite rapid (B. Liu, G. Deng, K. Wu, and J. B. Vincent, unpublished results). Once
Cr is brought into the cell by endocytosis, it must leave the endosome to enter the
cell cytosol. As Cr3+ is not readily reduced by any biological reducing agents, so
that it can be transported by divalent metal ion transporters (in a fashion similar to
Fe), it must be transported by another mechanism; this is another area requiring
further research [30].
A human study of chromium absorption as a function of Cr intake has often
been cited as evidence of an essential role for Cr; however, this single study
requires reproduction. Anderson and Koslovsky have reported an inverse relation-
ship between dietary chromium intake and degree of absorption observed in human
studies [31]. The data suggest that absorption of Cr varies approximately from 0.5
to 2.0% for Cr intakes of ~15–50 μg per day. This difficult to perform study is far
from definitive; for example, a distinct difference is found if the data are separated
into male and female subjects. For males, no statistical variation occurs for chro-
mium absorption as a function of intake, while an apparent inverse trend is observed
for the female subjects. However, these data are in striking contrast to this same
lab’s studies reported two years earlier [32]. Chromium absorption was determined
to be ~0.4% for free-living individuals; when Cr intake was increased by over
fourfold, urinary chromium excretion increased over fourfold while maintaining
~0.4% absorption of chromium for both males and females. The difference between
the two studies lies in the range of Cr intakes of ~15–50 μg per day for the former
and ~60–260 μg per day for the latter, suggesting that an inverse relationship
between Cr intake and absorption, if it exists, exists only at the lowest portion of
the range of intakes. The former study requires a careful examination in terms
of statistical analysis and propagation of error, in addition to reproduction,
before this study can be used as evidence for an essential role for Cr in humans
(or female humans).
Cr concentrations in the human urine and blood serum are proportional to Cr
intake [32,33], while human urine Cr concentrations do not correlate with serum
glucose, insulin, or lipid parameters or with age or body mass [32]. Additionally, in
rats, Cr concentrations in the liver and kidney correlate with Cr intake [34]. Urinary
Cr loss is increased in type 2 diabetic subjects [35,36], raising the question of
whether the increased Cr loss could result in a conditional Cr deficiency; however,
studies with model diabetic rats (alloxan-treated rats [37] and Zucker diabetic fatty
rats [38]) have shown that the increases in urinary Cr excretion are the result of
increases in Cr absorption (perhaps simply as a result of increased water consump-
tion). Thus, urinary Cr loss is controlled by absorption of Cr, and Cr apparently is
not a conditionally essential element.
178 Vincent

An increase in urinary Cr excretion has been reported for human subjects on


self-selected diets in response to a glucose challenge, while no effect was observed
for individuals taking a Cr supplement (200 μg Cr as CrCl3 for 3 months) [33].
Urinary Cr loss after a glucose challenge was found not to be predictable and
suggested to not reflect Cr status [33]. Yet, the extent of movement of chromium to
the urine in response to a glucose challenge did change, from an increase at normal
Cr intake to no increase when supplemented with Cr (the inverse of the expected
observation). Also in this study, the Cr intake of the individuals in the study was not
established. The results from humans on self-selected diets are consistent with stud-
ies of urinary Cr loss in subjects on diets supplemented with a variety of varying
carbohydrates [39]. The greater the increase in the amount of insulin in the blood in
response to the various carbohydrates, the more Cr was lost in the urine [39]. Thus,
Cr appears to be mobilized in response to insulin, rather than directly to glucose or
other carbohydrates. A range of responses to the carbohydrates was noted. Some of
the subjects who in response to the diets had the highest circulating blood insulin
levels had decreased abilities to mobilize Cr for excretion in the urine (within 90 min);
thus, a group of subjects with decreased carbohydrate tolerance appeared to have
decreased urinary Cr loss [39]. The Cr content of the self-selected diets of individuals
in the study was not determined, and the subjects do not appear to have been questioned
about whether they were consuming any Cr-containing supplements [39,40].
Urinary Cr excretion after a glucose tolerance test does not differ between con-
trol men or hyperinsulinemic men or differ between men on diets with differing
high amylase cornstarch contents [41]. Eight of 10 healthy individuals have been
found to have increased urinary Cr loss (ng Cr/min) for 4 hours after an oral glucose
tolerance test compared to the 4 hours before the test such that the mean Cr loss was
significantly greater after the test than before, while no mean effect was observed
for 13 diabetic subjects [42]. Finally, Morris and coworkers conducting hyperinsu-
linemic euglycemic clamp studies have shown that increases in blood insulin levels,
not specifically blood glucose levels, are responsible for a decrease in plasma Cr
and an accompanying increase in urinary Cr loss [43], consistent with their earlier
studies demonstrating increased urinary Cr loss after an oral glucose challenge [44].
Thus, humans appear to increase urinary Cr loss in response to an increase in blood
insulin concentrations (whether from a carbohydrate or insulin challenge) although
the magnitude of the change appears to be quite variable, including some individu-
als who may not respond potentially as a result of decreased glucose tolerance.
This increase apparently results from the increased movement of Cr bound to transferrin
as noted above.
Rats have been conclusively shown to increase Cr excretion in response to an
insulin or glucose challenge [27,28,45]. If Cr were essential and had a role under
physiological conditions in insulin sensitivity, this increase in urinary Cr loss in
response to insulin could potentially serve as a biomarker for Cr. However, studies
on rats on the purified diets containing as low as possible to very high Cr contents
(described in ref. [21]) show that the increase in rate of urinary Cr loss does not
correlate with Cr intake, even at the lowest Cr content [46]. At the highest Cr intake
and thus highest background rate urinary Cr loss, insulin did not stimulate an
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 179

increase in rate of Cr loss [46]. These results are very similar to those described
above in humans [33]; thus, insulin-stimulated Cr loss is not a biomarker for Cr
status, and the movement of Cr in response to insulin does not provide evidence for
its being essential.
One cannot help but notice that Cr appears to be set up in terms of transport to
play a role in glucose metabolism. In the bloodstream, Cr binds tightly to one site of
transferrin. While transferrin is kept only 30% saturated with iron and has similar
binding constants for both Fe3+ binding sites, Cr3+ binds more rapidly and more
tightly to the site that Fe3+ binds to more slowly; thus, transferrins appear primed to
carry Cr in addition to Fe. As transferrin movement is insulin-sensitive, Cr bound to
transferrin is delivered to tissues in an insulin-sensitive fashion; this transport of Cr
is primarily to the skeletal muscle [27,28], where most glucose is metabolized in
response to insulin. Cr is then rapidly removed from these tissues.

2.2.3 Total Parenteral Nutrition

Starting in the late 1970s, studies of patients on total parenteral nutrition (TPN)
have been used to support the proposal that chromium is an essential element
[47–50]. This stems from patients on TPN who developed impaired glucose uti-
lization or glucose intolerance and neuropathy or encephalopathy [47,48,51–55].
The symptoms were reversed by chromium infusion and not by other treatments,
including insulin administration alone. While limited to less than ten individual
cases, these studies have been interpreted as providing evidence of clinical symp-
toms associated with chromium deficiency that can be reversed by supplementa-
tion. Another patient on TPN who developed symptoms of adult-onset diabetes and
hyperlipidemia but died had low tissue chromium levels [56]. Additionally, the
effects of chromium supplementation on five patients on TPN requiring a substan-
tial amount of exogenous insulin have been examined. Three subjects displayed no
beneficial response while two showed a possible beneficial response to chromium
supplementation [57]. Subjects received TPN containing 10 μg Cr/day followed by
supplementation with an additional 40 μg Cr/day for 3 days and then restoration of
the normal TPN.
Curiously the development of symptoms that were reversible by chromium sup-
plementation does not correlate with serum chromium levels [49], indicating that
either serum chromium levels are not an indicator of chromium deficiency or that
another factor is in operation. Additionally, these incidences of diagnosed potential
chromium deficiency have been questioned recently as they lack consistent relation-
ships between the chromium in the TPN, time on TPN before symptoms appear,
serum chromium levels and symptoms [58].
The most notable features of these studies are the quantities of Cr administered.
In the cases where apparent deficiencies were reported, the TPN solutions provided
2–240 μg Cr/day. For comparison, all the Cr in the TPN is introduced into the blood-
stream, while only 0.5% of Cr in the regular diet is absorbed into the bloodstream.
Thus, 30 μg of Cr in a typical daily diet presents only ~0.15 μg Cr to the bloodstream.
180 Vincent

The TPN solutions are consequently providing 13–67 times the required amount of
chromium; thus, based on these data, the TPN solutions cannot be considered
Cr-deficient. Subjects were, in turn, treated with 40–250 μg Cr/day added to the
TPN solution to alleviate their conditions, clearly pharmacological doses, as the
largest dose provided 1.7×103 times more chromium than a standard diet.
Consequently, the results with the insulin-resistant TPN patients can only be considered
as providing evidence for a pharmacological role of chromium. The data are not
relevant for examining whether chromium is an essential element.
Not surprisingly, as TPN provides ten or more micrograms of chromium per day,
TPN patients are accumulating chromium in their tissues [59,60]. Calls are appear-
ing for the re-examination of the chromium levels in TPN solutions in terms of a
need to reduce recommended levels [61].
In summary, evidence to designate chromium an essential element does not exist.
While the possibility always exists that evidence could surface in the future to sup-
port a biological role for chromium, such assumptions cannot be taken into current
considerations. The next review of the status of chromium by the Committee on the
Scientific Evaluation of Dietary Reference Intakes of the National Academies of
Science (USA) must seriously consider revising its status.

3 Is Chromium Pharmacologically Relevant?

3.1 Rodent Disease Model Studies

Several rat models of type 2 diabetes have been utilized to examine the effects of
Cr(III) administration [5]. Three models have symptoms arising from mutations of
the leptin receptor: the JCR:LA-cp, Zucker obese and Zucker diabetic fatty rats.
Leptin is a hormone produced by adipocytes that signals the brain that the appetite
should be suppressed. Consequently, as the leptin signaling system is blocked at the
receptor, the JCR:LA-cp and Zucker obese rats become markedly obese and insulin-
resistant and possess somewhat elevated blood glucose levels and elevated levels of
blood insulin, triglycerides, and cholesterols. The Zucker diabetic fatty (ZDF) rats
have an additional, uncharacterized mutation that results in these rats developing
symptoms very comparable to type 2 diabetes in humans, including elevated blood
glucose levels, in addition to the high triglycerides and cholesterol levels. In con-
trast to the obese models, the ZDF rats have smaller body masses than healthy
Zucker rats. Some general statements for studies of Cr(III) complex administration
using these three models can be made. When Cr is administered at a young age, it
has no effect on body mass and food intake [62–69]. Cr administration generally
appears to have no effect on fasting blood glucose levels but to lower glycated
hemoglobin levels. (This might be explained by the data of Vincent and coworkers
[66], which show that while glucose levels tend to be lower in Cr-treated animals at
several instances during the administration period, that this effect is not significant;
however, glycated hemoglobin levels, which serve as a window to the average
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 181

exposure of red blood cells to glucose over 60–90 days, reflect a beneficial effect on
blood glucose over this time.) Cr appears generally to be beneficial to lipid metabolism,
lowering total cholesterol levels; however, effects on other lipid variables are incon-
sistent. Thus, in these rat models of diabetes and obesity-related insulin resistance,
Cr appears to have beneficial effects on insulin resistance, marginally beneficial
effects on blood glucose, and beneficial effects on the grossly elevated plasma lipid
levels. Unfortunately, only a tiny percentage of human type 2 diabetes cases are the
result of mutations in leptin or its receptor.
The Goto–Kakizaki rat is a non-obese model of type 2 diabetes; the origins of the
diabetes at a molecular level are not known. Two studies have examined the effects
of [Cr(pic)3] (1–100 mg/kg daily) for either 4 or 32 weeks [70,71]. Unfortunately
the reports do not indicate whether the dose is of Cr as the compound or of the com-
pound (in which case ~12.5% of the dose would be Cr). No effects were observed
on body mass, fasting blood glucose or insulin levels, or glucose or insulin areas
under the curve in a glucose tolerance test. For this model, Cr(III) appears to have
no appreciable effect.
The chemical streptozotocin when administered intravenously or intraperitone-
ally relatively selectively kills the beta cells of the pancreas, destroying nearly all
the body’s ability to produce insulin. Thus, rats treated with the chemical serve as
an excellent model of type 1 diabetes (not type 2 diabetes). To generate a better
model for type 2 diabetes studies, the addition of a high-fat diet has been utilized in
addition to the chemical treatments or streptozotocin has been given to newborn
rats, rather than adults. Four studies have examined the effects of Cr supplementa-
tion on these type 2 models where they found lower fasting glucose, total choles-
terol, and triglycerides concentrations [72–75]. Studies using just streptozotocin
have given inconsistent results but are also very different in design from one another
making interpretation difficult [5].
In summary, the results of the studies with rats undergoing modified streptozoto-
cin treatments (lower fasting glucose but not insulin and effects on lipids) are differ-
ent from those of the Goto–Kazizaki rats (no effects) that are in turn different from
the results from the leptin-receptor mutation models (lower fasting insulin but not
glucose and effects on lipids). No great dependence appears on dose (when the
doses are supranutritional), length of time of Cr administration or form of Cr. The
origin of the diabetes appears to make a significant difference on the potential ben-
efits of Cr administration.
Mouse models of diabetes with mutations to the genes for leptin, the ob/ob
mouse, and leptin receptor, the db/db mouse, have also been studied in terms of
effects of Cr(III) administration. Both these models display obvious obesity.
Unfortunately, not all the studies have used well-defined forms of Cr. The results of
these studies have been conflicting in terms of fasting blood glucose and cholesterol
concentrations, although glucose and insulin levels in glucose tolerance tests con-
sistently tend to be lower [76–84] (reviewed in [5]).
Thus, with one exception, Cr(III) treatment of rat and mouse models of type 2
diabetes have had beneficial effects, although the effects differ from one model to
the other. These differences in the models may be significant to the results observed
in human clinical trials.
182 Vincent

3.2 Clinical Studies

While human studies of the effects of chromium supplementation have failed to


observe effects in healthy subjects, clinical trials of the effects of chromium supple-
ments on type 2 diabetic subjects have failed to generate consistent results. A recent
review that included only studies that were placebo-controlled and used a chemi-
cally well-defined form of Cr identified 19 studies that met the criteria [5]. (Not
including well-defined studies eliminates Cr sources such as “Cr-enhanced yeast”).
Nine of the 19 reports reported no effects from supplementation; another may or
may not have seen significant changes depending on how the statistical analysis is
performed. Studies using 150–1000 μg Cr daily for 6 weeks to 16 months have
reported no effects from Cr, while studies using 200–1000 μg for 10 days to 6
months reported beneficial effects. Studies using over 100 subjects, that should have
more power to distinguish potential differences, reported no effects in one case and
beneficial effects from supplementation in the others. Several studies are quite
small, lacking the statistical power to potentially observe effects.
Similarly, no pattern was identified in terms of beneficial effects on particular
symptoms from Cr supplementation. Fourteen studies examined fasting blood glu-
cose levels. Five reported that levels dropped with supplementation while nine
observed no effect. Four studies observed no effect on fasting insulin levels while
levels were lower in three studies. Triglyceride levels were unaffected in four stud-
ies and lower in two studies. Glycated hemoglobin levels were reported to be lower
in four studies, but no change was reported in five studies. Effects on cholesterol
levels were slightly more consistent. Seven studies reported no lowering of total
cholesterol while three noted decreases. For HDL, six studies reported no effect,
while a single study reported an increase in levels; for LDL, five studies reported no
effects, while only a single study reported a decrease. In response to some type of a
glucose challenge, four studies observed no effects on glucose levels while three
saw positive effects; in terms of insulin response, one study had mixed results
depending on the time interval that Cr was administered, while another reported
positive effects. Behavior of the blood variables across the studies was simply found
to be too inconsistent to draw any firm conclusions. This inconsistent behavior
existed whether these studies are broken down by the compound used, the amount
of Cr, the number of subjects, or the length of the study [5].
Two thorough meta-analyses of the effects of Cr supplements on type 2 diabetic
subjects have been reported. Althius et al. [85] in 2002 performed a meta-analysis
on studies under a contract from the Office of Dietary Supplements of the National
Institutes of Health (USA). Using their criterion for inclusion (trials containing a Cr
treatment group and a control), the authors identified only four studies of subjects
with type 2 diabetes for analysis. The combined data from the studies, except those
from a study by Anderson and coworkers [86], showed no effect from chromium on
glucose or insulin concentrations. Thus, they concluded that the data on diabetics
were inconclusive. The authors also examined the effects of Cr supplements on
healthy subjects or subjects with impaired glucose tolerance (but not type 2 diabetes)
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 183

in 14 trials including 425 subjects; no associations between Cr administration and


glucose or insulin concentrations were found.
Another meta-analysis was reported by Balk et al. in 2007 [87], the most thorough
meta-analysis on Cr supplementation in terms of blood variables reported to that
date. Forty-one randomized controlled trials were identified that examined the
effects of chromium supplementation on glucose metabolism and lipids concentra-
tions in ≥10 non-pregnant adults (i.e., healthy and diabetic subjects) for ≥3 weeks.
However, almost half were determined to be of poor quality. Nine studies were
funded by the food or supplement industry, 18 were by non-industry sources, and 14
did not indicate the funding source. Ten studies used Brewer’s yeast, 15 studies used
CrCl3, 5 studies used Cr nicotinate and 15 studies used [Cr(pic)3]; some studies
compared multiple sources of Cr. No benefit from Cr supplementation was identi-
fied for healthy individuals [87]. Eighteen studies were identified that examined
type 2 diabetic subjects. Cr supplementation was found to statistically improve gly-
cemic control in type 2 diabetics. The effects were fairly small but significant over-
all. When broken down by Cr source, the effects were small but significant for
subjects on yeast and [Cr(pic)3] but not CrCl3. Most significantly, the authors
determined the results were not definitive because of the poor quality and heteroge-
neity of the studies. Overall Cr did not affect lipid levels, while [Cr(pic)3] lowered
glycated hemoglobin levels. However, lower glycated hemoglobin levels were only
observed in 3 interventions out of 14, two of which came from a single, large study
(that of Anderson and coworkers [86]). Amongst fasting glucose studies, a trend was
observed that industry-sponsored studies were more likely to observe beneficial
effects. The authors also expressed concerns that the Brewer’s yeast results sug-
gested that another component in the yeast may be having an effect because effects
were observed at lower doses of Cr. As a bottom line the authors concluded that Cr
supplementation ‘may have a modest effect’ on glucose metabolism in type 2 dia-
betics but that ‘the large heterogeneity and the overall poor quality limit the strength
of our conclusions’ and that more randomized trials are required [87]. The study
was supported by a contract from the Agency for Healthcare Research and Quality
(US Department of Health and Human Services).
Three studies meeting the appropriate criteria have appeared since the Balk et al.
meta-analysis. These are a small study by Lai [88] with Cr yeast with a 10 subject
treatment and 10 subject control that observed small effects on plasma glucose,
insulin, and glycated hemoglobin; a study with Cr yeast utilizing 57 subjects by
Kleefstra et al. [89] that observed no effects; and a study by Cefalu et al. [90] with
93 subjects that observed no effects with 1000 μg Cr daily as [Cr(pic)3]. These studies,
because of the participant size of the last two, would have significantly affected
the results of the meta-analysis if they could have been included, making any effect
of Cr on fasting glucose in type 2 diabetics even more questionable. One must
also note that any meta-analysis is likely to be biased toward the positive as studies
with negative results tend to be published less frequently than positive reports.
Basically, the results come down to the following: (i) clinical trials on Cr(III) complex
supplementation for healthy subjects observe no effects from treatment, (ii) clinical
studies on Cr(III) complex supplementation are equivocal for type 2 diabetic
184 Vincent

subjects, and (iii) the results of the trials with diabetic subjects are basically only
considered equivocal, rather than without observable effect, because of the results
of the single large, well-designed study by Anderson and coworkers [86]. This study
is unique in being the only study using subjects from China and needs to be
independently repeated.
In a review in 1998, Anderson [91] split studies on Cr supplementation of type 2
diabetics into two groups: subjects receiving ≤200 μg Cr daily and subjects receiv-
ing >200 μg Cr daily. Using all the studies identified with diabetic subjects to that
date, Anderson suggested that >200 μg Cr were required for diabetic subjects to
generate an observable effect. The effect appeared to be largest for [Cr(pic)3] where
this apparent effect was the result of only the single study by Anderson and cowork-
ers [86]). Subsequently, this requirement has commonly been cited. However, stud-
ies since 1998 have failed to follow the trend identified by Anderson.
Cefalu and coworkers [90,92] in a preliminary and then in a subsequent report
potentially may have found a relationship that might explain the different results
between populations in the various studies. In a double-blind, placebo-controlled
study, 93 subjects with a fasting plasma glucose level of at least 6.94 mmol L–1
received 1000 μg Cr daily as [Cr(pic)3] or placebo for 24 weeks [90]. Comparison
of the treatment and control groups found no effects on body mass, percentage body
fat, free fat mass, or abdominal fat deposits, fasting glucose, glycated hemoglobin,
or insulin sensitivity. Yet, effects were observed when the Cr-receiving subjects at
the end of the study were divided into responders (≥10% increase in insulin sensi-
tivity from baseline) and non-responders. At baseline, responders had lower insulin
sensitivity and higher fasting glucose and glycated hemoglobin levels than non-
responders. Thus, Cefalu and coworkers might potentially have identified predictors
for type 2 diabetic subjects that might preferentially respond to Cr treatment. These
results will need to be carefully tested in additional studies where the ‘responder’
group is identified before the Cr administration to establish whether a subsequent
difference is actually manifested.
According to the American Diabetes Association in its 2010 Clinical Practices
Recommendations, ‘Benefit from chromium supplementation in people with diabetes
or obesity has not been conclusively demonstrated and therefore cannot be recom-
mended’ [93]. The American Diabetes Association dropped any mention of chro-
mium in its 2011, 2012, and 2013 recommendations.
In December 2003, Nutrition 21, the major supplier of chromium picolinate,
petitioned the United States Food and Drug Administration (FDA) for eight qualified
health claims:
1. Chromium picolinate may reduce the risk of insulin resistance.
2. Chromium picolinate may reduce the risk of cardiovascular disease when caused
by insulin resistance.
3. Chromium picolinate may reduce abnormally elevated blood sugar levels.
4. Chromium picolinate may reduce the risk of cardiovascular disease when caused
by abnormally elevated blood sugar levels.
5. Chromium picolinate may reduce the risk of type 2 diabetes.
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 185

6. Chromium picolinate may reduce the risk of cardiovascular disease when caused
by type 2 diabetes.
7. Chromium picolinate may reduce the risk of retinopathy when caused by
abnormally high blood sugar levels.
8. Chromium picolinate may reduce the risk of kidney disease when caused by
abnormally high blood sugar levels [94].
After extensive review, the FDA issued a letter of enforcement discretion allow-
ing only one (No. 5) qualified health claim for the labeling of dietary supplements
[94,95]: ‘One small study suggests that chromium picolinate may reduce the risk of
type 2 diabetes. FDA concludes that the existence of such a relationship between
chromium picolinate and either insulin resistance or type 2 diabetes is highly uncer-
tain.’ The small study was performed by Cefalu et al. [96]. This study was a placebo-
controlled, double-blind trial examining 1000 μg/day of Cr as [Cr(pic)3] on 29 obese
subjects with a family history of type 2 diabetes; while no effects of the supplement
were found on body mass or body fat composition or distribution, a significant
increase in insulin sensitivity was observed after four and eight months of
supplementation.
This raises the question of why the discrepancy between human and rodent stud-
ies exists. Rodent studies observing beneficial effects generally provided rats
between 80 and 1000 μg Cr/kg body mass daily. Based on mass, this would corre-
spond to 5.2 to 65 mg Cr daily for an average 65 kg human. Even when corrected
for the increased metabolic rate of rats compared to humans, this range corresponds
to ~1 to 13 mg of Cr daily. Thus, human clinical trials may have only started to
approach the dose necessary to see a beneficial effect in humans. The amount of Cr
used in clinical trials needs to be increased before ruling out that Cr has no effect on
type 2 diabetic subjects. However, one cannot rule out that something is unique
about rodents that allows Cr to have beneficial effects. Unfortunately, studies of Cr
supplementation on farm animals are also equivocal and often use doses in propor-
tion to body mass even smaller than those used in human clinical trials [5,97].
Recently, Vincent [5] has proposed that in order to definitely determine whether
Cr supplementation has an effect on diabetics, human clinical trials should:
(1) be performed with sufficient power to be able to realistically observe effects, on
subjects whose baseline characteristics are well established, and for periods of
time of at least 4–6 months. Knowing baseline characteristics is particularly
important, given the possibility at the current dosages that only subjects with
the highest degrees of insulin resistance may be responsive to Cr.
(2) be performed with larger doses of Cr(III). Studies using JCR:LA-cp or ZDF
rats utilized 80–1000 μg Cr/kg daily corresponding to approximately 5.2–65
mg daily for a human (based on body mass). If corrected for the increased
metabolic rate of rats, this still correspond to ~1–13 mg daily. Studies are
needed using 5–7 mg Cr(III) daily for 4–6 months or longer.
(3) be carefully monitored for any deleterious effects, especially when using the
higher doses of Cr(III).
186 Vincent

3.3 Proposed Mechanisms of Action

3.3.1 Insulin Signaling

When many bioinorganic chemists or nutritionists think of a biological form of


chromium, glucose tolerance factor (or GTF) may be their first thought. As has been
reviewed many times recently [5,25,98], the studies postulating the existence of
GTF are flawed, and the material isolated from Brewer’s yeast and also called GTF
is an artifact of its isolation. The term GTF should be removed from the lexicon of
the chemistry and nutrition communities. What then can be said about the action of
chromium at a molecular level?
Given that Cr(III) appears to have pharmacological effects in increasing insulin
sensitivity and altering lipid metabolism in rodent diabetes models, Cr must interact
directly with some biomolecules(s) to generate these effects. To begin to elucidate
how Cr can affect insulin sensitivity at a molecular level, the effects of Cr on cultured
mammalian cells have been probed. However, research results are contradictory
such that the state of the field is not immediately clear (reviewed in [98]). Using the
lesson learned from toxicology studies of [Cr(pic)3] (see Section 4.2), some of the
discrepancies might be explained based on the stability of the Cr(III) complexes and
what form of Cr(III) is actually being presented to the cells (and whether this form
is biologically relevant); yet, this does not aid in elucidating the site of action of Cr.
Most of these studies have used adipocytes (or preadipocytes) or skeletal muscles,
cells known to incorporate Fe via endocytosis of transferrin. These cells should,
thus, intake Cr via transferrin endocytosis. Given that Cr-loaded transferrin can be
readily prepared, the physiologically relevant form of Cr, i.e., Cr transferrin, should
be used in cell culture studies to deliver Cr to the cells.
One result is nearly uniform across cell culture studies utilizing skeletal muscle,
adipocytes, or adipocyte-like cells – Cr enhances glucose uptake and metabolism in
a fashion dependent on insulin (see, for example, [99]). Numerous pathways by
which a Cr biomolecule could manifest itself in these effects have been proposed.
However, research results in in vitro and in vivo systems are contradictory, such that
the state of the field is not immediately clear (Table 1). Attention has been focused
on two sites of action in the insulin signaling cascade as the potential sites of Cr
action, insulin receptor (IR) and Akt.
The most thorough studies observing increased IR signaling from Cr(III) treat-
ment were reported by Brautigan and coworkers [100]. Preincubation of Chinese
hamster ovary (CHO) cells overexpressing IR with [Cr(pic)3], Cr histidine (actually
a complex mixture of numerous Cr-histidine complexes), or [Cr3O(propionate)6
(H2O)3]+ (Cr3) activated IR tyrosine kinase activity in the cells at low doses of insu-
lin. While the concentration dependence was only examined for Cr histidine, the
effect was concentration-dependent. Neither insulin binding to the cells nor IR
number was affected. Additionally, the addition of Cr did not inhibit dephosphoryla-
tion of the IR by endogenous phosphatases or added PTP1B (phosphotyrosine phos-
phatase 1B). Also, Cr apparently did not alter redox regulation of PTP1B (i.e., by trapping
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 187

Table 1 Selected studies of effects of chromium administration on insulin signaling pathway.a


Cell or organism Chromium compound Effect Refs.
Skeletal muscle CrCl3, [Cr(pic)3], Up-regulation of insulin receptor [152]
Cr peptide complexes mRNA levels
Insulin-resistant [Cr(pic)3] No effect on insulin receptor [120]
3T3-L1 and Akt mRNA levels
adipocytes
Chinese hamster [Cr(pic)3], Cr3, Activated IR kinase activity [100]
ovary cells Cr histidine
JCR:LA rat [Cr(pic)3] Increased insulin receptor, IRS-1, [63]
and Akt phosphorylation
and increased PI3K activity
3T3-L1 Cr(D-phe)3 Increased phosphorylation of Akt [153]
adipocytes but not insulin receptor
3T3-L1 [Cr(pic)3] No effect on phosphorylation of [113,115]
adipocytes insulin receptor, IRS-1, or Akt
KK/HIJ mice Milk powder enriched Increased IRS-1 tyrosine phospho- [154]
skeletal with trivalent Cr rylation, increased Akt activity,
muscle and decreased IRS-1 serine-307
phosphorylation
3T3-L1 Cr histidine Increased insulin-stimulated glucose [101]
adipocytes uptake and insulin-stimulated
tyrosine phosphorylation of IR
C2C12 skeletal Cr oligo-mannuronate Enhanced phosphorylation of IR, [155]
muscle cells PI3K, and Akt and AMPK
a
Table adapted from [5].

the oxidized inactive form or by preventing its reduction and reactivation). CrCl3
and Cr histidine were found not to activate the kinase activity of a recombinant frag-
ment of IR. The authors concluded that Cr inside the cell modified the receptor in
some manner, activating its kinase activity [100]. Subsequently, Brautigan, et al.
[101] demonstrated that Cr histidine stimulated tyrosine phosphorylation of IR in
3T3-L1 adipocytes in the presence of insulin but not of MAPK (mitogen-activated
protein kinase) or 4E-BP1, markers for activation of transcription and translation,
respectively, in the presence of insulin; glucose uptake in the presence of insulin
was also stimulated by Cr histidine. The effects of Cr histidine were also examined
in competition with those of vanadate [101]; the results were interpreted in terms of
Cr having an action involving IR activation and potentially in another action beyond
IR activation that increases GLUT4 transport.
Sreejayan and coworkers [81] using Cr(D-phenylalaninate)3 (Cr(D-phe)3) have
generated evidence for an association between Cr and Akt. Cr(D-phe)3 (5 or 25 μM
for 10 days) was found to increase insulin-stimulated glucose uptake by cultured
mouse 3T3-adipocytes. Treatment of the cells with 5 μM Cr for 0.5 to 4 hours or 0.1
to 100 μM Cr for 2 hours did not increase insulin-stimulated phosphorylation of IR
(Tyr1146) significantly, while under similar conditions insulin-stimulated Akt phos-
phorylation (Thr308) was increased significantly.
188 Vincent

To reconcile the heterogeneous results in the studies with cultured cells (Table 1),
the complexes need to be studied under uniform conditions – the same cells treated
in the same manner for the same period of time with the same Cr complex at the
same concentrations. Additionally the Cr compounds need to be examined over a
range of concentrations over varying periods of time with each of the cell types. The
stability of the Cr complexes in the culture media needs to be established. Only in
this manner will the actual Cr species in contact with the cells be established.
Similarly, the distribution, concentration, and form of the Cr in the cells needs to be
determined. Control experiments using just the ligands need to be performed to
determine if any effects arise from just the ligands. Without this type of comprehen-
sive treatment, progress in interpreting the body of cell culture experiments is going
to be difficult if not impossible as has already been found in toxicology studies (see
Section 4.2). Studies would probably be best performed if Cr-transferrin, the form
of Cr by which the metal is delivered to cells, were utilized.
One specific biomolecule has been proposed as the biologically active chromium-
binding molecule. This is the only biomolecule other than transferrin known to bind
Cr in vivo, low-molecular-weight Cr-binding substance (LMWCr or chromodulin).
This molecule occurs in the tissue, the bloodstream, and the urine and appears to
bind Cr in the tissues for its elimination from the body via the urine. The history of
studies of this molecule has been exhaustively reviewed [5,102] and is beyond the
coverage of this review. The inability of the organic portion of this Cr-peptide com-
plex to be characterized generated significant controversy, as the situation bore
similarity to the previous inability to characterize the organic component of GTF
[103]. Another important concern is that a Cr-loading procedure is necessary in the
purification of LMWCr, so that the peptide could be followed (by its Cr content)
through the isolation procedure; thus, the animal providing the tissue or body fluid
is usually administered a Cr(III) or Cr(VI) source or such a source is added to the
tissue homogenate or fluid [5,102]. Rupture of CrO42 −-treated mammalian cultured
cells resulted in Cr being bound to a low-molecular-weight species with spectro-
scopic properties similar to LMWCr [104]. This was interpreted in terms of LMWCr
being an artifact generated during isolation; however, the unnatural method of pre-
senting CrO42 − in high concentration to cultured cells also suffers from the types of
problems discussed above when using cultured cells. Thus, this study only shows
that apoLMWCr can potentially bind Cr in a cell extract and potentially bind Cr
tight enough to remove it from other biomolecules, consistent with the results of the
isolation procedures of LMWCr described above. The Cr environment of LMWCr
has been characterized by a variety of techniques including paramagnetic NMR,
EPR, X-ray absorbance, and variable temperature magnetic susceptibility [105,106].
The peptide component has recently been sequenced by mass spectrometry [107];
the sequence begins with four glutamate residues whose cyclizing blocked attempts
at Edman degradation sequencing. The peptide binds four chromic ions with identi-
cal binding constants and cooperativity as apoLMWCr (within experimental error)
[107]. LMWCr has been found to stimulate insulin-dependent glucose incorpora-
tion and metabolism in isolated rat adipocytes [99,104] and in vitro to stimulate
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 189

(or perhaps retard the deactivation of) the kinase activity of the insulin-activated
insulin receptor [108,109].
A mechanism for LMWCr in amplifying insulin signaling has been proposed
[110,111]. This proposal was put forward when Cr was thought to be essential; the
mechanism needs to be altered, so that it would be in vogue under conditions of Cr
supplementation, so that abnormally high concentrations of holoLMWCr are gener-
ated. In this mechanism, apoLMWCr is stored in insulin-sensitive cells. Responses
to increases in blood insulin concentrations result in activation of the insulin-
signaling cascade: insulin binds to its receptor bringing about a conformational
change that results in the autophosphorylation of tyrosine residues on the internal
side of the receptor, transforming the receptor into an active tyrosine kinase and
transmitting the signal from insulin into the cell. In response to this signaling, trans-
ferrin moves from the bloodstream into cells, carrying in part Cr3þ into the cells.
The Cr flux results in loading of LMWCr with Cr. The holoLMWCr then binds to
the insulin receptor, presumably assisting to maintain the receptor in its active
conformation and amplifying insulin signaling. This mechanism requires demon-
stration that it can (or cannot be) active in vivo to verify (or refute); clear demonstra-
tion that the IR is directly involved in increasing insulin sensitivity by Cr would
support this mechanism. As Cr is probably not an essential element, LMWCr could
be part of a Cr detoxification system as suggested by Yamamoto, Wada, and Ono
[112]; Cr supplementation, which leads to increased Cr concentrations in the body,
could lead to increased concentrations of holoLMWCr, capable in turn of affecting
insulin signaling. Studies need to determine the origin of LMWCr, i.e., what protein
is it made from and what enzymes are involved? Is the holoLMWCr biologically
active at physiological levels (suggesting a potential biological role for Cr) or is it
significantly active only when Cr concentrations are high? Does LMWCr interact
with the IR in vivo, or does it manifest its effects elsewhere?

3.3.2 Cholesterol and Fatty Acid Metabolism

Elmendorf and coworkers have examined the effects of CrCl3 and [Cr(pic)3] on 3T3-
L1 adipocytes [113–117] (however see [118,119]). In their first report [113], CrCl3
and [Cr(pic)3] were shown to increase GLUT4 transport to the plasma membrane in
the presence of insulin. Cr treatment did not affect IR, insulin receptor substrate-1
(IRS-1), PI3K, or Akt regulation but decreased plasma membrane cholesterol.
Subsequently, the effects of [Cr(pic)3] were shown to be dependent on the glucose
concentration of the media with the effects being observed at 25 mM, but not 5.5
mM [114]. [Cr(pic)3] activated AMPK (AMP-activated protein kinase) and improved
defects in cholesterol transporter ABCA1 trafficking and cholesterol accrual in the
high glucose treated cells [117].These researchers have postulated that Cr mani-
fested its effects via affecting the cholesterol homeostasis and the membrane fluidity
[113–117]. Yao and coworkers [120,121] determined that [Cr(pic)3] increased
glucose uptake and metabolism and GLUT4 transport in 3T3-L1 adipocytes; the effects
190 Vincent

were independent of insulin. Cr (60 nM) had no effect on IR or Akt phosphorylation


but was found to activate MAPK independent of its effect on GLUT4 translocation.
They also looked at the effects of Cr at both 25 and 5.5 mM glucose in their studies
described above; similar results were observed at both glucose concentrations in
contrast to Elmendorf and coworkers.
The use of exclusively [Cr(pic)3] in some of the studies examining membrane
properties generates some questions that may be related to differing results between
cell studies. While not particularly lipophilic, despite being neutral in charge [122],
the compound still appears to be able to partition to a significant degree to cell mem-
branes. This membrane incorporation of [Cr(pic)3] results, for example, in increased
membrane permeability [123]. Thus, some of the observations related to cholesterol
homeostasis may be specifically related to the use of [Cr(pic)3], its lipophilicity, and
its stability in cell culture media. Notable in this regard is a recent report showing
that [Cr(pic)3] associates with the lipid interface in reverse micelle model mem-
branes and that a similar association could explain the increased association of the
insulin receptor, phosphorylated IRS-1, and phosphorylated Akt in detergent-resistant
membrane microdomains [124].

3.3.3 Inflammation and Oxidative Stress

Jain and Kannan have shown that monocytes exposed to high glucose concentra-
tions have lower levels of the cytokine TNF-α (tumor necrosis factor-α) in the pres-
ence of 100 μM CrCl3 for 24 hours at 37°C [125]. Treatment with CrCl3 also
inhibited stimulation of TNF-α secretion in these cells by 50 μM H2O2. Lipid per-
oxidation and protein oxidation in the presence of H2O2 was also inhibited by CrCl3.
As increased TNF-α secretion may be associated with insulin resistance, Jain has
proposed in an interview that increased insulin sensitivity arising from Cr adminis-
tration may be mediated by lowering of TNF-α levels [126]. In a follow-up study,
CrCl3 in combination with estrogen lowered lipid peroxidation in high glucose-
treated monocytes [127]. The combination was also found to decrease interleukin-6
(IL-6) secretion. Cr was proposed to potentiate the effects of estrogen [127].
Curiously, another group has shown that Cr(III) treatment (350–500 ppm) results in
increased TNF-α production by macrophages (in the absence of high glucose con-
centrations) [128]. This activation by chromium (CrCl3) may be regulated by tyro-
sine kinases [129]. The results in the presence of high glucose could also point to an
association between reactive oxygen species and chromium, but these studies must
be considered extremely preliminary. Additionally, the fate of Cr in these cell cul-
ture studies needs to be examined. Subsequent studies in Zucker diabetic fatty [65]
and streptozotocin-induced diabetic rats [130] have found that Cr(III) administra-
tion can lower blood levels of TNF-α, IL-6, and C-reactive protein, although differ-
ences appeared to be observed depending on choice of Cr(III) complex administered.
Cr(III) administration has also been reported to lower blood levels of TNF-α in a
clinical trial of type 2 diabetic subjects, although again differences appeared to be
observed depending on choice of Cr(III) complex administered [131].
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 191

4 Is Chromium Toxic?

4.1 Chromate

Lay and coworkers [132,133] have proposed that chromate generated enzymatically
(i.e., from hydrogen peroxide or other species generated by enzymes) from Cr(III)
in the body could act as a phosphotyrosine phosphatase (PTP) inhibitor, in a similar
manner to vanadate, and that the site of action of Cr is at the PTPs. The proposal that
chromate could be involved in chromium action in vivo is based on the ability of
hydrogen peroxide to oxidize Cr(III) compounds to chromate, suggesting the appar-
ent beneficial effects of Cr actually stem from side effects of its toxicity [133]. To
demonstrate this, Lay and coworkers exposed chromium picolinate, CrCl3 and the
basic chromium carboxylate cation Cr3 to 0.10–1 mM hydrogen peroxide for 1–6 h
in 0.10 M HEPES buffer at pH 7.4. This resulted in the formation of chromate in
efficiencies of from 1% ([Cr(pic)3] for 6 h with 1 mM H2O2) to 33% (the cation for
6 h with 1 mM H2O2). The cation could also be oxidized with hypochloride or glu-
cose oxidase or xanthine oxidase (enzymes that produce H2O2). However, when one
considers the amount of Cr humans consume from their diet and from nutritional
supplements and the low % absorption and that cell concentrations of peroxide are
10–7 to 10–8 M while numerous reductants (such as ~5 mM ascorbate) are present,
the probability that cell concentrations of chromate could even approach the Ki of
chromate for phosphatases is negligibly small [5]. Similarly, toxicity from chromate
at these concentrations is unlikely. Given the enormous doses of Cr(III) complexes
shown to have no detrimental effects (see Section 4.2), this proposed mechanism of
toxicity from chromate generated from Cr(III) sources can be ignored.

4.2 Chromium Picolinate and Other Cr(III) Complexes

The potential toxicity of Cr picolinate, [Cr(pic)3], the most popular form of Cr sup-
plement over the last two decades, has been an area of intense debate, but consensus
has probably recently been reached (for recent reviews see [5,134,135]). In mam-
malian cell culture studies and mammalian studies in which the complex is given
intravenously [5,134], [Cr(pic)3] is clearly toxic and mutagenic, unlike other
commercial forms of Cr(III) supplements. The first study to raise concerns about
potential toxic effects, by Stearns and coworkers [136], demonstrated, using CHO
cells, that [Cr(pic)3] as a solid suspension in acetone or the mother liquor from the
synthesis of [Cr(pic)3] (before the compound precipitates from solution) caused
chromosomal aberrations. Subsequent studies have shown that the complex gives
rise to a variety of types of oxidative damage and is clastogenic [137–143]. This led,
for example, in fruit flies (Drosophila) to dominant female sterility, appreciable
delays in development of larvae and adults, and lower success rates in pupation
and eclosion; the Cr dosage in these studies was approximately equivalent to a
192 Vincent

human consuming one 200 μg Cr-containing supplement a day [144]. The ability of
[Cr(pic)3] to generate chromosomal aberrations in polytene chromosomes of the
salivary glands of Drosophila larvae was also examined; in the [Cr(pic)3]-treated
group, 53% of the identified chromosomal arms were positively identified as con-
taining one or more aberrations, while no aberrations were observed for the identi-
fied chromosomal arms of the control group [145]. No effects on Drosophila were
observed for other Cr(III) compounds examined [144,145]. However, when given
orally to mammals, [Cr(pic)3] does not appear to be toxic nor appear to be a mutagen
or carcinogen.
An NIH-commissioned study of the effects of up to 5% of the diet (by mass) of
male and female rats and mice for up to 2 years found no harmful effects on female
rats or mice or male mice and at most ambiguous data for one type of carcinogenic-
ity in male rats (along with no changes in body mass in either sex of rats or mice)
[146]. Despite numerous claims that [Cr(pic)3] is absorbed better than inorganic
forms of Cr used to model dietary Cr, CrCl3, Cr nicotinate (the second most popu-
lar form of Cr sold as a nutritional supplement), and [Cr(pic)3] are absorbed to a
similar degree in rats [24,147,148]. Only 1% of absorbed Cr from the supplement
is found in the bloodstream as [Cr(pic)3], suggesting that little of the intact mole-
cule is absorbed [149]. When ingested, the complex probably hydrolyzes near the
stomach lining, releasing the Cr, which is subsequently absorbed. The picolinate
ligands also alter the redox properties of the Cr center such that it is more suscep-
tible to undergoing redox chemistry in the body than hexaaqua Cr(III) [150,151].
The hydrolysis of the complex is probably fortuitous, releasing the Cr before the
intact [Cr(pic)3] complex can be absorbed to an appreciable level and potentially
enter into redox chemistry, in contrast to the cell studies where the very stable,
neutral complex could be absorbed intact. The message of these conflicting results
is that applying solutions of Cr(III) compounds to cultured cells in general does
not present Cr(III) to the cells in a comparable fashion to that in which Cr(III)
is presented to cells in the body; the difference may be crucial to the results and
interpretation of the study.
In summary, Cr(III) supplementation appears to be safe at levels currently used
in nutritional supplements and in pharmacology studies, in line with assessments by
the Food and Drug Administration (USA) and European Food Safety Authority.
However, as no benefit has been demonstrated for Cr supplementation of healthy
individuals, any potential risk from supplementation would appear to outweigh
potential benefits at the current time.

5 Concluding Remarks and Future Direction

At present Cr cannot be considered as an essential element as (i) nutritional data


demonstrating Cr deficiency and improvement in symptoms from Cr supplementa-
tion are lacking and (ii) no biomolecules have convincingly been demonstrated
to bind Cr and have an essential function in the body. No beneficial effects have
6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 193

convincingly been demonstrated from Cr supplementation by healthy humans.


Cr(III) supplementation appears to be safe at levels currently used in nutritional
supplements and in pharmacology studies. While studies with rodent models repro-
ducibly demonstrate beneficial effects from Cr supplementation at pharmacological
doses, the scientific literature for clinical trials in diabetic humans lacks consistent
and reproducible outcomes.
Future clinical studies need to be more carefully designed including the utiliza-
tion of an appropriate number of subjects and appropriate amount of administered
Cr, the use of well characterized Cr(III) compounds, and the examination of whether
particular subgroups of type 2 diabetic subjects are likely to benefit from chromium
supplementation. Further studies are required to investigate the mechanism and
mode of action of Cr(III) at the molecular level in enhancing insulin sensitivity and
potentially improving cholesterol metabolism.

Abbreviations and Definitions

AI adequate intake
AMPK AMP-activated protein kinase
CHO Chinese hamster ovary
Cr3 [Cr3O(propionate)6(H2O)3]+
Cr(D-phe)3 Cr(D-phenylalaninate)3
[Cr(pic)3] chromium picolinate
4E-BP1 4E-binding protein-1
ESADDI estimated safe and adequate daily dietary intake
FDA Food and Drug Administration
FEEDAP Panel on Additives and Products or Substances Used in Animal Feed
FTC Federal Trade Commission
GLUT4 glucose transporter type 4
GTF glucose tolerance factor
HDL high density lipoprotein
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
IL-6 interleukin-6
IR insulin receptor
IRS-1 insulin receptor substrate-1
LDL low density lipoprotein
LMWCr low-molecular-weight chromium-binding substance
MAPK mitogen-activated protein kinase
PI3K phosphatidylinositol 3-kinase
PTP phosphotyrosine phosphatase
PTP1B phosphotyrosine phosphatase 1B
TNF-α tumor necrosis factor-α
TPN total parenteral nutrition
ZDF Zucker diabetic fatty (rats)
194 Vincent

Acknowledgment The author wishes to thank the USDA for supporting his recent research on
the nutritional biochemistry of chromium (National Research Initiative Grant 2009-35200-05200
from the USDA Cooperative State, Research, Educational, and Extension Service).

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Chapter 7
Manganese in Health and Disease

Daiana Silva Avila, Robson Luiz Puntel, and Michael Aschner

Contents
ABSTRACT ............................................................................................................................. 200
1 INTRODUCTION ............................................................................................................. 200
1.1 Manganese Essentiality ............................................................................................. 201
1.2 Manganese Pharmacokinetics ................................................................................... 202
1.3 Manganese Biochemistry and Physiology ................................................................ 204
2 MANGANESE TRANSPORT .......................................................................................... 206
2.1 Manganese Uptake in Relation to Oxidative State.................................................... 207
2.2 Cellular Manganese Uptake ...................................................................................... 208
2.3 Cellular Manganese Efflux........................................................................................ 209
3 MANGANISM. A NEURODEGENERATIVE DISEASE ............................................... 210
4 SYMPTOMS AND SENSITIVE POPULATIONS ........................................................... 211
5 MANGANISM VERSUS PARKINSON’S DISEASE ....................................................... 211
6 MANGANESE IN THE ETIOLOGY OF OTHER NEURODEGENERATIVE
DISORDERS ..................................................................................................................... 212
6.1 Manganese and Amyotrophic Lateral Sclerosis........................................................ 212
6.2 Manganese and Alzheimer’s Disease ........................................................................ 213
6.3 Manganese and Huntington’s Disease ...................................................................... 213
7 MOLECULAR MECHANISMS OF TOXICITY ............................................................. 214
7.1 Dopamine Oxidation ................................................................................................. 214
7.2 Mitochondrial Dysfunction ....................................................................................... 215
7.3 Astrocytosis ............................................................................................................... 215
8 GENETIC SUSCEPTIBILITY .......................................................................................... 216
9 TREATMENT .................................................................................................................... 217

D.S. Avila • R.L. Puntel


Biochemistry Graduation Program, Universidade Federal do Pampa, Uruguaiana,
Rio Grande do Sul, Brazil
e-mail: avilads1@gmail.com; robsonunipampa@gmail.com
M. Aschner (*)
Department of Pediatrics and Pharmacology, The Kennedy Center for Research
on Human Development and The Molecular Toxicology Center, Nashville, TN 37232, USA
e-mail: michael.aschner@vanderbilt.edu

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 199
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_7, © Springer Science+Business Media Dordrecht 2013
200 Avila, Puntel, and Aschner

10 GENERAL CONCLUSIONS .......................................................................................... 218


ABBREVIATIONS .................................................................................................................. 219
ACKNOWLEDGMENTS........................................................................................................ 220
REFERENCES ........................................................................................................................ 220

Abstract Manganese is an important metal for human health, being absolutely


necessary for development, metabolism, and the antioxidant system. Nevertheless,
excessive exposure or intake may lead to a condition known as manganism, a neuro-
degenerative disorder that causes dopaminergic neuronal death and parkinsonian-like
symptoms. Hence, Mn has a paradoxal effect in animals, a Janus-faced metal.
Extensive work has been carried out to understand Mn-induced neurotoxicity and to
find an effective treatment. This review focuses on the requirement for Mn in human
health as well as the diseases associated with excessive exposure to this metal.

Keywords dopamine • essentiality • manganese • manganese enzymes • manganism


• mitochondria • neurodegenerative diseases • parkinson’s disease-related genes
• treatment

Please cite as: Met. Ions Life Sci. 13 (2013) 199–227

1 Introduction

Manganese, a group 7 metal in the periodic table, is the twelfth most abundant element
in the earth’s crust. It exists in a number of chemical and physical forms in the
atmosphere’s particulate matter and in water [1]. Mn does not occur naturally in a
pure state, and is found as both inorganic and organic compounds, the inorganic
form being the most common. Because the Mn outer electron shell can donate up to
7 electrons, it can occur in 11 different oxidation states, varying from −3 to +7 [2].
In living tissue, Mn has been found as Mn2+, Mn3+, and possibly as Mn4+, while
Mn5+, Mn6+, Mn7+, and other complexes of Mn at lower oxidation states, are not
observed in biological materials [3,4].
The versatile chemical properties of Mn have enabled its industrial usage in iron
and steel production, manufacture of dry cell batteries, production of potassium
permanganate and other chemicals, as oxidant in the production of hydroquinone,
manufacture of glass and ceramics, textile bleaching, as an oxidizing agent for
electrode coating in welding rods, adhesives, paint, matches and fireworks, and tan-
ning of leather. Organic compounds of Mn are also present in fuel additive, methyl-
cyclopentadienyl manganese tricarbonyl (MMT) as well as in several fungicides.
Moreover, considering that Mn is a paramagnetic metal, namely that it has unpaired
electrons in its outer d shell, it can also be detected with magnetic resonance
imaging (MRI), positron emission tomography (PET), and single-photon emission
computed tomography (SPECT) [1,5]. These techniques allow for the tracking of
Mn dynamics repeatedly in the same subject in vivo [1,6]. Mn can also interact with
7 Manganese in Health and Disease 201

fluorophore fura-2, by quenching and increasing its fluorescence, representing a


new methodological approach for in vitro kinetic studies. Thus, given its ubiquitous
nature and widespread use in both industrial and non-industrial processes, several
health organizations have expressed concern about the potential health effects of
occupational/environmental Mn exposure.
Mn is an essential element for humans, animals, and plants; it is required for
growth, development, and maintenance of health. Routes of Mn exposure are mainly
through dietary intake, dermal absorption, and inhalation. Accordingly, the primary
source of Mn intoxication in humans is due to occupational exposure as in miners,
smelters, welders, and workers in dry-cell battery factories [7–10], in which significant
neurological dysfunction has been associated with Mn exposure [11,12]. Indeed,
epidemiological studies of industrial workers have suggested a relationship between
elevated environmental Mn exposure and an increased risk for parkinsonian distur-
bances [13–17], an association that has also been supported by numerous laboratory
studies [18–24]. While the exact mechanisms underlying the neurotoxic effects of
Mn remain unclear, these studies collectively suggest that elevated environmental
exposures to Mn may be sufficient to exacerbate the emergence of neurological
diseases [23,24]. Thus, in the next sections of this chapter we will discuss some
details concerning Mn in health and disease.

1.1 Manganese Essentiality

Mn is an essential nutrient necessary for a variety of metabolic functions including


those involved in normal human development, activation of certain metalloenzymes,
energy metabolism, immunological system function, nervous system function,
reproductive hormone function, and in antioxidant enzymes that protect cells from
damage due to free radicals [25,26]. Mn also plays an essential role in regulation of
cellular energy, bone and connective tissue growth, and blood clotting. Mn is an
important cofactor for a variety of enzymes, including those involved in neurotrans-
mitter synthesis and metabolism [27]. Indeed, in the mammalian brain, small
amounts of Mn are required for brain development, cellular homeostasis, and for the
activity of multiple enzymes [28–30]. Additionally, Mn is believed to be involved in
the stellate process production in astrocytes, as well as in the metabolism of brain
glutamate to glutamine, a step carried out by glutamine synthetase (GS).
Taking into account the variety of enzymatic processes which require Mn, an
inadequate daily supply of the metal is associated with a variety of health repercus-
sions, ranging from generalized growth impairment, birth defects, reduced fertility,
and impaired bone formation, to altered metabolisms of lipids, proteins, and carbo-
hydrates [31,32]. However, few occurrences of Mn deficiencies have been reported
in humans, with symptoms including dermatitis, slowed growth of hair and nails,
decreased serum cholesterol levels, decreased levels of clotting proteins, increased
serum calcium and phosphorus concentrations, and increased alkaline phosphatase
activity [25,33,34]. In addition, several human diseases have been reported to be
202 Avila, Puntel, and Aschner

associated to low blood Mn concentrations, including epilepsy, Mseleni disease,


Down’s syndrome, osteoporosis, and Perthest disease [35], nevertheless, the role of
Mn deficiency in these diseases remains unclear. In general, highly severe deficiencies
in dietary Mn supply are necessary to observe clinical symptoms [36,37].
The U.S. Food and Drug Administration (U.S. FDA) suggests a Reference Daily
Intake (RDI) for Mn at 2 mg/day for adults (Federal Register 2007, 72 FR 62149),
although there is no consensus regarding the safe and adequate levels of this nutrient
for various age groups. This recommended dosage is based upon the U.S. National
Research Council’s (NRC), which established estimated safe and adequate dietary
intake (ESADDI) of 2–5 mg/day for adults [38]. Additionally, it is known that Mn
essentiality in humans varies depending of the life-stage and of the sex [39].
Accordingly, it is suggested by the National Academy of Sciences (NAAS) that an
adequate intake of Mn is 2.3 mg/day for adult men and 1.8 mg/day for adult women
[39,40]. The difference is accounted for by differential Mn absorption in men versus
women [41]; it has been attributed to lower serum ferritin concentrations in men as
compared to women [39,41]. Lactating or pregnant women are also thought to have
increased Mn requirement [39]. Moreover, life-stages are also known to influence
dietary Mn requirements. Accordingly, in newborns (less than six months of age)
adequate Mn intake is defined as 3 μg/day; at seven to twelve months of age, adequate
Mn intake increases to 600 μg/day [39]. In children one to three years of age, adequate
Mn intake approximates 1.2 mg/day, and in children four to eight years of age, the
adequate Mn intake increases to 1.5 mg/day.

1.2 Manganese Pharmacokinetics

Intricate regulation of Mn absorption and tissue specific accumulation is crucial for


the proper regulation of the activity of Mn-dependent enzymes. Thus, understand-
ing Mn’s essentiality and toxicity in the brain requires knowledge of its regulation
in the periphery. Three major factors have been postulated to modulate plasma Mn
levels. First, given that the main source of Mn is diet, tight regulation of gastrointes-
tinal absorption of Mn is crucial. Second, following Mn absorption and a concomi-
tant increase in plasma Mn levels, transport of Mn to target organs, including the
liver, is necessary to prevent Mn-induced toxicity in the periphery. Finally, Mn must
be eliminated from the plasma via shuttling to bile [42]. Thus, homeostatic controls
tightly restrict Mn absorption and regulate Mn excretion to maintain stable tissue
levels despite fluctuations in daily Mn dietary exposure. However, exposure to high
Mn concentrations, as might occur in occupational settings, may overwhelm homeo-
static controls and results in elevations in tissue Mn concentrations. Accordingly,
both pulmonary uptake and particulate transport via the olfactory bulb [42,43] can
lead to deposition of Mn within the striatum and cerebellum and inflammation of
the nasal epithelium [44].
It is generally accepted that Fe has a strong influence on Mn homeostasis, since
both metals share binding and uptake via the transferrin (Tf) transporter and the
7 Manganese in Health and Disease 203

divalent metal transporter-1 (DMT-1) (see more details in Section 2 of this chapter).
It is known that Mn ions (Mn3+) bind at the same location as ferric ions (Fe3+) on
the large glycoprotein molecule mucin, which is known to stabilize the ions, pre-
venting precipitation in the lumen of the gastrointestinal tract [45]. Moreover, both
metals are known to have an affinity for the intercellular metal binding molecule
mobilferrin [46].
Absorption of metal ions into enterocytes is known to take place via transmem-
brane transporters. Thus, during Fe deficiency the number of transporters in entero-
cyte membranes is increased in order to maximize Fe absorption [47]. This will
inevitably result in increased Mn absorption, particularly in the absence of Fe.
Indeed, in rodent models, Fe deficiency is associated with increased Mn absorption
across the gastrointestinal tract, as well as increased Mn deposition in the brain
[48,49]. Moreover, the absorption of Mn by the gastrointestinal tract is highly
dependent upon the quantity of ingested Mn and net accumulated levels in the
plasma. While Mn is transported by simple diffusion in the large intestine, Mn is
absorbed by active transport in the small intestine [42]. In contrast, Mn excretion into
bile is driven by concentration gradients leading to its flow from liver to bile [50].
About 3–5% of dietary Mn is absorbed in the gastrointestinal tract as Mn2+ and
Mn4+ [29]. Mn2+ is oxidized to Mn3+ by liver and plasma ceruloplasmin and trans-
ported through the blood [51,52]. Mn tends to form tight complexes with other
ligands [4]. Accordingly, a variety of plasma proteins or ligands have been impli-
cated as specific Mn carrier proteins, including transglutaminase, beta-globulin,
albumin, and Tf [53,54]. As a result, its free plasma and tissue concentrations tend
to be extremely low [55].
Intracellular Mn2+ is sequestered in the mitochondria of the brain and liver via
the Ca2+ uniporter [56,57]. Thus, mitochondria are the primary pool of intracellular
Mn; however, nuclei have also been implied (remains debatable) to preferentially
accumulate this metal [21,58,59]. In addition, it was recently shown that Mn2+ may
induce fragmentation of the Golgi apparatus, indicating a specific role of this com-
partment in maintaining Mn homeostasis [60]. The Golgi harbors the Ca2+/Mn2+-
ATPases of the secretory pathway (SPCAs) [61], which possesses a high-affinity
Mn2+ transport capacity [62]. This is also supported by in vivo studies reporting that
brain areas with high SPCA expression also show enhanced Mn2+ accumulation
upon continuous systemic MnCl2 infusion in mice [63], and by the observation that
a gain-of-function mutation in SPCA, which specifically enhances Golgi Mn2+
transport, improves survival of Mn2+-exposed cells [64]. Thus, failure of efficient
Mn2+ detoxification by saturating the SPCA-mediated removal via the Golgi may
result in enhanced Mn2+ accumulation in the mitochondria, thereby causing mito-
chondrial impairment [60].
Mn enters the brain from the blood either across the cerebral capillaries and/or
the cerebrospinal fluid (CSF). At normal plasma concentrations, Mn appears to
enter into the CNS primarily across the capillary endothelium, whereas at high
plasma concentrations, transport across the choroid plexus appears to predominate
[65,66], consistent with observations on the rapid appearance and persistent elevation
204 Avila, Puntel, and Aschner

of Mn in this organ [67]. Indeed, radioactive Mn injected into the blood stream is
concentrated in the choroid plexus within 1 hour after injection. Three days post-
injection it is localized at the dentate gyrus and CA3 of the hippocampus [68].
The concentration of Mn in the brain varies across brain regions. The highest Mn
levels are found in the globus pallidus in humans and in the hypothalamus in rats
[28,69–75]. Spectroscopy in rats has demonstrated that mitochondria in the basal
ganglia accumulate the highest amount of Mn [76,77]. Differential metal transporter
expression patterns and diffusion constants for Mn in various brain regions must
explain, at least in part, the asymmetry in Mn accumulation across brain regions
[78]. The preferential accumulation of Mn in basal ganglia is often associated with
a clinical disorder referred to as manganism, which is characterized by a set of
extrapyramidal symptoms resembling idiopathic Parkinson’s disease (IPD) (see
more details in Section 3 of this chapter). However, further characterization of the
absorption and elimination kinetics, as well as Mn uptake and export pathways is
necessary to better understand the basis of differential Mn accumulation across dif-
ferent brain regions.
The physiological half-life of Mn in the adult rat and primate brain is approxi-
mately 51 to 74 days [52,55,73,79]. The main excretion mechanism for Mn depends
on normal liver function. Indeed, blood Mn concentrations are increased during the
active phase of acute hepatitis as well as in post hepatic cirrhosis, and a significant
correlation exists between blood Mn and the activities of liver enzymes in patients
with hepatitis and cirrhosis [80,81]. Corroborating these observations, MRI has
consistently shown signal hyperintensity in the globus pallidus in cirrhotic patients
[82]. Furthermore, direct measurements in pallidal samples obtained from the
autopsies of cirrhotic patients revealed several-fold increases in Mn concentrations,
and histopathologic evaluations showed Alzheimer’s type II astrocytosis [83]. The
disorder is characterized as hepatic encephalopathy, and it is associated with cogni-
tive, psychiatric, and motor impairments, all of which are known to be also associ-
ated with manganism [84].
From physiologically based pharmacokinetic models, it has been proposed that
54
Mn clearance from the body follows biphasic elimination, with a short “fast” elim-
ination phase (with half-times of around a few days) followed by a longer “slow”
elimination phase. This elimination behavior was consistently observed with all
exposure routes. Thus, the availability of tracer studies for multiple exposure routes
permitted a comparison of dose route differences in elimination. Accordingly, the
faster clearance in monkeys and humans occurred from oral exposure, whereas the
slowest clearance occurred following intravenous (iv) administration [85].

1.3 Manganese Biochemistry and Physiology

As pointed out above, Mn is an essential nutrient necessary for a variety of functions.


Accordingly, it acts as an activator of the gluconeogenic enzymes pyruvate carboxylase
and isocitrate dehydrogenase, is involved in protecting mitochondrial membranes
7 Manganese in Health and Disease 205

through superoxide dismutase, and it activates glycosyl transferase, which is


involved in mucopolysaccharide synthesis [86], just to name a few. Mn it is also a
cofactor for many other enzymes, such as transferases, hydrolases, lyases, and
integrins. Nevertheless, additional investigation is needed to identify the complete
set of Mn-dependent enzymes in mammalian systems as for many of these activi-
ties, Mn is not the only metal that can act as a cofactor; iron, magnesium or copper
are readily able to substitute it. In addition, the majority of Mn-dependent enzymes
are found in bacteria and plants and only a few have been systematically studied
in mammalians.
Although Cu and Mg can substitute for Mn as a cofactor for some enzymes, there
is a subset of enzymes with roles in neuron and/or glial that are strictly dependent
upon the presence of Mn. These discrete Mn-binding proteins (manganoproteins)
include glutamine synthetase, superoxide dismutase 2 (SOD2), arginase, pyruvate
decarboxylase, and serine/threonine phosphatase [87–89].
Glutamine synthetase is the most abundant manganoprotein; it is predominantly
expressed in astrocytes, where it converts glutamate to glutamine. Because GS con-
tains four Mn ions per octamer [90], Mn has been proposed to regulate GS activity.
In fact, insufficient Mn increases glutamate trafficking, glutamatergic signaling, and
excitotoxicity [91]. Furthermore, it has been proposed that the increased suscepti-
bility to seizures observed in individuals with Mn deficiency may be due to dimin-
ished GS levels and/or activity [92].
Arginase regulates elimination of ammonia from the body by converting
L-arginine, synthesized from ammonia, to L-ornithine and urea as part of the urea
cycle. Moreover, in the brain, L-arginine is converted to nitric oxide by neuronal
nitric oxide synthetase. Proper regulation of arginase promotes neuronal survival by
impairing nitric oxide signaling [93,94].
Pyruvate carboxylase is an essential enzyme required for glucose metabolism that
interacts with Mn to generate oxaloacetate, a precursor of the tricarboxylic acid (TCA)
cycle [95]. Interestingly, in the brain, pyruvate carboxylase is predominantly expressed
in astrocytes [58,96]. Protein phosphatase-1 is essential for glycogen metabolism,
cell progression, regulation synthesis, and release of neurotrophins, which promote
neuronal survival and synaptic membrane receptors and channels [97].
Finally, SOD2 is a mitochondrial enzyme that detoxifies superoxide anions
through the formation of hydrogen peroxide. Although the concentration of Mn
within neurons is low (<10−5 M), their high mitochondrial energy demands is cor-
related with a propensity for increased SOD2 in neurons compared to glia [28,42].
Furthermore, loss of SOD2 activity increases the susceptibility to mitochondrial
inhibitor induced toxicity and causes oxidative stress [98].
Thus, Mn deficiencies, although not frequently reported in humans, may result in
several biochemical and structural defects [35,99,100]. Accordingly, taking into
account the Mn-dependent enzymatic processes, it is clear that inadequate daily
supply of this metal may be associated with a variety of health repercussions [31,32].
In contrast, excessive levels of Mn can accumulate in the brain and lead to neuro-
toxicity. Because the area of the CNS comprising the basal ganglia is very complex
and its function is dependent upon the precise function and balance of several
206 Avila, Puntel, and Aschner

neurotransmitters, it is not surprising that symptoms of manganism often overlap


with IPD. Indeed, evidence exists to support that persistent exposure to Mn may
predispose an individual to acquire dystonic movements associated with PD (more
details concerning Mn toxicity are provided in Section 3 of this chapter).

2 Manganese Transport

Considering the delicate relationship between essentiality and toxicity, Mn homeo-


stasis is vital for the optimal functioning of any organism. Although some research
has focused on mechanisms associated with the transport of Mn across the blood-
brain barrier (BBB), the exact identity of the carrier(s) involved in Mn trafficking
into the brain remains somewhat controversial. Over the past decades, active trans-
port [65] and facilitated diffusion [51,66] mechanisms have been described. More
recently, Mn transport has been ascribed to high affinity metal transporters of Ca
and Fe.
Several of these transporters include DMT-1, which belongs to the family of
natural resistance-associated macrophage protein (NRAMP) [47,101]; ZIP8, a
member of the solute carrier-39 [102]; Tf receptor (TfR) [51,103], which is known
to be responsible for Fe3+ uptake; voltage-regulated [104] and store-operated Ca2+
channels [105]; and the ionotropic glutamate receptor Ca2+ channels [106]. It was
also observed that distribution after intravenous injection of 54Mn is readily trans-
ported into the brain as the free ion [66]. Moreover, studies have also demonstrated
that a Mn-citrate complex could be transported either by a monocarboxylate trans-
porter (MCT) [107], organic anion transporter polypeptide (OATP) or ATP-binding
cassette (ABC) superfamilies [108].
Leak-pathways for Mn also likely exist, especially in areas lacking intact BBB,
namely the circumventricular organs [109] (Figure 1). While the relative contribu-
tion of each of these transporters and/or mechanism(s) remains unknown, it is likely
that optimal tissue Mn concentrations are maintained by the involvement of all of
them. As pointed out above, in plasma more than 80% of Mn 2+ is bound to
α 2 - macroglobulin and albumin. However, these complexes (Mn2+-albumin or
α 2-macroglobulin) may not take part in the transport across BBB (see Figure 1).
TRPM7, which is ubiquitously expressed in vertebrates and functions as an
active Ca2+-selective transporter and a serine/threonine protein kinase, is a putative
Mn transporter. The kinase activity is important for its metal transport function.
Specifically, the transporter operates by regulating intracellular Ca2+ levels and
Mg2+ homeostasis through the creation of an inward current, thus contributing to the
establishment of a cellular membrane potential. Physiological levels of Mg2+ and
Ca2+ are necessary for maintaining the permeability of TRPM7 to Mn2+, Co2+, and
Ni2+ [110]. Finally, the homomeric purinoreceptors, including P2X and P2Y, have
been invoked to transport Mn. These receptors are ATP-dependent and ubiquitously
expressed on endothelial cells [111–113]. Nevertheless, purinoreceptors have a
relatively lower affinity for Mn than for the other divalent metals they transport
(Ca > Mg > Ba > Mn) [110].
7 Manganese in Health and Disease 207

Mn transport into CNS


Blood –Brain Barrier

Blood compartment Brain Parenchyma

?
ZIP8 /ZIP14 Mn2+
Tf Mn3+
Citrate Mn2+
Free Mn2+
Leak pathways
DMT-1 Mn2+
Mn2+ -albumin

Physiological Mn
plasma levels

Figure 1 Mechanism of Mn transport across the BBB under physiological Mn exposure levels.
Transporters and relevant manganese oxidation states associated with Mn transport are demonstrated.
Mn bound to albumin is excluded from passing the BBB given its size. Arrow size depicts the relative
importance of each of the transporters in this process, bolder arrows representing more prominent
transport mechanisms. Please refer to the discussion for additional details. Since it has yet to be
determined whether ZIP8 functions to transport Mn across the BBB, the process has been annotated
with a question mark.

Although non-protein-bound Mn enters the brain more rapidly than Tf-bound Mn


[65,66], the question remains as to which form represents the predominant mechanism
of transport in situ. Analyses of transport mechanisms based on tracer techniques
employing bolus injections of Mn into the circulation cannot be easily interpreted.
Thus, while tracer studies represent a sensitive technique for quantifying Mn trans-
port, it must be noted that the information derived from such studies does not neces-
sarily reflect the chemically active or functional forms in which Mn exists and is
transported in vivo. This is due to the saturation of blood ligands for Mn and the
likelihood that Mn in the free form exists at concentrations in excess to those expected
under physiological conditions. Thus, transport kinetics under such conditions may
not necessarily mimic physiological conditions. In the next subsections, we briefly
discuss some aspects related to Mn transport in biological systems.

2.1 Manganese Uptake in Relation to Oxidative State

Mn can cross the BBB and blood-cerebrospinal fluid barrier (BCB) through several
carriers (see Figure 1) and in different oxidation states [42]. Indeed, emerging
reports have indicated that Mn2+ can be transported via DMT-1, the divalent metal/
208 Avila, Puntel, and Aschner

bicarbonate ion symporters ZIP8 and ZIP14, various calcium channels, the solute
carrier-39 (SLC39) family of zinc transporters, park9/ATP13A2, and the Mg trans-
porter hip14. Accordingly, DMT-1 belongs to a large family of metal transporters,
which are responsible for the transport of divalent metals ions, including Mn, Fe,
Cu, and Cd [114]. Thus, DMT-1 is involved in Mn accumulation in the brain. DMT-1
works as hydrogen ion symporter, transporting one hydrogen ion and one divalent
cation in the same direction. This protein is responsible also for exporting the Mn2+,
which is released into the cytoplasm [115]. Alternatively, Mn2+ ions may be directly
transported from the blood stream by crossing the cellular membrane through volt-
age-regulated or glutamate-activated ionic channels, which are usually responsible
for the transport of Ca2+ into the cell [116]. Finally, emerging experimental data has
indicated that huntingtin-interacting protein 14 and 14L (Hip14, Hip14L) mediates
transport of Mn2+ and other divalent metals (Mg2+, Sr2+, Ni2+, Ca2+, Ba2+, Zn2+) across
cell membranes [117,118]. Alternatively, Mn3+ entry via the TfR, which mediates
Fe3+ uptake, is also considered [88].

2.2 Cellular Manganese Uptake

A critical regulator of brain Mn levels is the DMT-1 (also referred to as the DCT, or
divalent cation transporter) which is known to shuttle both Mn2+ and Fe2+ ions, as
well as other divalent metals. This transporter belongs to the NRAMP gene family
[47,101]. Disruption of the orthologous DMT-1 gene in the rat or mouse, results in
significantly lower tissue levels and uptake of Mn and Fe in the brain [119–121].
Consistent with a role for DMT-1 in brain Mn uptake, nasal Mn absorption is also
significantly attenuated in b/b rats, and olfactory DMT-1 protein levels are signifi-
cantly elevated in Fe-deficient rats [122].
Notably, a recent study has shown that DMT-1 contributes to neurodegeneration
in an experimental model of PD [123]. These authors observed increased expression
of a specific DMT-1 isoform (DMT-1/Nramp2/Slc11a2) in the substantia nigra of
PD patients. Moreover, the authors also showed that the administration of 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, a dopaminergic toxin used in experi-
mental models of Parkinson’s disease) increased DMT-1 expression in the ventral
mesencephalon of mice, which was concomitant with Fe accumulation, oxidative
stress, and dopaminergic cell loss [123]. Additionally, DMT-1-mediated metal
transport across rat brain endothelial cells in culture has been reported to be pH-,
temperature-, and Fe-dependent [110,124,125].
The TfR is the major cellular receptor for Tf-bound Fe, but because Tf can also
bind trivalent Mn, TfR can also mediate Mn3+ transport by endocytosis. Mn3+ inter-
nalized through the endocytic pathway must be released from Tf and reduced to
Mn2+, which is transported through DMT-1 to the cytosol. The TfR is an active
transporter that is pH- and Fe-dependent [110]. Both in vivo and in vitro studies
have reported that Mn is efficiently transported via the TfR. For example, a spontaneous
mutation in a murine gene linked to the TfR, referred to as hypotransferrinemic,
7 Manganese in Health and Disease 209

results in a drastic serum TfR deficiency, impairs Mn transport, and disrupts Fe


deposition [126,127].
Additional cellular Mn transporters include the Mn-citrate transporters and
the Mn-bicarbonate symporters. Indeed, a small fraction of Mn has been found in
the plasma as citrate complex. Crossgrove and Yokel have demonstrated that a
Mn-citrate tridentate complex with a non-coordinated central carboxylate moiety is
a probable substrate for the anion transporter or a monocarboxylate transporter
[107]. Moreover, the members of the organic anion transporter polypeptide or ATP-
binding cassette superfamilies may transport Mn-citrate complexes [108]. The
Mn-bicarbonate symporters, ZIP8 and ZIP14, have also been identified as members
of the solute carrier-39, and are expressed on brain capillaries [102], although it has
yet to be determined whether these proteins are functional at the BBB [1,128].
Nevertheless, these symporters utilize a HCO3− gradient as the driving force for Mn
uptake across the plasma membrane.
Gitler and colleagues have recently reported that the park9 gene responsible for
early-onset Parkinsonism also transports Mn [118]. The park9 gene encodes a puta-
tive P-type transmembrane ATPase (ATP13A2) protein. Although the exact function
of park9 is unknown, it is generally thought to be a shuttle for cations, including Mn
across the cell membrane. Biochemical studies have demonstrated that the highest
and lowest park9 mRNA levels are localized within the substantia nigra and cere-
bellum, respectively [129]. Mn transport via voltage-regulated channels [104],
store-operated channels [105], ionotropic glutamate receptor channels [106] (all
Ca2+ channels), and choline transporters [130] has also been described.
Another possible mechanism for Mn transport involves dopamine transporters
(DAT). It is believed that DAT facilitates Mn transport into dopaminergic striatal
neurons and that Mn accumulates in the globus pallidus via axonal transport
[131,132]. As a result, blockage of the DAT in the striatum would attenuate Mn
accumulation in striatal neurons and would cause decreased Mn concentrations in
the globus pallidus [132] (see Figure 2). Nevertheless, while the tissue specific
expression of each of the aforementioned Mn transporters is yet to be determined, it
is likely that optimal tissue Mn levels are maintained through the involvement of all
the above and likely other unknown Mn transporters.

2.3 Cellular Manganese Efflux

Little information exists on the putative extracellular transport mechanisms of Mn.


In the intestine ferroportin-1 (Fpn) is located at the basal membrane and exports Fe
to the circulation [133,134]. Yin and colleagues [135] have implicated Fpn as a Mn
exporter. Using an inducible HEK293T cell model, they showed that Fpn expres-
sion reduced Mn-induced toxicity and decreased Mn accumulation. In addition, Mn
was also able to increase Fpn levels [136]. From these studies, it was concluded that
Fpn participates in Mn efflux (see Figure 2). In accordance to previous data, it was
recently shown that Mn is a substrate for Fpn, and that this export process is inhibited
210 Avila, Puntel, and Aschner

Mn2+

Mn2+
Mn2+ 2+
Mn

Mitochondria

TfR
Nucleus Golgi

HIP 14
DAT Mn3+

Mn2+
4

Mn2+ Mn2+
Mitochondria
Ca2+ channels

DMT-1
ZIP 4 / ZIP 8
Citrate
Mn2+ 2+ Mn2+
Mn

Mn2+/ HCO-3

Figure 2 Identified and putative Mn transporters. These illustrated Mn transporters have been
demonstrated to facilitate Mn trafficking (uptake, storage, efflux) between the extra- and intracellular
milieu. Each of these transporter proteins has also been implicated in the transport of other metals.

by a low extracellular pH and by incubation in a high K+ medium, indicating the


involvement of transmembrane ion gradients in Fpn-mediated Mn transport [137].
Interestingly, Fpn is expressed in tissues involved in both Fe and Mn homeosta-
sis, including the developing and mature reticuloendothelial system, the duodenum,
liver, and the pregnant uterus [138,139]. Fpn has also been identified in cells of the
central nervous system including those of the BBB, choroid plexus, neurons, oligo-
dendrocytes, astrocytes, and retina [133,140]. Nevertheless, it has yet to be deter-
mined whether Mn shares Fe exporters, such as Fpn, in this process. Moreover, the
role of Fpn in Mn homeostasis remains to be elucidated in each of the neural cell
types.

3 Manganism. A Neurodegenerative Disease

Manganism (also referred to as locura manganica) has been known for 150 years
and it is caused by the preferential accumulation of Mn in brain areas rich in dopa-
minergic (DAergic) neurons (caudate nucleus, putamen, globus pallidus, substantia
nigra, and subthalamic nuclei) [79,141,142]. Mn can readily oxidize catecholamines,
7 Manganese in Health and Disease 211

including dopamine (DA), altering homeostasis in these areas [70]. Perturbation in


striatal DA levels likely explains the biphasic syndrome experienced by patients
with manganism. An initial phase of increased DA production is associated with
psychotic episodes commonly observed in psychiatric patients [143]. As Mn poi-
soning progresses, catecholamine levels decrease, most likely due to the loss of
nigrostriatal DAergic neurons and, consequently, the parkinsonian-like symptoms
ensue [1,13,70]. Hence, in early stages of manganism, upon cessation of Mn expo-
sure, the symptoms might be reversed, whilst in patients with motor disturbances,
manganism is irreversible [144].

4 Symptoms and Sensitive Populations

The initial stages of manganism are characterized by psychiatric symptoms, including


emotional liability, mania, compulsive or aggressive behavior, irritability, reduced
response speed, hallucinations, feeding and sex disturbances, intellectual deficits,
humor changes, sex dysfunctions, as well as mild motor impairment. In established
manganism cases, the classic extrapyramidal symptoms (motor symptoms) become
prominent and include mask-like face, limb rigidity, mild tremors, gait disturbance,
cock-like walk, slurred speech, excessive salivation and sweating, and a disturbance
of balance, all of which are also observed in IPD [144–147].
Considering the routes and sources of exposure, affected populations commonly
include welders, miners, and people that work in a Mn-polluted environment [147–
149]; infants fed with Mn-containing formulas [109]; patients with hepatic enceph-
alopathy [150,151], and subjects fed with parenteral nutrition [152,153]. In addition,
subjects that have genetic pre-disposition have been recently considered as sensitive
populations, as described in Section 8.

5 Manganism versus Parkinson’s Disease

Idiopathic Parkinson’s disease is a progressive neurodegenerative disorder with a


slow onset, and compared with the familial forms of the disease, it is associated with
advanced age (>55 years of age). The four cardinal signs of IPD are tremor at rest,
bradykinesia (hypokinesia and akinesia), rigidity and postural instability [154–156].
The disease is characterized by loss of neurons [154] in the substantia nigra pars
compacta (SNpc) and decreased DA levels in the caudate and putamen [155].
Excessive brain Mn levels can also represent a risk factor for IPD [16,145,147–
149]. Case-controlled studies have revealed a strong correlation between
Mn-exposed populations and increased susceptibility to PD [16,145,147–149].
Manganism and PD share in their etiology common cellular mechanisms such as
preferential accumulation of Mn within mitochondria resulting in oxidative stress
[21,157] and selective DAergic neurotoxicity [1,158,159]. Parkinsonism in welders
212 Avila, Puntel, and Aschner

versus non-welders is clinically distinguishable only by the age of onset (46 versus
63 years, respectively) [149,160]. Furthermore, the prevalence of PD is higher
among welders versus age-standardized individuals in the general population
[149,160]. However, direct evidence that Mn exposure in welders is responsible for
this increased prevalence has not been reported yet.
Manganism commonly occurs in response to acute Mn exposures, whereas PD
likely reflects long-term exposure to relatively low Mn exposures [145,158].
Manganism features less frequent kinetic tremor, or no tremor versus patients with
PD [129,147–149]. Acute high Mn exposures also lead to dystonias and a “cock-walk”
with symptoms becoming progressive and irreversible [147,149]. In addition to affecting
the basal ganglia, manganism is also known to affect other brain regions, including the
cortex and hypothalamus and at the morphological level leading to neuronal loss and
reactive gliosis in the globus pallidus and substantia nigra pars reticulata (SNpr) in the
absence of Lewy bodies, which are a hallmark of PD [147,149,161,162]. Furthermore,
in manganism, damage to the striatum (caudate nucleus and putamen) and subthalamic
nucleus may occur, while the SNpc is generally spared whilst PD is predominantly
characterized by neuronal loss in the SNpc [161].
PD and manganism are analogous in several mechanistic ways. Mn-induced
neurotoxicity involves mitochondrial dysfunction, increase in endoplasmic reticu-
lum stress factors and oxidative stress, as also observed in PD patients studied
post-mortem [145,148,158,161]. Mn can cause the increase in fibril formation by
α-synuclein, thus inducing neuronal death. The α-synuclein aggregates, named
Lewy bodies, are one of the hallmarks of PD. Furthermore, there are several genetic
factors associating both disorders, which will be described in detail below (Section 8).

6 Manganese in the Etiology of Other Neurodegenerative


Disorders

6.1 Manganese and Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects


motor neurons (MNs) in the spinal cord and the cortex [163]. ALS patients gradually
loose MNs, and muscles weaken, ultimately leading to respiratory distress and death
[163]. A small percentage of ALS cases are hereditary, due to mutations in the Cu/Zn
superoxide dismutase (SOD1) gene. Expression of these mutant forms of SOD1 in
animal models leads to ALS-like phenotypes [164]. Oxidative stress is proposed to
be a central mechanism leading to Mn cell loss in ALS, other contributing mecha-
nisms include excitotoxicity, astrocytosis, mitochondrial impairment, calcium/mag-
nesium imbalance, and Mn toxicity [165–167]. Mn-SOD (SOD2), in contrast to its
orthologue, SOD1, does not contribute to the genetic predisposition to ALS [168],
but it may slow down ALS-like syndrome progression in mice, and its activity
has been shown to be reduced in the serum and the CSF of ALS patients [168].
7 Manganese in Health and Disease 213

Mn levels were found to be lower in blood cells, but were significantly increased in
the sera of ALS patients [169,170], supporting a role for Mn-mediated neurotoxicity
in ALS.

6.2 Manganese and Alzheimer’s Disease

Heavy metals, especially the essential metal Zn and the non-essential metal Al, have
been shown to play a role in amyloid-beta (Abeta) aggregation and toxicity, both of
which are characteristics of Alzheimer’s disease (AD) [171]. In a recent study,
Abeta precursor-like protein 1 (APLP1) was found to be the most up-regulated gene
in the frontal cortex of monkeys (Cynomologous macaques) chronically exposed to
Mn. This result was associated with cortical Mn accumulation [172], cortical neu-
ron degeneration, and apoptotic marker expression [172], consistent with previous
reports of cognitive impairment in those animals. Conversely, over-expression of
Abeta in mice led to Mn accumulation in the brain, suggesting that Abeta could play
a role in Mn homeostasis and toxicity [173]. Similar to ALS, despite Mn accumula-
tion, SOD2 antioxidant activity is reduced in AD, likely contributing to oxidative
stress [174]. Furthermore, Mn can also produce alterations related to AD without
the senile plaque formation. In cases of chronic Mn exposure, neuronal degenera-
tion in the globus pallidus is associated with the development of Alzheimer’s type II
astrocytosis, in which cells typically exhibit enlarged, pale nuclei, margination of
chromatin and, often, prominent nucleoli [175].

6.3 Manganese and Huntington’s Disease

Huntington’s disease (HD) is a progressive neurodegenerative disease with a preva-


lence of 5 in 10,000 people worldwide. HD is characterized by motor impairment,
cognitive deterioration, emotional disturbance, and psychiatric deficits, caused by
expansion in the glutamine-encoding triplet repeat by mutation in the HTT gene
[175]. Environmental factors have also been suggested to contribute to the residual
variation in age of onset, perhaps even more so than genetic factors [176]. In this
context, metals such as Mn may be involved in modulating and interacting with HD.
A study by Williams et al. [177] described a novel gene-environment interaction
between expression of mutant HTT and Mn. Specifically, acute Mn exposure of
cultured striatal cells unexpectedly decreased the vulnerability of mutant expressing
cells (STHdhQ111/Q111) to Mn cytotoxicity compared to wild-type (STHdhQ7/Q7) [177].
Furthermore, total intracellular Mn levels following Mn exposure in STHdhQ7/Q7
and STHdhQ111/Q111 cells were significantly lower in mutant than wild-type cells
[177,178]. Moreover, the mutant HTT–Mn interaction was corroborated in vivo
using the YAC128Q mouse model of HD; these mice accumulated less Mn in the
striatum than wild-type animals following subcutaneous Mn injections [179].
214 Avila, Puntel, and Aschner

7 Molecular Mechanisms of Toxicity

The cellular, intracellular, and molecular mechanism(s) underlying Mn neurotoxic-


ity are incompletely understood [21,109,141,142,158,161]. Nevertheless, it has
been demonstrated that Mn affects numerous biological activities, dependent upon
levels and routes of exposure, dosage, age of the exposed individual, and exposure
duration [6,180,181]. Mn is known to induce increased oxidative stress, a well-
established molecular mechanism of Mn-induced toxicity. Below, we discuss the
main mechanisms that are believed to mediate Mn-induced neurodegeneration.

7.1 Dopamine Oxidation

Dopamine (DA) is one of the most abundant catecholamines within the brain, con-
trolling locomotion, emotion, and neuroendocrine system. Chronic exposure to Mn
has been shown to cause the degeneration of nigrostriatal DAergic neurons leading
to symptoms that resemble PD [13,148,182]. However, Mn’s effects are dependent
upon the experimental conditions, form of Mn (oxidation state), route of adminis-
tration, and exposure duration [1,6,142,147,183]. Postnatal Mn exposure causes a
decline in pre-synaptic DAergic functioning, reduced DA transporter expression,
and DA uptake in the striatum, and a long-lasting decrease in DA efflux [184,185].
Conversely, in adult animal models, exposure to Mn inhibits DA neurotransmis-
sion and depletes striatal DA [29,144,183,186,187], thereby resulting in motor
deficits [161].
Although it is generally accepted that free radicals play a key role in mediating
Mn-induced DAergic neurodegeneration [188,189], the precise mechanism of
Mn-induced neurotoxicity remains unknown. One hypothesis invokes the ability of
Mn to enhance reactive oxygen species (ROS) generation, thus forming quinines
[82,190,191]. Indeed, the Mn-catalyzed autoxidation of DA involves redox cycling
of Mn2+ and Mn3+ in a reaction that generates ROS and DA-o-quinone, thereby lead-
ing to oxidative damage [82,190–192]. Thus, elevated rate of autoxidation of cyto-
plasmic DA induced by Mn may contribute to DAergic cell death secondary to the
formation of cytotoxic quinones and ROS [190,191].
Mn-induced DA oxidation is a complex process involving several steps in which
semiquinone, aminochrome intermediates, L-cysteine or copper and NADH are
implicated [158,182,193]. Mechanisms underlying semiquinone and aminochrome-
induced damage in the Mn-induced neurodegenerative process likely include: (i)
NADH or NADPH depletion; (ii) inactivation of enzymes by oxidizing thiol groups
or essential amino acids; (iii) formation of ROS; and (iv) lipid peroxidation. It is
noteworthy that neither Mn2+ nor Mn3+ can generate hydroxyl radicals from hydro-
gen peroxide and/or superoxide via Fenton-type or Haber-Weiss-type reactions,
while Mn2+ can scavenge and detoxify superoxide radicals [3,188].
7 Manganese in Health and Disease 215

7.2 Mitochondrial Dysfunction

As mentioned in Section 1.2, intracellular Mn preferentially accumulates in the


mitochondria, mainly as Mn2+ via the Ca2+ uniporter [21,157,194–196]. Elevated
intramitochondrial Mn interferes with oxidative respiration, leading to excessive pro-
duction of ROS, and consequently, mitochondrial dysfunction [21,157,194–196]. The
ability of Mn to enhance oxidative stress is due to the transition of its oxidative state
+2 to +3, which increases its pro-oxidant capacity [192]. Superoxide produced in the
mitochondrial electron transport chain may catalyze this transition through a set of
reactions similar to those mediated by SOD and thus lead to the increased oxidant
capacity of the metal [195]. Since Mn3+ has greater pro-oxidant potential than Mn2+,
its production in the mitochondria may accentuate oxidative damage [197].
Moreover, Mn can directly impair mitochondrial function by inhibiting the mito-
chondrial electron transfer chain [21,88,198], resulting in a reduced ATP production,
increased leakage of electrons, and increased O2· − production [199]. Although, Mn3+
is more potent at inhibiting complex I [3,197], Mn2+ is the predominant species
within cells and is largely bound to ATP [196,197]. Notably, Mn in biological media
in any of the oxidation states will spontaneously generate Mn3+. Interestingly, even
trace amounts of Mn3+ can cause formation of ROS [200]. The involvement of ROS
in Mn-induced mitochondrial dysfunction is also supported by observations on the
efficacy of antioxidants in attenuating its effects [201].
Mn also interferes with Ca2+ homeostasis in mitochondria by inhibiting its efflux
[202,203]. Oxidative stress generated by high Mn concentrations leads to the induc-
tion and opening of the mitochondrial permeability transition (MPT) pore, a Ca2+-
dependent process, resulting in increased solubility to protons, ions, and solutes, loss
of the mitochondrial inner membrane potential (Δψm), impairment of oxidative phos-
phorylation, and ATP synthesis and mitochondrial swelling [202,204,205]. Indeed,
Mn has been shown to decrease Δψm in a concentration-dependent manner, indicat-
ing that this Ca2+-dependent process likely mediates Mn neurotoxicity [204,206,207].
Apoptotic mechanisms secondary to changes in mitochondrial function have also
been implicated in Mn-induced neurotoxicity. Ca2+-induced MPT opening leads to the
activation of the Bcl-2 family of proteins, especially Bax/Bak, culminating with the
release of cytochrome c (Cyt c) [208,209]. Cyt c activates, via ERK (extracellular
signal-regulated kinases), the cysteine protease caspase-3, which mediates apoptosis,
chromatin condensation and DNA fragmentation [210]. Consistent with these obser-
vations, Mn exposure has been shown to lead to ERK and caspase-3 activation in
astrocytes [204]. Furthermore, DNA strand breakage at low Mn levels was reported,
thereby reinforcing the mitochondrial role in mediating its neurotoxicity [211].

7.3 Astrocytosis

Astrocytes make up approximately 50% of the human brain volume [212] and assume
many critical pathophysiological roles essential for normal neuronal activity, including
glutamate uptake, glutamine release, K+ and H+ buffering, volume regulation and
216 Avila, Puntel, and Aschner

membrane–membrane mediated trophic cell signaling [92,180]. Unlike neurons,


astrocytes concentrate Mn to levels at least 50-fold higher than the culture media,
thus functioning as the major homeostatic regulators and storage site for Mn
[213,214]. Primate models of Mn toxicity have shown astrocytic pathological alter-
ations (Alzheimer type II) [17,22,151] and exposure of cultured astrocytes to patho-
physiologically relevant concentrations of Mn led to a dose- and time-dependent
cell swelling, which appears to be a consequence of oxidative stress and changes in
MPT [215]. Increased accumulation of Mn in astrocytes has also been shown to
alter glutamate homeostasis and elicit excitatory neurotoxicity [27]. For example,
Mn decreases astrocytic glutamate uptake [180,181,216] and reduces the expression
of the astrocytic glutamate:aspartate transporter (GLAST) [27,217,218], leading to
increased extracellular glutamate levels. Additionally, expression of glutamine
transporters was downregulated in Mn-exposed cultured astrocytes [219], contribut-
ing to the disruption of the glutamate-glutamine cycling in the brain.
The inhibition of the Na+/K+-ATPase by reactive oxygen species also likely con-
tributes to Mn-induced astrocytic dysfunction [220]. Mn increases the uptake of the
amino acid L-arginine, a substrate for the inducible form of nitric oxide synthase
(iNOS), which can lead to ROS production as a consequence of nitric oxide produc-
tion [221,222]. ROS have also been shown to directly interfere with glutamate
uptake [223], possibly via oxidation of thiol groups in the transporter protein
[220]. Thus, Mn accumulation in astrocytes has the potential to lead to oxidative
damage in these cells as well as adverse effects on glutamate clearance from the
extracellular space.

8 Genetic Susceptibility

Recently, the association of mutations of PD-related genes and manganism has been
reported. DJ-1 (Park7), together with parkin (Park2) and Pink1 (Park6), form an E3
ubiquitin-ligase complex that is involved in α-synuclein degradation [224]. The
physiological functions of these proteins involve protection against oxidative stress.
Both mitochondrial dysfunction and oxidative stress can modulate the ubiquitin-
proteasome pathway and have been implicated as causative factors for the abnormal
accumulation of proteins in familial forms of PD [145]. Recessive inheritance of
PARK 2 mutations may also cause increased environmental sensitivity to Mn expo-
sure, as observed by Aboud et al. [225]. Using human induced pluripotent stem cells
(hIPCs) derived from a subject at genetic risk by PARK 2 mutation, the authors
found significant high reactive oxygen species levels and increased mitochondrial
fragmentation after Mn exposure in vitro [225].
Mutations in parkin are associated with early onset of PD, associated with
DAergic neurodegeneration, however, absent Lewy bodies formation [145]. The
parkin gene encodes an E3 ubiquitin ligase, which has cytoprotective properties.
Transient transfection with the parkin gene in SH-SY5Y cells inhibits Mn-induced
cell death [226]. Exposure to welding fumes containing Mn led to decreased Park2
7 Manganese in Health and Disease 217

protein levels in DAergic brain areas in rodents [227]. Loss of function and/or
decreased expression of parkin has been associated with overexpression of DMT-1
and linked to PD [123,227], as well as manganism [228]. Conversely, increased
Parkin expression levels have been shown to attenuate Mn-induced neurotoxicity,
likely by reducing its transport [226,228]. DJ-1 was also decreased in striatum of
rats exposed chronically to welding fumes [227]. Mutations in dj-1 account for
1–2% of early-onset cases of PD [229]. The protein encoded by this gene is
expressed in the brain, including neurons within the SNPc and striatum, areas pri-
marily affected in PD [230]. DJ-1 expression has been localized to the matrix and
intermembrane space of mitochondria [231] and it is thought to function as an anti-
oxidant protein [232]. Dj-1-knockout mice exhibit increased mitochondrial free
radical formation and inactivation of enzymes [232].
Leucine-rich repeat kinase 2 (LRRK2) or PARK8 is a cytoplasmic enzyme pres-
ent in DAergic neurons. Mutations in this gene causing increased kinase activity
lead to typical features of PD [233]. Kinases require the formation of an ATP-
divalent metal cation complex, and Mg2+ typically participates in this catalysis.
Recent studies in G2019S cells, where LRRK2 is mutated and shows increased
enzyme activity, have demonstrated that Mn2+ can displace Mg2+ at the active site
and increase the catalytic rate of the enzyme [234]. Because this mutation is present
in 22–41% of PD cases, changes in the enzyme activity caused by Mn may result in
a gain-of-function type mechanism of toxicity, leading to decreased cell survival
[234].
Furthermore, mutations in a putative Mn exporter gene SLC30A10 have been
recently described [235]. These mutations are associated with marked motor impair-
ment, including a Parkinsonian-like syndrome. This inherited autosomal recessive
mutation leads to hypermanganesemia, dystonia, polycythemia, and hepatic cirrho-
sis [236]. The hypermanganesemia associated with SLC30A10 mutation is extreme,
with patients having whole blood Mn levels of 1200–6400 nmol/L, compared with
normal whole blood Mn (<320 nmol/L) [236].

9 Treatment

Even though manganism has been studied for several years, treatment approaches
are still controversial. Some authors have shown that levodopa, a standard treatment
for PD, decreased Mn symptoms; however most of the studies do not indicate this
drug as efficient [148,149]. Hence, other drugs have been tested in non-human ani-
mals and some of these treatments may be used in future clinical trials.
Antiinflammatory agents, such as indomethacin and para-aminosalicilic acid,
reduced Mn-induced increase in oxidative stress (isoprostanes) and neuroinflamma-
tion (prostaglandin E2) [237–240]. Notably, indomethacin protected against pro-
gressive spine degeneration and dendritic damage in striatal medium spiny neurons
of mice exposed to Mn [239,240]. This protection is probably mediated by the tran-
scription factor NF-κB [241]. Using transgenic mice expressing a transcription
218 Avila, Puntel, and Aschner

factor fused to a green fluorescent protein (GFP), Moreno and coworkers showed
that Mn exposure increased NF-κB reporter activity and nitric oxide synthase 2
(NOS2) expression in both microglia and astrocytes, and that these effects were
prevented by supplementation with steroid 17β-estradiol [241]. Estrogen also
decreased neuronal protein nitration in treated mice and inhibited apoptosis in stria-
tal neurons co-cultured with Mn-treated astrocytes in vitro [241]. Furthermore,
tamoxifen, an estrogen-related compound, effectively reversed glutamate transport
inhibition in a Mn-induced model of glutamatergic deregulation, suggesting a
potential therapeutic modality in neurodegenerative disorders characterized by
altered glutamate homeostasis [242]. In agreement with this study, Xu et al. showed
that the pretreatment of rats with the NMDA (N-methyl-D-aspartate) antagonist
MK801 protected neurons from Mn-induced glutamate excitotoxicity [243].
Synthetic and natural antioxidants have been tested in Mn-induced neurotoxicity
models. The chain breaking antioxidant Trolox concomitantly administered to Mn
in developing pups showed neuroprotective effects by decreasing apoptotic activity,
isoprostane levels and p38 phosphorylation [244]. Ebselen and diethyl-2-phenyl-
2-tellurophenyl vinyl phosphonate are organochalcogens that also reversed the neu-
rotoxic effects of this metal. Natural products such as Melissa officinalis extract and
silymarin reduced oxidative stress associated to Mn exposure [245,246].
Several studies have addressed genetic factors that mediate Mn toxicity. Streifel
and coworkers used mice lacking NOS, postulating that they would be protected
from the neurotoxic effects of Mn [247]. They found that loss of NOS2 reduced
NO-induced peroxynitrite formation, thus attenuating Mn-related peroxynitrite
adduct formation in the striatal-pallidum and substantia nigra pars reticulata. These
mice showed attenuated alterations in neurobehavioral function and neurochemistry
in vivo and loss of NOS2 also prevented astrocyte-mediated neuronal apoptosis in
vitro [247]. Expression of parkin, an E3 ubiquitin ligase also linked to PD, protects
against Mn toxicity, as observed in SH-SY5Y cells [248]. Conversely, deletion of
parkin leads to an increase in DMT-1 levels, thus causing increase in Mn uptake
[248]. Furthermore, it was reported for yeast that expression of PARK9, a gene
linked to PD, protected cells from Mn toxicity [118].
In C. elegans, Benedetto et al. observed that Mn-induced DAergic neurotoxicity
requires the NADPH dual-oxidase BLI-3, suggesting that in vivo BLI-3 activity
promotes the conversion of extracellular DA into toxic reactive species, which, in
turn, can be taken up by DAT-1 in DAergic neurons, thus leading to oxidative stress
and cell degeneration [248]. BLI-3 knockout or inhibition may represent a novel
strategy for mitigating Mn neurotoxicity.

10 General Conclusions

Because of its paradoxal effects on human health, Mn exposure or intake has been
studied for quite some time. Several mechanisms have been proposed for man-
ganism, such as (i) dopamine oxidation; (ii) glial toxicity, particularly in astrocytes;
7 Manganese in Health and Disease 219

(iii) oxidative stress; (iv) mitochondrial dysfunction; (v) alteration in the expression
of PD-related genes. However, there are many questions that have yet to be clarified.
Treatment approaches have also been investigated, focusing on the mechanisms
that were described in this chapter. Remarkably, although Mn intake is necessary for
the normal functioning of the organism, it is necessary to regulate its environmental
and occupational exposures, as once excessive exposure occurred, it may lead to
neurological dysfunction for which effective treatment has yet to be developed.

Abbreviations

ABC ATP-binding cassette


Abeta amyloid β
AD Alzheimer’s Disease
ALS amyotrophic lateral sclerosis
APLP amyloid β precursor-like protein
ATP adenosine 5′-triphosphate
BBB blood-brain barrier
BCB blood-cerebrospinal fluid barrier
CA3 cornu Ammonis, region 3
CNS central nervous system
CSF cerebrospinal fluid
Cyt c cytochrome c
DA dopamine
DAT dopamine transporter type
DMT divalent metal transporter
ERK extracellular signal-regulated kinase
ESADDI estimated safe and adequate dietary intake
FDA Food and Drug Administration
Fpn ferroportin
GFD green fluorescent protein
GLAST glutamate:aspartate transporter
GS glutamine synthetase
HD Huntington’s Disease
Hip huntingtin-interacting protein
hIPCs human induced pluripotent stem cells
HTT huntingtin
iNOS nitric oxide synthase (inducible form)
IPD idiopatic Parkinson’s disease
LRRK2 leucine-rich repeat kinase 2
MCT monocarboxylate transporter
MMT methylcyclopentadienyl manganese tricarbonyl
MNs motor neurons
MPT mitochondrial transition pore
220 Avila, Puntel, and Aschner

MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
MRI magnetic resonance imaging
NAAS National Academy of Sciences
NADH nicotinamide adenine dinucleotide reduced
NADPH nicotinamide adenine dinucleotide phosphate reduced
NMDA N-methyl-D-aspartate
NOS nitric oxide synthase
NRAMP natural resistance-associated macrophage protein
NRC National Research Council
OATP organic anion transporter polypeptide
PARK Parkinson protein
PD Parkinson’s disease
PET positron emission tomography
RDI reference daily intake
ROS reactive oxygen species
SLC39 solute carrier-39
SNpc substantia nigra pars compacta
SNpr substantia nigra pars reticulata
SOD superoxide dismutase
SPCA Ca2+/Mn2+ ATPase of the secretory pathway
SPECT single-photon emission computed tomography
TCA tricarboxylic acid
Tf transferrin
TfR transferrin receptor
TRPM7 transient receptor potential cation channel, subfamily M, member 7

Acknowledgments M.A. acknowledges funding by R01ES10563 and P30ES00267; D.S.A.


acknowledges support by FAPERGS (ARD11/1673-7) and CNPq (Universal 476471/2011-7).

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Chapter 8
Iron: Effect of Overload and Deficiency

Robert C. Hider and Xiaole Kong

Contents
ABSTRACT.............................................................................................................................. 230
1 Introduction.............................................................................................................. 231
1.1 Aqueous Iron Solution Chemistry.............................................................................. 231
1.2 Iron-Dependent Proteins. The Nature of the Iron Binding Sites................................ 235
1.2.1 Heme-Containing Proteins............................................................................. 235
1.2.2 Iron-Sulfur Proteins........................................................................................ 236
1.2.3 Non-heme, Non-sulfur, Iron-Dependent Enzymes......................................... 237
1.2.4 Transport and Iron Storage Proteins............................................................... 238
1.3 Iron Transport............................................................................................................. 240
1.3.1 Cellular Iron Transport................................................................................... 241
1.3.2 Regulation of Iron Metabolism...................................................................... 245
1.4 Iron Physiology.......................................................................................................... 246
1.4.1 The Role of Hepcidin..................................................................................... 247
2 Iron Deficiency and Anemia............................................................................... 248
2.1 Iron Requirements of Man......................................................................................... 248
2.2 The Influence of Anemia on Human Physiology....................................................... 250
2.3 Dietary Sources of Iron.............................................................................................. 252
2.4 Iron Fortification........................................................................................................ 253
2.5 Oral Iron Supplementation......................................................................................... 253
2.6 Anemia of Chronic Disease....................................................................................... 254
3 Systemic Iron Overload....................................................................................... 255
3.1 Non-transferrin Bound Iron....................................................................................... 256
3.2 Hereditary Hemochromatosis.................................................................................... 256
3.2.1 HFE Hemochromatosis.................................................................................. 257
3.2.2 Juvenile Hemochromatosis............................................................................ 257
3.2.3 Ferroportin Disease........................................................................................ 257
3.2.4 Treatment by Iron Chelation.......................................................................... 258

R.C. Hider (*) • X. Kong


Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building,
Stamford Street, London, SE1 9NH, UK
e-mail: robert.hider@kcl.ac.uk; k0839118@kcl.ac.uk

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 229
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_8, © Springer Science+Business Media Dordrecht 2013
230 Hider and Kong

3.3 Transfusional Siderosis.............................................................................................. 258


3.3.1 The Hemoglobinopathies............................................................................... 258
3.3.2 Myelodysplastic Syndrome............................................................................ 262
3.4 Hereditary Disorders of Mitochondrial Iron Overload.............................................. 263
3.4.1 Sideroblastic Anemia..................................................................................... 263
3.4.2 Friedreich’s Ataxia......................................................................................... 263
3.4.3 Glutaredoxin-5 Deficiency............................................................................. 264
3.5 Animal Models of Iron Overload............................................................................... 264
3.6 Genetic Screening for Thalassemia............................................................................ 264
4 Iron-Selective Chelators with Therapeutic Potential...................... 266
4.1 Design Features.......................................................................................................... 266
4.1.1 Metal Selectivity............................................................................................ 266
4.1.2 Ligand Selection............................................................................................. 267
4.1.3 Hexadentate, Tridentate, and Bidentate Iron(III) Complexes........................ 267
4.1.4 Critical Features for Clinical Application: Molecular Size
and Hydrophobicity........................................................................................ 270
4.1.5 Toxicity of Chelators and Their Iron Complexes........................................... 270
4.2 Orally Active Iron Chelators in Current Use............................................................. 272
4.2.1 Tridentate Chelators....................................................................................... 272
4.2.2 Bidentate Chelators........................................................................................ 274
4.2.3 Use of Iron Chelators to Treat Diseases Other than Thalassemia.................. 276
5 Neuropathology and Iron................................................................................... 277
5.1 Alzheimer’s Disease................................................................................................... 278
5.2 Parkinson’s Disease.................................................................................................... 279
5.3 Pantothenate Kinase-2 Deficiency............................................................................. 279
5.4 Macular Degeneration................................................................................................ 279
5.5 Potential of Iron Chelators for the Treatment of Neurodegeneration........................ 280
6 The Role of Iron Chelation in Cancer Therapy....................................... 281
7 Iron and Infection................................................................................................... 282
7.1 Tuberculosis............................................................................................................... 283
7.2 Malaria....................................................................................................................... 283
8 Overview and Future Developments............................................................. 284
Abbreviations................................................................................................................... 285
References......................................................................................................................... 286

Abstract  Iron is a redox active metal which is abundant in the Earth’s crust. It has
played a key role in the evolution of living systems and as such is an essential element
in a wide range of biological phenomena, being critical for the function of an enormous
array of enzymes, energy transduction mechanisms, and oxygen carriers. The redox
nature of iron renders the metal toxic in excess and consequently all biological
organisms carefully control iron levels. In this overview the mechanisms adopted by
man to control body iron levels are described.
Low body iron levels are related to anemia which can be treated by various forms
of iron fortification and supplementation. Elevated iron levels can occur systemi-
cally or locally, each giving rise to specific symptoms. Systemic iron overload
results from either the hyperabsorption of iron or regular blood transfusion and can
be treated by the use of a selection of iron chelating molecules. The symptoms of
many forms of neurodegeneration are associated with elevated levels of iron in
certain regions of the brain and iron chelation therapy is beginning to find an
application in the treatment of such diseases. Iron chelators have also been widely
8  Iron: Effect of Deficiency and Overload 231

investigated for the treatment of cancer, tuberculosis, and malaria. In these latter
studies, selective removal of iron from key enzymes or iron binding proteins is
sought. Sufficient selectivity between the invading organism and the host has yet to
be established for such chelators to find application in the clinic.
Iron chelation for systemic iron overload and iron supplementation therapy for
the treatment of various forms of anemia are now established procedures in clinical
medicine. Chelation therapy may find an important role in the treatment of various
neurodegenerative diseases in the near future.

Keywords  anemia • iron • iron chelators • iron overload • iron toxicity •


neurodegeneration

Please cite as: Met. Ions Life Sci. 13 (2013) 229–294

1  Introduction

1.1  Aqueous Iron Solution Chemistry

Iron, element 26 in the periodic table, is the fourth most abundant element in the
Earth’s crust. It has the maximum value of nuclear binding energy of all elements
and so tends to accumulate as a result of star based fusion reactions. It is 1000 times
more abundant than both copper and zinc. Its position in the middle of the first tran-
sition metal row of the periodic table leads to incompletely filled d orbitals and thus
the possibility of various oxidation states, the most common being (II), d6, (III), d5,
and (IV), d4. Iron(IV) is highly reactive and is restricted to intermediates formed
during enzyme catalytic cycles, whereas iron(II) and iron(III) are widely distributed
in solution, in the solid phase, and bound to proteins.
When iron salts (both iron(II) and iron(III)) are dissolved in water, they undergo
hydrolysis (equation 1), the process effectively being the deprotonation of
coordinated water molecules.

3+ 2+
OH2 OH2
H 2O OH2 H2O OH
FeIII FeIII + H+
H2O OH2 H2O OH2 (1)
OH2 OH2

   1    

With iron(II) (ferrous iron), the hydrolysis of micromolar solutions tends to take
place at pH values greater than 7.0 (Figure 1). In contrast, with iron(III) (ferric iron),
the charge density on the metal ion is much higher, thereby polarizing the water
232 Hider and Kong

Figure 1  Iron(III) and iron(II) speciation plots; [iron]total, 1 μM; KH2O solubility products, iron(III):
2.79 × 10–39 M4, iron(II): 4.87 × 10–17 M3.

molecule more effectively than iron(II). As a result, dissociation of the proton from
hexa-aqua iron(III) (1) occurs more easily, hydrolysis initiating at much lower pH
values, typically pH 2.0 (Figure 1). Whereas it is possible to prepare a 10 μM iron(II)
sulfate solution at pH 7.0, the maximum concentration of iron(III) sulfate at this pH
values will be less than 10–18M. Hydroxide ions are also able to crosslink hydrated
iron(III) species forming dimers and oligomeric anions (2, 3) [1], finally generating an
insoluble polymer of ferrihydrate [2]. The condensation reaction occurs rapidly at

neutral pH values, the initial phase of oligomer formation producing species between
16 and 30 iron atoms. In the ­presence of suitable ligands, for instance HEIDI (4) and
citrate (5), these complexes can be stabilized [3]. As a direct consequence of this
high affinity for oxygen donors, iron(III) must be coordinated by ligands in order to
maintain appreciable aqueous solubility at most physiological pH values.
8  Iron: Effect of Deficiency and Overload 233

CO2H CO2H
HO
N CO2H HO2C
HO CO2H

4 5

The two most common geometries for iron(II) and iron(III) complexes are
octahedral for 6-coordinate complexes (1) and tetrahedral for 4-coordinate
complexes (6). Octahedral stereochemistry is more frequently observed. As iron(III)
possesses a smaller ionic radii than iron(II) (0.65 and 0.78 Å, respectively) and a
higher net charge (3+ and 2+), the charge density on the iron(III) ion is much greater
than that on the iron(II) ion (0.59 versus 0.27 eÅ–2). This large difference leads to
a markedly different ligand selectivity. Iron(III) favors coordination by charged
oxygen atoms such as hydroxide, phenoxide, phosphate, and carboxylates, whereas
iron(II) favors interaction with aromatic amines, imidazoles, and sulfhydryl groups
(see Table 1 [4] for a direct comparison of affinity constants with a range of ligands).
As a result of this difference in ligand selectivity, the addition of complexing
agents has a major influence on the FeIII/FeII reduction potential. tris-Phenanthroline
iron(II) is exceptionally stable, shifting the reduction potential between Fe(Phen)33+
and Fe(Phen)32+ in favor of the iron(II) complex (E0 = +1.1 volt). In contrast, desfer-
rioxamine-B (7), an iron(III)-selective siderophore binds iron(III) much more

HO O H
S N N
O
Fe S
S CH3
S O N
+
H3N HN O HO O
N
OH

6 7

tightly than iron(II) resulting with a reduction potential of −0.45 volt. The range of
reduction potentials for iron complexes spans the range +1.1 v to −0.8 v and it is
significant to note that a large proportion of this range is covered by iron-­proteins
(+0.4 v to −0.5 v) [5].
The redox activity of the FeIII/FeII pair can, depending on the coordination of
the metal, reduce molecular oxygen to form the superoxide anion (equation 2)
and reduce hydrogen peroxide to the hydroxyl radical (equation 3), the latter
equation corresponding to the Fenton reaction. As superoxide can be further

Fe II + O2  Fe III + O·2− (2)



Fe III + H 2 O2  Fe III + OH − + OH· (3)

234 Hider and Kong

Table 1  Stability constants for iron complexes.


Iron(III) Iron(II)
Ligand log Keq N pFeIII log Keq N pFeII
Catecholate O 43.8 3 15.5 13.5 2 6.0

O
Phenanthroline 14.1 3 <14.6 21.0 3 11.5

N N
Oxalate −
O O 18.5 3 <14.6 5.2 2 6.0

O O−
Acetohydroxamate O 28.3 3 <14.6 8.5 2 7.6

O
N
H
Citric acid (5) 18.2 2 16.7 4.4 1 6.1
Nitrilotriacetate + 24.0 2 18.1 12.8 2 7.1
O3C H CO3
N

CO3

EDTA O2C O2C 25.1 1 23.5 14.3 1 12.4


+
NH
+
NH
CO2 CO2
Desferrioxamine-B (7) 30.5 1 26.6 9.5 1 6.0
Histidine O 4.7 1 <14.6 10.4 2 6.0

N O
+
NH3
HN
The affinity constants refer to the cumulative constants in the following equation:
K1 K2 K3
Fe + L  FeL + L  FeL 2 + L  FeL 3. pFe is defined as concentration of uncoordinated iron
when [Fe]total = 10–6 M, [Ligand]total = 10–5 M at pH 7.4. Data taken from [4]. For pFeIII, a value of
<14.6 indicates that the ligand has a lower affinity than H2O at pH 7.4. For pFeII, a value of 6.0
indicates that the ligand does not bind iron(II) at pH 7.4.
N: maximum number of ligands in complex

converted to the highly damaging hydroxyl radical, rapid generation of these


species is damaging to cells [6]. If the reduction potential of an iron complex is
either high (> +0.2 v) or low (< −0.2 v) then redox cycling does not occur readily
because, under the former condition iron(II) is preferentially stabilized and under
8  Iron: Effect of Deficiency and Overload 235

the latter, iron(III) is preferentially stabilized. However with a ligand possessing


a reduction potential close to zero, for instance EDTA (Eo = +0.12 v) both the
iron(III) and iron(II) complexes are relatively stable and so the coordinated iron
can readily redox cycle between the two oxidation states.
As with ligand selectivity, the kinetic lability of a metal complex is heavily
dependent on the charge density of the cation, the higher the value, the slower the
rate of exchange. One method adopted to compare the lability of a series of metal
ions is to monitor the rate of exchange of water molecules on the corresponding
aqueous ions (equation 4). For ions such as Ca2+, which have a low charge density,

Fe(H 2 O)36+ + H 2 O*  Fe(H 2 O)5 ( H 2 O * ) + H 2 O (4)



the water-exchange rates approach the diffusion limit (k = 5 × 108 s–1); this renders
Ca2+ ideal for a rapid signalling role in cells. The value for iron(II) is 4.4 × 106 s–1
and for iron(III) it is 1.6 × 102 s–1, iron(III) exchanging much more slowly than
iron(II). Clearly, iron(II) is kinetically labile and is able to exchange rapidly
between binding sites. In contrast iron(III) is much less labile, the rate of
exchange between multidentate ligands being particularly slow; for instance the
half-life of the donation of iron(III) from FeIII∙EDTA (1mM) to desferrioxamine-B
(7, 1 mM) is 42 h [5].

1.2  I ron-Dependent Proteins. The Nature of the Iron


Binding Sites

Man has over 500 iron-containing metalloproteins [2].

1.2.1  Heme-Containing Proteins

In this protein class, iron is bound to a porphyrin molecule by the 4 aromatic nitrogen
atoms in octahedral fashion, the axial ligands are typically provided by the protein,
but can also provide a binding site for gaseous molecules such as dioxygen
(Figure 2). There are three main classes of heme proteins: oxygen carriers, as typified
by hemoglobin and myoglobin; activators of molecular oxygen, as typified
by peroxidases, catalases, and cytochrome P450s. Peroxidases and catalases oxidize
a range of compounds using H2O2 as a substrate; cytochrome P450s hydroxylate
a wide range of xenobiotic and endogenous substrates, including steroids, using
­oxygen as a substrate. Electron transport proteins, as typified by cytochromes a, b,
and c, are components of the respiratory chain, they accept electrons from reduced
donor molecules, transferring them to appropriate acceptors, thereby linking
substrate oxidation to cytochrome c oxidase [2].
236 Hider and Kong

Figure 2  Heme center


in hemoglobin.

Figure 3  2Fe-2S and 4Fe-4S


clusters:  S;  Fe.

1.2.2  Iron-Sulfur Proteins

In this protein class, iron is bound to sulfur in tetrahedral fashion. Two predominate
core structures are found in man, 2Fe-2S clusters and 4Fe-4S clusters (Figure 3).
Many of the proteins that contain these clusters are involved in electron transfer.
Thus ferredoxins are located in electron transfer chains and can also act as donors
to enzymes where they exchange electrons between redox centers which are
­physically separated. The valence state of 2Fe-2S clusters oscillates between 2+ and
3+ and with 4Fe-4S clusters, between 1+ and 2+. The activity of these proteins is
not limited to one-electron transfer and iron-sulfur clusters are found in dehydratases
and S-adenosylmethionine-dependent enzymes [2].
8  Iron: Effect of Deficiency and Overload 237

1.2.3  Non-heme, Non-sulfur, Iron-Dependent Enzymes

These enzymes fall into two broad categories, those containing either mononuclear
or dinuclear iron.
A large number of mononuclear non-heme iron enzymes are involved with the
activation of dioxygen to catalyze the hydroxylation of a wide range of substrates.
With the α-oxoglutarate-dependent enzymes, iron(II) is bound to the enzyme via
two histidines and an aspartate residue (Figure 4) and is involved with the hydrox-
ylation of amino acids and the demethylation of histones [6]. Pterin-dependent
hydroxylases fall into a related enzyme group and these are responsible for the
hydroxylation of aromatic amino acids [6]. The dinuclear iron proteins typically
contain a four helix bundle protein fold surrounding a (μ-carboxylato)diiron core.
The two iron atoms are typically separated by less than 0.4 nm and have one or more
bridging carboxylate ligands, together with bridging oxo or hydroxo ligands [7].
The diiron center is bound to the protein via imidazole and carboxylate functions
(Figure  5). Such centers are found in the H chains of ferritin, where iron(II) is
oxidized to iron(III) and deposited in a core of ferrihydrite and in ribonucleotide
reductase where the diiron center generates a tyrosyl radical which is utilized in the
conversion of ribosides to deoxyribosides [7].

Figure 4  Proposed mechanism of Fe(II)- and 2-oxoglutarate-dependent dioxygenase-catalyzed


reactions. In the example of asparaginyl hydroxylation, the reaction proceeds through a radical
mechanism involving an iron-oxo intermediate, which adopts both iron(III) and iron(IV) oxidation states
during the process. The decarboxylation of 2-oxoglutarate occurs simultaneously. Reproduced
with permission from [6]; copyright 2007, Nature Publishing Group.
238 Hider and Kong

Figure 5  The metal binding


sites of cytoplasmic and
nuclear iron(II)-dependent
enzymes. (a) ribonucleotide
reductase; (b) ferroxidase
center on the H-ferritin
subunit.

1.2.4  Transport and Iron Storage Proteins

In view of the potential redox activity of many simple iron complexes, the levels of
such labile iron forms are maintained at carefully controlled limits, both extracel-
lularly and intracellularly. In the extracellular compartments iron is almost entirely
transported between cells, tightly bound to transferrin. Within cells, ferritin acts as
an iron sink and is in equilibrium with the labile iron(II) pool, which depending on
the cell type, is limited to the range 0.5–2 μM [8].
Serum transferrin is a globular protein which possesses two high affinity iron(III)
binding sites (Figure 6a) [9]. Each site consists of two tyrosines, one histidine, one
aspartate, provided by the protein, together with a synergistically bound carbonate
anion. These ligands create an octahedral coordination sphere (Figure 6b) with a high
selectivity for iron(III) and an affinity of 10–20 M–1 (conditional stability constant at
pH 7.4) [9]. Serum transferrin is typically circulating in the blood at a level of 35 μM,
with between 25 and 35% of the iron binding sites occupied. Under such conditions
the levels of the so called non-transferrin bound iron are vanishingly small. Transferrin
delivers iron to cells which express transferrin receptors [10,11].
Ferritin is a protein composed of 24 homologous subunits, designed to create a
large aqueous enclosure for the storage of iron atoms. Each ferritin molecule can
accommodate up to 4500 iron atoms [12] (Figure 7). Ferritin is the major iron
storage protein for all mammalian tissues and consists of a mixture of two subunits
referred to as L- and H-subunits. In general, L-rich ferritins are characteristic of
8  Iron: Effect of Deficiency and Overload 239

Figure 6 (a) Human serum transferrin [9]; (b) coordination sphere of ferric ion in the N-lobe
binding site of human serum transferrin [11]. Reproduced with permission from [25]; copyright
2012, Elsevier.

organs storing iron for long periods (e.g., liver) and these molecules tend to possess
a high iron content. H-rich ferritins, which are characteristic of the heart, have a
major role in iron detoxification, they possess a ferroxidase site (see Figure 5b)
and tend to contain a lower iron content. Iron(II) enters the various pores of the
oligomeric structure (Figure 7b) and is rapidly oxidized to iron(III) either by the
growing core of ferrihydrite or at the ferroxidase site of the H-subunit [13]. Ferritin
molecules are constantly being synthesized and removed to lysosomes, where they
are degraded, thereby releasing the entrapped iron [14]. This iron enters the cytosolic
labile iron pool under the tight control of iron(II) transport proteins, which are
located in the lysosomal membrane.
240 Hider and Kong

Figure 7  The three-dimensional structure of the ferritin subunit and 24-mer. (a) 24-meric human
H-chain ferritin molecule viewed down the four-fold symmetry axis (PDB 1FHA). (b) The crystal
growth mechanism of core formation in ferritin [13]. Reproduced with permission from [13];
copyright 1981, Elsevier.

1.3  Iron Transport

In mammalian cells there is a labile iron pool which supplies iron to the mitochon-
drion for incorporation into heme and iron-sulfur cluster proteins (Figure 8). It is
also the source of iron for many cytoplasmic iron-dependent enzymes. As non-­
coordinated iron salts can catalyze the formation of toxic oxygen-containing radi-
cals, the levels of this labile iron pool must be tightly controlled. Cells must be able
to sense iron levels and regulate homeostasis in such a manner as to maintain non-
toxic levels. The concentration of this iron pool is determined by the rates of iron
uptake, utilization for incorporation into iron proteins, storage in ferritin and iron
export from the cell (Figure 8). The concentration of the pool falls into the 0.5–2
μM range and a likely structure is iron(II) bound to a single molecule of glutathi-
one (8) [15]. This structure ensures protection against autoxidation of iron(II) and
offers a mechanism for the introduction of iron into protein-bound iron sulfur clus-
ters via glutaredoxins [8]. The selection of iron(II) in preference to iron(III) for this
role is logical in view of the kinetic lability of iron(II) being 5 × 104 times higher
than that of iron(III).
8  Iron: Effect of Deficiency and Overload 241

Figure 8  Cytoplasmic iron fluxes. DMT1, divalent metal transporter-1; IRP, iron responsive
protein. FBXL5, F-box and leucine-rich repeat protein 5.

O
H
O
N N
H −
− O O
O +
S H3N

H2O OH2 O
II
Fe
H 2O OH2
OH2

1.3.1  Cellular Iron Transport

Within the circulation, transferrin-bound iron is the principle iron source for all
mammalian tissues; although under iron overload conditions, transferrin becomes
saturated and non-transferrin bound iron appears in the serum and is taken up by
cardiac and endocrine tissue by uncharacterized transporters.
242 Hider and Kong

1.3.1.1  Transport of Iron-Loaded Transferrin

There are at least three classes of transferrin receptors in mammalian cells; TfR1,
which is expressed on many cell types, TfR2, which is restricted to hepatocytes and
erythroid cells, and a third type, only located in epithelial kidney cells. The basic
mechanism of iron transport is the same for each class. Fe2-transferrin-TfR com-
plexes bind to clathrin-coated pits which are internalized into endosomes. The
endosome lumen is acidified to pH 5.5, whereupon both transferrin and TfR undergo
conformation changes to release transferrin-bound iron. A ferrireductase reduces
the released iron(III) to iron(II), which is a substrate for the divalent metal-ion
transporter 1 (DMT1) (Figure 9). Both apo transferrin and TfR are recycled to the
cell surface. It has been estimated that a transferrin molecule experiences over 200
such cycles during its life time [10].

1.3.1.2  Absorption of Dietary Iron

Inorganic iron present in the diet is predominantly iron(III) and this is reduced
on the surface of duodenal enterocytes by the ferrireductase DCYTB (Figure 10).
The resulting iron(II) enters the cell through the divalent metal transporter

Figure 9  Macrophage iron fluxes.


8  Iron: Effect of Deficiency and Overload 243

Figure 10  Duodenal enterocyte located on intestinal villae. Associated with the unidirectional
movement of iron.

which is proton-­coupled [16]. DMT1 has been shown to transport other divalent
metals, such as Zn(II), Mn(II), and Co(II), but the physiological relevance of this
property is unclear.
Heme iron is transported from the lumen of the duodenum by the transport
protein HCP1 [17]. Once absorbed, heme is degraded by heme oxygenase to form
iron(II) (Figure 10). Thus both DMT1 and HCP1 supply iron to the labile iron pool
(possibly as FeII. Glutathione, 8).
244 Hider and Kong

1.3.1.3  Mitochondrial Iron Transport

Mitochondria are major sites of iron consumption in mammalian cells as they


accommodate the enzymes involved in heme and iron-sulfur cluster synthesis. The
uptake of iron is dependent on membrane potential and facilitated by the transport
proteins mitoferrin-1 and mitoferrin-2 [18]. Mitoferrin-2 is widely distributed in
different tissues, whereas mitoferrin-1 is restricted to erythroid cells. Ferrochelatase,
the terminal enzyme in heme synthesis receives iron(II) directly from the inner
mitochondrial membrane and has been demonstrated to form a oligomeric complex
with mitoferrin-1 [19]. It has been suggested that frataxin shuttles iron(II) from the
same membrane, directing iron to enzymes involved in iron sulfur cluster
synthesis.

1.3.1.4  Ferroportin-Mediated Iron Efflux

Duodenal enterocytes, macrophages, hepatocytes, and CNS cells release iron in a


controlled manner by use of the iron efflux transporter ferroportin (FPN1). This
transporter was independently discovered by three groups and was originally termed
Ireg1 [20], ferroportin [21], and MTP [22]. The term ferroportin has been widely
adopted. The gene encodes a highly hydrophobic membrane protein with 10–12
transmembrane spanning domains, which bears little sequence identity with any
other transporter family. Indeed, ferroportin is specific for iron(II) and is the only
iron exporter identified to date. Ferroportin is located on the basolateral side of
duodenal enterocytes and in the cytoplasmic membranes of both macrophages and
hepatocytes (Figure 10).
For efficient transfer of freshly effluxed iron(II) to apo transferrin, ferroportin is
frequently closely coupled with a multi-copper ferroxidase, for instance, hephaestin
or ceruloplasmin, whereupon the iron(II) is rapidly oxidized to iron(III), without the
generation of oxygen-containing free radicals.

1.3.1.5  Iron Metabolism Facilitated by the Macrophage

Macrophages acquire iron via the transferrin receptor, hemoglobin, via


­erythrophagocytosis and the haptoglobin receptor and heme, sequestered by hemo-
pexin via the lipoprotein receptor, CD91 (Figure 9). Changes in the surface glyco-
proteins of red blood cells take place as they age. These modified proteins are
recognized by ­receptors on the macrophage surface and trigger phagocytosis.
Haptoglobin and hemopexin scavenge hemoglobin and heme respectively.
Both are released in the circulation as a result of hemolysis (Figure 9) [23]. On
entry to the cytoplasm, the heme moiety is degraded by heme oxidase and the
resulting iron(II) enters the labile iron pool. On exposure to the lumen of a pha-
golysosome, hemoglobin is degraded, releasing heme which is able to penetrate
the membrane also gaining access to heme oxidase. Iron(II) is exported from the
macrophage via ferroportin and the effluxed iron(II) is oxidized to iron(III) by
ceruloplasmin.
8  Iron: Effect of Deficiency and Overload 245

1.3.2  Regulation of Iron Metabolism

Iron homeostasis is regulated by balancing iron uptake with intracellular storage


and use. In mammalian cells, this is largely achieved at the level of protein syn-
thesis. Regulatory sequences in mRNA molecules are located in the noncoding
regions situated at 5’ and 3’ extremities of the coding regions. Location in the
5’ region is usually associated with initiation of translation, whereas location in
the 3’ region is associated with enhanced mRNA stability [24]. The regulatory
sequences relating to iron metabolism are termed iron responsive elements (IRE)
and their structural features are well established [25]. Iron responsive proteins
(IRP) bind to IREs and thereby control the translation of the related mRNA. An
IRE consists of an upper stem of five base-paired residues that assume a helical
structure which is separated from a lower stem of base-paired structures by an
unpaired cytosine, thereby creating a bulge in the structure (Figure 11). At the
top of the structure is a six membered loop which includes the sequence AGU.

Figure 11  The iron responsive element is an RNA stem-loop structure. The upper and lower
stems are composed of base-pairs which are held in a helical conformation. In the six-membered
loop AsafNAsafdasdasdasd, the C at position 1 of the loop forms a base-pair with the G at position
5. This C-G base-pair structures the loop, allowing the A, G, and U residues to form a region that
can form multiple hydrogen bonds with proteins. The upper and lower stems are separated by an
unpaired “bulge” C that confers flexibility on the structure by interrupting the helix. Reproduced
with permission from [25]; copyright 1997, Elsevier.
246 Hider and Kong

Figure 12  Mechanism of iron-regulatory protein 1 (IRP1) in the translational regulation of


(a) transferrin receptor and (b) ferritin.

Table 2  Known genes with IRE regions.


Gene name IRE position Function
H and L Ferritin 5’UTR Iron storage
Erythroid δ-amino-levulinate synthase 5’UTR Erythroid heme synthesis
Succinate dehydrogenase 5’UTR TCA cycle
m-Aconitase 5’UTR TCA cycle
Ferroportin 5’UTR Iron efflux
DMT1 3’UTR Iron uptake
Transferrin receptor 3’UTR Iron uptake
Cell cycle control gene (CDC14A) 3’UTR Cell cycle

These residues constitute the major recognition feature for IRPs, both the bulged
C and the AGU region fit into binding pockets of IRP molecules. The affinity
constant for this interaction falls in the range 10–30 pM [2,26].
In man there are two IPRs and when iron levels are low they both bind to mRNA.
When iron levels are adequate, IRP1 acquires a [4Fe-4S] cluster converting it to the
enzyme aconitase (Figure 8) and IRP2 is degraded by proteasomes. The binding of
a IPR to the transferrin receptor mRNA enhances the stability of RNA and hence
increases protein synthesis (Figure 12a). This in turn promotes receptor mediated
iron uptake. The effect on ferritin mRNA is to reduce protein synthesis (Figure 12b),
thereby increasing the availability of intracellular iron. A number of proteins with a
central role in iron metabolism are controlled in analogous fashion (Table 2).

1.4  Iron Physiology

While the body levels of other dietary metals can be regulated by excretion in the
feces and urine, humans do not possess the ability to remove excess iron from the
8  Iron: Effect of Deficiency and Overload 247

Figure 13  Iron homeostasis


in a normal adult man. Loss
is uncontrolled (epithelial
desquamation and in women,
menstruation).

body. Consequently, several proteins have evolved which regulate iron homeostasis,
their location being primarily in duodenal endothelial cells, hepatocytes and
­reticuloendothelial macrophages. It is essential for these cells to function in concert.
Regulation of total body iron is achieved by feedback mechanisms that operate on
iron absorption. Approximately 4 mg of iron circulates bound to transferrin (this is
about 0.1% of total body iron). Seventy five percent of body iron is in the form of
heme proteins, mainly hemoglobin, the remainder being located in cells either in
non-heme enzymes or ferritin. Although only 1–2 mg of iron is absorbed each day
by normal individuals, 20 mg is transferred daily to the bone marrow for erythropoi-
esis and is efficiently recycled by macrophages (Figure 13). There is no active
excretion mechanism for iron; loss is uncontrolled and results mainly from bleeding
and epithelial desquamation. It has been estimated that the average iron atom
­survives in a human for approximately ten years.

1.4.1  The Role of Hepcidin

Hepcidin is a 25 residue peptide containing four disulfide bridges (Figure 14). It has


an amphipathic nature and possesses a net positive charge. The structure is highly
conserved within mammals and fish [27]. Removal of the N-terminal pentapeptide
leads to loss of activity. Hepcidin regulates iron export from a range of cells by
248 Hider and Kong

binding to ferroportin, causing internalization and degradation of the iron-efflux


protein. The mechanism of this internalization is similar to that of other receptors
and involves hepcidin-induced phosphorylation and subsequent ubiquitination
of ferroportin. Thus, under conditions of high hepcidin serum levels, ferroportin
located in the duodenal epithelial cells and macrophages will be internalized and
such cells are unable to efflux iron. Under conditions of low hepcidin serum levels,
the opposite holds and thus, high iron fluxes enter the serum originating from both
duodenal epithelial cells and macrophages.

Figure 14  Human hepcidin.

Hepcidin is produced primarily in the hepatocyte, the synthesis being regulated


by both the iron and the inflammatory status of the organism. High concentrations
of fully saturated transferrin trigger the involvement of bone morphogenetic protein
(BMP), which in turn facilitates the phosphorylation of the transcription factor
SMAD4. SMAD4 controls hepcidin synthesis. Hepcidin synthesis is also regulated
by erythropoietic signals such as GDF15. In addition, hepcidin synthesis is regulated
by inflammatory stimuli, thus, interleukin 6 activates the JAK/STAT signaling
pathway which again stimulates the hepcidin promoter [28].
Thus, under conditions where man is replete in iron (high saturation of transferrin),
hepcidin synthesis is increased and there is an overall retention of iron by the
duodenum, hepatocyte, and macrophage. The converse also holds, thus conditions,
where a low transferrin saturation exists, will lead to the inhibition of hepcidin
production and a concomitant enhanced release of iron from the same group of
cells. Significantly, elevation of interleukin 6 occurs when man is infected by micro-
organisms and parasites. Such an action triggers hepcidin synthesis, reducing the
extracellular availability of iron and thereby enhancing the host defense [29].

2  Iron Deficiency and Anemia

2.1  Iron Requirements of Man

Healthy term infants with a normal birth weight are born with high hemoglobin
levels and sufficient iron stores to support growth for the first six months of life [30].
Babies are born with body iron loads, typically in the region of 80 mg/kg; the
corresponding value for adult men is 55 mg/kg. Thus, the low iron content in human
milk does not present a problem to the suckling infant and renders infection to be
less of a problem.
8  Iron: Effect of Deficiency and Overload 249

Figure 15  Daily iron requirements. Information taken from [31].

Preterm and low birth weight infants exhaust their iron stores at an earlier age
than normal children and thus, there is a greater chance of anemia with such children.
The transfer of iron from maternal blood to the fetus occurs mainly in the third
trimester (Figure 15). Thus premature infants are born with lower iron stores. At six
months, solids and cereals should be given to babies, with the goal of beginning
to provide appreciable levels of iron in the diet. Iron requirement for the rest of
life remains close to 1 mg/day (Figure 15) [31], only exceeding this requirement
during the adolescent growth spurt, with menstruating females and during pregnancy.
Iron requirements increase dramatically during the second and third trimesters of
pregnancy, reaching a requirement of 5–6 mg/day. Iron requirement during lactation
does not increase. It is estimated that most p­ regnant women in developing coun-
tries and between 30 and 40% of pregnant women in developed countries are
iron-deficient [32].
Blood donations (500 mL/year) require an additional 0.6–0.7 mg iron per day, a
significant addition to the normal adult requirement of 1.1 mg/day. In developing
countries intestinal parasitic infections can cause appreciable blood loss which
in turn leads to increased iron requirements. A list of causes of iron deficiency is
presented in Table 3.
250 Hider and Kong

Table 3  Causes of iron Inadequate dietary iron intake


deficiency. Single food diets in infancy
Dieting, fasting, and malnutrition
Diet with high content of inhibitors of iron absorption
Antacid therapy
Increased iron requirements
Rapid growth during adolescence
Menstruation
Pregnancy
Erythropoietin therapy
Trauma
Increased iron loss
Bleeding from gastrointestinal and genitourinary tracts
Intravascular hemolysis
Parasitic infestations
Intense exercise
Blood donation
Decreased absorption of iron
Diseases of the stomach and small intestine
Celiac disease
Infection by Helicobacter pylori
Crohn’s disease
Genetic defects
Mutation of DMT1 gene
Mutation of glutaredoxin-5 gene

2.2  The Influence of Anemia on Human Physiology

After birth, the erythron has the priority for transferrin-bound iron as compared to
other tissues. In general, erythrocyte production is unperturbed until the body iron
stores are depleted. When the stores become limiting, the saturation of transferrin
decreases and patients then show signs of iron-deficient erythropoiesis; protopor-
phyrin and zinc protoporphyrin appear in the erythrocytes and these changes are
followed by the onset of microcytosis.
Iron deficiency is known to be associated with decreased physical activity as
demonstrated by exercise tolerance and work performance [33]. These effects have
been thoroughly investigated in rodent models. As hemoglobin levels decrease,
so do those of myoglobin and the cytochromes. Thus, there is diminished oxygen
transport in the blood and diminished oxygen diffusion in muscle. There is also a
decrease in the mitochondrial iron-sulfur content which is associated with a decrease
of mitochondrial oxidative capacity. These changes have a major influence on
physical activity; the Harvard Stop Test score registers impaired performance even
with mild anemia (Figure 16a) [34], as does the worktime in workers performing a
sub maximal exercise test (Figure 16b) [35].
8  Iron: Effect of Deficiency and Overload 251

Figure 16  (a) Harvard Step Test score among a group of Guatemalan agricultural laborers [34].
(b) Work time in a group of Indonesia agricultural workers performing a submaximal exercise test
as a function of severity of anemia [35].

The adverse influence of anemia on mental behavior is more difficult to


demonstrate, but as 25% of the body oxygen is utilized by the brain, adverse effects
can be anticipated. Iron is required for the myelination of the spinal cord, for the
synthesis of chemical transmitters, particularly dopamine and serotonin and for
mitochondrial function. The influence of anemia on mental development has been
reviewed and details from 18 studies described [36]. Taken together, they support the
252 Hider and Kong

hypothesis that iron deficiency is associated with developmental delay. With longer
term intervention studies, again there is evidence of a causal link between iron
deficiency anemia and poor developmental performance [36].

2.3  Dietary Sources of Iron

There are two major classes of iron in the diet, heme iron and non-heme iron. The
former is derived from hemoglobin, myoglobin, and cytochromes and is found in
animal sources such as red meat and seafood. Non-heme iron is derived mainly
from plant sources such as lentils, beans, rice and cereals (Table 4). The absorption
of heme iron is relatively efficient, ranging from 15 to 35%; whereas the absorption
of non-heme iron is less efficient (2–20%) and can be inhibited by the presence of
other components in the diet. Heme iron, which is strongly chelated by protopor-
phyrin IX, is absorbed by the HCP-1 transporter (Section 1.3.1.2), and is not sub-
ject to competition from other iron-binding compounds which are present in the
lumen of the gut. In contrast, non-heme iron is absorbed in the iron(II) state by
DMT-1 (Section 1.3.1.2) and so is susceptible to the presence of iron chelating
ligands in the gut lumen.
Compounds such as phytates, polyphenols, and tannins, which are widely
distributed in plant foods, are capable binding to both iron(II) and iron(III), rendering
the iron non-bioavailable [37]. When such compounds bind iron(II), they autoxidize
the metal to iron(III). Phytate is present in legumes, rice, and cereals. Tannic acid is
present in tea and coffee. Their presence can inflict a powerful inhibitory influence

Table 4  Iron content in food.


Fe (mg per serving)
Food Heme Non-Heme % of daily requirement (male)
Liver sausage 85 g 9.4 – 52
Beef steak 85 g 3.2 – 18
Turkey 85 g 2.3 – 13
Chicken breast 85 g 1.1 – 6
Halibut 85 g 0.9 – 5
Iron fortified cereal 236 mL – 18 100
Soybean 236 mL – 8.8 48
Lentils 236 mL – 6.6 36
Spinach 236 mL – 6.4 35
White rice 236 mL – 3.2 18
Raisins 236 mL – 2.7 15
Egg 1 – 1 6
Brown bread 1 slice – 0.9 5
Taken from the US Department of Agriculture database.
236 mL ≡ 1 standard cup
8  Iron: Effect of Deficiency and Overload 253

of non-heme iron absorption. Conversely, the presence of vitamin C (ascorbic acid)


enhances the absorption of non-heme iron, the reduced form of the vitamin reducing
iron(III) to the more bioavailable iron(II) (equation 5).

HO O O O
HO O
+ Fe3+ + Fe2+ + H+
HO OH HO (5)
O
O O
AscH Asc

A typical iron intake for an adult male is 16–18mg/day, whereas for women it is
lower, typically 12 mg/day. Thus, for most situations there should be sufficient
iron in the diet, although the consumption of energy-restricted diets or those rich in
poorly bioavailable iron can contribute to inadequate iron absorption. Selected food
sources and their iron contents are presented in Table 4.

2.4  Iron Fortification

Iron fortification of commonly used foods is a practical and cost-effective strategy


to improve the iron nutrition of a large population. The concept has been widely
adopted in developed countries over the past 50 years, typically with the iron forti-
fication of breakfast cereals. The efficacy of iron fortification in the developing
countries has not been so successful and improved guidelines and materials are
required. The following iron preparations have been used; ferrous sulfate (promotes
oxidation of fats and so leads to rancidity), ferrous fumarate (likely to be similar to
ferrous sulfate), elemental iron (finely divided metallic iron), ferric pyrophosphate
and ferric EDTA. The bioavailability of the ferric salts and elemental iron is lower
than that of ferrous sulfate.
The World Health Organization (WHO) has issued recommendations for a range
of iron compounds to be used and the level to be adopted in developing countries.
Good efficiency has been reported from South Africa, Morocco, Vietnam, Chile,
Thailand, and Kenya [32]. In principle, adverse effects are possible with respect to
iron overloading diseases and in regions with a high prevalence of malaria, however,
this is less likely to occur with iron fortification when compared with iron supple-
mentation (Section 2.5).

2.5  Oral Iron Supplementation

Another approach to control iron deficiency is iron supplementation, which unlike


fortification delivers a relatively large dose of iron in the absence of food. The majority
of patients with iron deficiency anemia respond to oral iron therapy. A wide range of
iron preparations are available for oral application, the majority being iron(II) by
254 Hider and Kong

virtue of its greater bioavailability. Thus ferrous sulfate, ferrous fumarate, and ferrous
gluconate are widely marketed, indeed ferrous sulfate was introduced as a therapy
for anemia by Blaud in 1832 [38]. By virtue of its low cost and good bioavailability
ferrous sulfate is still widely used for oral therapy, however, its use is associated
with a range of gastrointestinal side effects that frequently lead to poor compliance.
There are “slow release” formulations available which can reduce side effects, but
typically they also reduce iron absorption when compared to the parent compound.
Another problem with iron(II) salts in oral formulations is that they can inhibit the
absorption of a wide range of other therapeutics, including antibiotics, L-dopa, and
thyroxine [39].
Up to 40% of patients have symptoms associated with the oral administration of
iron(II) salts. These symptoms can be avoided by utilizing iron(III) complexes,
although the bioavailability of simpler iron(III) salts is lower than the analogous
iron(II) salts due to the extreme low solubility of iron(III) hydroxide (Section 1.1).
However, if the iron(III) complex is sufficiently stable and water-soluble, then the
bioavailability can reach that achieved by ferrous sulfate. Iron(III) maltol [40] and
iron(III) polymaltose [41] are two such compounds reported to possess excellent
bioavailability, although there is controversy as to the effectiveness of such com-
pounds [42]. Presumably the iron complexes are reduced by DCYTB, thereby
generating iron(II) which is the main substrate for DMT1 (Section 1.3.1.2).

2.6  Anemia of Chronic Disease

The anemia of chronic disease is an acquired disorder of iron homeostasis which


may be associated with infection, malignancy, organ failure or trauma (Table 5).
The anemia is typically mild to moderate and the erythrocyte size being close to

Table 5  Incidence of anemia in various disease groups.


Disorder Percentage of cases with anemia
Infection
Tuberculosis 20
HIV 10–35
Trauma
Heart transplant 70
Intensive care 40
Autoimmune
Rheumatoid arthritis 15
Inflammatory bowel disease 10–70
Cancer
Palliative care 40–75
Chronic kidney disease 40
Heart failure 30
Aging (>65 years) 10–20
Data adopted from [43].
8  Iron: Effect of Deficiency and Overload 255

normal. Macrophages, which normally recycle iron (Section 1.3.1.5), retain it and


intestinal iron absorption is reduced under these pathological conditions [43]. An
association with proinflammatory cytokines was suspected for many years and it
has recently been established that this modified cellular behavior is associated with
elevated levels of hepcidin (Section 1.4.1) [44,45]. Expression of hepcidin that is
inappropriately high for the normal body iron status results in interruption of intestinal
iron absorption and macrophage iron recycling. Thus, there is less iron available for
erythropoiesis. IL-6 is the cytokine that induces hepcidin expression [45] and its
levels have been shown to correlate with the severity of anemia in rheumatoid
arthritis, cancer, and aging [43].
Treatment options for patients with anemia of chronic disease depends on the
severity of the anemia and since the anemia is derived from the innate immune
response to infection, there is little clinical support for the use of exogenous iron or
erythroid-stimulating agents, such as erythropoietin. In principle it is possible to
mobilize macrophage iron by chelation and to redistribute it to the transferrin iron pool
[46,47], however, to date no such compounds have been introduced into the clinic.
In contrast to the above general statements, in the specific cases of autoimmune
disorders and end-stage renal disease, iron treatment in conjunction with erythroid-­
stimulating agents can be effective [48,49]. With the use of erythropoietin, the
rate of iron mobilization from stores is unable to match the iron requirements of
the expanding red cell mass and consequently iron supplementation is required.
In many patients oral supplements will not provide an optimal supply of iron and
such patients are frequently intolerant to oral iron therapy, in such cases
intravenous iron preparations can be introduced. These preparations consist of a
carbohydrate ligand and either ferric hydroxide or ferric oxide cores. They are
designed to be non-­immunogenic and to be efficiently removed from the circulation
by macrophages, which catabolize the complex and render the iron available
for apo transferrin. Iron dextrin was the first such preparation to be used clini-
cally, but it has been associated with a wide range of side effects. However,
there are now much improved formulations available [50,51]. An advantage of
some of these preparations is that doses up to 1 g of iron can be administered in a
single infusion [51].

3  Systemic Iron Overload

Iron overload can be present in one of two general forms. Firstly, in situations where
erythropoiesis is normal but the plasma iron level exceeds the iron binding capacity
of transferrin (e.g., hereditary hemochromatosis); the resulting non-transferrin
bound iron (NTBI) is deposited in parenchymal cells of the liver, heart, and endocrine
tissues [52]. The second type results from increased catabolism of erythrocytes
(e.g., transfusional iron overload). In this situation iron initially accumulates in
reticuloendothelial macrophages, but subsequently spills over into the NTBI pool
and parenchymal cells. Parenchymal iron deposition leads to organ damage.
256 Hider and Kong

3.1  Non-transferrin Bound Iron

When transferrin is fully saturated, any iron entering the blood forms part of the
NTBI pool. NTBI, unlike transferrin, lacks an address system and tends to be
absorbed by highly vascular tissue, such as the heart and endocrine organs. As iron
accumulates in these organs, redox cycling leads to the generation of toxic oxygen
radicals. Transferrin also contributes to defence against infection by depriving
microorganisms of an iron supply [29]. Conversly, the presence of NTBI presents a
weakness, rendering the host more susceptible towards infection.
There are many small molecules in the blood which are capable of binding
iron(III), the principle oxidation state of iron in the serum. These include acetate,
phosphate, and citrate. Of these, citrate has the highest affinity for iron(III) [53] and
there are two dominating complexes under the conditions provided by serum,
[Fe(citrate)2] (9) and [Fe3(citrate)3] (10) [53,54]. At an iron concentration of 1 μM,
complex (9) dominates at physiological citrate levels (typically 100 μM), whereas
at a level of 10 μM iron the oligomeric iron citrate (10) begins to dominate [53].
Another important iron binding component of the serum is albumin, which is present
at a relatively high concentration (600 μM) [55,56]. Which of these three forms of
NTBI gains facile entry into parenchymal cells has not been established.

− O O
O
O O
O
O
O
O O O O
O O
Fe O O Fe Fe O
O O
O O O O
O O Fe
O
− OO
O
O− O O O−
O O

9 10

3.2  Hereditary Hemochromatosis

The term hereditary hemochromatosis covers a heterogeneous group of iron overload


disorders (Table 6). They are linked to gene mutations associated with hepcidin,
leading to low levels of hepcidin. This in turn leads to the hyperabsorption of dietary
iron, up to 8–10 mg per day. Over a period of time transferrin saturation gradually
increases from the normal value of 30% to 100%, which is associated with the
appearance of non-transferrin bound iron and the associated inappropriate tissue
distribution of iron [57]. In hereditary hemochromatosis, hepatic iron overload
can lead to fibrosis, cirrhosis, and carcinoma of the liver [58] and to non-­hepatic
symptoms including cardiomyopathy, diabetes, hypogonadism, and arthritis [59].
In contrast, the CNS does not develop iron overload.
8  Iron: Effect of Deficiency and Overload 257

Table 6  Hereditary iron overload disorders.


Type Gene Onset Clinical expression
HFE hemochromatosis 1 HFE Late Hepatic
Juvenile hemochromatosis 2A HFE2 Early Cardiac and endocrine
Juvenile hemochromatosis 2B HAMP Early Cardiac and endocrine
TfR2 hemochromatosis 3 TFR2 Late Hepatic
Ferroportin disease 4 SLC40AI Late Hepatic

The above symptoms tend to develop late in life, but the disease can be managed
by phlebotomy (each 500 mL of blood contains 250 mg of iron), the earlier the
diagnosis the better. Indeed, early diagnosed patients experience a normal life span.
In addition to phlebotomy patients should avoid iron supplementation and limit
consumption of red meat and alcohol.

3.2.1  HFE Hemochromatosis

HFE hemochromatosis is the most frequent form of hemochromatosis [60] and


there is a particularly high prevalence among North European caucasians (1 in 200).
The discovery of HFE was the first cloning success to contribute to our understanding
of iron metabolism [61]. This important finding was rendered possible by the
observation of Simon et al. that the hemochromatosis phenotype is frequently
associated with a particular human leukocyte antigen haplotype [62]. The HFE
protein is an atypical membrane-bound MHC class 1 molecule which interacts
with β2-microglobulin; its link with iron transport remains to be established. Many
patients bear a mutant HFE, with a C282Y substitution and HFEC282Y fails to
incorporate in the plasma membrane [63]. The frequency of HFEC282Y homozygosity
is 1:200, although the clinical penetrance of the disease is lower [58,64]. Additional
HFE mutations are also associated with hemochromatosis [65]. Significantly
ablation of HFE promotes a hemochromatotic phenotype in mice [66].

3.2.2  Juvenile Hemochromatosis

Juvenile hemochromatosis unlike HFE hemochromatosis is a relatively rare disease


mainly located in Southern Europe [67]. These patients rapidly accumulate iron and
are more likely to present with cardiomyopathy and endocrinopathy than with liver
disease (Table 6). This pathology is linked to the mutation of the gene responsible
for hemojuvelin (a bone morphogenetic protein receptor operating upstream of the
hepcidin pathway), which leads to the expression of extremely low levels of hepcidin.
A small subset of these patients possesses a mutation to the HAMP gene, which encodes
for hepcidin. Low hepcidin leads to enhanced iron absorption (Section 1.4.1).

3.2.3  Ferroportin Disease

Ferroportin disease is characterized by moderate to severe iron overload. It is more


frequent than Types 2 and 3 hemochromatosis and is caused by mutations to the
258 Hider and Kong

ferroportin gene. Affected individuals express high hepcidin levels and the disease
displays phenotypic heterogeneity [68].

3.2.4  Treatment by Iron Chelation

Phlebotomy is an extremely efficient method of removing iron. Treatment of


severely iron-loaded hemochromatosis patients typically involves weekly phlebotomy;
which can lead to removal of up to 1g of iron every month. Iron chelation therapy
cannot achieve such high excretion rates but does have the advantage of reducing
NTBI levels (vide infra). Patients suffering from juvenile hemochromatosis may
rapidly develop cardiac complications and such people can benefit from a combination
of phlebotomy and chelation therapy. In principle, orally active iron chelators could
find application in the treatment of hemochromatosis, and deferasirox (vide infra)
has been reported to show good efficacy [69].

3.3  Transfusional Siderosis

The management of anemias associated with ineffective erythropoiesis (for instance


thalassemia, sickle cell disease, hemolytic anemia, and myelodysplastic syndromes)
requires frequent blood transfusion. Blood contains plenty of iron (0.5 mg mL–1)
and this accumulates in the tissues as man does not actively excrete iron (Section 1.4).
In addition, ineffective erythropoiesis inhibits hepcidin expression which leads
to the stimulation of dietary iron absorption. Overall this pathology leads to trans-
fusional siderosis, the formation of non-transferrin bound iron and the inappropriate
iron loading of liver, cardiac, and endocrine tissue [70].

3.3.1  The Hemoglobinopathies

Thalassemia and sickle cell anemia are the most common worldwide monogenic
diseases. They occur at their highest frequency in countries of the developing world
and it has been estimated that there are 250 million carriers. 300,000 children are
born each year with major hemoglobin disorders [71]. The high incidence of these
disorders reflects their association with malaria resistance which has developed over
thousands of years [72]. More than 3,000 million people live in malarious areas and
currently the disease is responsible for at least two million deaths each year. Any pro-
tective mechanism against this dominating infection that develops within the native
population will be gradually amplified and this has been the situation with thalas-
semia and sickle cell anemia where a range of mutations have gradually accumulated
within the population. The correspondence between the distribution of malaria in the
Old World before major control programs were established and the distribution of
thalassemia is striking (Figure 17) [73]. The cellular mechanisms related to this type
of protection are complex but increasingly well understood [73,74].
8  Iron: Effect of Deficiency and Overload 259

Figure 17 (a) Distribution of malaria in the Old World before major control programs were
established. (b) Distribution of α- and β-thalassemia [73].
260 Hider and Kong

Figure 18  Changes of human hemoglobin with development. There is a switch from hemoglobin-­F
to adult hemoglobin-A together with a small amount of hemoglobin-A2 in the immediate post
natal period [75]. Hemoglobins; F = α2γ2; A = α2β2; A2 = α2δ2. Reproduced with permission from
[75]; copyright 2001, Blackwell Science Ltd.

Table 7  Annual births with Thalassemia dominated symptoms


major hemoglobin disorders. Hb Bart’s hydrops (α°/α°) 5,000
Hb H disease (α°/α+) 10,000
β-Thalassemia major (β°) 23,000
Hb E/β-Thalassemia 19,000
Total 57,000
Sickle cell anemia dominated symptoms
SS disease 217,000
S/β-Thalassemia 11,000
HbC/S 55,000
Total 283,000
Reproduced with permission from [76]; copyright
2006, World Health Organization.

Hemoglobin production in man is complicated. The type of hemoglobin produced


in fetal life is altered after birth (Figure 18) as a result of the sequential suppression
and activation of individual genes [75]. Clearly, there are a number of possible
hemoglobin disorders that can result from this genetic framework and a breakdown of
these disorders as monitored by the annual number of births is given in Table 7 [76].
8  Iron: Effect of Deficiency and Overload 261

The thalassemias are characterized by a reduced rate of synthesis of one or more


globulin chain(s); α°- and β°-thalassemias are associated with no production of
α- or β-chains, respectively, α+- and β+-thalassemias are associated with a reduced
production of α- or β-chains. These conditions all lead to an imbalanced globin
chain production, which is associated with ineffective erythropoiesis and shortened
red blood cell survival. Because thalassemias occur in populations in which hemo-
globin variants are also common, some children inherit thalassemia from one parent
and a hemoglobin variant from the other. These interactions produce disorders of
varying severity. Thus, sickle cell/β-thalassemia may be as severe as sickle cell ane-
mia and hemoglobin E/β-thalassemia can produce similar pathology to that of thal-
assemia major. Hemoglobin E is inefficiently synthesized and hence produces a
mild form of β-thalassemia [75].

3.3.1.1  Thalassemia

Although the α-thalassemias are more common than the β-thalassemias, severe
forms lead to intrauterine death and so do not pose a major burden on health
care. It is the β-thalassemias and their combination with hemoglobin E that
produce severe anemia. These diseases are particularly common throughout
the Indian subcontinent and South East Asia. Over 180 different mutations have
been identified amongst the β globin chains of β-thalassemia patients. The bulk
consists of single base changes which lead to different degrees of reduction in
the synthesis of α-chains, thereby leading to the clinical diversity β-thalassemia.
The reduction of α-chain production leads to an excess of α-chains, which are
unstable and tend to precipitate, preventing the normal maturation and survival
of erythrocytes [77].
β-Thalassemia major (β°) is associated with the total absence of β-subunits
and consequently is fatal in the absence of regular blood transfusion, which in
turn is associated with systemic iron overload. With untreated patients, death
generally occurs in the second decade of life as a result of infection or heart dis-
ease [78]. Iron chelation therapy prevents the development of iron overload and
as a result, the life style of thalassemia patients has dramatically improved over
the past 50 years. Desferrioxamine (DFO, 7) a natural siderophore was intro-
duced in the clinic by SephtonSmith in 1962 [79]. It scavenges and binds iron(III)
extremely tightly, leading to the formation of a stable non-toxic iron complex
(Figure 19) [80] which is excreted via the bile. Unfortunately DFO is not orally
active and has to be administered parenterally over prolonged time periods, as it
is rapidly cleared by the kidney [81]. Never-the-less it has been a remarkably
successful pharmaceutical and has extended the lives of thousands of
β-thalassemia major patients.
There is a wide pathological diversity of β-thalassemia patients due to the large
number of different mutations, for instance the inheritance of a severe mutation
from one parent and a mild mutation from the second parent or the inheritance of
β-thalassemia from one parent and an abnormal Hb from the other [82]. As a group,
262 Hider and Kong

Figure 19 Ferroxamine,
the iron complex of
desferrioxamine [80].

these patients are described as suffering from β-thalassemia intermedia. Management


depends on the severity of the disease and may involve transfusion and chelation or
in some cases, chelation therapy alone (vide infra) [82].

3.3.1.2  Sickle Cell Disease

Expression of sickle cell disease, like β-thalassemia is highly variable, ranging from
mild phenotypes (mostly SC and Sβ+ genotypes) to severe disease (mostly SS and
Sβ° genotypes). Transfusion therapy is a key component of patient management and
is an effective treatment for chest syndrome, heart failure, and stroke. Monthly
transfusions decrease the risk of recurrent stroke. Straight transfusion is used when
the Hb level is less than 8 g dL–1 and exchange transfusion is recommended with
normal Hb levels [83]. The target percentage of HbS in patients receiving regular
transfusions is 30% [84]. Both straight and exchange transfusion lead to iron
overload and although the endocrinopathology is less marked in sickle cell anemia
patients when compared with β-thalassemia patients [85], iron overload should be
treated by chelation (vide infra).

3.3.2  Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a diverse group of hematological disorders


that lead to ineffective production of myeloid blood cells with a risk of transformation
to leukemia. This bone marrow stem cell disorder is associated with ineffective
erythropoiesis and leads to anemia. Diagnosis of MDS is made typically between 60
and 75 years, diagnoses are rare in children. Many MDS patients become dependent
on blood transfusion and develop transfusional iron overload [86] which is
­asso­ciated with cardiac problems [87]. Although this iron overload can be treated with
8  Iron: Effect of Deficiency and Overload 263

desferrioxamine, the demanding continuous parenteral treatment leads to difficulties


with compliance, particularly with this typically elderly group of patients [86].
Treatment with orally active iron chelators has good potential (vide infra).

3.4  Hereditary Disorders of Mitochondrial Iron Overload

Several iron loading disorders are characterized by mitochondrial accumulation in


specific tissues, without systemic iron overload. They result from mutations in pro-
teins involved in either heme or iron-sulfur cluster biosynthesis. These two meta-
bolic pathways occur in the mitochondria and under normal conditions consume the
majority of cellular iron.

3.4.1  Sideroblastic Anemia

Sideroblastic anemias are a group of disorders that are associated with the forma-
tion of a large number of ringed sideroblasts in the marrow. Sideroblasts are eryth-
roblasts (nucleated red blood cells) which contain precipitates of non-heme iron
aggregates deposited within the cristae of mitochondria [68]. X-linked sideroblastic
anemia (XLSA) is the most common form of sideroblastic anemia which results
from defects in 5-aminolevulinate synthase, a key enzyme in heme biosynthesis.
Management of this disease frequently requires blood transfusion which leads to
iron overload [68].

3.4.2  Friedreich’s Ataxia

Friedreich’s ataxia is an autosomal recessive neurodegenerative disorder linked to


the functional inactivation of frataxin (FXN) [88,89]; a mitochondrial protein which
has a critical role in the assembly of iron-sulfur clusters [90]. An expanded GAA
triplet repeat is found in both alleles of the FXN gene. This triplet repeat leads to
decreased RNA transcription and lower levels of frataxin. The degree to which
transcription is suppressed is proportional to the length of the GAA repeat; with
frataxin levels, 5% of normal, being associated with long GAA repeats and the
association of higher frataxin levels (30% of normal) with shorter repeats [91].
Thus, patients with shorter GAA repeats generally have a less severe phenotype.
Frataxin deficiency is associated with decreased ATP synthesis and elevated
mitochondrial iron levels. The tissues most severely influenced are cardiac [92],
gastrointestinal and brain [93]. Treatment with orally active iron chelators can
be beneficial (vide infra).
264 Hider and Kong

3.4.3  Glutaredoxin-5 Deficiency

Glutaredoxin-5 (Grx 5) is a protein cofactor essential for the iron-sulfur cluster


assembly pathway [94]. Its absence is causatively linked to microcytic anemia with
a sideroblastic-like phenotype [95]. The disease requires regular blood transfusion
which leads to systemic iron overload. The pathogenic mechanism involves inhibi-
tion of heme synthesis in erythroid precursor cells via accumulation of apo-IRP1
that represses 5-aminolevulinate synthase (ALAS) mRNA translation [96].

3.5  Animal Models of Iron Overload

Over the past two decades genetic analysis of patients with inherited iron homeostasis
disorders and the analysis of mutant mice, rats, zebra fish, and fruit flies has greatly
facilitated the present understanding of iron metabolism [97]. These animal models
are excellent systems for investigating iron homeostasis and for the identification of
new therapeutic agents. Although iron metabolism in zebra fish and the fruit fly is
not understood in detail, iron metabolism in rodents is similar to that in man. Models
have been developed which simulate iron deficiency anemia, sideroblastic anemia,
various forms of defective iron transport, hemochromatosis, and Friedreich’s ataxia
(Table 8) [21,98–100,104–120].
The first mutant to provide a useful insight into iron transport was the hypotrans-
ferrinaemic mouse (hpx) [98,101], although the mk mouse was discovered in 1970
[102] and the Belgrade rat in 1969 [103]. One of the benefits from this work will be
the accurate diagnosis of human iron deficiency and iron overload disorders; for
instance, the clinical approach to hemochromatosis has been strongly influenced by
diagnostic testing [68].

3.6  Genetic Screening for Thalassemia

The improved understanding of the pathophysiology of the thalassemias has led to


improved symptomatic treatment by blood transfusion and chelation therapy.
However, this therapy is expensive and complicated due to the increasing problem
of obtaining blood that is free from HIV and hepatitis.
Because thalassemia and sickle cell heterozygotes are relatively easy to identify,
these diseases are suitable for investigation by prenatal diagnosis and termination of
pregnancies of homozygote babies. In the 1970’s with the advent of fetal blood
sampling, it became possible to undertake such diagnosis by monitoring hemoglobin
chain synthesis [121] and later by DNA analysis [122,123]. Screening was soon
established in several countries, for instance Sardinia and Cyprus, with remarkable
success (Figure 20) [124]. There has now been success in reducing the number of
babies born with thalassemia major in Greece, Italy and United Kingdom [121].
Table 8  Mutations in iron metabolism genes.
Protein Mutation Loss of function Human disease
Mutations causing anemia
Transferrin hpx (mice) [98] Severe anemia, iron overload Atransferrinemia
in non-­hematopoietic tissues
Transferrin receptor 1 Tfr knockout (mice) [99] Embryonic death with severe anemia
Zebra fish (chianti) [120] Embryonic death with severe anemia
Divalent metal transporter 1 mk (mice) [100] Severe iron deficiency anemia
Belgrade rat [100] Severe iron deficiency anemia
Zebra fish (chardonnay) [119] Iron deficiency anemia
Hephaestin sla (mice) [104] Microcytic anemia
Steap 3 (ferroxidase) Steap 3 knockout (mice) [112] Impaired ferric reductase activity in transferrin endosome
Mutations causing hemochromatosis
8  Iron: Effect of Deficiency and Overload

Hfe protein Hfe (mice) [105] Increased body iron, decreased hepcidin Hemochromatosis (Type 1)
Hemojuvelin Hjv (mice) [106,107] Increased body iron, decreased hepcidin Hemochromatosis (Type 2A)
Hamp 1 (hepcidin synthesis) Hamp 1 (mice) [108] Increased body iron, absence of hepcidin Hemochromatosis (Type 2B)
Transferrin receptor 2 Tfr 2 (mice) [109] Increased body iron, decreased hepcidin Hemochromatosis (Type 3)
Ferroportin Slc40al [110] Heterozygous mice have iron loading of Hemochromatosis (Type 4)
Flat iron mouse Kupffer cells and low transferrin
Zebra fish (weissherbst) [21] Decreased hepcidin
Smad 4 Smad 4 (mice) [111] Increased body iron, decreased hepcidin Hemochromatosis
Mutations causing altered mitochondrial metabolism
5-Aminolevulinic acid synthase Alas2 knockout (mice) [113] Increased mitochondrial iron
Alas2 zebra fish (sauternes) [117] Embryonic lethal (day 12) microcytic anemia
Ferrochelatase Fech (mice) [114] Microcytic anemia and porphyria Porphyria
Frataxin Fxn knockout (mice) with human Progressive neurodegeneration and cardiac pathology Friedreich’s ataxia
mutant constructs [115]
Mitoferrin 1 Mfrn 1 knockout (mice) [116] Profound anemia, Embryonic death Sideroblastic anemia
265

Glutaredoxin 5 Grxs zebra fish (shiraz) [118] Microcytic anemia Sideroblastic anemia
Zebra fish (frascati) [120] Anemia _
266 Hider and Kong

Figure 20 Number of births of new cases of β-thalassemia in Sardina since a screening and prenatal
diagnosis program was developed in the mid-1970s [124]. Reproduced with permission from [124];
copyright 1998, Elsevier.

4  Iron-Selective Chelators with Therapeutic Potential

4.1  Design Features

Desferrioxamine-B (DFO) (7), the most widely used iron chelator in hematology
over the past 40 years, has a major disadvantage of being orally inactive [125]. In
order to identify an ideal iron chelator for clinical use, careful design consideration
is essential; a range of specifications must be considered such as metal selectivity
and affinity, kinetic stability of the complex, bioavailability, and toxicity. This field
has recently been reviewed [126,127].

4.1.1  Metal Selectivity

Chelating agents can be designed for either the iron(II) or iron(III) oxidation state.
High-spin iron(III) is a tripositive cation of radius 0.65 Å, and forms most stable
bonds with charged oxygen atoms, such as those found in DFO (7). In contrast, the
iron(II) cation, which has a lower charge density, prefers chelators containing nitrogen
such as 1,10-phenanthroline. Ligands that prefer iron(II) retain an appreciable affin-
ity for other biologically important bivalent metals such as copper(II) and zinc(II)
ions. In contrast, iron(III)-selective ligands, typically oxyanions and notably
hydroxamates and catecholates, are generally more selective for tribasic metal
cations over dibasic cations and as most tribasic cations, for instance aluminium(III)
and gallium(III), are not essential for life, iron(III) provides the best target for
8  Iron: Effect of Deficiency and Overload 267

‘iron chelator’ design under biological conditions. An additional advantage of


high-­affinity iron(III) chelators is that, under aerobic conditions, they will chelate
iron(II) and rapidly autoxidize it to the corresponding iron(III) species [128].

4.1.2  Ligand Selection

Catechol moieties possess a high affinity for iron(III). This extremely strong inter-
action with tripositive metal cations results from the high electron density of both
oxygen atoms. However, this high charge density is also associated with the high
affinity for protons (pKa values of 12.1 and 8.4). Thus the binding of cations by
catechol has marked pH sensitivity. The hydroxamate moiety possesses a lower
affinity for iron than catechol, but the selectivity of hydroxamates, like catechols,
favors tribasic cations over dibasic cations. Due to the lower value of the proton-
ation constant (pKa ~9 ), competition with hydrogen ions at physiological pH is less
pronounced than for that of catechol ligands.
Hydroxypyridinones (Figure 21) combine the characteristics of both hydroxa-
mate and catechol groups, forming 5-membered chelate rings in which the metal
is coordinated by two vicinal oxygen atoms. The hydroxypyridinones are mono-
protic acids at pH 7.0 and thus, like hydroxamates, form neutral tris-iron(III)
complexes. 3-Hydroxypyridin-4-ones are highly selective for tribasic metal cat-
ions over dibasic cations as indicated by the low reduction potential of iron com-
plexes (−620 mV versus NHE). 8-Hydroxyquinoline binds iron(II) more tightly
than 3-­hydroxypyridin-4-ones as indicated by its higher redox potential of the
iron complex (−150 mV versus NHE). Never-the-less it is capable of scavenging
iron under biological conditions, forming a 3:1 complex with iron(III) at pH 7.0.
Although aminocarboxylates and hydroxycarboxylates bind iron(III) they are
less selective, frequently possessing appreciable affinities for calcium(II) and
magnesium(II), in addition to zinc(II) and copper(II).

4.1.3  Hexadentate, Tridentate, and Bidentate Iron(III) Complexes

The coordination requirements of high spin iron(III) are best satisfied by six donor
atoms ligating in an octahedral fashion to the metal center, the affinity for the ligand
generally decreasing in the sequence; hexadentate > tridentate > bidentate
(Figure  22). The overall stability constant trends for bidentate and hexadentate
ligands are typified by the bidentate ligand N,N-dimethyl-2,3-dihydroxybenzamide
(DMB) and the hexadentate congener MECAM (Figure 21) where a differential of
6 log units in stability is observed (40.2 versus 46).
Although MECAM binds iron(III) more tightly than its bidentate analogue
DMB, other hexadentate catechols, for instance, enterobactin (Figure 21), bind
iron(III) even more tightly. The smaller the conformational space of the free ligand,
the higher the stability of the complex; as the difference between the flexibility of the
ligand and its corresponding iron complex decreases, so does the entropy difference.
268 Hider and Kong

Bidentate Ligands

OH

O
OH

N
O NMe2 H OH

N,N-Dimethyl-2,3- Acetohydroxamic acid


dihydroxybenzamide (DMB)
O
OH

N N
OH

8-Hydroxyquinoline 3-Hydroxy-1,2-dimethylpyridin
-4(1H)-one (Deferiprone)

Tridentate ligands
HOOC

OH N N

N N
N CH3
HO
S COOH OH

Desferrithiocin Desferasirox (ICL670)

Hexadentate ligands
OH

O
OH OH

NH OH
OH O
HN
OH O
H O
N O O

O HN O O NH
HO
N O
HO O
HO H O
HO
HO OH
MECAM Enterobactin

Figure 21  Iron(III) chelating agents.


8  Iron: Effect of Deficiency and Overload 269

O O O
O O O N O O
Fe Fe Fe
O O N O O O
O O O

Bidentate ligand Tridentate ligand Hexadentate ligand


3:1 complex 2:1 complex 1:1 complex

Figure 22  Schematic representation of chelate ring formation in metal-ligand complexes.

Table 9 pFe3+ values of Iron(III) chelator pFeIII


selected iron(III) chelators.
Bidentate
Dimethyl-2,3-dihydroxybenzamide DMB 15
Acetohydroxamic acid 13
3-Hydroxypyridinone (Deferiprone) 20.5
8-Hydroxyquinoline 20.6
Tridentate
Desferrithiocin 20.4
Desferasirox 22.5
Hexadentate
Desferrioxamine-B (7) 26.6
MECAM 28
Enterobactin 31.5
pFeIII = −log[Fe3+], when [Fe3+]total = 10–6 M, [Ligand]total
= 10–5 M at pH 7.4.
The structures of the chelators are given in Figure 21.

Thus enterobactin (log stability constant = 48) can be considered to possess a degree
of preorganisation in contrast to MECAM which does not [129].
Under biological conditions, a comparison standard which is generally more
useful than the stability constant is the pFe3+ value [130]. pFe3+ is defined as the
negative logarithm of the concentration of the free iron(III) in solution. Typically
pFe3+ values are calculated for total [ligand] = 10–5 M, total [iron] = 10–6 M at pH
7.4. The comparison of ligands under these conditions is useful, as the pFe3+ value,
unlike the stability constants log K or log β3, takes into account the effects of ligand
protonation and denticity as well as differences in metal-ligand stoichiometries.
Comparison of the pFe3+ values for hexadentate and bidentate ligands reveals that
hexadentate ligands are far superior to their bidentate counterparts under typical in
vivo conditions. The values for DMB, MECAM, and enterobactin being 15, 28, and
31.5, respectively (Table 9) [130].
270 Hider and Kong

The formation of a complex will also be dependent on both free metal and free
ligand concentration and as such will be sensitive to concentration changes. The
degree of dissociation for a tris-bidentate ligand-metal complex is dependent on the
cube of [ligand] whilst the hexadentate ligand-metal complex dissociation is only
dependent on [ligand]. Hence the dilution sensitivity to complex dissociation for
ligands follows the order hexadentate < tridentate < bidentate. It is for this reason
that the majority of natural siderophores are hexadentate compounds and can there-
fore scavenge iron(III) efficiently at low metal concentrations [131]. In general,
pFe3+ values follow the trend hexadentate > tridentate >bidentate as exemplified by
the examples in Table 9.

4.1.4  C
 ritical Features for Clinical Application: Molecular Size
and Hydrophobicity

In order to achieve efficient oral absorption, the chelator should possess appreciable
lipid solubility which may facilitate the molecule to penetrate the gastrointestinal
tract (partition coefficient between n-octanol and water greater than 0.2) [132].
Molecular size is also a critical factor which influences the rate of drug absorption
[133]. Indeed, it has been proposed by Lipinski et al. that the molecular weight
should not exceed 500 in order to achieve efficient oral absorption [134]. This
molecular-weight limit provides a considerable restriction on the choice of chelator
and may effectively exclude hexadentate ligands from consideration; most sidero-
phores, for instance DFO (7) and enterobactin (Figure 21) have molecular weights
in the range 500–900. In contrast, bidentate and tridentate ligands, by virtue of their
much lower molecular weights, tend to possess higher absorption efficiencies.

4.1.5  Toxicity of Chelators and Their Iron Complexes

The toxicity associated with iron chelators originates from a number of factors;
including inhibition of metalloenzymes, lack of metal selectivity, redox cycling of
iron complexes between iron(II) and iron(III), thereby generating free radicals, and
the kinetic lability of the iron complex leading to iron redistribution.
Enzyme inhibition: In general, iron chelators do not directly inhibit heme-­containing
enzymes due to the inaccessibility of porphyrin-bound iron to chelating agents.
In contrast, many non-heme iron-containing enzymes such as the lipoxygenase and
aromatic hydroxylase families and ribonucleotide reductase are susceptible to chelator-
induced inhibition [135]. Lipoxygenases are generally inhibited by hydrophobic
chelators, therefore, the introduction of hydrophilic characteristics into a chelator
tend to minimize such inhibitory potential [136]. Stereochemistry can also limit
chelator access to the metal binding center and the introduction of a rigid side chain
close to the chelating center of the molecule can reduce inhibitory properties [137].
Thus, careful control of the bulk and shape of iron chelators leads to minimal inhibitory
influence of many metalloenzymes.
8  Iron: Effect of Deficiency and Overload 271

Metal selectivity: An ideal iron chelator should be highly selective for iron(III) in
order to minimize chelation of other biologically essential metal ions which could
lead to deficiency with prolonged usage. Many ligands that possess a high affinity
for iron(III) also have appreciable affinities for other metals such as zinc(II), this
being especially so with carboxylate- and nitrogen-containing ligands. However,
this factor is less of a problem with the bidentate catechol, hydroxamate, and
hydroxypyridinone ligand groups, which possess a strong preference for tribasic
over dibasic cations. In principle, competition with copper(II) could be expected to
be a problem, however under most biological conditions this is not so, as copper is
extremely tightly bound to chaperone molecules [138].
Iron-complex structure and redox activity: In order to prevent free radical produc-
tion, iron should be coordinated in such a manner as to avoid direct access of oxy-
gen and hydrogen peroxide, and to possess a redox potential which cannot be
reduced under biological conditions. Most hexadentate ligands with oxygen con-
taining ligands such as DFO are kinetically inert and reduce hydroxyl radical pro-
duction to a minimum by failing to redox cycle.
Chelators that are capable of binding both iron(II) and iron(III) at neutral pH
values have potential to redox cycle. This is an undesirable property for iron scaveng-
ing molecules, as redox cycling can also lead to the production of reactive oxygen
radicals (Figure 23). Significantly, the high selectivity of siderophores for iron(III)
over iron(II) renders redox cycling under biological conditions unlikely. Thus the
iron complexes of enterobactin and desferrioxamine are extremely low, namely −750
and −468 mV (versus NHE) [130]. In similar fashion, iron-­deferiprone has a low
redox potential (−620 mV versus NHE) [139]. Iron complexes with redox potentials
above −200 mV (versus NHE) are likely to redox cycle under aerobic conditions.
Kinetic lability of iron complexes: Hexadentate ligand iron complexes tend to be
inert, the rate of dissociation of the complex being vanishingly small at neutral pH
values. This renders such molecules ideal scavengers of iron. In contrast, bidentate
and tridentate ligands are less kinetically stable and are able to dissociate at appre-
ciable rates, thereby possibly facilitating iron redistribution. Such a property is

Figure 23  Redox cycling


of an iron complex.
272 Hider and Kong

undesirable for most therapeutic applications, where efficient iron excretion is


required. In order to avoid appreciable redistribution of iron in mammalian body
tissues, chelators possessing a high iron(III) affinity are required; generally a pFe
value ≥20 appears to be sufficient to minimize the redistribution of iron.

4.2  Orally Active Iron Chelators in Current Use

Iron chelation therapy prevents the development of iron overload and as a conse-
quence the life style of thalassemia major patients has been dramatically improved
with the application of DFO (7) (Section 3.3.1.1). However, DFO is not an ideal
therapeutic chelator due to its oral inactivity and rapid renal clearance (plasma half-­
life of 5–10 min) [140]. In order to achieve sufficient iron excretion, it has to be
administered subcutaneously or intravenously for 8–12 h/day, 5–7 days/week [141].
Patient compliance with this regimen is frequently poor. Furthermore, NTBI
(Section  3.1) is present in such patients whenever the plasma DFO level is low,
rapidly reappearing on the cessation of DFO perfusion (Figure 24) [142]. As DFO
is typically infused for 5 nights, this only provides protection for 40h per week; that
is approximately 25% of the time. As transferrin is saturated in most of these
patients, NTBI is present for 75% of the time and therefore has the possibility of
gradually loading the heart and endocrine tissue with iron, even in well chelated
patients. A large proportion of patients treated with DFO suffer from adverse car-
diovascular events [143].

4.2.1  Tridentate Chelators

Unlike hexadentate and bidentate molecules, it is difficult to design tridentate


ligands which only possess oxygen anion coordination sites [144], the central ligand
typically being nitrogen (Figure 22).

Figure 24  The effect of


DFO infusion at 50 mg/
kg/24h (intravenous) on
NTBI is shown in a single
patient with thalassemia
major both on starting
the DFO infusion and on
stopping the infusion
at 48 hours [142].
8  Iron: Effect of Deficiency and Overload 273

Desferrithiocins: Desferrithiocin (DFT) (11) is a siderophore isolated from


Streptomyces antibioticus. It forms a 2:1 complex with iron(III) at neutral pH using
a phenolate oxygen, a carboxylate oxygen, and a nitrogen atom as ligands [145].
It possesses a high affinity for ferric iron (pFe3+ = 20.4). Long term studies of DFT in
normal rodents and dogs at low doses have shown toxic side effects, such as reduced
body weight and neurotoxicity [146]. However, a range of synthetic analogues
of DFT have been prepared in an attempt to design molecules lacking renal and
neurotoxicity [147] and two such molecules have been identified, namely deferitrin
(12) and FBS0701 (13). Deferitrin (12), was found to be highly effective when given
orally to rats and primates. Histopathological analysis indicated some nephrotoxicity
but much less than that arising from DFT [148]. Phase I clinical trials demonstrated

OH HO OH

N CH3 N CH3
N
S COOH S COOH

11 12

good oral absorption, however the compound was not progressed beyond Phase II
clinical trials due to nephrotoxicity. FBS0701 (13) also binds iron(III) with high
affinity and in contrast to deferitrin, demonstrated no observable toxicity at a pre-
dicted dose level range in preclinical studies [149]. The compound has entered clini-
cal trials sponsored by Ferrokin Biosciences [150], where it has been shown to be
well tolerated and to possess favorable pharmacokinetics [151]. FBS0701 is cur-
rently in Phase II clinical trials.
Triazoles: Triazoles have been investigated as potential ligands by Novartis [152].
These molecules chelate iron(III) with two phenolate oxygens and one triazolyl
nitrogen. The lead compound deferasirox (14) possesses a pFe3+ value of 22.5
and is extremely hydrophobic, with a log Poctanol/water value of 3.8. As a result, it can
penetrate membranes easily and possesses good oral availability. Indeed, when
orally administered to hypertransfused rats, deferasirox promotes the excretion of
chelatable iron from hepatocellular iron stores four to five times more effectively
than DFO [153].

HOOC
O O
O O
OH N N
N CH3 N
S HO
COOH OH

13 14
274 Hider and Kong

By virtue of a high proportion of both the free ligand and the 2:1 iron complex
binding to albumin (greater than 98%), the ligand possesses low toxicity despite its
strong lipophilic character. The extreme hydrophobicity of this chelator necessitates
formulation in dispersion tablets, containing the disintegrants, SDS, povidone, and
crospovidone. Thus, deferasirox is typically given once daily each morning as a
dispersed solution in water, half an hour before breakfast. Deferasirox has been
demonstrated to be efficient at removing liver iron from regularly transfused patients
[154] but is apparently less effective at removing cardiac iron [155]. Deferasirox
(14) forms a 2:1 iron complex which possesses a net charge of 3– and a molecular
weight over 800. Should such a complex form intracellularly, it is possible that the
iron will remain trapped within the cell. The redox potential of the 2:1 iron complex
is −600 mV (versus NHE) confirming that deferasirox is highly selective for iron(III)
and that it will not redox cycle under biological conditions. As with all therapeutic
iron chelators there are side effects associated with deferasirox [156], kidney toxic-
ity being particularly prevalent [157].

4.2.2  Bidentate Chelators

On the basis of selectivity and affinity, particularly considering pFe3+ values,


3-hydroxypyridin-4-one (Figure 21) is the optimal bidentate ligand for the chelation
of iron(III) over the pH range of 6.0–10.0 and to date is the only bidentate class to
have been subjected to extensive clinical study.
Dialkylhydroxypyridinones: The 1,2-dimethyl derivative (deferiprone, Ll, CP20)
(15) is marketed by Apotex Inc. Toronto, Canada, as FerriproxTm. Deferiprone was
first reported as a potential orally active iron chelator in 1984 [158] and demon-
strated to be active in man in 1987 [159]. It was licensed for use in India in 1994 and
in Europe in 1999, receiving full marketing authorization in 2002. The FDA pro-
vided approval for its use in 2011. There are numerous reports indicating the com-
parative effectiveness of desferrioxamine and deferiprone [160]. A particularly
important property of deferiprone is its ability to penetrate cells, coordinate iron,
forming a neutral complex, which is also capable of permeating membranes. Thus,
iron can be readily removed from iron-loaded cells including those of cardiac tissue
(Figure 25) [161]. This ability extends to the clinical situation [162,163], where it
has been demonstrated that deferiprone therapy is associated with significantly
greater cardiac protection than DFO in patients with thalassemia major [143,164].
Unfortunately, the dose required to keep a previously well chelated patient in
­negative iron balance with deferiprone is relatively high, in the region of 75–100 mg
kg–1 day–1. One of the major reasons for the limited efficacy of deferiprone in clini-
cal use is that it undergoes extensive metabolism in the liver. The use of deferiprone
has a range of associated side effects [165], the most important being a low inci-
dence of reversible agranulocytosis [166].
8  Iron: Effect of Deficiency and Overload 275

Figure 25  Schematic representation of the penetration of deferiprone [LH]0 through the plasma
membrane. The bidentate ligand scavenges loosely bound intracellular iron, forming the 3:1 complex,
which also carries zero net charge. Efflux as the iron complex leads to iron excretion.

O
OH
O O O
OH OH OH OH
N
H
OH N
N CH3 N H3C N CH3
CH3 OH CH3 CH3 CH3 O

15 16 17 18

High iron affinity hydroxypyridinones: In order to further improve chelation


efficacy, considerable effort has been applied to the design of novel hydroxypyridi-
nones with enhanced pFe3+ values [167]. Novartis synthesized a range of bidentate
hydroxypyridinone ligands, which possess an aromatic substituent at the 2-position.
The lead compound (16) was found to be orally active and highly effective at remov-
ing iron from both the iron-loaded rat [168] and marmoset [169]. In similar fashion,
Hider and coworkers have demonstrated that the introduction of either a
l′-hydroxyalkyl group (17) [170] or an amido function (18) [171] at the 2-position
of 3-hydroxypyridin-4-ones enhances the affinity for iron(III) over the pH range
5–8. These changes result in an increase in the corresponding pFe3+ values due to
the reduced competition with hydrogen ions; thus the 2-amidopyridin-4-one (18)
has a pFe value of 21.7 as compared with that of the analogous deferiprone (15)
which possesses a pFe value of 20.5. In practical terms this means that at pH 7.4
(18) binds iron over ten times more tightly than deferiprone.
These novel high pFe3+ HPOs show great promise in their ability to remove iron
under in vivo conditions. Detailed dose-response studies suggest that chelators with
high pFe3+ values scavenge iron more effectively at lower doses when compared
276 Hider and Kong

with simple dialkyl substituted hydroxypyridinones and so in principle can be used


at the lower dose of 20 mg kg–1. A number of related compounds are currently
undergoing preclinical evaluation.
Combined therapy with desferrioxamine and hydroxypyridinones: By virtue of its
small size and ability to penetrate cells, deferiprone has the capability of efficiently
scavenging excess iron. However due to its bidentate nature, the ability of deferi-
prone for iron(III) at neutral pH values is highly concentration-dependent and at rela-
tively low concentrations (<5 μM) the iron deferiprone complex will donate iron to
competing ligands [172]. If deferiprone is used together with a high affinity hexaden-
tate chelator such as desferrioxamine, the deferiprone iron complex will readily
donate iron to the kinetically more stable desferrioxamine [173]. Indeed, deferiprone
enhances plasma NTBI removal in the presence of desferrioxamine [174]. Early
clinical studies indicated that such combination therapy is effective at increasing iron
excretion [175]. These observations led to more extensive clinical investigations
using deferiprone and desferrioxamine in sequential fashion and resulted with
beneficial effects in survival, iron removal and cardiac disease [176,177].

4.2.3  Use of Iron Chelators to Treat Diseases Other than Thalassemia

4.2.3.1  Sickle Cell Anemia

Transfusion has been introduced for the treatment of sickle cell anemia, due to its
beneficial effect in the treatment of crises and in the reduction of the incidence of
stroke [178] (Section 3.3.1.2). Again an effective orally active iron chelator permits
regular transfusion of such patients without the worry of inducing an associated iron
overloaded state. There have been a number of trials comparing the efficacy of dif-
ferent chelators and the outcome of these studies reviewed [179]. At the present
time there is no clear preference for a particular chelator. In a multicenterd study of
iron overload [180], three patient groups have been compared (transfused thalas-
semia patients, transfused sickle cell patients and non-transfused sickle cell
patients). There were more endocrine problems in the transfusion-dependent thalas-
semia patients (56.3%) than in both transfused sickle cell patients (13.1%) and non-­
transfused sickle cell patients (7.8%) and it has been suggested that this difference
may relate to the different NTBI levels and hence speciation (Section 3.1).
Generally, NTBI levels in thalassemia patients are higher than those of sickle cell
patients [181]. This finding could also explain the low incidence of cardiac disease
in transfused sickle cell patients [180]. There was a relatively poor compliance
observed with deferasirox in the sickle cell anemia group [182].

4.2.3.2  Myelodysplastic Syndromes

Many patients with myelodysplastic syndromes (MDS) become dependent on blood


transfusions (Section 3.3.2) and so treatment by an orally active iron chelator is
appropriate. The use of deferiprone is not ideal in view of the risk of agranulocytosis
8  Iron: Effect of Deficiency and Overload 277

which may be higher in this patient group [183]. In contrast, deferasirox has found
useful application in removing excess iron from transfused MDS patients [184,185].
As cardiac problems are the most frequent serious complications associated with
iron overload in MDS patients [86], procedures designed to maintain NTBI levels at
a low level should be adopted. Daily administration of deferasirox is thus likely to
provide a useful option.

4.2.3.3  Friedreich’s Ataxia

Friedreich’s ataxia involves the accumulation of iron in mitochondria; cardiac,


gastrointestinal and brain tissues being most severely affected (Section 3.4.2).
In principle, an iron chelator which can readily cross membranes, gain access to
the mitochondrial matrix, scavenge inappropriately deposited iron and efflux from
the mitochondrion as a stable iron complex, should find application in the treatment
of this disease. Deferiprone is one such compound (Figure 25) as demonstrated by
Cabantchik and coworkers [46,186]. Treatment of Friedreich’s ataxia patients with
deferiprone for 6 months led to a decrease in iron levels that had specifically
accumulated in dentate nuclei of the brain and also improved gait and control of the
gastrointestinal tract [93]. This preliminary clinical study has now been extended to
a multicenter investigation.

5  Neuropathology and Iron

Neurodegeneration is a complicated multifaceted process which leads to many chronic


disease states. A broad classification can be achieved on the basis of neuropatho-
logical changes:
(i) Disorders characterized by the accumulation of abnormal proteins leading to a
selective loss of neurons; examples include Alzheimer’s disease (Aβ amyloid
plaques), Parkinson’s disease (Lewy bodies), Huntington disease (Huntingtin
aggregates), and Pick’s disease (Pick bodies).
(ii) Disorders resulting from dysfunction of motor neurons; examples include
multiple sclerosis, amyotrophic lateral sclerosis, Friedreich’s ataxia, and
progressive supranuclear palsy.
Significantly, all these disease states have been associated with elevated levels of
iron in the brain resulting from a loss of control of iron homeostasis in particular
areas of the brain [187]. The control of neuronal cytosolic iron(II) levels is achieved
by an interplay of fluxes centered on membrane transporters, mitochondria, lyso-
somes, and ferritin (Figure 8) together with neuromelanin which acts as an addi-
tional storage site for iron and other transition metals [188]. The concentration of
iron in the cerebrospinal fluid, and hence the interstitial fluid, ranges between 0.2
and 1.1 μM, whereas transferrin levels are low, typically 0.25 μM [189]. Thus, CSF
iron levels will frequently exceed the binding capacity of transferrin and in view of
the relatively high levels of citrate, form complexes 9 and 10.
278 Hider and Kong

The levels of this non-transferrin bound iron will influence cytosolic levels of iron(II).
Abnormal iron accumulation induces the oxidation of reduced glutathione in human
neuronal cells [190] and this in turn, by virtue of the involvement of glutathione in
iron sulfur cluster synthesis [8,191], can lead to a reduction in the respiratory
Complex I activity (Complex I contains 8 Fe-S clusters). Reduction of Complex I
activity can result in vicious cycles of increased oxidative stress, increased iron
accumulation, and decreased GSH content [189]. Significantly, defects in mitochondrial
electron transport have been reported for Alzheimer’s disease, Parkinson’s disease,
Huntington disease, and Friedreich’s ataxia [192].

5.1  Alzheimer’s Disease

Alzheimer’s disease is the most common form of dementia, there being over 24 million
sufferers at the present time and by 2040 it has been estimated that there will be
80 million worldwide. Central to the disease is the formation of amyloid plaques
which predominately consist of Aβ amyloid peptide, a 42 membered peptide resulting
from the breakdown of a membrane-bound protein (APP) (Figure 26) [193].
There is continual production of Aβ42 in normal individuals, but in Alzheimer’s
disease patients, proteosome processing becomes less efficient and the peptide
accumulates both in the membrane and as soluble oligomers. The latter aggregate to
form amyloid plaques (Figure 26). The oligomers and the membrane bound Aβ42
molecules possess an iron binding site [194,195] which endows the peptide with

Figure 26  Amyloidogenic processing of amyloid precursor protein (APP). APP is cleaved to
produce membrane bound Aβ42 peptide, which is in equilibrium with water soluble oligomers of
Aβ42 peptide. The oligomers aggregate to form amyloid plaques.
8  Iron: Effect of Deficiency and Overload 279

pro-oxidant activity [196,197]. It has recently been proposed that APP possesses
ferroxidase activity, which is inhibited in Alzheimer’s disease, thereby facilitating
neuronal iron accumulation [198,199]. Brain iron accumulation has been demon-
strated in Alzheimer’s disease patients by both MRI [200] and ICP-MS [201]. This
sequence of events could lead to mitochondrial toxicity as outlined in Section 5.1.
Clearly there is potential for iron chelation therapy, both in prophylactic and therapeutic
treatments of Alzheimer’s disease (Section 5.5).

5.2  Parkinson’s Disease

Parkinson’s disease, like Alzheimer’s disease, tends to be more common in the


elderly, about 4% of those over 80 years experience symptoms. Although stiffness
and tremor are common symptoms, there are many features of the disease [202], for
instance the nigrostriatal and caudate nucleus dopaminergic neurons are adversely
effected leading to cell death and neuronal loss. Genetic factors, environmental
factors and aging can all influence the onset of the disease [203]. Lewy bodies
appear and there is multiple evidence of both damage induced by free radicals
[204,205] and for an increased nigral iron content [206,207]. In particular, there are
reduced glutathione levels in the nigral brain region and the mitochondrial Complex
I is inhibited [205].
As with Alzheimer’s disease there is clear potential for iron chelation therapy
(Section 5.5).

5.3  Pantothenate Kinase-2 Deficiency

Pantothenate kinase-2 (PANK 2) deficiency is a relatively uncommon autosomal


recessive trait which is characterized by progressive Parkinson-like rigidity, together
with mental and emotional retardation (previously termed Hallervorden-Spatz syn-
drome) [208]. Onset is in late childhood, death typically occurring within 10 years.
The globus pallidus and substantia nigra possess elevated levels of iron, mostly
located in microglia and macrophages giving rise to the ‘the eye of the tiger’ MRI
brain image [209,210]. PANK 2 is the first enzyme involved in the biosynthesis of
coenzyme A and is located in the mitochondria. Patients with PANK 2 deficiency
suffer from a coenzyme A deficiency and a related elevation of cysteine [211] which
has been suggested to increase iron levels by chelation [212].

5.4  Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blind-


ness in the developed nations, in people aged 65 and older [213]. Oxidative stress
and free radical damage have been implicated in the pathogenesis of AMD [214]
280 Hider and Kong

and iron is a likely oxidant, as AMD-affected maculars possess a higher iron content
than healthy age-matched maculars, as demonstrated by Perl’s stain [215]. Whereas
iron accumulation is observed in AMD retinas, it is unclear whether iron is directly
involved in AMD pathogenesis or is simply a byproduct of AMD pathology.
However, there are several lines of evidence that link iron with AMD pathogenesis;
for instance, the levels of bone morphogenetic protein 6 (BMP 6), a major regulator
of systemic iron (Section 1.4.1), is increased in advanced AMD eyes [216] and
hepcidin-knockout mice experience age-dependent increases in retinal iron fol-
lowed by retinal degeneration [217]. It is significant that retinal iron accumulation
resulting from Friedreich’s ataxia and PANK 2 disease is associated with retinal
degeneration [218].

5.5  P
 otential of Iron Chelators for the Treatment
of Neurodegeneration

A critical prerequisite for a chelator capable of treating various forms of neurode-


generation is to be orally active and to readily permeate the blood brain barrier or in
the case of the eye, the blood-retinal barrier. Thus, the chelator should have a molec-
ular weight <500, be sufficiently hydrophobic to partition in membranes and yet be
sufficiently hydrophilic to achieve water solubility at pharmacological levels [219].
Of the three chelators currently used in the clinic, only deferiprone possesses these
properties (Figure 25), which accounts for why it has been so extensively investi-
gated for the treatment of neurodegenerative diseases [93,220–223]. A range of
8-hydroxyquinoline derivatives, including clioquinol (19) [224], VK-28 (20) [225],
and an iron-binding peptide (21) [226], have also been investigated.

OH OH
N
N
N
Cl

S
I N N
OH OH NAPCSIPE

19 20 21

Deferiprone (15), VK-28 (20), and clioquinol (19) have each demonstrated
neuroprotective action in various models of Alzheimer’s disease [227–229] and
clioquinol has been investigated in a related clinical trial [230]. A similar situation
exists with Parkinson’s disease, where a number of successful studies have been
achieved in various animal models with both 8-hydroxyquinolines [225,231]
and deferiprone [220]. Deferiprone is currently under clinical investigation for
efficacy and safety in Parkinson’s disease [232,233]. Deferiprone has also been
demonstrated to have a beneficial effect in the clinical treatment of age-dependent
retinal degeneration [221,234] and PANK 4 disease [222,223].
8  Iron: Effect of Deficiency and Overload 281

Various strategies have been investigated in an attempt to enhance the transfer of


chelators across the BBB. In principle pendant glucose molecules can be attached to
molecules in order to enhance their ability to permeate the BBB. Due to the critical
requirement of the brain for glucose, the BBB is endowed with a high density of GLUT
1 hexose transporter protein. With this strategy in mind, glucose conjugates (22) and
(23) have been synthesized [235,236], but to date have not been demonstrated to cross
the BBB [237]. Another approach involves the attachment of chelators to nanoparticles
[238]. Chelator hybrid molecules are also under current investigation in a range of
neurodegenerative models, for instance, radical scavengers (24) [239] and monoamine
oxidase inhibitors (25) [240]. This field has recently been reviewed [241].

O
OH

N OH O
O O O
HO HO
OH
HO HO NH HO
N
O OH R

22 23

O
OH

N
N
N

HO
N
OH

24 25

6  The Role of Iron Chelation in Cancer Therapy

The importance of iron in tumor cell growth has been discussed over the past 50
years [242], high levels of dietary iron having been linked to increased tumor devel-
opment [243,244]. More recently an iron regulatory gene signature has been linked
to the incidence of breast cancer [245], and both gastrointestinal and liver cancer
have been specifically associated with dietary iron [246]. Cell proliferation is depen-
dent on a plentiful supply of iron and iron chelators can interfere with the cell cycle,
282 Hider and Kong

causing G1/S arrest by removing iron from ribonucleotide reductase [247] and
deoxyhypusine hydroxylase [248]. Many chelating agents have been investigated
for their potential to selectively inhibit tumor cell growth, in particular desferriox-
amine [249], spermidine catecholamides [250], and PIH analogues [247]. At the
present time it has not been possible to achieve an acceptable selective toxicity
against tumor cells in the clinic.

N N
O O

HN HN
N OH N OH
N N
N
NH
N
S N
OH

26 27 28

Pyridoxal isonicotinoyl hydrazine (PIH) derivatives (26) show potential in this


regard and Richardson and his coworkers have thoroughly investigated this group
of chelators. The β-napthol derivative (27) has been reported to be a particularly
effective antiproliferative agent [251], as have the closely related thiosemicarba-
zones (28) [252]. The precise role of iron in the cell-cycle control remains unclear,
but the posttranscriptional regulation of a cyclin-dependent kinase inhibitor may also
feature in this network of iron-dependent reactions [253].

7  Iron and Infection

Iron is essential for the growth of almost all microorganisms and thus bacteria
and fungi have evolved strategies to scavenge iron from the soil, fresh and marine
water, and living organisms. One of the most common strategies is siderophore
production. Siderophores are low molecular weight compounds (500–1500 dal-
tons) which possess a high affinity and selectivity for iron. There are over 500
different siderophores 270 of which have been structurally characterized [131];
desferrioxamine (7), desferrithiocin (11), and enterobactin (Figure 21) are typi-
cal examples.
Pathogenic bacteria and fungi have developed the means of survival in animal
tissue. They may invade the gastrointestinal tract (Escherichia, Shigella, and
Salmonella), the lung (Pseudomonas, Bordatella, Streptococcus, and
Corynebacterium), skin (Staphylococcus) or the urinary tract (Escherichia and
Pseudomonas). Such bacteria may colonize wounds (Vibrio and Staphylococcus)
and be responsible for septicaemia (Yersinia and Bacillus). Some bacteria survive
for long periods of time in intracellular organelles, for instance Mycobacterium.
Because of this continual risk of bacterial and fungal invasion, animals have
8  Iron: Effect of Deficiency and Overload 283

d­ eveloped a number of lines of defence based on immunological strategies, the


complement system, the production of iron-siderophore binding proteins and the
general “withdrawal” of iron [254].
There are two major types of iron-binding proteins present in most animals that
provide protection against microbial invasion – extracellular protection is achieved
by the transferrin family of proteins and intracellular protection is achieved by
ferritin (Section 1.2.4). Under normal conditions transferrin is about 25–40% saturated,
which means that any freely available iron in the serum will be immediately
scavenged – thus preventing microbial growth. Most siderophores are unable to
remove iron from transferrin, although some, for instance aerobactin, can compete
[255,256]. Mammals also produce lactoferrin, which is similar to serum transferrin
but possesses an even higher affinity for iron [257]. Lactoferrin is present in secre-
tory fluids, such as sweat, tears and milk, thereby minimizing bacterial infection.
Ferritin is present in the cytoplasm of cells and limits the intracellular iron level to
approximately 1 μM. Siderophores are unable to mobilize iron from ferritin. In
addition to these two classes of iron binding proteins, hepcidin is involved in con-
trolling the release of iron from absorptive enterocytes, iron-storing hepatocytes,
and macrophages (Section 1.4.1). Infection leads to inflammation and the release of
interleukin-6 (IL-6) which stimulates hepcidin expression. In humans, IL-6 produc-
tion results in low serum iron, making it difficult for invading pathogens to infect.
In addition to these “iron withdrawal” tactics, mammals produce an iron-­
siderophore binding protein, siderochelin [258]. Siderocalin is a potent bacteriostatic
agent against E. coli. As a result of infection, it is secreted by both macrophages and
hepatocytes, enterobactin being scavenged from the extracellular space. Indeed, mice
are highly susceptible to E. coli infections when the siderocalin gene is “knocked-
out” [259]. Siderocalin binds a wide range of tris-catecholate siderophores.

7.1  Tuberculosis

Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis infects one-­


third of the world population. Despite the development of new drugs, the incidence
of TB continues to increase. Increased iron status enhances tuberculosis infection
[260]. Siderophore molecules used by Mycobacterium for iron acquisition are
potential therapeutic targets, as are synthetic iron chelators which are capable of
outcompeting siderophores for iron.

7.2  Malaria

Many metabolic components of the erythrocytic malaria parasite are dependent


on iron, including ribonucleotide reductase, and mitochondrial function. Several
­studies have indicated that the erythrocyte labile iron pool is the iron source for
284 Hider and Kong

malaria parasites (Section 1.3) [246]; a finding somewhat confirmed by the


observation that iron chelators suppress the growth of P. falciparum in human
erythrocytes. A wide range of chelators have been demonstrated to inhibit the
growth of erythrocytic malaria parasites under in vitro conditions [261,262],
however with the exception of desferrioxamine, extension to animal studies has
proved to be less impressive [262].
There have been a number of clinical investigations centered on the use of
desferrioxamine, where it has displayed an appreciable antimalarial activity but
failed to effect a cure [246].

8  Overview and Future Developments

The development of iron biochemistry has been dramatic over the past 50 years.
A large range of iron-dependent enzymes have been characterized, the chemistry of
electron transfer proteins, containing iron-sulfur clusters, is beginning to be under-
stood and oxygen-binding iron centers are very well characterized. The highly con-
trolled transport of iron throughout multicellular organisms and the mechanism of
intracellular distribution is now understood, although there is one tissue where iron
distribution is far from clear: the brain. Transferrin levels are much lower in CNS
than in blood and there are a number of brain proteins which appear to be influenced
by iron levels and yet currently have no known function, for instance amyloid
precursor protein and α-synuclein. Clearly, this is an area that deserves intensive
investigation, particularly as the progression of many forms of neurodegeneration
appears to be enhanced by elevated iron levels.
Over the same period iron chelators have emerged as an important therapeutic
class. For many years the orally inactive desferrioxamine was the only iron chelator
available for clinical use, but during the past twenty years two other chelators have
been introduced, deferiprone and deferasirox. Both are orally active and this has
rendered the treatment of iron overload to be more “patient friendly” thereby
enabling clinicians to investigate the use of iron chelation for diseases other than
systemic iron overload, for instance Parkinson’s disease and macular degeneration.
There are more iron chelators under development and it is likely that in years to
come there will be a selection of iron chelators available for clinical use that will
cover ranges of both iron affinity and membrane penetrative ability. Studies are in
progress to design lysosomotrophic and mitochondrotrophic chelators.
Improved therapeutic control of a wide range of anemias has been accomplished
over the past 50 years and in particular the development of a range of relatively
nontoxic parential iron preparations has proved to be highly beneficial. Surprisingly,
the most common oral supplement for the treatment of iron deficiency anemia is
ferrous sulfate, which was first introduced to medicine in 1832. There is a very clear
requirement for the introduction of a replacement oral therapy due to the various
toxic side effects of ferrous sulfate. Hopefully one or more such replacements will
be developed in the near future.
8  Iron: Effect of Deficiency and Overload 285

Abbreviations

ALAS 5-amino levulinate synthase


AMD age-related macular degeneration
APP amyloid precursor protein (Fig. 26)
AscH– ascorbic acid
ATP adenosine 5′-triphosphate
BBB blood brain barrier
BMP bone morphogenetic protein
CD91 lipoprotein receptor
CNS central nervous system
CSF cerebrospinal fluid
DCYTB duodenal cytochrome b
DF desferrithiocin
DFO desferrioxamine
DMT1 divalent metal-iron transporter 1
EDTA ethylenediamine-N,N,N′,N′-tetraacetic acid
FBXL5 F-box and leucine-rich repeat protein 5
FDA Food and Drug Administration
FPN1 ferroportin
FXN frataxin
GDF15 growth differentiation factor 15
GLUT 1 glucose transporter 1
Grx 5 glutaredoxin-5
HAMP hepcidin synthesis gene
Hb hemoglobin (Fig. 16)
HCP1 heme carrier protein 1
HEIDI 2,2′-(2-hydroxyethylazanediyl)diacetic acid
HFE hemochromatosis protein
HIV human immunodeficiency virus
HPO hydroxypyridinone
ICP-MS inductively coupled plasma mass spectrometry
IL-6 interleukin 6
IRE iron responsive elements
IRP iron responsive proteins
JAK/STAT janus kinase/signal transducer and activator of transcription
MDS myelodysplastic syndrome
MECAM N,N′-(5-((2,3-dihydroxycyclohexa-1,3-dienecarboxamido)methyl)-
1,3-phenylene)bis(methylene)bis(2,3-dihydroxybenzamide)
MHC major histocompatibility complex
MRI magnetic resonance imaging
MTP metal transporter protein
NHE normal hydrogen electrode
NTBI non-transferrin bound iron
286 Hider and Kong

PANK 2 pantothenate kinase-2


PIH pyridoxal isonicotinoyl hydrazine
SDS sodium dodecyl sulfate
SMAD 4 tumor suppressor gene
SS sickle cell disease
TB tuberculosis
TfR1 transferrin receptor 1
TfR2 transferrin receptor 2
WHO World Health organization
XLSA X-linked sideroblastic anemia

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Chapter 9
Cobalt: Its Role in Health and Disease

Kazuhiro Yamada

Contents
ABSTRACT ............................................................................................................................. 296
1 INTRODUCTION ............................................................................................................. 296
1.1 Cobalt and Vitamin B12 Deficiency ........................................................................... 296
2 COBALAMIN, VITAMIN B12 .......................................................................................... 297
2.1 Biochemistry of Cobalamin ...................................................................................... 297
2.2 Cobalamin Binding Proteins and Transporting System ............................................ 298
2.2.1 Overview of the Cobalamin Absorption and Delivering System .................. 298
2.2.2 Absorption of Cobalamin .............................................................................. 298
2.2.3 Intracellular Processing of Cobalamin .......................................................... 300
2.3 Cobalamin-Dependent Enzymes in Mammals .......................................................... 303
2.3.1 An Overview of the Cobalamin-Dependent Enzymes .................................. 303
2.3.2 Methylmalonyl-Coenzyme A Mutase and Related Metabolism in Mammals ....... 303
2.3.3 Methionine Synthase and Related Metabolism in Mammals ........................ 306
3 VITAMIN B12 DEFICIENCY AND DISEASE................................................................. 310
3.1 Vitamin B12 Deficiency.............................................................................................. 310
3.2 Methylmalonic Aciduria ........................................................................................... 311
3.3 Hyperhomocysteinemia............................................................................................. 311
3.4 Megaloblastic Anemia .............................................................................................. 312
3.5 Cobalamin Neuropathy ............................................................................................. 312
3.6 Other Diseases Related to Vitamin B12 Deficiency ................................................... 312
3.7 Animal Models .......................................................................................................... 313
4 NON-CORRINOID COBALT ........................................................................................... 314
4.1 Non-corrinoid Cobalt-Containing Proteins ............................................................... 314
4.2 Overload of Cobalt .................................................................................................... 315
5 IMPLICATIONS AND FUTURE DEVELOPMENT ....................................................... 315
ABBREVIATIONS .................................................................................................................. 316
ACKNOWLEDGMENT .......................................................................................................... 317
REFERENCES ........................................................................................................................ 317

K. Yamada (*)
Department of Biochemistry, Uniformed Services University of the Health Sciences,
4301 Jones Bridge Road, Bethesda, MD 20814, USA
e-mail: kazuhiro.yamada@usuhs.edu

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 295
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_9, © Springer Science+Business Media Dordrecht 2013
296 Yamada

Abstract The primarily function of cobalt in humans is based on its role in cobalamin
(Cbl, vitamin B12). Therefore, this chapter will focus on the physiological roles
of Cbl and the importance of cobalt in human health. Cbl acts as the cofactor for
two enzymes, i.e., methylmalonyl-CoA mutase and methionine synthase, in humans.
Both enzymes are important for health. In addition, unlike other water-soluble
vitamins, there is a unique absorption, delivery, and activation system for Cbl in
mammals. Therefore, this chapter will also review the literature on the Cbl
transporting system, which is crucial for Cbl function.

Keywords cobalamin • methionine synthase • methylmalonyl-CoA mutase


• vitamin B12

Please cite as: Met. Ions Life Sci. 13 (2013) 295–320

1 Introduction

1.1 Cobalt and Vitamin B12 Deficiency

Cobalt (Co) is an essential (mineral) micronutrient for humans. Historically, however,


obvious nutritional Co deficiency has not occurred in humans. This could imply that
cobalt is not an essential factor for human health, per se. In contrast, Co deficiency has
been identified in some ruminant mammals, including sheep, goat, and cattle. Ruminants
raised in areas where cobalt is scarce show reduced food intake and growth retardation.
This problem has been historically recognized by dairy farmers. Later, this pheno-
menon was realized to be vitamin B12 (B12) deficiency rather than simple Co deficiency,
and that it was related to the synthesis of cobalamin (Cbl) by microorganisms in
the stomach. Moreover, scientists working in veterinary nutrition had identified an
“animal protein factor”, that is, a nutritional growth factor that is exclusively present
in animal foods, B12 being the strongest animal protein factor [1]. These observations
indicate that the primary function of cobalt in mammals is in the form of Cbl. B12 is an
essential micronutrient for humans, and Cbl is its functional unit. Therefore, this
chapter focuses on the function of Cbl as the primary role for cobalt in humans.
The Recommended Dietary Allowance of Cbl for the adult human is 2.4 μg per
day, which is the lowest of all nutrients. Cbl and the related substances, corrinoids,
can only be produced by certain microorganisms. Cbl found in food is originally
from these bacteria, in which the complex molecule is synthesized using at least 25
genes in the Cob operon [2]. The B12 content in food is very low. Its greatest abun-
dance is in meat, fish, and milk products; it is generally absent from fruits and veg-
etables, but nori, an edible green and purple seaweed, is a non-dairy product that
contains a significant amount of B12 [3]. It is therefore an important potential source
of B12 for strict vegetarians. However, the bioavailability of B12 in seaweed is still
controversial because seaweed may contain pseudovitamin B12, which is an inactive
B12 analog [3]. Humans require an authentic form of Cbl, and a human possesses an
ingenious system for the precise selection and absorption of Cbl.
9 Cobalt: Its Role in Health and Disease 297

Cbl acts as the cofactor for two enzymes present in humans, i.e., methionine
synthase and methylmalonyl-CoA mutase. One of the best-characterized human
diseases caused by B12 deficiency is megaloblastic anemia, also known as pernicious
anemia. It is thought that the inactivation of methionine synthase is responsible for
this disease. Dysfunction of Cbl-dependent enzymes can be caused by inadequate
intake of B12. However, it can even occur in the presence of adequate amounts of
B12, due to inheritance of gene mutations of Cbl-dependent enzymes or failure of the
Cbl delivery system. Thus, functioning of these proteins is also essential for proper
Cbl function in humans.

2 Cobalamin, Vitamin B12

2.1 Biochemistry of Cobalamin

Cbl has been called nature’s most beautiful cofactor [4] and was identified as the
anti-pernicious anemia factor from liver in 1948 [5,6]. Since then, many studies on
the chemical properties of Cbl have been reported. The structure was solved by
Hodgkin et al. using X-ray structural analysis in 1956 (Figure 1) [7]. As the structure
shows, Cbl is a large and complex molecule. The characteristic tetrapyrrole ring is
called the corrin ring, and compounds containing the corrin ring are called corri-
noids. Unlike iron in the porphyrin ring of heme, whose tetrapyrrole ring is similar
to the corrin ring, cobalt in Cbl is not interchangeable with other metals. Cobalt
cannot be released from the ring unless the ring is broken.
To investigate the chemical properties of cobalt bound to Cbl, cobaloxime can be
used as a model compound, although it does not itself possess B12 activity. Cbl con-
tains a nucleotide loop connected to the D ring of the corrin ring, and the dimethyl-
benzimidazole (DBI) base in the tail of the nucleotide loop is coordinated to the
cobalt atom (Figure 1). The DBI coordinating side of the corrin ring is referred to
as the lower axial position. Cobinamide and cobamide are corrinoids that lack the

R CONH2

H2NOC B CONH2
A
N
H2NOC N
Figure 1 Structure of Co N
N C
cobalamin. Vitamin B12 H2NOC D

(cyanocobalamin,
CONH2
Coα-[α-(5,6-demethylbenzi- O
midazolyl)]-Coβ-cyano- N

cobamide) has the CN NH N


group in the upper ligand. HO
Methylcobalamin (R = CH3-), O
cyanocobalamin, R= CN-
O methylcobalamin, R= CH3-
and adenosylcobalamin
O P O adenosylcobalamin, R= 5’-deoxyadenosyl-
(R = 5′-deoxyadenosyl-)
O- HO
are the cofactor forms.
298 Yamada

nucleotide loop and the DBI moiety (compared to Cbl), respectively. In the cob(III)-
alamin state (which indicates a +3 oxidation state of cobalt), Co in Cbl is six-coordinate.
It has an upper ligand, e.g., methyl, 5′-deoxyadenosyl, water, or cyano groups, for
methylcobalamin (CH3-Cbl), adenosylcobalamin (AdoCbl), aquacobalamin, or
cyanocobalamin (CN-Cbl), respectively. CH3-Cbl and AdoCbl are the cofactor forms
for methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), respectively.
CN-Cbl is a largely artificial form of Cbl, produced and purified industrially, that
can be converted to active cofactor forms in the body. In the strictest sense, B12 is
CN-Cbl, as this is the commercially most available form. Cobalt in the corrin ring can
be reduced to the +2 or +1 states, called cob(II)alamin and cob(I)alamin, respectively.
Cob(II)alamin contains five-coordinate cobalt without any upper ligand. Cob(I)-
alamin is four-coordinate, so neither the DBI moiety nor an upper ligand are coordinated
to Co. Cob(I)alamin is known as a hypernucleophilic species [8]. Other variants of
cobamide can be found in nature. For example, the DBI moiety can be replaced by
adeninyl, 2-methyladeninyl, 5-hydroxybenzimidazolyl, or methoxybenzimidazolyl
groups in pseudoB12, in factor A, factor III, or 5′-methoxybenzimidazole cobamide,
respectively. For mammalian nutrition, Cbl is the most active form of B12; other
cobamides show very weak or no B12 function.

2.2 Cobalamin Binding Proteins and Transporting System

2.2.1 Overview of the Cobalamin Absorption and Delivering System

The famous discovery of B12 as the anti-pernicious anemia factor was conducted by
Minot and Murphy [9]; they treated pernicious anemia patients with large amounts
of animal liver. Liver contains quantities of B12 that are approximately 10 times
greater than in other meats. The B12 content in calf liver, which is one of the richest
B12-containing foods, is ~50 μg/100 g. In general, however, it is not necessary for
normal adults to eat large amounts of liver.
To take advantage of the precious nutrient Cbl, a specific transportation system
is available immediately after intake of food. In all steps for Cbl transportation,
there are specific proteins, whose function is essential for proper delivery and pro-
cessing of Cbl. Hence, dysfunction of any of these proteins caused by gene muta-
tions may result in functional B12 deficiency. While the discovery of the Cbl
absorption and transportation system is an old story, a new era has emerged as genes
coding for the proteins responsible for intracellular processing of Cbl have recently
been identified.

2.2.2 Absorption of Cobalamin

The pioneer work on absorption of Cbl was done by Castle. In 1936, Castle and
Ham reported that administration of a digested mixture of beef meat and gastric juice
to pernicious anemia patients provided an effective cure [10]. The substances in
9 Cobalt: Its Role in Health and Disease 299

Figure 2 Crystal structures of Cbl binding proteins and Cbl binding forms. (a) Intrinsic factor-
Cbl complex (PDB 2PMV). (b) Transcobalamin-Cbl complex (PDB 2BB5). (c) CblC protein, the
MMACHC gene product (PDB 3SC0). Proteins are shown in the ribbon models and Cbl is illus-
trated in the stick mode. Figures are generated by PyMol [127].

beef meat and gastric juice were named the external and internal factors, respectively.
Later, both factors were identified: the external factor was shown to be Cbl and the
intrinsic factor to be a glycoprotein secreted from the stomach. The glycoprotein
was later called intrinsic factor (IF). There is another Cbl-binding protein, haptocor-
rin (also known as R-binder), present in the digestive tracts of humans. However, IF
shows the highest affinity for Cbl, and a lower affinity for other corrinoids. Binding
of IF is quite selective for the lower ligand of corrinoids, which contain the imidazo-
lyl group as lower ligand [11]. The crystal structure of the IF-Cbl complex is shown
in Figure 2a [12].
This complex may provide the initial selection of Cbl as B12 because there are
many other B12 analogues, such as the aforementioned pseudoB12 contained in sea-
weed. After release of Cbl from food protein or from the Cbl-protecting protein
haptocorrin, Cbl is captured by the IF. In the ileum, the Cbl-IF complex is recog-
nized by cubam, the receptor for the IF-Cbl complex consisting of a heterodimer of
amnionless and cubilin [13,14]. Mutation of genes for cubam may cause the
Gräsbeck-Imerslund syndrome [15,16], which is characterized by malabsorption of
Cbl with normal IF production and function. If a patient has an autoantibody against
IF due to autoimmune destruction of gastric parietal cells by an atrophic gastritis,
300 Yamada

Cbl binding to IF or interaction between the IF-Cbl complex and the IF receptor
can be inhibited, potentially resulting in malabsorption of Cbl. Once formed, the
complex of the IF-Cbl-IF receptor is absorbed by endocytosis.
IF is degraded in the lysosome and Cbl passes through the cytosol of the
ileal epithelial cell to the bloodstream. Cbl is released from ileal cells by MRP1, a
multidrug resistance protein [17], although an alternative pathway has been
proposed [18]. In blood, Cbl binds with either transcobalamin (TC) or to haptocorrin.
The crystal structure of the complex of TC-Cbl is shown in Figure 2b [19 ].
The Cbl-TC complex is then captured by the TC receptor on target cells.

2.2.3 Intracellular Processing of Cobalamin

Cbl must undergo cellular transport and activation by proteins before it can
bind to Cbl-dependent enzymes. Patients with hereditary B12 deficiency due to defects
in Cbl utilization have been identified. To date, nine complementation groups for
impaired cellular Cbl metabolism, CblA-CblG, CblJ, and mut, have been identi-
fied. (The CblH complementation group could be a subclass of the CblD comple-
mentation group [20]). All genes corresponding to each group have been identified
in the last 10 years. With the exception of the MS and MCM proteins, comple-
mentary genes were identified prior to isolation of the protein because the com-
plementary proteins could not be detected in cell extracts, even when 57Co-labeled
Cbl was used [21,22]. The functions of most of these gene products are still under
active investigation.
Cellular Cbl processing is summarized in Figure 3. The Cbl-TC complex is rec-
ognized by the TC receptor on the cell surface and absorbed into the cell by endo-
cytosis. Cbl is released from TC in the lysosome, and then it is transferred to cytosol.
From lysosome to cytosol, there are two genes, LMBD1 and ABCD4, which are
responsible for the CblF and CblJ complementation groups, respectively. These
genes encode membrane proteins. The CblF protein (the LMBD1 gene product) is
identified as a lysosomal membrane exporter of Cbl [23]. The protein shows signifi-
cant homology to the lipocalin-1 interacting membrane receptor: a cell receptor for
internalization of lipocalins. Lipocalins are small proteins carrying hydrophobic
molecules in body fluids. The CblJ protein (the ABCD4 gene product) is an ABC
transporter, and it is suggested that ATPase activity of the protein is required for Cbl
processing [24].
The CblC protein, the gene product from MMACHC (methylmalonic aciduria,
cblC type, and homocystinuria) [25], may have a role in accepting Cbl from these
membrane proteins [26], although there is no evidence for the direct interaction of
two membrane proteins and the CblC protein. The crystal structure of the CblC
protein has been solved [27] (Figure 2c). Unlike IF and TC, the lower ligand of Cbl
is dissociated upon binding to the CblC protein. On the one hand the conformation
of Cbl in solution and upon binding to IF and TC is called the DBI-on form, and on
the other that of Cbl in the CblC protein is referred to as the DBI-off form. The first
observation of the DBI-off form of Cbl was in the Cbl-binding domain of MS [28].
While both MCM and MS have the signature amino acid sequence motif, DxHxxG
9 Cobalt: Its Role in Health and Disease 301

CN
TC•Cbl complex
Co
DBI
TC receptor

cytosol
e
som
lyso TC
endocytosis
degradation
CblF
CN 1
BD
L M
Co
4
DBI
B CD
CblJ A

mitochondrion CblC
CN
MMACHC
Co CblG
DBI

mtr MS
DBI

Co
Ado

CblB
Co

Ado
MMAB
Co
DBI CblD
DBI

MMADHC His
Ad

DB
o

I
Co

CblA inactivation CblE


MMAA Co mtrr MSR
DBI

His
Ado
DBI

Co Co
CH3
DBI

Ado
His
mut Co
MCM
DBI

His

Figure 3 Cellular cobalamin metabolism. Nine complementation groups, CblA~G, CblJ, and
mut, are shown in bold letters and the responsible genes in light letters (see text for details).

for the DBI-off form of Cbl binding, the CblC protein does not contain the motif.
CblC exhibits the catalytic function for the reductive de-cyanation of CN-Cbl in
the presence of methionine synthase reductase (MSR), a dual flavoprotein similar to
the P450 reductase family (see Section 2.3.3), and NADPH [26]. MSR cannot be the
302 Yamada

physiological partner protein for the reduction, so the reducing protein has yet to
be identified.
Mutations in LMBD1, ABCD4, and MMACHC genes cause both methylmalonic
aciduria and homocysteienemia because their impairment causes dysfunction of
both MCM and MS. In contrast, the phenotype of patients in the CblD complemen-
tation group varies; it can cause methylmalonic aciduria (MMA) or homocystei-
enemia, or both. Hence, the function of the CblD protein must be down-stream of
that of the CblC protein, and the CblD protein controls the fate of Cbl; to be trans-
ported into the mitochondrion or to remain in the cytosol. The gene, MMADHC
(methylmalonic aciduria, cblD type, and homocystinuria), corresponding to the
CblD group, was cloned in 2008 [29]. The biochemical structure and function of
CblD have not yet been reported. Because a reductive partner is required for the
reduction of CN-Cbl bound to the CblC protein, the CblD protein might have
reductase activity in addition to controlling the branching point of the Cbl delivery
pathway (Figure 3).
In mitochondria, Cbl has to be processed to become the active cofactor form,
AdoCbl. MCM is the product of the mut gene, and the mut0 and mut– groups are
subtypes of patients that represent complete and partial deficiency of MCM enzyme
activities, respectively. Since MCM strictly requires AdoCbl for its activity, it was
thought that CblA and CblB proteins should have both adenosyltransferase activity
and Cbl reductase activity. The CblB protein, which is the product of the MMAB
gene, shares homology with bacterial ATP-cob(I)alamin adenosyltransferase [30].
Crystal structures of human ATP-cob(I)alamin adenosyltransferase [31] and the
bacterial homologue with Cbl [32] have been solved.
The four-coordinate cob(II)alamin already mentioned facilitates the reduction
of cob(II)alamin in catalysis because the enzyme needs to generate cob(I)alamin
for the reaction. The purified CblB protein can produce AdoCbl from cob(II)-
alamin and ATP in the presence of MSR and NADPH [33]. MSR cannot be the
physiological reductase because the CblE complementation group is indepen-
dent from MCM function. The physiological reductase has yet to be identified.
The CblA protein, which is the product of the MMAA gene [34], was initially
postulated to be the Cbl reductase. However, the enzyme property has recently
been proposed as a molecular chaperone. The protein is similar to members of
the GTPase family of metal insertion proteins. The protein function was pro-
posed using bacterial MCM and MeaB, an orthologue of MMAA in bacteria, as
a model (see the next section).
In cytosol, Cbl finally binds to MS. MS is encoded in the mtr gene, which cor-
responds to the CblG complementation group [35–37]. For MS, CH3-Cbl is the
active cofactor, but the non-methyl form of Cbl, i.e., cob(II)alamin, can also bind
and act as cofactor because MS can regenerate the active cofactor. For this reaction,
an electron donor is required. The reducing equivalent is supplied from NADPH via
MSR. The gene encoding MSR is identified as mtrr, which corresponds to the CblE
complementation group [38] (see the next section).
9 Cobalt: Its Role in Health and Disease 303

2.3 Cobalamin-Dependent Enzymes in Mammals

2.3.1 An Overview of the Cobalamin-Dependent Enzymes

There are two enzymes that use Cbl as a cofactor in humans, MCM and MS. AdoCbl
is utilized by MCM and CH3-Cbl is the active cofactor for MS. Binding of Cbl to
both enzymes is as the DBI-off form [28,39], which is shown in Figure 4a and 4b.
The biochemical properties of these Cbl-dependent enzymes have been reviewed
recently in this series [40]. This section will therefore focus on the physiological
aspects of these enzymes.

2.3.2 Methylmalonyl-Coenzyme A Mutase and Related


Metabolism in Mammals

MCM catalyzes the rearrangement of the carbon backbone in the conversion of


methylmalonyl-CoA to succinyl-CoA (Figure 5). MCM requires AdoCbl to cata-
lyze this reaction. Although there are more AdoCbl-dependent enzymes known in
the microbial world, MCM is the only AdoCbl-dependent enzyme in mammals. All
AdoCbl-dependent enzymes catalyze difficult reactions that require the production
of reactive radical species.
The production of reactive radical species is accomplished using AdoCbl as the
radical generator [41] (Figure 5). AdoCbl undergoes homolytic cleavage of the
Co-C bond and yields a 5′-deoxyadenosyl radical and cob(II)alamin. The radical
abstracts a hydrogen atom from the substrate to form deoxyadenosine. The radical
on the substrate rearranges the carbon backbone, and then the radical on the product
is returned to the cofactor. Because of the high reactivity of the radical intermediate,
AdoCbl-dependent enzymes are sensitive to inactivation due to unexpected side
reactions. Auxiliary reactivating proteins have been studied using AdoCbl-
dependent diol dehydratase, a bacterial enzyme. The reactivation factors for diol
dehydratase from Klebsiella oxytoca have been identified and characterized [42–
46]. The overall reaction of the reactivation consists of the replacement of the inac-
tivated cofactor binding to the inactive holoenzyme by the authentic cofactor,
AdoCbl, in the presence of ATP. The molecular chaperone function of the reactivat-
ing factor can be found in other bacterial AdoCbl-dependent enzymes, such as glyc-
erol dehydratase and ethanolamine ammonia-lyase, which bind AdoCbl in the
DBI-on form.
Banerjee and colleagues have reported that MeaB, which is a G-protein and
shares homology to the human MMAA protein, acts as the molecular chaperone
for MCM [47–49]. Three auxiliary functions of MeaB for MCM have been
proposed: (i) “Editing”, MCM-MeaB-GTP prevents binding of cob(II)alamin to
apoMCM using the binding energy of GTP by MeaB. (ii) “Gating”, MCM-MeaB-GTP
Figure 4 Protein structures of methylmalonyl-CoA mutase and methionine synthase. (a) The cobalamin
binding domain of human MCM (monomer) is shown in red. The substrate binding domain is
illustrated in cyan with transparency. Cbl is shown in stick mode (PDB 2XIQ). (b) The cobalamin
binding domain of E. coli MetH (methionine synthase). The cap structure is shown in transparency
(PDB 1BMT). (c) The overall structure of the MCM dimer. The color scheme is the same as in
panel (a). One of two subunits is shown transparent. (d) Structures of bacterial methionine synthase.
The substrate homocysteine and folate binding domains of T. maritima MetH are shown in yellow
and green, respectively (PDB 1Q8J). The Cbl binding domain (red) and the AdoMet binding
domain (blue) of E. coli MetH are drawn in the reactivation conformation (PDB 1K7Y). -- Proteins
and Cbl are illustrated in ribbon models and in the stick mode, respectively. Figures are generated
by PyMol [127].
9 Cobalt: Its Role in Health and Disease 305

S CoA
O H
-
OOC H 2
H H
methylmalonyl-CoA
S CoA
O H
-
OOC
1 Ado H H Ado
AdoCbl Cbl
II
Cbl
II
3
MCM MCM MCM
O
S CoA
-
OOC H
H H

O
H S CoA 4
-
OOC H
H H
succinyl-CoA

Figure 5 Reaction mechanism of methylmalonyl-CoA mutase. (1) Unfavorable equilibrium of the


homolytic cleavage of AdoCbl. (2) A hydrogen atom abstraction forms the substrate, methylmalonyl-
CoA. (3) Rearrangement of the carbon skeleton and the radical migration. (4) A hydrogen
atom abstraction by the product radical from 5′-deoxyadenosine. Ado• = 5′-deoxyadenosyl radical,
AdoCbl = adenosylcobalamin, CblII = cob(II)alamin, MCM = methylmalonyl-CoA mutase.

introduces AdoCbl to apoMCM (cofactor loading) with GTP hydrolysis.


(iii) “Rescue”, MCM-MeaB-GTP displaces the inactive cofactor, Cbl without the
5′-deoxyadenosyl group. Crystal structures of the MeaB protein from Methylobacterium
extorquens AM1 [50] and the human MMAA protein [51] have been reported.
The crystal structure of MCM has been solved using Propionibacterium shermanii
MCM [39], and the structure of the human enzyme was recently solved (Figure 4c)
[51]. The overall structure of the catalytic subunits of both MCM is similar. However,
there are important structural differences between the bacterial and human proteins;
for example, the bacterial MCM forms a heterodimer, while the mammalian enzyme
is a homodimer. Biochemical study of the structure of the human MCM and MMAA
proteins reveals that the basic role of the molecular chaperone in bacterial systems
is almost adaptable to the human system, although the detailed mechanism could
be different [51].
Under physiological condition, the mammalian MCM protein exists mostly as
apoprotein, which lacks the AdoCbl cofactor. Even when sufficient amounts of Cbl
(25 μg CN-Cbl/kg diet) were added to the diet of rats, the ratio of holo- to total
MCM is less than 5% [52]. The low ratio of holoMCM has been observed in sheep
[53] and fruit bat’s tissues [54]. During B12 deficiency, MCM activity is lower,
due to the lack of cofactor. Interestingly, the amount of MCM protein in the liver
of the B12-deficient rats is increased [52]. While the physiological significance of
apoMCM is unknown, it stands in stark contrast to that of the apoMS protein, which
is quite unstable.
306 Yamada

Valine, isoleucine,
odd-chain fatty acids

-
HCO3
O O O O O O
1 2 3 -
- - O
S CoA O S CoA O S CoA S CoA
CH3 CH3 O

ATP ADP
propionyl-CoA (S )-methyl- (R )-methyl- succinyl-CoA
malonyl-CoA malonyl-CoA

4
HS CoA
TCA cycle
O O
HO OH
CH3

methylmalonic acid

Figure 6 Propionyl-CoA metabolism: (1) Biotin-dependent propionyl-CoA carboxylase. (2)


Methylmalonyl-CoA racemase. (3) Methylmalonyl-CoA mutase. (4) (S)-Methylmalonyl-CoA
hydrolase.

MCM is involved in propionyl-CoA catabolism to succinyl-CoA, which includes


branched-chain amino acid and odd-chain fatty acid metabolisms (Figure 6).
Propionyl-CoA is combined with HCO 3− in the presence of ATP, forming
( S )-methylmalonyl-CoA by the reaction catalyzed by propionyl-CoA carboxylase,
a biotin-dependent enzyme. The product, (S)-methylmalonyl-CoA, is converted to
(R)-methylmalonyl-CoA by methylmalonyl-CoA racemase. (R)-methylmalonyl-CoA
is the only substrate capable of binding in the substrate-binding pocket to MCM.
Dysfunction of MCM elevates the level of methylmalonic acid in blood and urine,
which are known as methylmalonic anemia and MMA, respectively. Methylmalonic
acidemia is organic acidemia, which is often diagnosed in the early neonatal period,
and this symptom is one of the hallmark characteristics of B12 deficiency.

2.3.3 Methionine Synthase and Related Metabolism in Mammals

The reaction catalyzed by MS is shown in Figure 7. Overall, the reaction consists of


the transfer of the N5-methyl group of methyltetrahydrofolate (CH3-H4folate) to the
thiol group of homocysteine. CH3-Cbl acts as the intermediate for this reaction,
which is divided into two-steps: (i) the methyl group of CH3-Cbl is donated to
homocysteine, forming methionine and cob(I)alamin; (ii) the hypernucleophilic
cob(I)alamin accepts the methyl group from CH3-H4folate to regenerate CH3-Cbl.
This methyl transfer reaction proceeds by the ping-pong mechanism [55].
9 Cobalt: Its Role in Health and Disease 307

H R
O H N
- N
O O HN
O-
O
N N
COO-
NH NH2
H +
O H S NH3
R=
H4 folate Homocysteine

CH3 H R
O N 2 1
CH3 -Cbl · MS
HN
N COO-
CH3 +
S NH3
NH2 N N
H methionine

CH3-H4folate
CblI · MS
AdoHcy

e- e- + AdoMet
CblII · MS

Figure 7 Reaction catalyzed by methionine synthase. The solid lines (reactions 1 and 2) show the
catalytic cycle of the enzyme. The broken line indicates inactivation of the enzyme. The reductive
methylation is shown with the dotted line. Three different methyltransfer reactions are catalyzed by
methionine synthase: (1) Transmethylation from CH3-Cbl to homocysteine. (2) Transmethylation
from CH3-H4folate to cob(I)alamin. (3) Reductive methylation of cob(II)alamin to regenerate the
CH3-Cbl cofactor. Methionine synthase is a multi-modular protein, which consists of four domains:
The homocysteine, folate, cobalamin, and AdoMet binding domains in a single polypeptide from
the N-terminus (Figure 4d). The enzyme orchestrates the domain arrangements for each reaction.

Because of the reactivity of the intermediate, cob(I)alamin loses an electron once


in approximately every 2000 turnovers [56,57]. Since the oxidized cofactor, cob(II)-
alamin, is inactive, the enzyme needs to reactivate the cofactor. MS is reactivated by
reductive methylation, in which one electron and a methyl donor are required to
reconstitute CH3-Cbl. The reactivating methyl donor is S-adenosylmethionine
(AdoMet). MSR, a dual flavoprotein similar to the P450 reductase family, and
NADPH are needed for the physiological electron donor for mammalian MS
[58,59]. Free CH3-Cbl can successfully bind to apoMS, forming holoMS. However,
aquacobalamin is much less effective to form the holoenzyme. When aquacobala-
min is reduced to cob(II)alamin in the presence of MSR and NADPH, the cofactor
can be efficiently loaded into apoMS [59,60]. MSR demonstrates the holoMS syn-
thase function, which was proposed for bacterial MS holoenzyme formation [61,62].
Biochemical properties and structural features of methionine synthase have been
studied using the MetH protein from Escherichia coli, a homologue of human MS.
Although this is a bacterial protein, the amino acid sequence of E. coli MetH [63]
shares very high homology (55% in identity) to that of human MS. Thus, E. coli
308 Yamada

MetH provides a model to understand the biochemical properties and catalytic function
of human MS. E. coli MetH has four domains; the N-terminus MetH contains the
homocysteine, folate, Cbl, and AdoMet binding domains [64]. Crystal structures of
the Cbl and AdoMet domains from E. coli have been solved [28,55,65] as have the
homocysteine and folate-binding domains from Thermotoga maritima MetH, which
is similar to E. coli MetH and human MS [66] (Figure 4d).
The structure of the Cbl-binding domain from E. coli MetH was the first solved
for any Cbl-binding proteins [28]. Upon binding to MetH, Cbl undergoes a large
conformational change; the DBI base is replaced by the His residue from the pro-
tein and coordinated to the cobalt atom. Even though the nucleotide loop would
appear to have no function because Cobinamide, which lacks the nucleotide loop,
cannot act as cofactor [57]. The ribose moiety of Cbl could act as a spacer[67],
and the phosphodiester group is necessary for catalytic function [57]. Cobinamide
methylphosphate, the Cbl analogue, which lacks ribose and the DBI moieties but
has the phosphodiester group, shows catalytic function. The analogue is, however,
easily released from the enzyme, whereas the Cbl-MS holoenzyme complex is
quite stable.
Protein structures of each domain of bacterial MS have been solved (Figure 4d),
however, the full-length protein structure has yet to be determined. The structural
analysis of bacterial MS implies large domain rearrangements during the catalytic
turnover of MS because the Cbl-binding domain has to interact with three indepen-
dent binding domains for each substrate to accomplish the methyl transfer reaction.
Cbl plays an important role not only for the methyl transfer reaction, but also for
domain rearrangement during catalysis through the His ligand of the protein
[68–70]. Because of the high homology between E. coli MetH and human MS, the
properties and reaction mechanisms are likely to be quite similar. For the reactiva-
tion of E. coli MetH, reduced flavodoxin is required as the electron donor. MSR is
homologous to the P450 reductase family of enzymes, which contains FMN and
FAD as prosthetic groups. Although the FMN domain in MSR is homologous to
flavodoxin, reduced-flavodoxin is unable to reactivate oxidized human MS [59],
indicating that specific protein-protein interactions are important.
MS is involved in folate and methionine metabolism, which is quite complex
(Figure 8). The dominant form of folic acid in blood is CH3-H4folate, which is
absorbed into cells. MS is the only enzyme capable of metabolizing CH3-H4folate in
mammalian cells. Thus, the reaction catalyzed by MS is the first step for folate
metabolism. The product, tetrahydrofolate (H4folate), is important as a carrier for a
C1 unit, which is a functional group consisting of a single carbon atom, such as
formyl or methylene groups.
The C1 unit on 10-formyltetrahydrofolate is used in purine synthesis and the
5-methyl group of thymidine monophosphate (dTMP) is derived from methylene-
tetrahydrofolate (CH2-H4folate). Thus, the folate C1 unit is important for de novo
synthesis of nucleic acids, precursors of DNA and RNA. H4folate is also important
for glycine, serine, and histidine metabolism. Thus, cellular storage of reduced
folates, except CH3-H4folate, is often referred to as the “functional folate pool”.
CH3-H4folate is produced by the reaction catalyzed by methylenetetrahydrofolate
9 Cobalt: Its Role in Health and Disease 309

transmethylation
cysteine CH3-X X polyamine
synthesis

cystathionine AdoHcy AdoMet


4 Ado 3 2 ATP
serine homocysteine methionine

1
CH3-H4folate CH3-H4folate H4folate
intracellular fluid (cytosol)

6
extracellular fluid (blood)

serine
glycine
5 CHO-H4folate 8

purine base
CH2-H4folate
H2folate synthesis

dUMP 7 dTMP

Figure 8 Folate and methionine metabolism. (1) Methionine synthase. (2) Methionine adenosyl-
transferase. (3) Adenosylhomocysteine hydrolase. (4) Cystathionine-β-synthase. (5) Methylene-
tetrahydrofolate reductase. (6) Serine hydroxymethyltransferase. (7) Thymidylate synthase.
(8) Dihydrofolate reductase.

reductase (MTHFR). This reaction is important to produce methionine using the C1


unit as the methyl source for the reaction of MS. Mammalian MTHFR activity is
strongly inhibited by AdoMet [71]. While S-adenosylhomocysteine (AdoHcy) is not
an activator for MTHFR, the enzyme’s activity is restored because AdoHcy com-
petes with the AdoMet binding site on MTHFR. This negative feedback is important
for the regulation of methionine production.
Inactivation of MS can happened due to the lack of Cbl, dysfunction of MSR,
or upon nitrous oxide (N2O) exposure. N2O, known as laughing gas, is used for
anesthetic purposes. It is known that long-time exposure to N2O can cause mega-
loblastic anemia. N2O exposure inactivates MS but not MCM [72]. The mecha-
nism of inactivation was studied using E. coli MetH. The analysis revealed that
inactivation is based on the irreversible chemical modification of the MS protein
[73–75]. To fully recover MS activity in tissues caused by N2O inactivation, it
takes approximately 48 hr [76 ]. The long recovery time to restore MS activity
is also observed in B12-deficient rats. In B12-deficient rat liver, MS activity is
lowered and MS protein levels are decreased while MS mRNA levels are not
changed [77]. Mammalian apoMS is quite unstable compared to holoMS [57,78].
Hence, administration of Cbl to B12-deficient rats does not allow for a quick recov-
ery due to lack of apoMS. To restore enzyme activity in both N2O-exposed and
B12-deficient rats, it takes a period of time necessary to synthesize new MS protein.
This is different from MCM, which recovers activity quickly because the MCM
apoenzyme is stable in mitochondria [52].
310 Yamada

Dysfunction of MS influences not only methionine production but also folate


metabolism. The MS-deficient mouse (Mtr−/− mouse) is embryonic lethal even when
various nutritional diets were fed, indicating the fundamental importance for this
enzyme [79]. Methionine is an essential building block for proteins. Moreover, it is
generated from AdoMet, which is the major methyl donor for the biological methyl
transferase reaction and is a substrate for polyamine synthesis. For folate metabo-
lism, MS introduces the reduced folate into the cellular “functional folate pool”.
When MS is inactivated, production of H4folate and methionine is decreased. The
reduced-methionine level lowers AdoMet synthesis. Since AdoMet is a strong inhi-
bitor for MTHFR, MTHFR increases its activity, forming CH3-H4folate. Additionally,
accumulation of homocysteine leads to increased AdoHcy, which competes with the
AdoMet binding site of MTHFR. However, the product, CH3-H4folate, can only be
utilized by MS, which is being inactivated, resulting in the accumulation of CH3-
H4folate. This is called the “methyl(folate)-trap hypothesis” [80,81]. Reduced folate,
such as H4folate, is trapped as the CH3-H4folate form, and thus the “functional folate
pool” is depleted.
While administration of folic acid can increase the cellular folate pool, it does
not provide the fundamental solution. Methionine supplementation also masks
B12-deficient symptoms, such as Cbl neuropathy [82,83] and testicular damage [78].
In this case, the “methyl-trap hypothesis” provides the following explanation:
Dietary methionine provides a source of AdoMet, which inhibits MTHFR activity, thus
CH3-H4folate is not produced and folate can be retained in the “functional folate
pool”. It seems that the methyl-trap hypothesis rationally explains the mechanism,
although it is still controversial. There is a report that demonstrates that amounts of
dNTPs, precursors of DNA, are normal [84] and the net amount of CH3-H4folate is
not increased during B12 deficiency [85].

3 Vitamin B12 Deficiency and Disease

3.1 Vitamin B12 Deficiency

We know how to diagnose B12 deficiency and how to effectively treat it. Even though
vitamin deficiency and the corresponding disease can be treated effectively, we still
do not understand which exact biochemical mechanism underlies most cases.
Despite that administration of Cbl is effective to megaloblastic anemia and Cbl
neuropathy, we still do not know what causes these diseases. That is so because Cbl
is not the direct trigger for megaloblastic anemia and Cbl neuropathy, as well as for
other symptoms of Cbl deficiency, even though they are well-established symptoms
of B12 deficiency. The secondary effects of B12 deficiency, i.e., metabolic imbal-
ances, could be responsible. Cbl-dependent enzymes, especially methionine syn-
thase, are important to maintain a healthy metabolism. There are still many things
remaining to identify the exact biochemical mechanisms. To that end, the typical B12
deficiency symptoms will be discussed.
9 Cobalt: Its Role in Health and Disease 311

3.2 Methylmalonic Aciduria

Methylmalonic aciduria is a characteristic symptom caused by defects of MCM


(Figure 6). Accumulated methylmalonyl-CoA is degraded to MMA and CoA by
enzymatic hydrolysis. (S)-methylmalonyl-CoA hydrolase has been purified from
rat liver [86]. Shimomura et al. [87] have reported purification and characteriza-
tion of rat liver 3-hydroxyisobutyryl-CoA hydrolase, which is involved in valine
metabolism. They observed that the enzyme showed the ability to hydrolyze
methylmalonyl-CoA and that it showed properties similar to the previously purified
(S)-methylmalonyl-CoA hydrolase.
When MCM is inactivated, propionyl-carnitine in blood is increased.
Accumulation of methylmalonyl-CoA can produce odd chain fatty acids. Although
odd-chain fatty acids may cause some symptoms of B12 deficiency, the exact bio-
chemical mechanism has not been established. There are several reasons for dys-
function of MCM, such as inadequate intake of B12, dysfunction of the Cbl absorption
or cellular processing systems or due to MCM gene mutations. If methylmalonic
aciduria were caused by low intake of B12, or impaired AdoCbl synthesis, adminis-
tration of AdoCbl would rescue the phenotype. Null mutations of MCM result
in neonatal lethality, if not properly treated. Protein restriction treatment is most
often chosen. Carnitine administration is effective, but the utility as a long-term
treatment is unknown.

3.3 Hyperhomocysteinemia

During the last two decades, homocysteine metabolism has been actively studied by
many researchers because hyperhomocysteinemia, the elevated level of homocysteine
in blood, was reported to be an independent risk factor for cardiovascular disease.
Epidemiological studies showed a relationship between hyperhomocysteinemia and
many other diseases, such as schizophrenia [88], Alzheimer’s disease [89], and
osteoporosis [90].
Homocysteine is a substrate for three enzymes, MS, betaine-homocysteine
methyltransferase (BHMT), and cystathionine β-synthase (CBS) (Figure 8). Although
BHMT is a liver-specific enzyme, MS is ubiquitously expressed in nearly all human
tissues. CBS catalyzes the reaction forming cystathionine from homocysteine and
serine. While homocysteine is not a direct substrate for MTHFR, the enzyme cata-
lyzes the reduction of CH2-H4folate to produce CH3-H4folate, which is the other sub-
strate for MS. Thus, dysfunction of MTHFR also causes hyperhomocysteinemia.
Affinity for homocysteine is highly variable; enzymes in the methionine re-
cycling pathway, i.e., MS and BHMT, show high affinity (low Km values) for homo-
cysteine, while CBS in the catabolic pathway has low affinity. Thus, dysfunction of
CBS causes higher levels of homocysteine in blood than do MS and MTHFR. Folic
acid fortification in food has been conducted and has succeeded in lowering plasma
homocysteine concentrations. However, recent epidemiological reports disprove
312 Yamada

“homocysteinemia as the risk factor for cardiovascular disease” [91–94]. Even so,
elevated-levels of homocysteine certainly indicate the failure to regulate folate and
methionine metabolism. It should be noted that lowering of homocysteine by folic
acid fortification may not be the solution because it fails to solve the disruption of
the methionine cycle due to B12 dependency.

3.4 Megaloblastic Anemia

Folate deficiency also causes megaloblastic anemia (formerly pernicious anemia).


It is thought that the cause is impaired DNA synthesis due to decreased de novo
dTMP synthesis, which results from a depleted “functional folate pool” due to dys-
function of MS, i.e., the “methyl-trap” (see Section 2.3.3, Figure 8). “Functional
folate” deficiency can explain the observed symptoms. Because CH2-H4folate is
essential for dTMP production as a methyl donor, dTMP production from the folate
cycle would be reduced, which results in decreased DNA synthesis.
Administration of folate to a B12-deficient patient with megaloblastic anemia
could be effective because it increases the folate pool. In addition, methionine sup-
plementation can alter the metabolic balance because it contributes to the produc-
tion of AdoMet, which is an inhibitor for MTHFR. While the methyl-trap hypothesis
is consistent with this mechanism, it is still controversial. Although B12 was initially
identified as the anti-pernicious anemia factor, the fundamental biochemical basis
of megaloblastic anemia is still unknown.

3.5 Cobalamin Neuropathy

Cbl neuropathy is an abnormality of the peripheral nervous system found in B12-


deficient patients. N2O-exposed patients show Cbl neuropathy, suggesting that
dysfunction of MS is related to this disease. Although Cbl neuropathy is caused
by impaired transmethylation [82,83], the cause is not yet fully understood.
Interestingly, despite the fact that this symptom responds to Cbl administration,
significant numbers of Cbl neuropathy patients show serum Cbl contents in the
lower normal range. Thus, it is suggested that serum MMA and homocysteine levels
are better indicators for biochemical diagnosis of Cbl neuropathy [95–97].

3.6 Other Diseases Related to Vitamin B12 Deficiency

A relationship between pernicious anemia and pregnancy has been suspected since
the mid-1930’s. However, the results of early clinical trials using Cbl treatment
were mixed, so this idea remains controversial. In 1962, Watson [98] and Sharp and
9 Cobalt: Its Role in Health and Disease 313

Witts [99] reported the relationship between serum B12 content and sperm maturation.
The effect of B12 deficiency on sperm maturation in humans can be reproduced in
dietary B12-deficient rats [100] and in the N2O-exposed rat model [101]. Both rat
models show catastrophic testicular damage, including aplasia of sperm and
spermatids. The testicular damage of the B12-deficient rat can be prevented by
methionine supplementation to the B12-deficient diet, indicating that dysfunction of
MS is responsible [78].
B12 deficiency in the elderly remains a significant concern. Clinical reports
indicate that B12 deficiency is related to dementia [102,103]. This could be due to
malnutrition or dysfunction of the Cbl absorbing system caused by autoimmunity.
Even in patients who are not B12-deficient, it is known that administration of B12
is an effective way to correct the circadian rhythm [104,105]. CH3-Cbl is the most
effective [106], so MS might be the responsible target. The biochemical basis of this
observation has not yet been clearly demonstrated.

3.7 Animal Models

Whole animal models are useful for understanding the functions of nutrients. B12-
deficient animal models have been reported that allow us to understand the role of
Cbl in human health. It is especially difficult to establish a B12-deficient animal
model using classical nutritional methods (i.e., by which animals are fed B12-
deficient diets) because Cbl shows biological activity in a very small amount.
Despite this difficulty, there are many potential animal models that allow investiga-
tion of dietary B12 deficiency, including monkeys, pigs, rats, mice, fruit bats, etc..
Co-deficient sheep are also used as B12-deficient animal models.
Some animals, such as rats, show coprophagia, and therefore need very careful
handling to prevent fecal recycling. Furthermore, the biological half-life of Cbl is
quite long, and is estimated to be approximately one year [107]. These obstacles
hamper investigation of Cbl function in normal animals. Since N2O is known as
specific inhibitor for MS, N2O-exposed animals have been frequently used as
MS-impaired animal models. Recently, an experimental autoimmune gastritis
mouse model was developed as a megaloblastic anemia model [108,109], although
a dietary B12 deficiency- or an N2O exposure-induced megaloblastic anemia animal
model has not been reported. For Cbl neuropathy, it has been reported that monkeys
[110,111], fruit bats [82], and pigs [83] develop Cbl neuropathy when B12-deficient
diets were fed or animals were exposed to N2O.
Genetic engineering and developmental biology have permitted the production
of transgenic and targeted gene-disrupted mice by many laboratories. These should
allow for investigations beyond that allowed by classical nutritional models. Gene-
disrupted mice targeted for proteins in the Cbl transporting system have been
reported only for TC receptor and megalin [112]. Targeting of cellular Cbl-delivering
and -processing protein genes have not been reported, to date. As described above, gene
disruption of Mtr (gene for MS) is embryonic lethal [79]. Dietary supplementation,
314 Yamada

such as with methionine, betaine, and/or, folic acid, did not rescue the phenotype.
Elmore et al. reported that intercross mating of a heterozygous Mtrr-deletional
mouse model could not produce homozygous Mtrr-deficient mice [113], indicating
that function of both MS and MSR are absolutely necessary for development.
However, a hypomorphic mouse model, i.e., a reduced-function MSR model, has
been reported [113]. The Mtrr gene in this mouse model is interrupted by
β-galactosidase/neomycin phosphotransferase gene, a “gene trap” which provides
the marker for screening. Therefore, the Mtrrgt/gt homozygote mouse can produce
the fusion protein of the FMN domain of MSR and β-galactosidase/neomycin phos-
photransferase. While the intact FMN domain of the fusion protein has a function,
the Mtrrgt/gt mouse shows disrupted methionine metabolism [113]. Although the
hypomorphic mouse provides a model for reduced function of MS and MSR,
phenotypes for B12-deficient symptoms, megaloblastic anemia, and neurological
abnormality, are not yet characterized.
A MCM knock-out mouse model has also been reported [114]. The knock-out
mouse shows increased MMA in urine and neonaternal lethality, which resembles
human mut0 patients (complete deficiency of MCM enzyme activities). The MCM
knock-out mouse was used to produce a humanized MCM-deficient mouse model, in
which the MCM gene from human mut0 patients (due to the missense mutation at
Arg403) was introduced [115]. Because the mouse mimics human MCM deficiency
both at the phenotypic and genotypic levels, this model allows evaluation of possible
treatments for MCM-deficient human patients. Effect of depletion of enzymes
involved in the propionyl-CoA pathway on methylmalonyl-CoA metabolism has also
been reported using Caenorhabditis elegans and RNA interference techniques for
gene knock-down [116]. While C. elegans is a non-vertebrate, the well-characterized
genetic and genomic information could provide a model for further investigation.

4 Non-corrinoid Cobalt

4.1 Non-corrinoid Cobalt-Containing Proteins

Non-corrinoid cobalt-containing enzymes are found in bacteria [117]. For example,


Co-dependent nitrile hydratase [118] is important for acrylamide production [119].
In this enzyme, the cobalt atom is placed in the active site with the unique post-
translational modification on the cysteine residues [120]. Whereas homologous
genes for nitrile hydratase are found in certain eukaryotes, such as Monosiga brevi-
collis [121,122], it is not present in the human genome.
In mammals, function of non-corrinoid cobalt in enzymes are rarely reported.
Methionine aminopeptidase has been purified from porcine liver and characterized
[123]. Activity of the mammalian methionine aminopeptidase can be stimulated in
the presence of Co2+ ion. However, the enzyme is also active in the presence of
other divalent ions such as Mg2+, and therefore does not exclusively require Co2+
for activity.
9 Cobalt: Its Role in Health and Disease 315

4.2 Overload of Cobalt

While non-corrinoid cobalt-specific proteins are not found in humans, there are
many reports using CoCl2. For example, CoCl2 is often used as a simulative hypoxia-
inducing reagent for cell culture [124]. Co2+ can substitute Fe2+ in the porphyrin ring
of heme. This alters the heme protein conformation to the deoxygenated form by
mimicking the lowered affinity for oxygen. Thus, it can cause hypoxia, resulting in
activation of hypoxia response genes, such as the erythropoietin gene. Nickel also
shows the same effect. Amounts necessary to see these effects in cell culture are
massive (~100 μM). Toxicity of high doses of CoCl2 has been recently reviewed [125].
Moreover, toxicity of nanocompounds containing cobalt are a novel concern [126].

5 Implications and Future Development

Roles of Cbl in health and disease were reviewed because the biological function of
cobalt is predominantly as Cbl. Functions of Cbl in humans have been studied by
many researchers since the isolation of Cbl. However, genes for intercellular trans-
porting proteins were only recently discovered. These absorbing, transporting, and
activating proteins are important for the function of Cbl because B12 is contained in
very low amounts in food.
Proper function of Cbl requires many auxiliary proteins. Currently, analyzing the
functions of transporting and activating proteins involved with Cbl is under investi-
gation in many laboratories. There might be even more intercellular Cbl-processing
proteins that remained so far undiscovered, for example a mitochondrial transporter.
These efforts should help to clarify the biochemical roles of Cbl-binding proteins in
the near future. For these studies, understanding of protein-protein interactions is
likely the key.
Understanding the contributions of Cbl for human health remains elusive because
the action of Cbl may not be the direct cause for B12-deficient symptoms, with the
exception of methylmalonic aciduria and hyperhomocysteinemia. Although it might
be possible that completely new physiological functions of Cbl are discovered at
this moment, the characterized function of Cbl is its cofactor role for the two
enzymes discussed. Dysfunction of Cbl-dependent enzymes disrupts normal metab-
olism, and the impaired metabolism could be causal for disease. Especially, dys-
function of MS causes disruption of many cellular processes. Hyperhomocysteinemia
could be the excellent biomarker for disturbed folate and methionine metabolism,
which could indicate the patient’s health is at risk. However, it is difficult to estab-
lish links to certain diseases, as we have observed between hyperhomocysteinemia
and cardiovascular disease.
Folate and methionine metabolism is important for supplying substrates for other
processes, such as methyl groups and precursors for DNA and RNA, and for providing
necessary amounts. Moreover, many nutrients affect global metabolism and the
requirements of metabolism could vary between individual persons. Such complexity
316 Yamada

precludes a definitive explanation for even well recognized characteristics of B12


deficiency after many years of research. Using combinations of current experimental
techniques and animal models, however, it should be possible to reveal the biochemical
mechanisms underlying B12 deficiency and disease, and to provide rational explanations
for influences of gene mutations and nutrients intake for the diseases. Such knowledge
would contribute to our overall wellness and quality of life.

Abbreviations

ABCD4 ATP-binding cassette transporter, D subfamily 4


AdoCbl adenosylcobalamin
AdoHcy S-adenosylhomocysteine
AdoMet S-adenosylmethionine
ATP adenosine 5′-triphosphate
B12 vitamin B12
BHMT betaine-homocysteine methyltransferase
Cbl cobalamin
CBS cystathionine β-synthase
CH2-H4folate methylenetetrahydrofolate
CH3Cbl methylcobalamin
CH3-H4folate methyltetrahydrofolate
CN-Cbl cyanocobalamin
DBI dimethylbenzimidazole
dNTP 2′-deoxynucleoside 5′-triphosphate
dTMP thymidine 5′-monophosphate
FAD flavin adenine dinucleotide
FMN flavin mononucleotide
GTP guanosine 5′-triphosphate
H4folate tetrahydrofolate
IF intrinsic factor
LMBD1 LMBR1 (limb region 1 homolog) domain containing 1
MCM methylmalonyl-CoA mutase
MMA methylmalonic acid
MMAA methylmalonic aciduria, CblA type
MMAB methylmalonic aciduria, CblB type
MMACHC methylmalonic aciduria, CblC type, and homocysteinuria
MMADHC methylmalonic aciduria, CblD type, and homocysteinuria
MRP1 multidrug resistance protein
MS methionine synthase
MSR methionine synthase reductase
MTHFR methylenetetrahydrofolate reductase
N2O nitrous oxide
NADPH nicotinamide adenine dinucleotide phosphate (reduced)
TC transcobalamin
9 Cobalt: Its Role in Health and Disease 317

Acknowledgment The author acknowledges Dr. C. L. Elmore (US Food and Drug Administration)
for reading and editing the English.

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Chapter 10
Nickel and Human Health

Barbara Zambelli and Stefano Ciurli

Contents
ABSTRACT ............................................................................................................................. 322
1 INTRODUCTION: THE DOUBLE FACE OF NICKEL
IN BIOLOGICAL SYSTEMS ........................................................................................... 322
2 NICKEL HAZARD FOR HUMAN HEALTH .................................................................. 324
2.1 Nickel-Induced Carcinogenesis ................................................................................ 325
2.1.1 The Carcinogenic Potential of Nickel ........................................................... 325
2.1.2 Molecular Mechanisms for Nickel-Induced Neoplastic
Transformation .............................................................................................. 326
2.2 The Different Faces of Nickel Allergy ...................................................................... 332
2.2.1 Nickel Effects on Immune Response ............................................................ 332
2.2.2 The Impact of Nickel-Induced Dermatitis .................................................... 334
3 NICKEL-DEPENDENT INFECTIOUS DISEASES ........................................................ 336
3.1 Nickel-Dependent Enzymes in Pathogenic Microorganisms .................................... 336
3.1.1 Glyoxalase I .................................................................................................. 336
3.1.2 Acireductone Dioxygenase ........................................................................... 337
3.1.3 Urease ............................................................................................................ 338
3.1.4 [NiFe]-Hydrogenase ...................................................................................... 339
3.2 Molecular Regulation of Nickel Homeostasis in Pathogenic Microorganisms............. 340
3.2.1 Nickel Membrane Transporters ..................................................................... 341
3.2.2 Nickel Molecular Chaperones and Metallo-Chaperones .............................. 342
3.2.3 Nickel Sensors ............................................................................................... 343
3.3 Nickel-Obligate Microorganisms with Severe Impact on Human Health................. 344
3.3.1 Helicobacter pylori as a Nickel-Dependent Pathogen: A Possible
Correlation between Nickel Intake and Cancer Development ...................... 344
3.3.2 Nickel Homeostasis and Intracellular Parasitism: Eukaryotic
and Prokaryotic Pathogens ............................................................................ 346

B. Zambelli • S. Ciurli (*)


Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology,
University of Bologna, Bologna, Italy
e-mail: barbara.zambelli@unibo.it; stefano.ciurli@unibo.it

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 321
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_10, © Springer Science+Business Media Dordrecht 2013
322 Zambelli and Ciurli

4 NICKEL ESSENTIALITY IN ANIMALS AND HUMANS ........................................... 348


4.1 Effects of Nickel Depletion in Higher Organisms .................................................... 348
4.2 Influence of Nickel Availability on Gut’s Microflora ............................................... 349
5 CONCLUSIONS AND OUTLOOK.................................................................................. 350
ABBREVIATIONS .................................................................................................................. 351
REFERENCES ........................................................................................................................ 352

Abstract This review focuses on the impact of nickel on human health. In particular,
the dual nature of nickel as an essential as well as toxic element in nature is
described, and the main forms of nickel that can come in contact with living systems
from natural sources and anthropogenic activities are discussed. Concomitantly, the
main routes of nickel uptake and transport in humans are covered, and the potential
dangers that nickel exposure can represent for health are described. In particular,
the insurgence of nickel-derived allergies, nickel-induced carcinogenesis as well
as infectious diseases caused by human pathogens that rely on nickel-based enzymes
to colonize the host are reviewed at different levels, from their macroscopic aspects
on human health to the molecular mechanisms underlying these points. Finally, the
importance of nickel as a beneficial element for human health, especially being
essential for microorganisms that colonize the human guts, is examined.

Keywords human health • nickel • nickel allergy • nickel carcinogenesis • nickel


homeostasis

Please cite as: Met. Ions Life Sci. 13 (2013) 321–357

1 Introduction: The Double Face of Nickel


in Biological Systems

Nickel is the 24th most abundant element regarding the natural abundance in the
Earth’s crust [1]. This metal exists in nature either in insoluble particles, which are
components of fumes and dusts, like nickel sulfides (NiS, Ni3S2), oxides (NiO), and
silicates, or in water-soluble nickel compounds, such as nickel acetate, nickel
chloride, and nickel sulfate. Natural sources of nickel include dusts from volcanic
emissions and weathering of rocks and soils [2]. Soluble and insoluble nickel
compounds are also found in soils and in waters [3]. In water, nickel ions are generally
divalent, present as the greenish hexa-hydrated [Ni(H2O)6]2+ ion.
The unique physical and chemical properties of nickel – low thermal and electri-
cal conductivities, high resistance to corrosion and oxidation, excellent strength and
toughness at elevated temperatures, and capability of being magnetized – make this
metal and its compounds suitable materials for many applications widely found in
modern industry [4]. Human activities, such as emission of nickel-containing fuel,
industrial nickel production and utilization of nickel compounds, concur to the
environmental release of nickel and to pollution by nickel and its products.
10 Nickel and Human Health 323

Human exposure to nickel occurs primarily via inhalation, ingestion, and dermal
absorption [5]. Insoluble particulate nickel enters the vertebrate cells by phago-
cytosis, whereas nickel carbonyl is soluble in lipids and permeates the plasma
membrane. Soluble nickel is transported into cells of vertebrate organisms by
diffusion or through calcium channels and/or divalent cation transporters (DMT-1),
involved in iron uptake [6]. Transport of nickel in blood plasma is mediated by
binding to albumin and some small ligands, such as amino acids (e.g., histidine)
and small peptides [7,8]. The Ni2+-L-histidine complex is the major form of nickel
transport across the cell membrane, and the Ni2+-albumin complex is the form for
systemic transport [9].
Exposure to nickel compounds yields a variety of adverse effects on humans.
Nickel immune reaction, as a form of dermatitis, is one of the most common aller-
gies in the modern world [10]. In addition, chronic nickel exposure can produce
serious respiratory, cardiovascular, and kidney diseases. Some alterations in immune
response in animal models have been observed as a result of nickel contact [11].
Nickel induces the production of reactive oxygen species (ROS), like the superox-
ide radical (O2−·), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) by
several cells, such as in neutrophils and monocytes. This ultimately causes apoptosis
in a number of cellular types, including human neutrophils and T-cells [12,13]. High
exposure to nickel impairs the normal homeostasis of essential metal ions, decreasing
the levels of calcium, magnesium, manganese, and zinc in different tissues [14] and
possibly interfering with normal iron cofactor binding to specific proteins [15–17].
Finally, the most serious concerns of nickel for human health is the nickel-induced
teratogenicity and carcinogenesis, documented by the International Agency for
Research on Cancer (IARC) in 1990 [18].
Despite its poisoning potential, nickel plays a fundamental role in living
organisms, revealing its double faced nature of both as an essential and toxic ele-
ment [19]. The importance of nickel for plants, bacteria, archaea, and unicellular
eukaryotes is well documented. In these organisms, the choice of nickel as cata-
lyst for important biological reactions is related to its flexible coordination
geometry, which makes this metal a very versatile element for many biological
applications [1]. Nickel is a necessary component in the active site of several
essential metallo-enzymes in bacteria and lower eukaryotes. So far, eight micro-
bial nickel-containing enzymes have been identified, which include urease,
hydrogenase, CO dehydrogenase, acetyl-CoA synthase, methyl-CoM reductase,
Ni-superoxide dismutase, acireductone dioxygenase, and glyoxalase I, while a
few other possible nickel-dependent enzymes are emerging [20]. The majority of
known nickel-dependent enzymes have been structurally determined and nickel
ions have been demonstrated to play an essential role in their enzymatic cataly-
sis. In higher eukaryotes, the only known nickel-depending enzyme is plant
urease. Some plant species that live in serpentine soils have evolved to hyperac-
cumulate nickel ions, creating complex systems of metal detoxification and
homeostasis that constitute appealing systems for phytoremediation of contami-
nated environments [21].
324 Zambelli and Ciurli

So far, no nickel-containing enzyme or cofactor is known in higher animals.


However, this metal has been included in the group of “possibly essential elements”
for animals and humans as early as the 1970s [19]. Many experiments on animal
models showed that nickel could be beneficial, if not essential, for optimal repro-
ductive function, bone composition and strength, energy metabolism, and sensory
function. The reasons of this essentiality remain obscure, even if some hypotheses
have been suggested [5, 22].
The double face of nickel, being both a poison and an essential micronutrient,
implies that nickel-dependent organisms have developed tightly regulated systems
for nickel handling, delivery it into the correct cellular location, and avoiding its
dangerous potential. Bacteria that rely on nickel ions for environmental colonization
and growth represent a good model to study nickel metabolism and homeostasis in
nickel-dependent organisms. Due to the relative paucity of known nickel-dependent
enzymes, the study of intracellular nickel homeostasis may represent a paradigm to
study the general handling of dangerous and essential metal ions in vivo.
In this chapter we review the present knowledge on the impact that nickel ions
have on human health, focusing on the dangerous potential of nickel, leading to
allergy, carcinogenesis, and infectious diseases, and the proofs of its essentiality, and
on what is currently known on the molecular mechanisms underlying these points.

2 Nickel Hazard for Human Health

The most diffuse hazardous health effects caused by nickel exposure are nickel-
induced carcinogenesis and allergy. They are both mediated by active changes in
metabolic pathways that underlie inflammation, stress response, oxidative stress,
cell proliferation, and cell death [23]. As no protein specific for nickel homeostasis
is known in mammals, one would not expect a specific nickel-mediated change of
gene expression and metabolism. Indeed, many of the nickel effects on cells are
triggered by non-specific interactions of nickel ions with macromolecules and gen-
eral formation of reactive compounds that mediate cellular damage. Furthermore,
the cellular response to nickel is related to signal transduction cascades such as
second messengers, protein kinases, phosphatases or transcription factors, which
are involved in general metal ion response. Notably, nickel exposure produces a
rather specific pattern of gene expression. Nickel-driven alteration of transcription
of genes involved in oxygen deficiency has been extensively studied in vitro for its
relevance to nickel carcinogenesis [23].
On the other hand, the activation of the inflammatory response, with the induc-
tion of genes for chemokines and cytokines correlated with nickel-induced allergy
and asthma, has been studied mostly in vivo [23]. Additionally, few proteins with
nickel-binding motifs have been identified and the effect of nickel binding to these
proteins can be related to the specific cellular response to nickel exposure. A sche-
matic representation of nickel uptake routes, intracellular distribution, and major
effects on human cells metabolism, which will be discussed in detail in the following
sections, is presented in Figure 1.
10 Nickel and Human Health 325

Figure 1 Schematic representation of known nickel uptake routes, intracellular distribution, and
its major effects on cellular metabolism in humans. ROS = reactive oxide species; HIF = hypoxia-
inducible factor; HRE = hypoxia-responsive enhancer; GSH = reduced glutathione; NF-κB = nuclear
factor κ-B; AP-1 = activating protein 1; TGF-β = transforming growth factor β; NF-AT = nuclear
factor of activated T cells; DMT-1 = divalent cation transporter 1; NDRG-1 = N-myc downstream
regulated gene 1, DAN = differential screening-selected gene aberrative in neuroblastoma.

2.1 Nickel-Induced Carcinogenesis

2.1.1 The Carcinogenic Potential of Nickel

The propensity of nickel workers to develop cancers in the respiratory tract was firstly
reported in 1933 [24]. Subsequent epidemiological studies and carcinogenic assays in
animals corroborated the carcinogenicity of nickel compounds, which is now generally
accepted [18]. Epidemiological studies have reported an increased risk of lung and
nasal cancers among nickel mining, smelting, and refinery workers [25]. For many
years, it was believed that only water-insoluble nickel components of fumes and dusts,
like nickel sulfides and oxides, were carcinogenic. Subsequent epidemiological studies
indicated instead that aerosols of water-soluble nickel compounds, such as nickel ace-
tate, nickel chloride and sulfate, are also carcinogenic in vivo, although with a lower
potential [24]. Nickel present in endoprostheses, bone-fixing plates and screws, and
other implanted medical devices made of metal alloys, has been suspected, but not
proven, to be the cause of sporadic local tumors. There is no epidemiological evidence
on possible cancer risk from general environmental and dietary nickel exposures.
326 Zambelli and Ciurli

In animal models, nickel compounds induce tumors at virtually all sites of


applications. Rats are more susceptible to nickel poisoning effects than mice, hamsters,
or rabbits. The reasons for these differences may reflect different abilities of the phago-
cytes to ingest and metabolize nickel-containing particles, the different mechanisms of
nickel uptake, transport, distribution, and retention in the animal body, and the differ-
ences in the capacity of antioxidant systems among animals of different species and
strains [23]. This suggests that genetic predispositions, including metabolic variations of
different species and strains of animals, may also play an important role in nickel carci-
nogenesis. Similar genetic predispositions possibly also occur in human populations.
The routes of administration that were shown to produce tumors include inhala-
tion, intramuscular, intrarenal, intraperitoneal, intraocular, and subcutaneous applica-
tions [23]. The injection of crystalline Ni2S3 or NiS into experimental animals resulted
in a high incidence of tumors in the application sites, while no tumor was induced in
animals treated with soluble NiSO4. In rats, intraocular and intramuscular injections
generated the highest tumor incidences, yielding tumors in all animals, often readily
metastasized to the lung and other organs [23]. Similarly, only water-insoluble nickel
compounds were found to be carcinogenic in rats via the inhalation route, while solu-
ble NiSO4 was not found to be carcinogenic [26]. Notably, intrarenal nickel injection,
beside causing kidney tumors in rats, also caused erythrocytosis due to induction of
erythropoietin, a part of the hypoxia-mimicking effect of nickel [23].
Insoluble nickel compounds are generally considered stronger carcinogens than
soluble compounds. The difference of their carcinogenic activity is related to the
faster clearance of soluble nickel ions from the tissues, which in turn retain insolu-
ble nickel particles longer [5]. These data indicate that prolonged exposure to a
nickel carcinogen is critical for tumor development. Nickel compounds also trans-
form human cells in vitro [27]. Crystalline nickel particles, phagocytized by cul-
tured cells, exist in intracellular vacuoles with internal acid pH that facilitates nickel
solubilization, creating local high concentrations of soluble nickel inside the cell.
Nickel-filled vacuoles easily migrate and reach the cellular nucleus, potentially
exerting their effect on DNA and nuclear proteins. On the contrary, exposure of cells
to water-soluble salts results in low nuclear and high cytosolic nickel contents [28].
Nickel compounds have been shown to produce a significant synergistic effect,
when co-administrated with other carcinogens, in enhancing cell transformation
both in vitro and in vivo. On the other hand, essential metals such as manganese,
magnesium, and zinc, co-administered with Ni3S2 to animal models, reduced local
tumor incidence in a dose-dependent manner. Magnesium was the strongest and
zinc was the weakest inhibitor. Separate administration of the essential metals did
not produce this effect [23].

2.1.2 Molecular Mechanisms for Nickel-Induced Neoplastic


Transformation

The molecular basis of nickel carcinogenesis remains elusive, as data obtained by


different groups are difficult to reconcile, and a general consensus on the mechanisms
has still to be reached. To be defined carcinogenic, a compound should provoke two
10 Nickel and Human Health 327

effects fundamental for tumor genesis, that is, heritable changes in gene expression
and cell proliferation. In most instances, carcinogenic compounds provide the first
condition by interacting with DNA and DNA-binding proteins, changing DNA
structure and inducing mutations in its sequence, usually during replication. Often,
these sequence changes alter the expression level or function of tumor suppressor
genes or oncogenes. However, nickel does not behave like a typical mutagen,
because it does not show high affinity for DNA nor displays mutagenic potential in
most assays on bacteria, fruit fly, mammalian cells, and whole animals [29–33].
Therefore, alternative routes for nickel to change the gene expression levels and
cellular phenotypes have been described. Nickel has been found to act at the DNA
level, mostly through epigenetic mechanisms. Nickel promotion of tumors, on the
other hand, occurs mostly at the protein level, and involves DNA-binding proteins
such as transcription factors, metal-binding proteins, and proteins participating in
important cellular pathways. The result is a change of the general cellular metabolism
and a deregulation of cellular homeostasis inducing carcinogenic transformations in
the cells.

2.1.2.1 Effects of Nickel on DNA Structure and Gene Expression

(i) Genotoxic effects. Nickel compounds have been reported to be mildly clasto-
genic, causing extensive DNA damage and chromosome aberrations, particularly in
the heterochromatic region of the genome [5]. Nickel generates oxides and reactive
species that produce DNA-protein cross-links and oxidative DNA damages [34].
This mechanism is typical of several transition metals that can generate reactive
oxygen species in biological fluids at physiological pH. However, this effect cannot
fully explain the high carcinogenic potential of nickel: this metal is a weaker gen-
erator of redox-active species, but it is as good a carcinogen as chromium, which is
very active in ROS production [35]. In addition, highly redox-active metals, such as
copper, which also binds DNA more avidly than nickel, are only weakly or not
carcinogenic [36]. The ability of carcinogenic metals to facilitate DNA damage
through inhibition of DNA-repair enzymes or binding to histones can also explain
its genotoxic activity [37,38].
(ii) Epigenetic effects. Further evidence from epidemiological, animal, and cellular
studies shows a role of epigenetics in nickel carcinogenesis, in addition to genetic-
based mechanisms. One major requirement for nickel carcinogenicity is the pro-
longed action on the target tissue, performed by compounds with limited solubility
and long retention in biologic fluids [24], which is typical of tumor promoters acting
through epigenetic mechanisms, rather than tumor initiators, which are usually
mutagenic. Indeed, nickel was found to synergistically increase the tumorigenic
potential of several carcinogenic agents.
The nickel-induced epigenetic changes include silencing of genes for DNA
repair and tumor suppressors, mostly occurring through nickel-driven DNA meth-
ylation, which can modify the chromatin structure and eventually the genetic
expression. In DNA of higher eukaryotes the methylation of CpG dinucleotides is
an important modification that leads to modulation of gene expression. In general,
328 Zambelli and Ciurli

increased cytosine methylation represses transcription, and it is thus more abundant


in the heterochromatin regions of chromosomes [39]. Exposure to nickel com-
pounds enhances DNA methylation, leading to inactivation of gene expressions
[40]. The nickel-induced silencing of a tumor suppressor gene, occurring through
promoter hypermethylation, has been associated with cellular transformations in
vivo [41]. Although the mechanisms by which nickel induces DNA hypermethyl-
ation are presently unknown, a possible model includes the ability of Ni2+ to substi-
tute Mg2+, normally bound to DNA, seeding chromatin condensation and triggering
de novo DNA methylation by methylases that recognize newly generated hetero-
chromatic and unmethylated DNA [40]. This property is related to the same charge
and very similar ionic radius of Ni2+ (69 pm) and Mg2+ (71 pm).
In addition to DNA methylation, nickel epigenetic effects are related to the ability
of nickel to affect the global levels of histone modifications, implicating global
deregulation of gene expression. Indeed, acetylation in the N-terminal tail of histone
proteins is a well-known mechanism to regulate the chromatin transcriptional state,
important to control the access of regulatory proteins to DNA. Conversely, histone
methylation results in more pronounced chromatin and gene silencing. Nickel
decreases the acetylation levels of histones H2A, H2B, H3 and H4, and increases
H3K9 dimethylation, and the ubiquitination of H2A and H2B [42]. The higher meth-
ylation of the histones occurs through the nickel-induced inhibition of specific Fe2+
and α-ketoglutarate-dependent demethylases belonging to the family of Jmjc-domain
containing histone demethylases (JHDM), such as JHMD1 and JMJD1A [43,44].
The inhibited acetylation of histone H4 is thought to occur through the direct interac-
tion of the metal ion with His18 of the histone itself, which would prevent its interac-
tion with the specific acetyl-transferase enzyme [45]. Low levels of histone acetylation
in nickel-exposed cells can also result from low levels of acetyl-CoA, a universal
donor of acetyl groups, due to the nickel-dependent inhibition of pyruvate dehydro-
genase kinase that converts pyruvate into acetyl-CoA [42]. The increase in H2A/H2B
ubiquitination has been correlated to nickel up-regulation of the ubiquitin-conjugating
enzyme H6 (UbcH6) E2 ligase or the inhibition of an unidentified histone de-ubiqui-
tinating activity [45]. Moreover, nickel induces the truncation, at the N-terminus of
the histone H2A, and, both at N- and C-termini, of the histone H2B, possibly through
the activation of specific nuclear proteolytic enzymes belonging to the calpain family
[46]. The importance of deregulation of histone modifications by nickel has been
demonstrated by observing that nickel-transformed cells, treated with the histone
deacetylase inhibitor, namely trichostatin A, revert the phenotype to that of untrans-
formed cells along with the gene expression profile, suggesting that histone acetyla-
tion might be inhibited during nickel-induced transformation [47].
In addition to changing gene expression levels, epigenetic effects also promote
cell proliferation: recent studies have demonstrated that the unlimited proliferative
character of a nickel-transformed cell line could be reversed by induction of senes-
cence. The senescence gene is located in the donor X chromosome, and its expres-
sion is regulated by DNA methylation. These data indicate that transformation by
nickel may involve DNA methylation and silencing of a senescence gene in the X
chromosome [48].
10 Nickel and Human Health 329

2.1.2.2 Effects of Nickel on Proteins, Transcriptional Regulators,


and Metabolic Pathways

(i) Disruption of calcium homeostasis. Nickel blocks calcium channels and disturbs
intracellular calcium homeostasis. This results in the experimentally observed rapid
proliferation of nickel-transformed cells in low-calcium media [49]. Since cytoplas-
mic calcium levels regulate expression of genes associated with cell growth,
differentiation, and apoptosis, derangement of calcium regulation would impact the
entire cellular metabolism [49]. In particular, nickel was shown to increase intracel-
lular calcium levels: nickel likely uses calcium channels to enter the cells, and
induces calcium release from intracellular stores, possibly through a cell surface
receptor. Therefore, changes of calcium homeostasis invoked by nickel exposure may
change the cellular expression induced by other signalling pathways, eventually
leading to malignant transformation [23].
(ii) Oxidative damage. Soluble and insoluble nickel compounds can be redox-active at
physiological pH, although to a lesser extent than iron and copper complexes, and
they can generate reactive oxygen species. This is possible when the redox couple
Ni3+/Ni2+ is formed, which usually only occurs when nickel is bound by certain natural
ligands like peptides and proteins, especially those forming square planar nickel com-
plexes. Reactions of such complexes with O2 or H2O2 yield the hydroxyl radical ·OH
and other radicals. The oxidation of water-insoluble nickel sulfides may involve both
nickel and sulfur and lead to generation of not only nickel-, but also sulfur-derived
ROS and other reactive intermediates (e.g. the sulfite anion). This enables the sulfides
to produce a wider spectrum of oxidative damage than other nickel compounds and
may be responsible for their high carcinogenic activity. In addition, nickel can deplete
some important antioxidant ligands, such as ascorbate and reduced glutathione (GSH).
Nickel is capable of depleting intracellular ascorbate through catalytic oxidation and
hydrolysis of both ascorbic and dehydroascorbic acid, and inhibition of ascorbic acid
transporters [23]. GSH depletion is likely the result of a cellular response to the ROS
species generated by nickel. Coherently, resistance to nickel toxicity is usually associ-
ated to high levels of GSH [50]. Finally, the enzymatic components of cellular antioxi-
dant defence, such as superoxide dismutase, catalase, glutathione peroxidase, and
glutathione reductase, are also affected by nickel exposition [23].
The nickel-induced oxidative stress can activate some transcriptional pathways
through some oxidation-sensitive transcription factors. ROS created by nickel exposi-
tion result in lipid peroxidation, whose products can create adducts with DNA, thus
altering gene expression. Protein oxidation, leading to protein fragmentations and
cross-linking with other molecules (e.g., with DNA) and oxidative DNA and chroma-
tin damage are also consequences of ROS generated by nickel. The presence of
cross-links in chromatin may lead to morphologic aberrations of chromosomes.
In vitro, nickel was found to promote DNA cleavage by H2O2 predominantly at the
cytosine, thymine, and guanine bases [51]. ROS attack on DNA’s sugar moiety
produces apurinic sites in DNA and mediates in vitro hydrolysis of 2′-deoxyguanosine.
The depurination occurs concurrently with DNA strand scission and fragmentation.
330 Zambelli and Ciurli

Some compounds generated by oxidative stress, like 8-oxoguanine, may also misdirect
DNA methylation and perturb orderly binding of transcription factors to DNA.
(iii) Activation of hypoxia signalling. Nickel exposure produces a rather specific
pattern of gene expression, which involves the same activation pathways of the
response to hypoxia [52], and in particular the activation of the HIF-1 transcription
factor. This protein exists as a HIF-1α/HIF-1β (ARNT) hetero-dimer, with the α
subunit being the regulatory unit, formed in response to low oxygen tension in the
cells. Under normal oxygen concentrations, HIF-1α is virtually undetectable in
most cells [53]. In these conditions, the protein interacts with the tumor suppressor
protein VHL, a part of the ubiquitin-ligase complex that induces the ubiquitination
of HIF-1α and its rapid degradation. The structural basis for specific interaction of
HIF-1α and VHL is provided by the introduction of the hydroxyl group at the C4
position of Pro402 and Pro564 [54], which facilitates hydrogen bonding with
Ser111 and His115 in VHL. On the other hand, hydroxylation of Asp803 prevents
complex formation between HIF-1α and the transcriptional co-activators CBP and
P300, providing a second mechanism by which HIF-mediated transcription is regu-
lated [55]. The hydroxylation reactions are carried out by specific hydroxylases that
employ both Fe2+ and ascorbate as cofactors to split dioxygen into two oxygen
atoms, one of them converted into hydroxide. Ascorbate is a reducing agent needed
to avoid iron oxidation, and to maintain the metal ion bound to the enzyme as Fe2+.
Hypoxia signalling reduces the HIF-1α hydroxylation, therefore stabilizing HIF-1α
and allowing it to join HIF-1α. The heterodimer translocates into the nucleus, where
it binds the hypoxia-responsive enhancers (HREs) and recruits the co-activator
acetyltransferase P300 [56].
Similarly to hypoxia, nickel was found to be a strong stabilizer of the HIF-1α
protein and an activator of HIF-dependent transcription, inhibiting its enzymatic
hydroxylation [56]. This likely occurs through a depletion of intracellular ascorbate
that follows nickel-driven oxidation and/or uptake inhibition [57]. This results in the
inactivation of the hydroxylases, followed by the induction of HIF-1 and activation
expression of hypoxia-inducible genes [56]. The activation of the hypoxic signal-
ling pathway and the switch of cellular metabolism to a state that mimics permanent
hypoxia may be a part of nickel-induced carcinogenesis [42]. Indeed, hypoxia is a
common state in tumors because transformed cells grow faster than the blood ves-
sels providing them with oxygen. This state can activate genes that enable cells to
overcome nutritive deprivation, to escape from the hostile metabolic microenviron-
ment, and to stimulate angiogenesis. Additionally, cellular responses to hypoxic
stress include inhibition of cell proliferation and, when cell damage is irreversible,
apoptosis. Therefore, imitation of the state of hypoxia by nickel may provide the
conditions for the selection of cells that have altered energy metabolism, changed
growth control and/or have become resistant to apoptosis. A result of nickel-induced
hypoxia response is the induction of numerous genes involved in glucose transport
and glycolysis, coding for carbonic anhydrase IX, ceruloplasmin, erythropoietin,
inducible nitric oxide synthase, vascular endothelial growth factor (VEGF), and
many others [23].
10 Nickel and Human Health 331

(iv) Additional alterations of other signalling pathways. Exposure of cells to nickel


also induces a change of gene expression that yields cells with spectra of expressed
genes similar to cancer cells. The proteins responsible for this effect are some tran-
scription factors, involved in different metabolic pathways, such as oxidative stress
defence (NF-κB, AP-1), inflammatory response (NF-κB, TGF-β), apoptosis (p53),
angiogenesis (ATF-1), calcium homeostasis (NF-AT), all activated by nickel [23].
Nickel also induces activation of the K-ras oncogene, and inhibits tumor suppressor
genes, such as Rb, p16, FHIT, Zac-1, and Gas-1 [23].
(v) Interaction with proteins. The gene coding for the Cap43 protein (also called
N-myc downstream-regulated gene 1 (NDRG1)) is induced by nickel through the
hypoxia pathway [58]. This protein is usually expressed at low levels in normal tissues,
but it is over-expressed in a variety of cancers, including lung, brain, melanoma,
liver, prostate, breast and renal cancers, and has been often used as a marker of
tumor progression [59]. The physiological function of this protein is not clear so far, but
likely it acts as a tumor suppressor protein. The fact that this protein is ubiquitously
expressed and highly conserved in all multicellular organisms, and that its expression
is regulated by different central pathways that respond to several stress and growth
signals, such as cellular proliferation, differentiation, growth arrest, neoplasia, tumor
progression and metastasis, hypoxia, heavy metal response, favors a central role in
cellular metabolism [60]. Impairing of calcium homeostasis, also triggered by nickel,
induces Cap43/NDRG1 production. Interestingly, three repeats of the GTRSRSHTSE
sequence are found in the C-terminal tail of Cap43/NDRG1, which are not present
in the other family members, and have been postulated to serve as nickel binding
motifs, and can bind one nickel ion each [61,62]. It is noteworthy that these repeats
fall into a region that in all NDRG family members is predicted to be unfolded [63],
therefore possibly involved in regulation through interaction with a partner or cofactor
such as nickel. The fact that the 30-amino acid fragment of the C-terminal tail of the
nickel-induced Cap43/NDRG1 is able to bind up to three nickel ions could shed
new light onto the complex mechanism of nickel toxicity, in particular in relation to
the physiological role exerted by the stress protein genes up-regulated by metal
exposure, suggesting a possible role of Cap43/NDRG1 as a detoxification agent and
a feedback mode of nickel sensing. The crystal structure of a homologue, NDRG2,
only expressed in adult skeletal muscle and brain, has been recently reported, and the
protein was thought to serve as molecular interactor for regulating cell proliferation
and cell differentiation [64].
Beside Cap43/NDRG1 protein and histones, nickel has been reported to form
complexes with other proteins, possibly significantly altering their conformations,
interactions, functionality, and eventually cellular homeostasis, triggering the
observed adverse effects. At physiological pH, the strength of Ni2+ interactions with
proteins depends on the type of amino acid residues, their positions relative to each
other, and their accessibility in the protein molecule. The greatest affinity for Ni2+ is
shown by histidine and cysteine residues in proteins. One of these proteins, named
DAN, possessing a Ni2+ binding motif at the C-terminal region, shows a tumor sup-
pressor activity. Interaction of DAN with Ni2+ can impair its activity triggering the
332 Zambelli and Ciurli

tumorigenic phenotype [65]. Similarly, cullin-2, a component of the complex that


serves HIF-1α ubiquitination, features three Ni2+ and Co2+ binding sites [66]. Metal
binding prevents ubiquitination, therefore contributing to the typical hypoxia
response. Nickel can also bind the iron regulatory protein 1 (IRP-1), a central regu-
lator of iron homeostasis [67]. Replacement of one Fe2+ with Ni2+ in the 4Fe-4S
cluster inactivates the protein enzymatic activity and may contribute to the nickel-
induced hypoxic signalling. The ability of Ni2+ and Cu2+ to bind neuromedin C, a
neuropeptide, may represent the overlap between the metabolisms of these two metal
ions, and may imply that Ni2+ is able to impair copper homeostasis in the brain [68].

2.2 The Different Faces of Nickel Allergy

2.2.1 Nickel Effects on Immune Response

The immune response triggered by nickel exposure is generally important, and


responsible of allergic contact dermatitis (ACD), one of the most significant conse-
quence of occupational exposure to nickel. The first event in nickel immunological
response is a silent sensitization phase, which is initiated upon the first contact with
the antigen, leading to the generation of allergen-specific T-cells, and lymphocyte
activation. Upon re-exposure to the allergen, an elicitation phase occurs, resulting in
clinically apparent inflammation. In this second phase, the T-cells exert cytotoxic
functions and secrete inflammatory mediators, such as chemokines and cytokines,
to amplify the inflammatory response and produce eczematous skin reaction [69].
As many ACDs, nickel allergy requires two events for being established both
for sensitization and elicitation, that is (i) the activation of antigen-specific T-cells,
and (ii) a non-specific proinflammatory microenvironment necessary for the
development of a hypersensitivity response, also called innate immune signal [70].
In humans, nickel ions can trigger both these events, directly activating proinflam-
matory pathways [70].

2.2.1.1 How Nickel Activates Antigen-Specific T-Cells

T-cells are the major effectors in Ni2+ hypersensitivity. These cells are usually acti-
vated when a peptide, recognized as non-self, is bound to a major histocompatibility
complex (MHC) protein of the membrane of an antigen-presenting cell (APC), and
interacts with a T-cell receptor (TCR) [71]. This is the signal that activates the lym-
phocytes and subsequently the immune cascade. Ni2+ ions, as many other immuno-
logically active low molecular chemicals, are defined as haptens, that is antigens
generally invisible to the immune system by themselves, becoming visible only
when bound to proteins or peptides [72]. While, generally, hapten recognition by
T-cells requires covalent hapten attachment to MHC-associated peptides, transition
metal ions such as Ni2+ do not form stable covalent protein modifications, they
10 Nickel and Human Health 333

rather produce geometrically highly defined coordination complexes with reversible


binding [73].
During the immune response, Ni2+ has been found to be reversibly bound to a
particular MHC-II molecule, called HLA-DR52c, and an unknown MHC-bound
peptide via a pH-sensitive interaction, consistent with metal ion coordination to
amino acid side chains of histidine or acidic residues [74]. The structure of HLA-
DR52c in complex with a self-peptide deriving from the Tu elongation factor (pTu),
has been reported [75]. Although the HLA-DR52c/pTu complex is not able to bind
Ni2+, this structure is important to understand how Ni2+ can be presented in this
complex. In particular, four residues (His81, Asp55, Gln70, and Gln74) have been indi-
cated as being potentially involved in metal coordination. However, these are not
sufficient to allow Ni2+ binding, coherently with the observation that a specific pep-
tide is needed to form the Ni/HLA-DR52c/peptide complex [75]. How the metal ion
influences TCR binding to the Ni/HLA-DR52c/peptide complex is still unknown.
It has been proposed that the metal ion binds to the interface between MHC and
TCR proteins, sterically influencing the interaction [76]. Based on current biochem-
ical data, hypothetically the metal ion can bind the MHC and become the
ligand of T-cells at least in four different ways [77]: (a) Ni2+ could bind the MHC
only; (b) Ni2+ can bind both the presented peptide and the MHC molecule; (c) Ni2+
can interact with the presented peptide only; (d) Ni2+ can induce a conformational
change of the peptide and/or of the MHC, which are therefore recognized as neo-
antigens. A further possibility is that Ni2+ ions interfere with the processing of
self-proteins in the APC and the exposure of cryptic self-peptides, which can be
recognized as non-self by TCR [78].
Besides activating T-cells in a Ni/MHC/peptide ternary complex, Ni2+ appears to
serve as a direct and peptide-independent linker between TCR and MHC, bearing
certain similarities to superantigen-mediated activation [79]. Potential Ni2+ contacts
both in the TCR and in the specific MHC molecule are responsible for Ni2+ reactiv-
ity independently from protein processing by antigen-presenting cells and peptide
binding to MHC. This new type of Ni2+-induced TCR-MHC crosslinking might
explain the high frequency of Ni-reactive T cells in the human population [79].

2.2.1.2 How Nickel Activates the Innate Immune Signal

Ni2+ is the only allergen for which a direct mechanism of innate immune activation
has conclusively been demonstrated and unravelled at the molecular level. In vitro
and in vivo experiments showed that treatment of human endothelial cells with Ni2+
triggers rapid expression of the surface molecules VCAM1, ICAM1, and E-selectin
and monocyte-attracting chemokine MCP-1 [80–82], required for leukocyte
adhesion and inflammation. On the other hand, expression of lymphocyte-attracting
cytokines such as IP10, Mig, MDC, PARC, and TARC, occurs at later stages and
correlates well with the infiltration of T-cells into the dermis and epidermis [82].
Molecular analysis revealed that, in humans, Ni2+ activates the IKK2/NF-κB and
the MAPK/p38 signalling pathways, both regulating gene expression leading to
334 Zambelli and Ciurli

inflammation [83,84]. These activations occur through Ni2+ interaction with the
membrane toll-like receptor 4 (TLR4), in combination with its co-receptor MD2, which
induces receptor dimerization probably bridging two TLR4 monomers. Coherently,
human cells expressing TLR4 and MD2 including macrophages, fibroblasts, and
dendritic cells were able to induce a proinflammatory response upon Ni2+ treatment
[85]. The non-conserved His456 and His458 residues in human TLR4 are critically
required for Ni2+-induced proinflammatory gene expression, possibly acting as specific
ligands for the metal ion [85]. Notably, mouse TLR4, which lacks these histidine
residues, is not able to produce the proinflammatory pathway in response to Ni2+
exposition. Activation of innate immunity in mice rather needs co-stimulating adjuvants,
such as the bacterial cell wall component lipopolysaccharide (LPS).
Another pathway responsible for Ni2+-induced ACD is the death receptor-
mediated or extrinsic apoptosis pathway. It was reported that Ni2+ transcriptionally
represses expression of cFLIP, a cellular antagonist of the pro-apoptotic caspase-8,
in both primary human keratinocytes and endothelial cells [86], which, as a result,
are strongly sensitized to apoptosis. Coherently, there is evidence for an increased
occurrence of death ligand-mediated keratinocyte apoptosis in the course of Ni2+-
induced ACD in sensitized individuals [87]. Enhanced cell death of keratinocytes is
predicted to impair the barrier function of the skin. Hence, Ni2+-mediated cFLIP
down-regulation might tip the balance towards increased apoptosis of certain skin
cell populations, which successively may augment the severity of the ACD response
during the elicitation phase by increasing the efficient concentration of Ni2+ arriving
in the epidermis [70]. In addition, nickel is able to induce apoptosis in a number of
immune cells, including human neutrophils and T-cells, through the mitochondrial
pathway that activates caspase-3, likely as a response to Ni2+-induced oxidative
stress [12,13]. The production of ROS by Ni2+ exposition also acts in concert with
the mechanisms described above to produce and amplify the inflammatory response.
In particular, ROS act as signalling molecules and are recognized as important
inducers of the proinflammatory response [69].

2.2.2 The Impact of Nickel-Induced Dermatitis

The immunological effects of nickel described above are responsible for allergic
contact dermatitis (ACD), which is the most spread dermatitis over the world and
is constantly increasing, reaching 20% of the human population. It was discovered
for the first time in 1930 [88]. It is caused by Ni2+ ions solubilized from nickel-
containing alloys by sweat and other body fluids that serve as sensitizing allergen
and come in contact with skin. Although the risk of occupational disability is an
issue for a relatively large group of professionals, such as metal workers, cashiers,
or hairdressers, the major problem associated with Ni2+-induced contact hyper-
sensitivity is the wide presence of this metal ion in modern industrial products, so
that it is very common to come in contact with the allergen. Ni2+ is released from
coins, earrings, watches, belt buckles, bras, mobile phones, dental and orthopedic
10 Nickel and Human Health 335

implants, and cardiovascular stents [70]. In Europe, currently around 65 million


individuals are sensitized to Ni2+ [89]. Legislative interventions limiting the amount
of Ni2+ release from products intended for prolonged skin contact, such as the
Danish nickel regulation in 1990 [90] and the EU directive 94/27/EG in 2001 (also
known as “Nickel Directive”), result in decreasing sensitization rates, but preva-
lence of contact eczema to Ni2+ are still considerably high [91]. In its classic
description, nickel ACD involves the hands and forearms mainly after occupational
exposure. Sensitized individuals generally experience a predictable localized
response following cutaneous exposure to nickel, including erythema, vesicle for-
mation, scaling, and pruritus [92]. A nickel-free diet remains controversial in the
treatment of ACD. In addition, inhaled nickel in ambient air might be a risk factor
for nickel sensitization [93]. The most important way of preventing nickel allergy
is the avoidance of exposure. Rubber gloves are inefficient for prevention of nickel
contact, as metal ions penetrate through them. Polyurethane coating of metal items
can be beneficial in protection from nickel contact, as are barrier creams containing
sodium EDTA [94].
Nickel is present in most dietary items, and food is considered to be a major
source of exposure to nickel for the general population. Certain foods, such as green
beans, broccoli, peas, canned vegetables and spaghetti, canned fruit, dried fruit,
nuts, cocoa, and chocolate are routinely found to be high in nickel content. Nickel
present in the diet of a nickel-sensitive person can provoke systemic reactions,
called systemic contact dermatitis (SCD). SCD is manifested as generalized eczem-
atous reactions, maculopapular rash and vasculitis-like skin, along with systemic
symptoms such as headache, malaise, diarrhea, fever, and arthralgia [95]. The exact
mechanisms of systemic contact dermatitis are still not known. The eczema flare at
sites of previous exposure suggests T cells resting at the area or homing the site
upon systemic allergen exposure [96]. Circulating immune complexes together with
non-specific cytokine release by the hapten stimulation can be responsible for the
generalized reactions [95,96].
In patients with SCD, adherence to a low-nickel diet and avoidance of local
exposure to metal objects result in the disappearance of skin symptoms [92].
Another therapeutic approach, studied for both ACD and SCD, is nickel hypo-
sensitization, that is the procedure allowing sensitized patients affected by ACD to
safely make contact with nickel containing objects, and those with SCD to freely eat
nickel-containing foods. This aim is pursued through oral administration of cap-
sules containing nickel to ACD and SCD patients. For ACD, the clinical studies,
performed with oral supplementation of high nickel doses, are still preliminary, as
they have been conducted with a limited number of subjects and for a limited period
of treatment, but have given promising results [97]. In the case of SCD, the studies
of oral hypo-sensitization with very low doses of nickel for a prolonged time led to
an improvement of the symptoms and a complete remission in 50% of the patients.
No side effects are observed during and after the treatment. The hypo-sensitization
treatment exerts its clinical effects through a modulation of the allergic immune
reaction specific for nickel [98].
336 Zambelli and Ciurli

3 Nickel-Dependent Infectious Diseases

Nickel in the active site of several enzymes is responsible for the catalysis of impor-
tant biological reactions, such as urea hydrolysis, hydrogen metabolism, methane
formation, CO/CO2 inter-conversion, superoxide metabolism, and detoxification of
methylglyoxal [99]. For many bacteria, archaea and unicellular eukaryotes these
nickel-dependent processes render possible the colonization of environments that
are inhospitable and hostile. These include parts of human and animal bodies, where
these nickel-obligate pathogens grow and survive in virtue of nickel-catalyzed
reactions. Consistently, some nickel-dependent enzymes are virulence factors for
pathogenic organisms. As no nickel-dependent enzyme is known in vertebrates,
catalyzed nickel-dependent processes and mechanisms of nickel delivery into specific
enzymes can be regarded and employed as possible selective targets to control the
pathogenesis of nickel-obligate organisms.

3.1 Nickel-Dependent Enzymes in Pathogenic Microorganisms

The biological role of nickel is essentially defined by its use as a cofactor of enzymes
found in all phyla of life (plants, fungi, eubacteria, and archaea). All known nickel
enzymes involve the transformation and/or production of gases (ammonia, carbon
monoxide, carbon dioxide, methane, dihydrogen, and dioxygen) involved in the
geo-biological cycles of carbon, nitrogen, and oxygen. The nickel ion in the active
sites of these enzymes exhibits a very large flexibility in metal coordination and
redox chemistry, spanning coordination numbers from 4 to 6 and oxidation states
from +1 to +3, with potentials ranging from +900 to −600 mV [100]. In these
enzymes, nickel often is inserted in a multinuclear metal cluster that also includes
modified amino acids and/or exogenous ligands [20].
The importance of nickel enzymes for human health is related to the fact that,
among eight known nickel-dependent enzymes, four (glyoxalase I, acireductone
dioxygenase, hydrogenase, and urease) are present in pathogenic microorganisms
and are often essential for their growth and pathogenesis. For example, infections
by urease-dependent organisms represent a widespread source of diseases, ranging
from tuberculosis to urinary tract infections, hepatic coma, and kidney stones [101].
The glyoxalase enzyme of the protozoan parasites has been regarded as a potential
chemotherapeutic target against eukaryotic pathogens, such as Trypanosoma or
Leishmania [102].

3.1.1 Glyoxalase I

Glyoxalase (Glx) I and GlxII catalyze the conversion of methylglyoxal, a toxic species
that forms covalent adducts with DNA, to lactate (Figure 2) [103]. For a long
time, all GlxI enzymes were believed to be Zn2+ enzymes, like the human GlxI.
10 Nickel and Human Health 337

Figure 2 Reaction catalyzed by GlxI and GlxII, and schematic structure of the nickel-containing
active site.

However, it was later shown that some GlxI enzymes are more active with Ni2+
in vitro. These include the GlxI from some bacteria, such as the pathogen
Pseudomonas aeruginosa, and trypanosomatids, responsible for several diseases,
such as Chagas’ disease and skin sores called leishmaniasis. It is not clear whether
this metal ion is functional for GlxI in vivo [104]. A single Ni2+ ion in an octahedral
coordination environment composed by 2 His, 2 Glu, and two water molecules,
acts as a Lewis acid catalyst and remains in the 2+ oxidation state throughout the
isomerization reaction (Figure 2). On the other hand, the inactive Zn2+ enzyme is
five-coordinated [103].

3.1.2 Acireductone Dioxygenase

Acireductone dioxygenase (ARD) catalyzes the incorporation of two oxygen atoms


into the substrate acireductone, the penultimate intermediate of the methionine salvage
pathway (Figure 3) [105,106]. The latter is a ubiquitous process that regulates
methionine levels, thus playing an essential role in several biosynthetic processes.
In Klebsiella ATCC 8724, this enzyme is a monomeric protein, showing a classic
jellyroll structural motif. The active site, relatively well exposed to the solvent, con-
tains Ni2+ in an octahedral high-spin coordination center, involving three His, one
Asp, and two water molecules in equatorial positions (Figure 3). The Ni2+-bound
enzyme catalyzes the so-called “off-pathway” reaction, leading to the formation of
relatively large amounts of carbon monoxide. Peculiarly, this enzyme can also bind
Fe2+ in the same coordination site. Fe2+ binding stabilizes a different protein form
that catalyzes the “on-pathway” reaction, leading to production of methionine. The
Ni2+- and Fe2+-bound forms differ constitutionally only in the identity of the metal
ion bound. Interestingly, this difference is sufficient to induce significant structural
338 Zambelli and Ciurli

Figure 3 Reaction catalyzed by acireductone dioxygenase, and schematic structure of the nickel-
containing active site.

changes resulting from differential packing of a compact hydrophobic core of the


protein, accompanied by extensive changes in secondary structure and ordering of
nearby structural features [107].

3.1.3 Urease

Urease is key to the global nitrogen cycle as it catalyzes the hydrolysis of urea, produced
by vertebrates, to ammonia and carbamate, which then spontaneously decomposes to
give a second molecule of ammonia and bicarbonate (Figure 4) [108].
The subsequent hydrolysis of the reaction products induces an overall pH
increase that has negative implications both in human and animal health, as it is
used by several pathogens, such as Helicobacter pylori, to colonize hostile acid
habitats. In addition, this reaction represents a nitrogen source for several organisms
that infect humans. Therefore, urease is a virulence factor for pathogens in the
animal gut, urinary tract, and stomach.
Microbial ureases are generally heteropolymeric proteins with a quaternary
structure (αβγ)3. In some bacteria, such as those of the genus Helicobacter, the trimer
is of the type (αβ)3, with the β subunit corresponding to the fused β and γ subunits
normally found in other bacteria, and presents a higher level of oligomerization that
leads to an enzyme with a quaternary structure ((αβ)3)4. On the other hand, plant
ureases are hexameric proteins α6, each α subunit being highly homologous to each
(αβγ) assembly of microbial ureases [109].
Several structures of ureases are available. In all cases, the active site contains
two Ni2+ ions bridged by the carboxylate group of a carbamylated lysine and by a
hydroxide ion (Figure 4). Each Ni is also coordinated by two histidines and one
water molecule, whereas Ni(2) is further bound to an aspartate. This results in a
penta-coordinate Ni(1) and hexa-coordinate Ni(2). In the resting state of the enzyme
from Bacillus pasteurii, the active site accommodates a fourth water molecule,
completing a tetrahedral cluster of solvent molecules [110]. The access to the active
site is regulated by a flexible helix-loop-helix segment, with the position of amino
acids involved in the catalysis being critically affected by the flap movement [111].
10 Nickel and Human Health 339

Figure 4 Reaction catalyzed


by urease, and schematic
structure of the nickel-
containing active site.

The structures of B. pasteurii urease (BPU), in the native hydrated form and
complexed with several inhibitors of different chemical classes, suggest a structure-
based reaction mechanism for urease, and highlight the importance of both metal
ions in the reactivity of the enzyme [109]. The structures of BPU bound with borate
[112], a substrate analogue, and with diamidophosphate [110], a transition state
analogue, are particularly significant to understand the reaction mechanism
[109,113].

3.1.4 [NiFe]-Hydrogenase

Hydrogenase enzymes catalyze the formation of hydrogen gas from protons and
electrons and/or the reverse reaction, oxidizing H2 as a source of reducing power,
and coupling it to the reduction of various terminal electron acceptors (e.g., O2,
NO3−, SO42 −, CO2, and fumarate), in energy-conserving pathways (Figure 5). They
can act as sensors for the availability of hydrogen gas [114].
The [NiFe]-hydrogenases are a class of hydrogenase enzymes that contain nickel
and iron in the active site and are widely distributed in bacteria and archaea, includ-
ing some pathogens [115]. A [NiFe]-hydrogenase is required for efficient coloniza-
tion by H. pylori and Salmonella enterica serovar Typhimurium, a food poison,
Campylobacter jejuni, a bacterium closely related to Helicobacter, as well as many
340 Zambelli and Ciurli

Figure 5 Reaction catalyzed


by [NiFe]-hydrogenases, and
schematic structure of the
nickel-containing active site.

enteric bacteria (e.g., E. coli, Shigella, and Yersinia species) [20,116]. These infectious
organisms use the hydrogen produced by other microorganisms in the body, a
resource not used by the human host, as a supply of energy for their respiratory
pathway. This permits the growth and maintains efficient virulence during animal
infections [116].
[NiFe]-hydrogenases are usually composed of two subunits. The smaller β sub-
unit contains three closely spaced FeS clusters (two [4Fe-4S] and one [3Fe-4S]),
linearly arranged, that transfer electrons to and from the active site. The active site
is constituted by a hetero-nuclear bimetallic [NiFe] center, buried at the bottom of a
hydrophobic channel in the larger α subunit. The nickel-containing hydrogenases
can be classified in two different categories, differing by ligation of the [NiFe] cluster
found in the active site to either cysteine or seleno-cysteine.
In the oxidized form of the enzyme, the Ni and Fe atoms are bridged by two
cysteines and a third, non-protein ligand, possibly a sulfide or an oxide (Figure 5).
The Ni atom is bound to two additional cysteines, forming the equatorial plane of a
distorted square pyramidal coordination geometry with the O/S bridging atom being
the fifth axial ligand. In [NiFeSe]-hydrogenase, selenium, in the form of seleno-
cysteine, is a ligand to Ni. The Fe atom resides in a pseudo-octahedral coordination
geometry, additionally bound to CO, and two CN–.
In the structure of the reduced enzyme, the non-protein bridging ligand in the
bimetallic center is absent (Figure 5), leading to the conclusion that activation of
[NiFe]-hydrogenases by H2 involves the removal of such a bridge, thereby causing
the opening of binding sites on both Ni and Fe atoms. The subsequent steps of the
catalytic mechanism have not been fully elucidated to date, but they likely include
nickel, iron, or cysteine thiolates as potential substrate-binding sites and redox
centers [115].

3.2 Molecular Regulation of Nickel Homeostasis


in Pathogenic Microorganisms

The essentiality of nickel for nickel-dependent organisms, together with the envi-
ronmental scarcity of this metal ion, led nickel-obligate pathogens to evolve a tight
system for nickel homeostasis with mechanisms that correlate Ni2+ availability with
10 Nickel and Human Health 341

Figure 6 Schematic representation of bacterial Ni2+ homeostasis, intracellular transport, and


utilization. ABC = ATP-binding cassette; NiCoT = nickel cobalt transporter; RcnA = resistance to
cobalt and nickel A; CznA = cadmium-zinc-nickel resistance A; MCR = methyl coenzyme-M
reductase; ADR = acireductone dioxygenase; GlxI = glyoxalase I.

the expression of proteins involved in nickel transport and utilization. In particular,


attaining sufficiently high intracellular nickel concentrations to meet the demand
of the nickel enzymes and, at the same time, protecting the cell by the poisoning
potential of nickel excess, requires (i) an efficient nickel uptake/efflux system, ii)
molecular chaperones and metallo-chaperones, and (iii) nickel sensors (Figure 6).

3.2.1 Nickel Membrane Transporters

Ni2+ ions should enter into the cytoplasm in order to be inserted into the active site
of nickel-dependent enzymes. On the other hand, nickel excess must be extruded
from the intracellular location, in order to prevent nickel-related danger at toxic
metal concentrations. Therefore, the metal ions must be able to pass through the
cytoplasmic membrane entering or exiting the cell according to the intracellular
nickel abundance. Low levels of nickel can travel through the plasma membrane by
way of multiple, possibly nonspecific, systems. However, in many of the microor-
ganisms that require this metal ion, dedicated nickel uptake transporters have been
342 Zambelli and Ciurli

identified that belong mainly to two different classes, the NikABCDE import pumps
and the nickel/cobalt permeases (NiCoT). The former, firstly characterized in
E. coli, belongs to the family of ATP-binding cassette (ABC) transporters, and cou-
ples the translocation of a substrate to the hydrolysis of ATP. NikB and NikC are
trans-membrane proteins that form a nickel pore. NikD and NikE are proteins that
bind to and hydrolyse ATP [117]. NikA is a periplasmic protein that binds one
nickel per protein in the context of a nickelophore. The nature of this complex is
controversial, but recent structural data have identified two free histidines coordi-
nating Ni2+ in the metal binding site of NikA, suggesting that Ni2+-(L-His)2 is the
nickelophore recognized by the periplasmic transporter [118]. This transport system
has been found in several pathogenic organisms, such as Brucella suis, Vibrio para-
hemolyticus, Helicobacter hepaticus, Yersinia sp., and Staphylococcus aureus
[119]. The NiCoT family is composed by monomeric single component permeases
with eight trans-membrane helices, found in many bacteria, such as the pathogen H.
pylori, as well as several archaea and fungi. While some family members are spe-
cific for Ni2+, other permeases transport both Ni2+ and Co2+, with the preference of
one metal over the other [20].
In gram-negative bacteria, some nickel-specific importers have been found in the
outer membrane. In particular, in H. pylori FecA3 and FrpB4 have been suggested
to be involved in TonB-dependent transport of nickel [20].
The most common mechanism for metal resistance is metal efflux, made by
exporters that, in several organisms, have been predicted and/or demonstrated to
pump nickel out of the cytoplasm and of the periplasm. Only in a few cases the
transporters are specific for nickel [120]. In particular, nickel exporters have been
identified for H. pylori and E. coli. In H. pylori, CznABC is proposed to be a novel
system that pumps Cd2+, Zn2+, and Ni2+ across both the inner and outer membranes
[121]. In E. coli, RcnA is a membrane transporter with six trans-membrane seg-
ments, with only limited homology with other transporters families, involved in
specific export of Ni2+ and Co2+ [122].

3.2.2 Nickel Molecular Chaperones and Metallo-Chaperones

Nickel activation pathways imply nickel delivery into the buried cavity of specific
enzymes, usually synthesized as apo-proteins undergoing activation through metal
ion incorporation in a post-translational regulation mechanism of the enzymatic
activity. Some of these pathways, usually involving a multistep, tightly regulated
mechanism with several accessory proteins, have been extensively studied in bacteria
and in some cases they even overlap (e.g., hydrogenase and urease pathways) [99].
The functional, biochemical and structural properties of these chaperones have
been investigated in the case of urease (UreDEFG proteins), hydrogenase
(HypABCDEF and SlyD), carbon monoxide dehydrogenase (CooCTJ), acetyl-CoA
decarbonylase synthase (AcsF), and superoxide dismutase (SodX and CbiXhp)
[99]. No accessory protein is known for acireductone dioxygenase and for gly-
oxalase so far.
10 Nickel and Human Health 343

Even though many aspects of these processes remain largely obscure, some
general aspects of the roles performed by the accessory proteins are maintained
throughout the investigated systems. In particular, (i) nickel storage, (ii) nickel
delivery, and (iii) nucleotide triphosphate hydrolysis have been found in several
nickel-driven enzyme activations.
These three functions are carried out by specific protein domains, organized in a
modular fashion, either in a single protein or in separated chaperones. The nickel
storage role is normally carried out by a His-rich pattern: this motif may constitute
a whole protein, such as in the case of Hpn or HspA, two nickel accumulators found
in Helicobacter pylori [123–125], or may be located at the C- or at the N-terminus
of a specific chaperone, as in the case of several UreE [126] and of UreG from
Mycobacterium tuberculosis [127], Streptomyces coelicolor and Glycine max [128]
for urease, HypB from Bradyrhizobium japonicum [129] and SlyD from Escherichia
coli [130] for hydrogenase, and CooJ from Rhodospirillum rubrum [131] for CO
dehydrogenase.
On the other hand, the nickel delivery function is performed using nickel binding
sites on specific metallo-chaperones, such as UreE for urease [109,132–134], HypA,
and possibly HypB, for hydrogenase [20], and CooJ for carbon monoxide dehydro-
genase [131]. Finally, hydrolysis of nucleotide triphosphates is required to complete
the biosynthesis of the enzyme with a reaction catalyzed by homologous P-loop
GTPases: HypB intervenes in the activation of both hydrogenase and urease [135],
AcsF plays its function in the activation of acetyl-CoA decarbonylase synthase
[136], CooC is involved in the assembly of carbon monoxide dehydrogenase [137],
while UreG is the GTPase essential for urease assembly [109].
UreG from many organisms, including the pathogenic bacteria M. tuberculosis
and H. pylori, has been reported to be an intrinsically disordered protein, existing in
a flexible behavior in vitro in the absence of other protein partners [127,138–141].
This observation reveals that, at least for urease, protein disorder plays a role in the
protein-protein interaction network leading to enzyme activation, and represents a
further post-translational mechanism to regulate the Ni2+-dependent enzymatic
activity. Furthermore, intrinsic disorder has been found also in some portions of
other urease proteins, such as in the C-terminal sequence of UreE involved in the
protein interaction with metal ions [134,142], which in turn regulates UreE interac-
tion with UreG [142], and in the C-terminal sequence of UreF, which is disordered
in the free form of the protein and becomes correctly folded upon UreF-UreD
interaction [143].

3.2.3 Nickel Sensors

The crucial players of nickel homeostasis networks are specific nickel-responsive


transcriptional regulators, which specifically bind promoters of genes involved in
Ni2+ homeostasis, such as nickel importers or efflux pumps, nickel storage proteins,
nickel chaperones and nickel-dependent enzymes. These proteins couple specific
metal ion binding with a change in their DNA binding affinity and/or specificity,
344 Zambelli and Ciurli

thus translating the concentration of a certain metal ion into a change in transcriptional
response. Several nickel sensors, belonging to different families of regulators, have
been discovered and characterized, including NikR (NikR family), RcnR (RcnR/
CsoR family), NmtR (ArsR/SmtB family), KmtR (ArsR/SmtB family), SrnRQ
(ArsR/SmtB family), and Nur (Fur family) [144].
These proteins exist as homo-dimers or homo-tetramers and usually act as tran-
scriptional repressors and negatively control mRNA synthesis, preventing RNA
polymerase from initiating the transcription at the promoter. Ni2+ ions bind in regu-
latory sites, acting (i) as co-repressors, increasing the affinity of the protein repres-
sor for the DNA operator sequences: this is the case of proteins that regulate genes
for metal ion efflux, storage, trafficking, and tolerance (RcnR, NmtR, KmtR); (ii) as
inducers, decreasing the affinity of the repressor for the DNA operator sequences,
eventually leading to transcriptional activation of genes for membrane uptake sys-
tems (NikR, SrnRQ, Nur). Only in the case of NikR from H. pylori the metal-
responsive transcriptional regulator act as pleiotropic regulator, exerting a dual
control, both as activator and repressor, on different promoters [144].
Nickel-protein interactions, selectively driven by the coordination chemistry and
geometry of metal binding sites, usually octahedral or square planar, are propagated
away from the specific metal binding site through changes in protein structure and/
or dynamics, along the protein backbone, resulting in a modification of the DNA
binding affinity of the protein. For example, binding of metal ions to their specific
coordination sites within the ArsR/SmtB family drives an allosteric change, with the
stabilization of a protein conformer with low affinity for DNA, and decreases the
internal dynamics of the protein backbone, leading to the unavailability, in energetic
terms, of the conformer that features high affinity for DNA [145]. On the other
hand, in case of HpNikR, the presence of bound Ni2+ [146,147] does not induce, by
itself, the stabilization of a specific conformer, and increases the protein dynamics
unlocking inter-domain motions, supporting the view that the likely mechanism of
interaction of the protein with its operator DNA sequence involves a selection of the
correct conformation coupled with an induced fit mechanism facilitated by the pres-
ence of bound Ni2+ [148,149]. These events produce a finely tuned metabolic
response driven by Ni2+ ions, including the coordinated control of the entire machin-
ery of metallo-enzyme synthesis and activation, as well as the systems of homeosta-
sis that involve competitive Ni2+ uptake, intracellular accumulation, and extrusion.

3.3 Nickel-Obligate Microorganisms with Severe Impact


on Human Health

3.3.1 Helicobacter pylori as a Nickel-Dependent Pathogen: A Possible


Correlation between Nickel Intake and Cancer Development

Helicobacter pylori is a Gram-negative bacterium and the principal causative agent


of many acute and chronic gastric pathologies, including peptic ulcer. It is a major
factor of risk for the insurgence of gastric carcinomas and lymphomas [150];
10 Nickel and Human Health 345

accordingly, the WHO classified the bacterium as a class 1 carcinogen in 1994.


After the colonization, untreated H. pylori infections persist for the entire life of the
host because of the inability of the human immune response to efficiently counter
the bacterium [151]. H. pylori is widespread worldwide, and infects up to 50% and
80% of adults in industrialized and developing countries, respectively.
Consistent with the hostility of the human stomach as a habitat for bacterial
growth, H. pylori has set up a number of adaptive mechanisms, allowing the bacte-
rium to promptly respond to environmental stresses, such as mild to strong acidity,
fluctuating nutrient availability and osmolarity, oxygen tension and host immune
responses. These adaptive responses rely on transcriptional regulatory networks that
control coordinated expression of tolerance and virulence genes in space and time
[152]. Key virulence determinants for the processes of bacterial colonization include
two essential nickel enzymes, that is, Ni2+-dependent urease, which allows buffering
of the acidic gastric environment, and a [NiFe]-hydrogenase, which allows con-
sumption of energy-yielding hydrogen, freely available in the gastric niche. These
enzymes were shown to be required for full colonization of mouse stomach.
The importance of Ni2+ ions in H. pylori physiology is emphasized by the num-
ber and diversity of proteins involved in nickel homeostasis, which control the
activity of nickel enzymes at the cellular level with three different processes: (i)
regulation of transcription and expression of genes encoding metallo-enzymes and
metal trafficking proteins, performed by the pleiotropic Ni2+ sensor NikR; (ii) spe-
cific delivery of the metal ion cofactors to the protein active sites and metal-binding
pockets, performed by the urease and hydrogenase chaperones; and (iii) intracellu-
lar metal ion accumulation and storage, performed by the outer membrane trans-
porters FrpB4 and FecA3, the Ni2+ permease NixA, the Ni2+ efflux system CznABC,
the cytoplasmic Ni2+ accumulators Hpn, Hpn-like, and HspA. Due to their impor-
tance for bacterial pathogenesis, these processes all represent possible targets for
the development of alternative antibacterial strategies. Coherently, ureG-negative
mutants (which can synthesize apo-urease but are unable to incorporate Ni2+ into the
active site) are deficient in colonizing the gastric mucosa of nude mice, thereby
highlighting a link between Ni activation of urease and host colonization [153].
As urease is one of the most abundant enzymes in H. pylori, representing up to
10% of the bacterial protein content, it is likely that the demand for nickel is high in
this microorganism. Similarly to other pathogens, metal ion starvation triggers the
expression of toxins and metal ion scavengers in H. pylori, allowing the pathogen to
compete with the host for these essential nutrients. Accordingly, infections by H.
pylori have been epidemiologically linked to certain forms of anemia and impaired
iron metabolism in hosts, proving that bacterial infection can significantly impact
on the balance of human metal ion homeostasis [154]. Nickel is naturally abundant
in many types of food [5], therefore, it is plausible that H. pylori enters in contact
with the necessary amount of Ni2+ to satisfy its metabolic demand in the stomach.
The absence of known enzymes that require nickel as an essential cofactor in higher
eukaryotes suggests that there is no competition for nickel between the host and the
bacterium. However, the flux of nickel is probably not continuous and occurs in
successive batch-like conditions rather than a constant stream of metals. In addition,
nickel availability is also a function of pH, which fluctuates widely within the gastric
346 Zambelli and Ciurli

compartments. As a consequence, H. pylori must be able to accumulate Ni2+ when


relatively high exogenous concentrations are available. This intracellular nickel res-
ervoir, most likely bound to Ni2+ storage proteins, would in turn be available for
urease or/and hydrogenase maturation when Ni2+ concentrations are limited. This
was demonstrated in a recent study, aimed to correlate Ni2+ levels introduced with
diet, and colonization of H. pylori [155]. Even in Ni2+-depleted diet, wild-type H.
pylori is able to colonize the host stomach because it can store Ni2+ ions in the intra-
cellular environment, relying on Hpn and Hpn-like Ni2+ accumulators. If these
proteins are mutated, the colonization levels of Ni-depleted animals are statistically
lower than colonization levels of Ni-fed animals.

3.3.2 Nickel Homeostasis and Intracellular Parasitism: Eukaryotic


and Prokaryotic Pathogens

Some nickel-dependent prokaryotic and eukaryotic microorganisms infect the


human body as intracellular parasites. The most important eukaryotic pathogens are
Trypanosomatids, such as Trypanosoma cruzi and Leishmania spp., which are pro-
tozoa causing Chagas’ disease and leishmaniasis, respectively. Their life cycle
includes an intracellular stage in the mammalian host. These organisms produce a
Ni2+-dependent glyoxalase I, which is essential for pathogen survival as it serves to
detoxify methylglyoxal, a toxic product of glycolysis and other metabolic pathways
(see Section 3.1) [156]. The use of Ni2+ ions for glyoxalase activity in trypanosoma-
tids is exceptional, as in other eukaryotes the enzyme cofactor is zinc. This reflects
distinctive substrate selectivity between the enzyme of the pathogen and human
host, indicating that the enzyme could be a potential chemotherapeutic target against
the protozoan parasite [157].
One of the most important intracellular infective bacteria worldwide is
Mycobacterium tuberculosis, the etiological agent of tuberculosis. This bacterium
establishes infection in adult humans primarily in the lungs by infecting macro-
phages, where it can remain asymptomatic or can become active manifesting the
clinical symptoms. Currently, the WHO estimates that one-third of the world’s pop-
ulation is latently infected with tuberculosis, with 9.27 million new cases and 1.76
million deaths annually [158]. Upon infection, M. tuberculosis transfers into a bac-
tericidal phagosome that is subsequently fused to a lysosome, creating a vacuole
characterized by an acidic (pH 5.5), highly nitro-oxidative, nutrient-limiting, and
possibly hypoxic microenvironment. In this hostile environment, M. tuberculosis
relies on its exceptional metabolic flexibility and ability to adapt, replicate and/or
persist within changing and adverse microenvironments. Initially, it has been proposed
that M. tuberculosis Ni2+-dependent urease contributes to alkalize the mycobacterium-
containing vacuole in macrophages, promoting a more favorable environment for
the intracellular persistence of the bacilli. However, a recent study indicated that
urease activity does not influence the general bacterial fitness in vitro. In addition,
the alkalizing effect of the M. tuberculosis urease activity has only been found in
resting macrophages, while it is not present in the more acidic microenvironment of
10 Nickel and Human Health 347

the phagolysosomal compartment of activated macrophages, suggesting that the


alkalizing effect provided by the mycobacterial urease activity is somehow modest
[159]. On the other hand, the observation that M. tuberculosis urease expression and
activity increase upon nitrogen deprivation [160], suggests that urea may be a poten-
tial source of nitrogen for M. tuberculosis. Consistently, this bacterium assimilates
urea, as major nitrogen source, in an urease-dependent manner, this process gener-
ating ammonia, which is one of the key precursors for the biosynthesis of essential
amino acids such as glutamate [159].
The apparent dispensability of urease activity in a mouse model suggests that
other readily available nitrogen sources within the host cell could bypass the need
for M. tuberculosis to metabolize urea, with a general functional redundancy,
metabolic versatility, and compensatory mechanisms that characterize M. tuber-
culosis ability to adapt to virtually any microenvironment encountered in its host, in
which carbon and nitrogen sources may vary qualitatively and quantitatively [159].
In addition, the ability of M. tuberculosis to infect extra-pulmonary sites may
require the urease activity for bacillus persistence or replication at specific sites
of infection within the host. In this case, the ability to utilize urea as a source of
nitrogen could be critical at specific sites of infection where other sources of
nitrogen are limited [159].
The mycobacterial urease activity may also have a role in evading the immune
response of the host in response to the bacterial infection. Indeed, it is known that
exogenous NH4Cl blocks phagosome-lysosome fusion and promotes phagosome-
endosome fusion in mouse mononuclear phagocytes [161,162]. This suggests that
ammonia production by M. tuberculosis may partly slow down the phagolysosome
formation, providing the bacterium with less hostile environmental conditions. In
addition, M. bovis was demonstrated to attenuate the MHC-II molecule expression
in infected macrophages, with consequent inhibition of CD4+ T-cell activation and
general suppression of the host immune system [163,164].
The ability of the mycobacterial urease to alkalize the microenvironment has
been exploited to develop a novel vaccine candidate, based on M. bovis urease,
consisting of a urease-inactive M. bovis bacterium that expresses the Listeria mono-
cytogenes listeriolysin [165]. In the absence of the mycobacterial urease activity, the
mild acidic pH of the bacillus-containing vacuole provides an ideal pH environment
for listeriolysin perforation of the vacuole membrane. The phagosome lysis pro-
motes mycobacterial translocation into the macrophage cytoplasm, leading to cel-
lular apoptosis and stronger immune responses that provide better protection upon
M. tuberculosis reinfection.
The importance of metal ion homeostasis for M. tuberculosis is underlined by the
observation that its genome encodes an atypically large number (at least twelve)
metal sensors of the ArsR-SmtB family [144], allowing to speculate that these pro-
teins enable the pathogen to respond rapidly to metal fluxes in the phagosome [166].
Notably, two of these metal-dependent transcription factors, named NmtR and
KmtR, specifically respond to Ni2+ and Co2+ concentrations and regulate the expres-
sion of membrane proteins – an ATPase efflux pump (NmtA) and a cation diffusion
facilitator (CDF), respectively – that control metal ion efflux from the cell [166].
348 Zambelli and Ciurli

The reason of having two Ni2+/Co2+ sensors for metal ion efflux systems has been
explained with the different affinity observed for these two proteins and their cog-
nate metal ions, which allows a fine modulation of transcriptional response [166]. In
particular, KmtR appears to have higher affinity for Ni2+ and Co2+, and it is therefore
able to detect the basal level of the two metal ions, thus de-repressing the gene of
CDF transport at low metal concentrations. Only a higher cytoplasmic metal ion
threshold is able to bind NmtR, which presents a lower affinity for Ni2+ and Co2+ as
compared to KmtR, allowing the expression of the NmtA efflux pump [166].
These data support the significance of these metal ions for this pathogen as well as
the bacterial ability to respond to metal fluxes, depending on two levels of Ni2+ or
Co2+ sensing.

4 Nickel Essentiality in Animals and Humans

Nickel is classified as a “possibly essential element” for animals and humans since
the 1970s [19]. Nickel deficiency in humans has never been reported, as, in general,
human nickel intake greatly exceeds the requirements, which have been estimated
between 5 and 50 μg per day [22].

4.1 Effects of Nickel Depletion in Higher Organisms

The essentiality of Ni2+ for higher eukaryotes, firstly proposed in 1920s, is still
debated [5]. The importance of this metal for animals and humans has been
tested evaluating the effect of nickel deficiency in animals. Rats grown in the
absence or in low-abundance of nickel exhibit very severe consequences, as
depressed growth, low hemoglobin, red blood cell counts and activity of several
liver and kidney enzymes, as well as the presence of urea, ATP, and glucose in
serum, and also glucose, glycogen, and triglycerides in the liver [167]. The
growth dependence on nickel was more significant in the second depleted gen-
eration, which also showed anemia, manifested in decreased hemoglobin and
hematocrit values [5]. Nickel deprivation during reproduction in rats increased
perinatal mortality [168], while in breeding goats it significantly decreased the
success of first insemination and conception rate and increased the number of
breeding attempts to achieve pregnancy and the abortion rate [169]. This effect
seems to be related to the ability of nickel to be involved in cyclic nucleotide-
gated (CNG) channel functions [170]. CNG channels are located in a number of
organs, including the central nervous, urogenital, and reproductive systems. The
CNG channels also have an important role in kidney function and, thus, in
sodium metabolism [170].
Additionally, nickel deficiency impairs the absorption of iron from the intestine
and the concentrations of several metals, such as iron, copper, and zinc, were also
10 Nickel and Human Health 349

decreased in the liver of nickel-depleted animals [171,172]. Nickel deficiency also


results in lowered specific activities of many enzymes involved in carbohydrate and
amino acid metabolism. It was suggested that the essentiality of nickel for higher
organisms is linked to its modulation of gene expression in eukaryotic cells [58].
Nickel has been proposed to be also involved in membrane stability or in lipid
metabolism [173].

4.2 Influence of Nickel Availability on Gut’s Microflora

The wide presence of Ni2+-dependent enzymes in prokaryotes and unicellular


eukaryotes raises the possibility that nickel, not required by the human and
animal body itself, is rather needed for the normal development of the gut’s
microflora, which impacts host metabolism in a variety of ways and is respon-
sible for several metabolic and cardiovascular diseases [174]. The gut microbi-
ota comprises the largest microbial community in the human body and represents
one of the highest cellular densities in natural ecosystems, reaching 1011 to 1012
cells/mL of luminal content, and includes species of all kingdoms of life,
Eukaryotes, Bacteria, and Archaea [175]. The bacterial composition is made
mostly by the Firmicutes and by the Bacteroidetes phyla and includes
Proteobacteria and Actinobacteria [176]. This population comes in contact
with all the nutrients that are ingested through diet, creating a symbiotic equi-
librium with the host for the utilization of micronutrients, including metal ion
cofactors. In particular, in the human intestinal tract, there are large populations
of bacterial cells with the potential to bind and sequester metals that enter the
body, such as the ones from the Lactobacillus group [177]. Therefore, it has
been postulated that gut microorganisms may play a role in protecting the host
from the toxic potential of metal ions ingested with diet, preventing their entry
to the body from the intestine, and this could also be the case with Ni2+ ions
[177]. Accordingly, while Ni2+ ions administered by other routes, such as inha-
lation or dermal exposure, show a carcinogenic potential, there is no epidemio-
logical evidence on possible cancer risk from dietary nickel exposures, and this
may be due to the protective effect of the gut microflora.
Besides this function, it is plausible that Ni2+-dependent enzymes play impor-
tant roles also for the intestine community, and that Ni2+ homeostasis in some of
these microorganisms is important for developing a health-promoting gut micro-
flora. Indeed, early work reported that several bacterial species found in the
human and animal intestine show consistent urease activity [178]. Among them,
some species of the genus Bifidobacterium, such as B. bifidum and B. longum, are
noteworthy [179], because of the known probiotic effects of some types of
Bifidobacteria, that for decades have been added to food to promote healthy effects
[180]. Other urease-producing species, coming from the genus Lactobacillus, such
as L. fermentum, are interesting as they codify an acid urease which possesses an
optimum of pH at 3–4 [178].
350 Zambelli and Ciurli

Figure 7 Reaction catalysed by methyl coenzyme-M reductase, and schematic structure of the
nickel-containing active site.

Methanogenic archaea compose an important component of the microorganisms


living in human and animal guts, where they use dihydrogen as reductant to produce
CH4 [181]. In humans, Methanobrevibacter smithii is the main actor of the metha-
nogen population. Methanogens have been proposed to play a key role in the patho-
genesis of irritable bowel syndrome and chronic constipation [181]. The last step of
methane formation is catalyzed by methyl coenzyme-M reductase [182]. The reac-
tion involves methane formation concomitantly with the formation of a disulfide
bond between coenzyme M and coenzyme B (Figure 7). The active site of this
enzyme contains coenzyme F430, a Ni tetrapyrrole hydrocorphin with the metal in
the Ni(I) oxidation state in the active form. Structural data on the active site has been
obtained for the inactive oxidized Ni(II) state (Figure 7). This correlates the presence
of nickel in the diet with the healthy methanogenic activity in the guts.

5 Conclusions and Outlook

The wide use of nickel in many modern technologies and in objects of common use
increases the amount of nickel release and accumulation into the environment and the
possibility for humans to come in contact with this metal. Indeed, nickel-containing
objects, such as coins, are the cause of severe occupational health problems, whose
social costs have to be considered worldwide.
The double nature of nickel, that is, acting both as a toxic and a beneficial element
for human health, was proposed already in the early 1900s. The requirement of
nickel as a cofactor in the active sites of enzymes has been recognized in 1975 and
since then several studies have been conducted, which were aimed to describe
10 Nickel and Human Health 351

the molecular mechanisms of the effects of nickel on all forms of life, including
higher organisms.
However, the rationale of nickel carcinogenesis and allergy in humans, as well as
the cascade of events involving metal ion-dependent gene regulation and protein-
protein interactions leading to nickel homeostasis in eukaryotes, is yet largely
unknown. A full understanding of the molecular aspects of the effects of nickel
on human health represents therefore an important challenge and a future task for
bioinorganic chemistry, with the potential to have a significant impact for the human
population.

Abbreviations

ABC ATP-binding cassette


ACD allergic contact dermatitis
acetyl-CoA acetyl-coenzyme A
APC antigen-presenting cell
Ard acireductone dioxygenase
ARNT aryl hydrocarbon receptor nuclear translocator
BPU Bacillus pasteurii urease
CDF cation diffusion facilitator
CNG cyclic nucleotide-gated
DMT-1 divalent cation transporter
EDTA ethylenediamine-N,N,N′,N′-tetraacetate
Glx glyoxalase
GSH glutathione (reduced)
GTP guanosine 5′-triphosphate
HIF hypoxia-inducible factor
HRE hypoxia-responsive enhancer
IKK Iκ-B kinase
IRP iron-regulatory protein
LPS lipopolysaccharide
MAPK mitogen-activated protein kinase
MHC major histocompatibility complex
NDRG N-myc downstream regulated gene 1
NF-κB nuclear factor κ-B
NiCoT nickel/cobalt permease
ROS reactive oxygen species
SCD systemic contact dermatitis
TCR T-cell receptor
TLR toll-like receptor
VEGF vascular endothelial growth factor
VHL von Hippel-Lindau tumor suppressor
352 Zambelli and Ciurli

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Chapter 11
Copper: Effects of Deficiency and Overload

Ivo Scheiber, Ralf Dringen, and Julian F.B. Mercer

Contents
ABSTRACT ............................................................................................................................. 360
1 INTRODUCTION ............................................................................................................. 360
2 COPPER BIOCHEMISTRY AND HOMEOSTASIS ....................................................... 361
2.1 Copper-Dependent Enzymes ..................................................................................... 361
2.1.1 Cytochrome c Oxidase .................................................................................. 362
2.1.2 Copper/Zinc Superoxide Dismutase ............................................................. 362
2.1.3 Ceruloplasmin ............................................................................................... 362
2.1.4 Lysyl Oxidase ................................................................................................ 363
2.1.5 Tyrosinase...................................................................................................... 363
2.1.6 Dopamine-β-Monoxygenase and Peptidylglycine α-Amidating
Monoxygenase .............................................................................................. 364
2.2 Cellular Copper Homeostasis.................................................................................... 364
2.2.1 Copper Uptake............................................................................................... 364
2.2.2 Copper Sequestration and Storage ................................................................ 366
2.2.3 Intracellular Copper Trafficking.................................................................... 367
2.2.4 Copper Efflux ................................................................................................ 368
2.3 Systemic Copper Homeostasis .................................................................................. 369
3 COPPER DEFICIENCY DISORDERS ............................................................................ 371
3.1 Nutritional Copper Deficiency .................................................................................. 371
3.1.1 Anemia .......................................................................................................... 371
3.1.2 Neuropathies ................................................................................................. 372
3.1.3 Connective Tissues and Vascular System...................................................... 372
3.1.4 Immune System ............................................................................................ 373
3.1.5 Copper and Development .............................................................................. 374

I. Scheiber
Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic
R. Dringen
Centre for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany
J.F.B. Mercer (*)
Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences,
Deakin University, Burwood, Victoria 3125, Australia
e-mail: jmercer@deakin.edu.au

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 359
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_11, © Springer Science+Business Media Dordrecht 2013
360 Scheiber, Dringen, and Mercer

3.2 Genetic Copper Deficiencies .................................................................................... 374


3.2.1 Menkes Disease and Occipital Horn Syndrome............................................ 374
3.2.2 Distal Hereditary Peripheral Neuropathy ...................................................... 375
4 COPPER OVERLOAD DISORDERS............................................................................... 375
4.1 Excessive Copper Intake ........................................................................................... 375
4.2 Genetic Copper Overload .......................................................................................... 375
4.2.1 Wilson Disease .............................................................................................. 375
4.2.2 Copper-Associated Infantile Cirrhosis .......................................................... 376
5 NEUROPATHOLOGY AND COPPER............................................................................. 376
5.1 Alzheimer Disease .................................................................................................... 376
5.2 Parkinson Disease ..................................................................................................... 377
5.3 Huntington Disease ................................................................................................... 378
5.4 Motor Neuron Disorders ........................................................................................... 378
5.5 Prion Diseases ........................................................................................................... 379
6 OVERVIEW AND FUTURE DEVELOPMENTS ............................................................ 380
ABBREVIATIONS .................................................................................................................. 380
ACKNOWLEDGMENT .......................................................................................................... 381
REFERENCES ........................................................................................................................ 381

Abstract Copper is an essential trace metal that is required for the catalysis of
several important cellular enzymes. However, since an excess of copper can also
harm cells due to its potential to catalyze the generation of toxic reactive oxygen
species, transport of copper and the cellular copper content are tightly regulated.
This chapter summarizes the current knowledge on the importance of copper for
cellular processes and on the mechanisms involved in cellular copper uptake,
storage and export. In addition, we will give an overview on disturbances of copper
homeostasis that are characterized by copper overload or copper deficiency or
have been connected with neurodegenerative disorders.

Keywords ATP7A • ATP7B • copper chaperones • copper homeostasis • Ctr1


• glutathione • Menkes disease • metallothioneins • Wilson disease

Please cite as: Met. Ions Life Sci. 13 (2013) 359–387

1 Introduction

Copper became widely bioavailable about 2–3 billion years ago with the advent of
an oxygen atmosphere that allowed for the conversion of Cu+ to the more soluble
Cu2+ ion [1]. Because of the ready interconversion between these two oxidation
states, copper has become an essential element for all organisms that have an oxida-
tive metabolism. In humans, it represents the third most abundant essential transi-
tion metal [2]. As a cofactor of several enzymes and/or as structural component,
copper is involved in many physiological pathways. Furthermore, copper is associated
with important biological processes including angiogenesis, response to hypoxia
and neuromodulation.
11 Copper: Effects of Deficiency and Overload 361

In recent years the importance of copper in human health has become increasingly
recognized, and it has moved from a minor element, affected in a few rare conditions,
to one that may be of vital importance in the pathology of several important neuro-
logical diseases. This recognition has largely come about as a consequence of the
rapid advances in understanding of the molecular basis of copper homeostasis.
This chapter will review some of the biological roles of copper and the diseases
that are known, or plausibly proposed to involve defects in copper homeostatic
mechanisms. Particular emphasis will be placed on the neurological disorders.

2 Copper Biochemistry and Homeostasis

2.1 Copper-Dependent Enzymes

Copper is an essential cofactor and/or a structural component in a number of impor-


tant enzymes of plants and animals (Table 1). In general, these enzymes are involved
in redox reactions [3]. The relatively high redox potential for the Cu2+/Cu+ system is
utilized by many enzymes for oxidation reactions, for example superoxide by
superoxide dismutase and catechols by tyrosinase. Among others, copper-depen-
dent enzymes participate in biological processes such as energy metabolism (e.g.,
cytochrome c oxidase), antioxidative defence (e.g., Zn,Cu-superoxide dismutase)
and iron metabolism (e.g., ceruloplasmin) [2].
On the basis of their optical and electron paramagnetic resonance (EPR) fea-
tures, copper-dependent enzymes are classified as type 1 (blue copper site), 2 or 3
copper enzymes [3]. Most copper enzymes contain only one type of copper center,
but in some (e.g., ceruloplasmin, cytochrome c oxidase) more than one type can be found.
Type 1 copper sites, also known as blue copper sites, exclusively function in single
electron transfers [3,4]. Type 2 copper sites lack unique features in their UV/Vis and
EPR spectra and catalytically activate enzyme substrates by direct interaction rather

Table 1 Mammalian copper-dependent enzymes.


Enzyme Function
Cytochrome c oxidase Oxidative phosphorylation
Cu,Zn superoxide dismutase (SOD1) Superoxide detoxification, signaling
Ceruloplasmin (Cp) Ferroxidase
Lysyl oxidase (LOX) Crosslinking of collagen and elastin
Tyrosinase Melanin synthesis
Dopamine-β-monoxygenase (DβM) Norepinephrine synthesis
Peptidylglycine α-amidating enzyme (PAM) Activation of peptide hormones
Copper amine oxidase Deamination of amines
Hephaestin Ferroxidase
Coagulation factors V and VIII Blood clotting
362 Scheiber, Dringen, and Mercer

than being involved in electron transfer [3]. In contrast to type 1 and 2 sites, type 3
copper sites are binuclear. These copper sites are constituted of two closely spaced
coupled copper ions, each of them coordinated by three histidines, which can be
reversibly bridged by dioxygen. The function of type 3 copper sites is the activation
and transport of oxygen [3].

2.1.1 Cytochrome c Oxidase

Cytochrome c oxidase is a member of the super family of heme-copper containing


oxidases. It is embedded in the mitochondrial inner membrane where it catalyzes
the electron transfer from reduced cytochrome c to dioxygen in the final step of
mitochondrial oxidative phosphorylation [5]. Mammalian cytochrome c oxidase
is a multimeric protein complex consisting of 13 subunits, encoded by both the
mitochondrial and nuclear genome. Biogenesis of the functional holoprotein is a
complicated process that requires several specific proteins, so-called assembly
factors, including Cox17, Sco1, and Sco2 [6,7]. Cytochrome c oxidase defi-
ciency is one of the most common causes of respiratory chain defects in humans.
Pathological features range from metabolic acidosis, weakness and cardiomyopathy
to neurodegeneration [6,7].

2.1.2 Copper/Zinc Superoxide Dismutase

The members of the ubiquitous family of superoxide dismutases (SODs) convert


superoxide to dioxygen and hydrogen peroxide for further disposal by catalase and
glutathione peroxidase. Superoxide is produced during the reduction of dioxygen
that occurs in respiration and during autoxidation of catecholamines as well as its
metabolites. Excess amounts of superoxide can lead to the formation of highly reac-
tive oxygen species (ROS) that would damage cellular constituents [8].
Three distinct types of SOD are found in mammals: copper/zinc superoxide dis-
mutase (Cu/Zn-SOD; SOD1), manganese superoxide dismutase (Mn-SOD, SOD2)
and extracellular superoxide dismutase (EC-SOD; SOD3) [9]. SOD1 is a homodi-
meric protein located largely in the cytosol. SOD2 contains manganese as metal
cofactor while both SOD1 and SOD3 contain catalytic copper and structural zinc
ions in their active sites [10]. Mutations in SOD1 have been linked to the motor
neuron disease, amyotrophic lateral sclerosis (ALS) [11].

2.1.3 Ceruloplasmin

Ceruloplasmin (Cp) belongs to the family of multicopper oxidases. Cp contains 6


copper atoms per molecule: three type 1 copper sites, a single type 2 copper ion and
a binuclear type 3 copper site. Cp has a ferroxidase activity and has a critical role in
iron homeostasis [12]. The majority of Cp is synthesized by hepatocytes and
11 Copper: Effects of Deficiency and Overload 363

secreted into circulation [12]. In the human central nervous system and testes a
glycosylphosphatidylinositol (GPI)-anchored form of Cp that is generated by alter-
native splicing has been identified in astrocytes and Sertoli cells, respectively [13].
During biosynthesis copper insertion into apo-Cp takes place late in the secretory
pathway [14]. In hepatocytes the copper transporting ATPase ATP7B and the
Niemann-Pieck C1 protein are required for proper metallation of Cp [15,16].
Cp is an acute phase response protein whose synthesis and secretion can be
strongly increased during pregnancy, inflammation, infection, and in diseases such
as diabetes, cancer as well as cardiovascular diseases [12]. Copper deficiency does
not affect the rates of biosynthesis and release of Cp by hepatocytes but results in an
increase of unstable apo-Cp in the plasma leading to lowering of Cp protein and
oxidase activity [17].
Aceruloplasminemia is an autosomal recessive disorder resulting from a loss
of function mutation in the Cp gene [18]. Due to the importance of Cp in iron
homeostasis, the lack of functional Cp in affected individuals is accompanied by
excessive iron accumulation in most tissues leading to neurological symptoms
such as retinal degeneration, mild dementia, dysarthria, dystonia as well as dia-
betes mellitus [18].

2.1.4 Lysyl Oxidase

Lysyl oxidase (LOX) has a crucial role in the formation, maturation, and stabiliza-
tion of connective tissue by catalyzing the cross-linking of elastin and collagen [19].
Copper incorporation into LOX propeptide takes place in the trans-Golgi network
(TGN) where it is delivered by the copper transporting ATPase, ATP7A [20].
Accordingly, LOX activity is low in patients suffering from Menkes disease (MD)
(Section 3.2.1), which is caused by mutations in the ATP7A gene and patients have
marked connective tissue dysfunctions [21,22]. Dietary copper status also affects
LOX activity, but does not alter tissue levels of the LOX protein [19].

2.1.5 Tyrosinase

Tyrosinase is the key enzyme in the biogenesis of melanin pigments. In mammals,


tyrosinase is mainly expressed in melanocytes and retinal pigment epithelium cells
where it is localized to specialized organelles known as melanosomes [23]. One of
the important functions of tyrosinase is the catalysis of the hydroxylation of
L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), the rate-limiting step in the
biosynthesis of melanins and dopamine, and its subsequent oxidation to DOPA
quinone [24]. During trafficking from TGN to melanosomes, tyrosinase loses its
copper and must be reloaded within melanosomes to sustain its activity.
For both the TGN and melanosome compartments, copper loading into tyrosinase
depends on the copper transporting ATPase ATP7A [25]. Consequently, mutations
364 Scheiber, Dringen, and Mercer

in ATP7A such as found in patients with MD and in the mottled mouse mutants
cause hypopigmentation, a feature found also in nutritionally copper deficient
animals [21].

2.1.6 Dopamine-β-Monoxygenase and Peptidylglycine α-Amidating


Monoxygenase

Dopamine-β-monoxygenase (DβM) and peptidylglycine α-amidating monoxygen-


ase (PAM) belong to a small class of copper proteins found exclusively in animals
[26]. DβM catalyzes the oxidative hydroxylation of dopamine to norepinephrine
and thus plays an important role in the metabolism of these catecholamines [26].
PAM exclusively catalyzes the C-terminal α-amidation of various glycine-extended
propeptides, a post-translational modification essential for the bioactivity of diverse
physiological regulators including peptide hormones, neurotransmitters, and growth
factors [27]. Due to the physiological importance of PAM, lack of functional PAM
in mice is embryonic lethal [28]. Proper metallation of DβM and PAM is essential
for their activity. In MD patients plasma catechol levels are altered and levels of
amidated peptides are low, reflecting DβM and PAM deficiency, respectively
[29,30]. Thus, copper loading of both, DβM and PAM, is likely to depend on
ATP7A. In support of this view, PAM activity is compromised in cells lacking func-
tional ATP7A, although expression levels of PAM are normal [30,31].

2.2 Cellular Copper Homeostasis

Given the requirement for copper on one hand and the potential toxicity of copper
on the other, cells have evolved mechanisms to regulate cellular copper concentra-
tions. Many of the components involved in cellular copper homeostasis are well
known at the molecular level. These include transporters that mediate the uptake
and efflux of copper, biomolecules that sequester and store copper and specialized
proteins called copper chaperones that guide copper to copper-dependent enzymes
and to organelles (Figure 1, Table 2).

2.2.1 Copper Uptake

Members of the copper transporter receptor (Ctr) family that were first described for
Saccharomyces cerevisiae [32], play a key role in the uptake of copper in eukaryotic
cells. In humans, the two Ctr-members hCTR1 and hCTR2 have been identified
[33]. CTR1 is a 190 amino acid transmembrane protein and is considered as the
major contributor to high-affinity copper uptake in mammalian cells [34].
Monovalent copper is thought to be the copper species transported by CTR1 [35].
CTR2 has been localized to late endosomes and lysosomes and may play a role in
copper recycling after degradation of copper enzymes [36].
11 Copper: Effects of Deficiency and Overload 365

Figure 1 Cellular copper homeostasis. Copper uptake is predominately mediated by the copper
transporter receptor 1 (Ctr1). Since Ctr1 has been reported to transport Cu+, this substrate is pro-
vided by a cuprireductase and/or by chemical reduction with reducing agents such as ascorbate. In
cells, copper is sequestered as GSH complex or stored in metallothioneins. The delivery of essen-
tial copper to copper-containing enzymes is mediated by specific copper chaperones, such as by
CCS to SOD1, by Cox17 to cytochrome c oxidase or by Atox1 to ATP7A. In many cell types
ATP7A transports copper into the TGN for incorporation into LOX and other secreted copper-
dependent enzymes. In hepatocytes, ATP7B supplies copper to ceruloplasmin. Elevated cellular
copper concentrations induce a reversible translocation of ATP7A to the plasma membrane and
ATP7B to subapical vesicles that allows direct export of copper.

Table 2 Proteins involved in mammalian copper homeostasis.


Protein Function
Copper transporter receptor 1 (Ctr1) Copper uptake
Copper transporter receptor 2 (Ctr2) Copper uptake
Divalent metal transporter 1 (DMT1) Copper uptake
Copper chaperone for superoxide dismutase (CCS) Intracellular copper trafficking
ATOX1 Intracellular copper trafficking
Cox17 Intracellular copper trafficking
Glutathione (GSH) Intracellular copper trafficking, storage,
and detoxification
Metallothionein (MT) Storage and detoxification
ATP7A Copper export
ATP7B Copper export

The extracellular N-terminus of hCTR1 contains two histidine-rich regions and two
methionine motifs [33]. Mutation analysis revealed that deletion of the first methio-
nine motif and/or of the His-rich regions has almost no effect on copper transport
activity of hCTR1 [37,38]. In contrast, mutation or deletion of methionine residues
366 Scheiber, Dringen, and Mercer

in the second methionine motif had a strong inhibitory effect on hCTR1-mediated


copper uptake [37]. Structural studies suggest that CTR1 is active as a trimer, forming
a pore for the passage of copper across the lipid bilayer [39].
Mammalian Ctr1 is ubiquitously expressed; however, expression levels are
tissue-specific, being highest in the liver, kidney, and intestine. In some tissues the
expression level of Ctr1 depends on the copper status and is influenced by the physi-
ological state, such as pregnancy and lactation [40]. Ctr1 plays an essential role in
embryonic development as deletion of Ctr1 in mice is embryonic lethal, most likely
due to an insufficient supply of the developing embryo with copper [41].
In cultured cells, Ctr1 is typically observed at the plasma membrane and in cyto-
plasmic vesicles and specific localization depends on the cell type [42]. In some
cells, addition of copper stimulates the endocytosis of the protein which is likely to
be a homeostatic control mechanism to prevent excessive copper uptake and poten-
tial copper toxicity [36]. There has been some controversy about Ctr1 localization
in polarized cells such as the intestinal enterocytes, but in this cell type, the current
evidence supports an apical location which is expected if the protein plays a role in
copper uptake from the diet [43].

2.2.2 Copper Sequestration and Storage

The accumulation of copper in the cytosol induces a risk for copper-mediated oxi-
dative damage and binding of copper to essential biomolecules. However, under
physiological conditions the concentration of free copper within the cell has been
calculated to be around 10–18 M which amounts to less than one free copper ion per
cell [44]. Such low concentrations of free copper are maintained by binding of cop-
per to metallothioneins (MTs) and ligands of low molecular mass such as glutathi-
one (GSH). MTs and GSH also represent the major molecules involved in the
intracellular sequestering and storing of excess copper. In addition, mitochondria
have been suggested to contribute to the cellular copper buffering capacity [45].
The tripeptide GSH is the most abundant low-molecular-weight thiol in cells,
being present in millimolar concentrations [46]. GSH is essential for the detoxifica-
tion of reactive oxygen species, maintains the cellular thiol reduction potential in a
strongly reduced state and is involved in redox regulation and signalling [46]. In addi-
tion, GSH has been linked to the transport and the detoxification of metal ions includ-
ing copper [47]. Indeed, the majority of cytosolic copper is bound to GSH [48] and
copper in the form of a Cu(I)-GSH complex is believed to be a major contributor to
the copper exchangeable pool in the cytosol [49]. In addition, GSH can participate in
cellular copper homeostasis by regulating the activities of copper transport proteins
such as ATP7A and ATP7B via glutathionylation/deglutathionylation [50].
Metallothioneins constitute a heterogeneous family of low-molecular-weight,
cysteine-rich proteins found in all eukaryotes [51]. In addition to a presumed role in
zinc and copper homeostasis, MTs have been implicated in the detoxification of
non-essential metals such as cadmium, protection against ROS, maintenance of the
intracellular redox balance, regulation of cell proliferation and apoptosis, as well as
11 Copper: Effects of Deficiency and Overload 367

in neuroprotection [51–56]. Four mammalian MT isoforms exist that are denoted


MT-1 to MT-4. The predominant isoforms MT-1 and MT-2 are ubiquitously
expressed in almost all organs and tissues, being most abundant in liver and kidney
[51]. The expression of MT-3 and MT-4 is confined mainly to the brain and stratified
epithelium, respectively [57,58]. MT-3 and MT-4 are constitutively expressed,
while MT-1 and MT-2 are both basally expressed and inducible by various stressors,
including heavy metals, oxidative stress and pro-inflammatory cytokines [51]. The
expression of MTs is induced by an excess of copper [59]. The excess hepatic cop-
per found in patients with Wilson disease (WD), and in the toxic milk mouse model
of this disease, is bound to MTs [60,61]. Since MTs are capable of binding excess
cellular copper, an increase in the cellular MT content confers resistance against
copper-induced toxicity [62]. Hence, the rise in MT levels reflects an adaptation of
cells to copper overload conditions.

2.2.3 Intracellular Copper Trafficking

The intracellular trafficking of copper is mediated by a group of proteins termed


copper chaperones. These specialized proteins shuttle copper to specific cellular
targets thus avoiding toxicity to other cellular components [63].
Antioxidant protein 1 (Atx1) is a small cytoplasmic copper-binding protein orig-
inally identified as a copper-dependent suppressor of oxygen toxicity in yeast strains
lacking both SOD1 and SOD2 [64]. In yeast, Atx1 shuttles copper to the copper
transporting P-type ATPase Ccc2 for subsequent transport into the secretory path-
way and delivery to the ferroxidase Fet3p needed for high affinity iron uptake [65].
The human homologue of Atx1, termed HAH1 or ATOX1, is a 68 amino acid pro-
tein that shares 47% amino acid identity Atx1 [64]. Atox1 has been demonstrated to
bind and transfer Cu(I) to the N-terminal metal-binding domains (MBDs) of the
copper transporting P-type ATPases, ATP7A and ATP7B [66–69]. Mutations in
ATP7B that lead to an impairment of protein-protein interactions with ATOX1 have
been identified in patients suffering from WD [67]. This finding highlights the
importance of specific protein-protein interactions in the transfer of copper from
ATOX1 to the copper transporting P-type ATPases as well as the significance of
ATOX1 in cellular copper homeostasis.
The copper chaperone for superoxide dismutase (CCS) is involved in the matura-
tion of SOD1 by inserting copper into this enzyme [70]. Although SOD1 from most
species can be activated independently of CCS, maximal SOD1 activity in the
majority of organisms relies on the presence of CCS [71]. CCS is primarily local-
ized to the cytosol and has a similar cellular distribution as its target protein [70].
The expression level of CCS depends on the cellular copper content [72]. Copper
deficiency has been demonstrated to result in an increase in CCS protein abundance
due to a lowered rate of proteosomal degradation of CCS [73].
Biogenesis of cytochrome c oxidase requires the assembly of 13 subunits into a
multimeric protein complex and the concomitant insertion of cofactors, including
three copper ions, two heme a groups, one zinc ion and a magnesium ion [6].
368 Scheiber, Dringen, and Mercer

Formation of the CuB and CuA site in the mitochondrial encoded subunits Cox1 and
Cox2 takes place within the mitochondrial intermembrane space (IMS), and thus
requires both the delivery of copper into this mitochondrial compartment as well as
the insertion of copper into the two copper centers. A number of proteins have been
identified so far to be involved in the insertion of copper ions into mammalian cyto-
chrome c oxidase [63]. The initial event in the transfer of copper to Cox1 and Cox2
is the Cox17-mediated transfer of copper to the Sco proteins Sco1 and Sco2 and
Cox11. This is followed by the subsequent insertion of copper into the nascent CuA
and CuB sites [63]. Cox17, initially identified in a yeast mutant displaying a respira-
tory defect, is essential for the metallation of eukaryotic cytochrome c oxidases
[74]. Cox17 is a small cysteine-rich, hydrophilic protein localized in the IMS and in
the cytosol of cells [74,75].
Although this dual localization suggests a role of Cox17 in the shuttling of cop-
per from the cytosol into the IMS, the primary function of Cox17 is the transfer of
copper to Sco1, Sco2, and Cox11 within the IMS [75]. Sco proteins are required for
the formation of the CuA site of cytochrome c oxidase [76]. In humans, the two
homologs hSco1 and hSco2 contribute to this process by transferring copper to the
binuclear copper center and by acting as thiol-disulfide oxidoreductases [77].
Consistent with their critical role in the formation of the binuclear CuA center, muta-
tions in Sco1 and Sco2 cause severe cytochrome c oxidase deficiencies [6].

2.2.4 Copper Efflux

Cellular copper efflux in mammals relies on the function of two proteins, ATP7A
and ATP7B. Mutations in ATP7A cause the human copper deficiency disorder MD
(Section 3.2.1) [78–80] and mutations in ATP7B cause the human toxicosis disorder
WD [81,82]. These proteins belong to the protein family of P1B-type ATPases, which
have key functions in metal homeostasis in most organisms [83]. P1B-type ATPases
are a subgroup of P-type ATPases that use the energy of ATP hydrolysis to transport
heavy metals across cellular membranes [83]. In addition to their critical function in
the efflux of excess cellular copper, ATP7A and ATP7B shuttle copper to the secre-
tory pathway for incorporation into copper-dependent enzymes such as tyrosinase,
PAM, DβM, LOX, and Cp (reviewed in [84,85]). The importance of these proteins
in the maintenance of copper homeostasis is illustrated by the severe clinical pheno-
types manifest by MD and WD (Sections 3.2.1 and 4.2.1) [86,87].
Human ATP7A and ATP7B are large multispanning membrane proteins that
share 50–60% amino acid sequence homology [85]. Their overall structure consists
of a cytosolic amino-terminus, eight transmembrane helices, an ATP-binding
domain an actuator domain, and a cytosolic carboxyl-terminus [88]. The N-terminal
tail of human ATP7A and ATP7B harbors six metal binding domains (MBDs), each
capable of binding one Cu+ ion [89]. Only the MBDs closest to the membrane,
MBD5 and MBD6, are important for efficient copper transport [90–92] while
MBD1-4 primarily function in the regulation of the catalytic activity in response
to copper [88]. The eight transmembrane helices (TMs) of ATP7A and ATP7B
are involved in the formation of the copper translocation channel [88]. Specific
residues within TM6-TM8 contribute to the intramembrane copper coordination
11 Copper: Effects of Deficiency and Overload 369

sites required for copper transmembrane transport. Mutation of the cysteines in a


conserved CPC motif in TM6 and mutation of Met1393 in TM8 have been shown to
result in an impaired catalytic activity of human ATP7B and murine ATP7A [93].
The ATP-binding domain of both ATP7A and ATP7B, located between TM6 and
TM7, comprises a nucleotide-binding domain (N-domain) and a phosphorylation
domain (P-domain) containing the DKTG, TGDN, and GDGxND signature motifs
of P-type ATPases [88]. The invariant Asp residue in the DKTG sequence motif of
the P-domain is crucial for the catalytic cycle of P-type ATPases [94]. In the case of
ATP7A and ATP7B, it accepts the γ-phosphate from ATP upon binding of ATP to
the N-domain and copper to the intramembrane copper sites [89]. The formation of
this phosphorylated intermediate induces conformational changes that allow the
copper ion to be released on the other side of the membrane. The catalytic cycle is
closed by the hydrolysis of the aspartyl phosphate bond and the return of the enzyme
to its initial state. The dephosphorylation step is facilitated by the actuator domain
(A-domain) linked to TM4 and TM5. This domain harbors the TGE signature motif
of the P-type ATPases that is strictly required for their phosphatase activity [89].
Consequently, mutations of the TGE motif in ATP7A and ATP7B result in hyper-
phosphorylated and catalytic inactive proteins [92,94].
ATP7A continuously recycles between the TGN and the plasma membrane,
whereas ATP7B traffics between the TGN and a cytosolic vesicular compartment
[95,96]. When copper levels are normal, both ATP7A and ATP7B have steady state
localization at the TGN, where they transport copper from the cytosol to the TGN
lumen for incorporation into copper-dependent enzymes. A rise in cytosolic copper
levels induces a shift in the steady state distribution from the TGN to the plasma
membrane and/or to a distinct cytosolic vesicular compartment in close proximity
to the plasma membrane (reviewed in [84,85]). Redistribution of ATP7A and ATP7B
back to the TGN occurs when cellular copper levels return to normal [95,96]. The
ability of ATP7A and ATP7B to efflux copper is linked to their ability to undergo
copper-induced redistribution [84]. Mutations that impair the copper-dependent
trafficking of these proteins have been associated with MD and WD [91,97,98].

2.3 Systemic Copper Homeostasis

The key features of systemic copper homeostasis are shown in Figure 2. Overall
balance of copper in the body is achieved by regulation of the rate of uptake of cop-
per in the small intestine and efflux of copper from the liver in the bile. Most dietary
copper is absorbed in the small intestine [2] and current evidence suggests that the
copper transporter Ctr1 is responsible for the apical uptake of copper. However, the
role and cellular localization of Ctr1 in this process has been somewhat controversial.
Some studies have reported that Ctr1 is basolateral in the enterocytes and the apical
copper entry into intestinal cells was thought to be mediated by a copper transport
system other than Ctr1 [99]. Nose et al. have shown that intestinal-specific knock
out of Ctr1 in mice produces a severe copper deficiency and death at about 3 weeks of
age showing that Ctr1 is a major component of the dietary uptake of copper [100].
Intestinal epithelial cells generated from these mice hyperaccumulated copper,
370 Scheiber, Dringen, and Mercer

Figure 2 Systemic copper homeostasis. The overall status and distribution of copper is regulated
primarily by the concerted action of CTR1 and ATP7A/B. Uptake of dietary copper occurs in the
small intestine where copper is taken into the intestinal enterocytes by CTR1 and is effluxed into
the circulation by ATP7A. Most of the newly absorbed copper is taken up by the liver, which
excretes excess copper in the bile, mediated by ATP7B. Copper crosses the blood brain barrier via
ATP7A, and this protein also plays a key part in supply of copper to fetus. ATP7B is responsible
for supplying copper to the milk.

whereas all organs tested suffered from a severe copper deficit [100]. Despite the up
to 10 times higher copper levels in these cells compared to that of intestinal epithe-
lial cells from control animals, activities of copper-dependent enzymes were
strongly reduced and levels of the copper chaperone for superoxide dismutase dra-
matically increased. Thus, it appears that Ctr1 in the enterocytes is required for
copper to be bioavailable.
In a subsequent paper Nose et al. demonstrated that Ctr1 is located on the apical
surface of enterocytes consistent with its role in uptake of dietary copper [43]. The
amount of Ctr1 on the apical surface of the enterocytes was increased when a low
copper diet was supplied to the animals, consistent with this being part of the
systemic homeostatic regulation of copper uptake which has been demonstrated in
humans by Turnlund et al. [101]. It is unclear why reports of the cellular localization
of Ctr1 have been so contradictory, however, it seems probable that problems with
the antibodies in use could be responsible [43].
The copper efflux protein ATP7A is responsible for the transport of copper across
the basolateral surface of the enterocyte, and its intracellular location is altered by
changes in dietary copper. In mice, it has been shown that increasing dietary copper
causes ATP7A to traffic from the TGN to vesicles close to the basolateral surface of
the enterocyte [102]. Presumably this process increases the copper delivery to the
portal circulation, allowing the excess copper to be eliminated by biliary excretion,
which is the major path of copper elimination from the body [2]. ATP7A has a role
in the distribution of copper around the body and in the maintenance of safe copper
levels in many cell types. It is expressed in most tissues, including intestine, skeletal
muscle, placenta, brain, heart, and kidney, but its expression in liver is very low
[78,80,103]. In contrast, ATP7B is abundantly expressed in the liver and at lower
11 Copper: Effects of Deficiency and Overload 371

levels in kidney, placenta, brain, lung, and heart [81,82]. ATP7B is the transporter
responsible for the regulation of biliary excretion of copper, and excess copper in
the hepatocyte stimulates trafficking of this protein from the TGN, where it supplies
copper to ceruloplasmin, to vesicles close to the apical membrane of the hepatocyte
that abuts the biliary caniliculus [95]. This copper-induced trafficking of ATP7B is
the principle homeostatic mechanism for removing excess copper from the body.
The difference in expression patterns between ATP7A and ATP7B correlates
well with the observed alterations in body copper homeostasis seen in MD and WD.
Inactivation of ATP7A in MD results in systemic copper deficiency due to dimin-
ished copper export from the intestine into the portal blood and defects in copper
efflux from other tissues such as the blood brain barrier and the kidney [104,105],
while failure of biliary copper excretion by ATP7B in WD leads to copper overload
in liver and other tissues [106].

3 Copper Deficiency Disorders

3.1 Nutritional Copper Deficiency

Copper deficiency in humans can occur through multiple mechanisms [107,108]. It


has been observed in premature and low-birth-weight infants who are born with low
hepatic copper stores [109], in individuals receiving total parenteral nutrition with-
out adequate copper supplementation [110], in malnourished infants [111], and in
persons with malabsorption syndromes [112]. Low copper intakes in the diet can
result in marginal copper deficiency and a significant proportion of the population
consuming a typical Western diet is estimated to have inadequate copper intake
[113]. Secondary copper deficiency can occur as consequence of high dietary intake
of zinc and this has occurred even in patients with WD [114] after pharmacological
treatments with copper chelating agents such as D-penicillamine or tetrathiomolyb-
date and following gastrointestinal surgery [115].
The clinical symptoms of copper deficiency in humans are numerous [107,116].
Early and common signs of acquired copper deficiency are hematological manifes-
tations such as anemia, leukopenia, neutropenia, and pancytopenia [117]. Bone
abnormalities including osteoporosis, bone fractures, and bone malformation have
often been observed in copper-deficient low-birth-weight infants and young chil-
dren [118]. Acquired copper deficiency may manifest with neurological symptoms,
the clinical presentation resembling that of myeloneuropathy observed in vitamin
B12 deficiency [119].

3.1.1 Anemia

Although the link between copper deficiency and anemia was first proposed in
the 19th century and followed up by a series of studies in the early 20th century,
copper deficiency remains an under-recognized cause of anemia in humans [120].
372 Scheiber, Dringen, and Mercer

Severe copper deficiency is relatively rare, but it is nevertheless a significant cause


of reversible refractory anemia and leukopenia, particularly neutropenia, and is
often misdiagnosed as myelodysplastic syndrome (MDS). The link between copper
and iron metabolism has been known for many years, but the detailed molecular
basis of this link is being clarified [121]. Nevertheless, the reason for anemia in
copper deficiency is complex and remains somewhat enigmatic [121,122]. The
common view is that copper deficiency leads to reduced activity of the multicopper
oxidases, ceruloplasmin and hephaestin, which are required to oxidize iron(II) to
iron(III) for binding to transferrin. But as Prohaska elegantly discusses, this common
hypothesis does not explain all the available evidence and other factors, perhaps
in the bone marrow, are probably responsible [122]. Similarly, the mechanism of
neutropenia remains unknown, however, it is possible that copper deficiency results
in the inhibition of differentiation and self-renewal of CD34(+) hematopoietic
progenitor cells [123].

3.1.2 Neuropathies

The first evidence of a link between copper and a neurological disease was provided
by the discovery that a demyelinating disease in sheep known as swayback was due
to copper deficiency [124]. More recently a similar condition which resembles
motor neuron disease, but resulting from chronic copper deficiency, has been
described in humans [119]. In a review of 55 patients, Jaiser and Winston identified
risk factors that include upper gastrointestinal surgery, zinc overload and malab-
sorption syndromes [119]. All of these factors result in reduced copper absorption.
There have been a number of case reports of neurological complications following
gastric bypass surgery for obesity [125,126]. The prevalence and incidence of
copper deficiency following gastric banding surgery for obesity was found to be
about 9.6% and many of these patients showed hematological changes. These
results suggest that frequent monitoring of the copper status of gastric banded
patients is warranted [119].
The mechanistic basis of demyelination due to copper deficiency is not under-
stood, however, reduced cytochrome oxidase activity is a possible cause. Interestingly,
older mice lacking axonal prion protein (PrPc, see Section 5.5) develop late-onset
peripheral nerve demyelination [127] and since PrPc binds copper, this mechanism
could be involved in the demyelination due to copper deficiency. Interestingly, the
neurologic deficits caused by copper deficiency are clinically indistinguishable from
the neuropathology in vitamin B12 deficiency [123].

3.1.3 Connective Tissues and Vascular System

Clear evidence for the importance of copper for correct formation and function of
connective tissues and the vascular system is seen in patients with MD and in a
marked form in the allelic variant, occipital horn syndrome (OHS) [21]. MD and
11 Copper: Effects of Deficiency and Overload 373

OHS patients have defective cross-linking of collagen and elastin due to low activity
of the copper-dependent lysyl oxidase [128]. OHS is a milder variant of MD, and
features predominately defects in connective tissue; patients have skin and joint
laxity and can die from aortic aneurysms. In addition, cardiovascular complications
appear to be connected to copper deficiency as MD patients are at higher risk of
congenital heart defects [129]. Low activity of lysyl oxidase has been proposed to
underlie the osteoporosis found in MD patients, as well as copper-deficient animals
[21]. However, a recent observation that SOD1-deficient mice developed osteoporo-
sis suggests another mechanism [130]. Nutritional copper deficiency has been found
to result in metabolic bone disease including osteoporosis in children of low birth
weight, which can be mistaken for child abuse [131].
Both osteoporosis and cardiovascular defects have long been known to occur in
copper-deficient domestic animals. In the 1930s there were reports of sudden death
in cattle (“falling disease”), attributed to cardiac failure [132]. Skeletal abnormali-
ties have been widely reported in animals (reviewed in [ 133]), but evidence
for copper deficiency in human cases of osteoporosis remains unresolved [134,135].
A relative new contributor to the copper effects on both the vascular and connective
tissue systems is extracellular SOD3. This enzyme receives its copper from ATP7A
which is found to be highly expressed in the vascular system [136]. Hypertension
induced by angiotensin II was more pronounced in a mouse mutant with a mutation
in ATP7A and SOD3 is an important modulator of oxidative stress-dependent
hypertension [137]. Another link of copper and cardiovascular disease is provided
by the finding of high levels of ATP7A expression in macrophages from murine
atherosclerotic lesions, and down-regulation of the copper transporter reduced the
amount of low density lipoprotein oxidation [138].

3.1.4 Immune System

In 1981 Prohaska and Lukasewycz reported that copper-deficient mice had an


impaired humoral-mediated immune response [139]. Earlier data had demonstrated
that copper-deficient rats were more sensitive to Salmonella infection [140]. A number
of studies in the 1980s showed that copper-deficient farm animals had impaired immune
responses. Boyne and Arthur demonstrated that neutrophils from copper-deficient
cattle had reduced ability to kill Candida albicans [141]. Babies with MD are susceptible
to infection [142]. However, the exact role of copper in immune function is still
unclear but more recent studies have begun to shed light on the mechanisms. Copper
deficiency was shown to reduce interleukin production in T-lymphocytes [143] and
secretion of interleukin-1α is dependent upon copper [144].
Copper has been used as an antimicrobial agent throughout recorded history and
its ability to kill microorganisms is presumably related to the generation of hydroxyl
radicals via Fenton chemistry [145]. This same mechanism is most likely responsible
for the killing of pathogens by macrophages. These cells were found to increase
their copper uptake in response to interferon-γ stimulation and most significantly
ATP7A was induced to traffic to the phagosomal compartment. It was proposed that
374 Scheiber, Dringen, and Mercer

ATP7A pumps copper into this compartment where it generates hydroxyl radicals,
thus, killing the ingested microorganism, but further studies are needed to establish
whether this mechanism applies to a wider range of microbial killing [146].

3.1.5 Copper and Development

Early evidence for the importance of copper in mammalian development was found
in sheep grazing on copper-deficient soils in Australia. Lambs from copper-deficient
mothers were born with a disease termed swayback, characterized by paralysis of
the hind limbs, convulsions, and blindness [124]. This disorder is a defect of myelin-
ation, which has been found in copper-deficient humans (see Section 3.1.2). Copper
supplementation of the mothers prevented this disorder [124]. Copper deficiency
during embryonic and fetal development can result in numerous gross structural and
biochemical abnormalities which can result from impaired free radical defense or
connective tissue abnormalities [147]. Mutations in ATP7A result in fetal copper
deficiency since this copper transporter is required for movement of copper across
the placenta [148]. Babies with MD have characteristic dysmorphic features and
other abnormalities at birth due to copper deficiency during gestation [21].
Mice with mutations in the Menkes gene orthologue display a range of develop-
mental effects ranging from fetal death, neonatal death or longer term survival with
connective tissue abnormalities depending on the degree of impairment of the activ-
ity of ATP7A [149]. The critical importance of copper in embryogenesis was dem-
onstrated by the death of mouse embryos at midgestation of development following
ablation of the gene for the copper transporter Ctr1 [41]. Copper is also critical
during postnatal development. Adequate copper supplies in milk are essential,
and mutations in the copper transporter ATP7B, which is involved in secretion of
copper into milk [150], result in death of suckling mice [151]. Recent studies on the
conditional knockout of ATP7A have further reaffirmed the vital importance of this
transporter and hence, copper for the developing mammal [152,153]. In humans, copper
deficiency due to inadequate nutrition is mostly manifest in babies, particularly
premature infants. The lack of copper can result in anemia, neutropenia, osteoporosis,
and neurological problems [107].

3.2 Genetic Copper Deficiencies

3.2.1 Menkes Disease and Occipital Horn Syndrome

As noted previously, severe copper deficiency is a hallmark of the X-linked reces-


sive diseases MD and occipital horn syndrome; both are caused by genetic defects
in the copper transporting ATPase, ATP7A [22]. The clinical features of MD include
severe progressive neurological degeneration, connective tissue abnormalities,
muscular hypotonia, and hypopigmentation of skin and hair [21]. Many of the clini-
cal symptoms of acquired copper deficiency and MD can be attributed to a decrease
in the activities of copper-dependent enzymes. Thus, hypopigmentation of skin and
11 Copper: Effects of Deficiency and Overload 375

hair results from reduced tyrosinase activity and abnormalities of bone and connective
tissue are principally due to lowered LOX activity [21,22,154,155]. While the exact
mechanism of copper deficiency myelopathy is not known [119], neurological
degeneration in MD is suggested to be primarily caused by a decrease in the activity
of neuronal cytochrome c oxidase [87]. Other pathogenic mechanisms probably
contribute to the extensive neurodegeneration in MD, for example, copper is known
to be neuroprotective against glutamate excitotoxicity [156].
OHS is primarily a connective disorder, due to specific effects of the specific
mutations of ATP7A on LOX activity. OHS is mostly caused by splice site muta-
tions that allow the production of a small amount of otherwise normal ATP7A.
Possibly, when ATP7A is present in only small amounts, it is primarily located on
the plasma membrane rather than the TGN, thus explaining the specific reduction in
LOX which receives copper in the TGN [157].

3.2.2 Distal Hereditary Peripheral Neuropathy

Two novel mutations in ATP7A were recently found to cause a form of distal heredi-
tary motor neuropathy [158]. Although affecting the same copper transporter that is
mutated in MD, the clinical phenotype is remarkably distinct. The disease primarily
causes gradual die-back of the long motor neurons in the legs. The reason for the
specific death of the axons of the motor neurons is unclear, but possibly involves
reduction in the protective effect of copper against glutamate excitotoxicity as noted
for MD. The long axons may be specifically sensitive to the subtle trafficking
defects found for the mutant ATP7As [158].

4 Copper Overload Disorders

4.1 Excessive Copper Intake

Acute copper toxicity has been described for individuals that accidentally or with
suicidal intention ingested high doses of copper [159]. For copper doses up to 1
gram, gastrointestinal symptoms predominate. With higher doses, nausea, vomiting,
headache, diarrhea, hemolytic anemia, gastrointestinal hemorrhage, liver and kid-
ney failure as well as death may occur [159].

4.2 Genetic Copper Overload

4.2.1 Wilson Disease

Chronic copper toxicity with predominant effects on the liver is a feature of the
autosomal recessive disorder WD, as well as the probable genetic overload disorders,
Indian childhood cirrhosis and idiopathic chronic toxicosis [86,160]. WD is caused
376 Scheiber, Dringen, and Mercer

by mutations in the copper transporting ATPase ATP7B that is responsible for


biliary excretion of copper. As a result copper accumulates in the liver to such high
levels that hepatocytes die and release copper that accumulates in the central
nervous system resulting in neurological abnormalities. Thus, WD can manifest as
a liver or neurological condition [21]. The disease is fatal unless measures are taken
to remove the excess copper from the body, either by copper chelation or blocking
intestinal uptake of copper with zinc [86,160].

4.2.2 Copper-Associated Infantile Cirrhosis

Indian childhood cirrhosis and idiopathic chronic toxicosis are severe chronic liver
diseases that are characterized by excessive hepatic copper accumulation in infancy
(as opposed to WD in which the copper accumulation occurs later in life) [160]. The
etiology of these rare and usually fatal diseases has been hypothesized to be a com-
bination of an unknown genetic defect affecting the copper metabolism and high
dietary copper intake [160–162].

5 Neuropathology and Copper

Impairment of copper homeostasis can lead to neurodegeneration, as exemplified by


both MD and WD [21] and as previously noted, severe nutritional copper deficiency
can lead to motor neuropathies (Section 3.1.2). Alterations of copper homeostasis
have also been associated with neurodegenerative diseases such as prion diseases,
Alzheimer disease (AD), Parkinson disease (PD) or Huntington disease (HD) but
the exact role of copper in these important neurological diseases remains unclear.

5.1 Alzheimer Disease

Alzheimer Disease is an irreversible and progressive disease that causes memory


loss and psychiatric disturbance. Aside from age, other risk factors include genetic
factors, gender and environmental factors [163]. The pathological hallmarks of AD
are the extracellular senile plaques and the intracellular neurofibrillary tangles in
brain [164]. The principal constituents of senile plaques are amyloid-β (Aβ) pep-
tides of 40 and 42 residues, which are generated form the integral membrane amy-
loid precursor protein by the consecutive action of β- and γ-secretase [163].
There is strong evidence that AD involves disturbances of copper homeostasis in
the brain. The senile plaques in AD brains are strongly enriched in copper and this
accumulation of copper could deplete adjacent neurons [165]. Results on copper
concentrations in the brain of AD patients have been somewhat contentious due to
11 Copper: Effects of Deficiency and Overload 377

technical issues, a recent study has shown a reduction of about 50% in copper in the
amygdala and hippocampus in AD brains [166]. Studies of compounds which affect
copper distribution in the brain as potential therapeutics for AD have shown promis-
ing results [167]. Although copper is depleted in some brain regions in AD patients,
the amyloid plaques have been shown to accumulate the metal [168], suggesting
that the binding of copper to the plaques is actually depleting copper from other
brain regions [169]. Copper has been shown to precipitate Aβ peptides in vitro and
it has been suggested that copper triggers the formation of senile plaques [169]. In
support of this view, Aβ deposition begins within the glutamatergic synapse [170],
where both copper [171,172] and Aβ [173] are released during synaptic transmis-
sion. However, although accumulation of Aβ peptides in the form of senile plaques
is the most prominent feature of AD, it is now widely accepted that soluble oligo-
meric Aβ species are the most toxic form of Aβ peptides [169]. Since Aβ can medi-
ate the reduction of Cu2+ to Cu+, copper may promote the toxicity of such Aβ
oligomers through the formation of ROS [174].
While the enhancement of Aβ toxicity by copper in vitro suggests a detrimental
role of copper in AD, the observed lower copper contents in the brain of AD patients
[166,175] and mouse models for AD [176,177] as compared to controls rather argue
for a copper deficit contributing to the neurodegeneration in AD. Copper supple-
mentation in a mouse AD model improved the survival of these animals [176].
Improved cognitive functions were also observed in another mouse model of AD
following administration of Cu(gtsm) as copper source [178]. However, intake of
copper had no effect on cognition in patients with mild AD in a phase 2 clinical trial
[179]. Mechanistically, copper deficiency may exacerbate disease progression by
influencing amyloid precursor protein processing and Aβ metabolism [170]. In
addition, copper deficiency may also influence the activity of copper-dependent
enzymes. In this regard low activities of cytochrome c oxidase [180] and SOD1
[181] have been reported for the AD brain. Therapeutic strategies aiming to restore
the normal copper distribution in AD brain are currently under investigation [174]
with promising results [182,183].

5.2 Parkinson Disease

Parkinson disease is characterized by a complex motor disorder known as


Parkinsonism that manifests with resting tremor, bradykinesia, rigidity, and postural
instability and is the second most common neurodegenerative disease in humans
[184]. The pathological hallmarks of PD are the loss of neuromelanin-containing
dopaminergic neurons in the substantia nigra pars compacta and the presence of
intracellular inclusions, called Lewy bodies consisting of the protein α-synuclein
[184]. The majority of cases are idiopathic with less than 10% of PD having a strict
familial etiology. The underlying mechanisms of idiopathic PD are not fully under-
stood. Among others mitochondrial dysfunction, oxidative stress, and inflammation
have been suggested in the pathogenesis of PD [184].
378 Scheiber, Dringen, and Mercer

There is accumulating evidence for dyshomeostasis of copper in PD. Parkinsonism


is frequently present in patients with neurological WD [185] and copper has been
demonstrated to accelerate aggregation of α-synuclein [186]. While the total copper
content in brains of PD patients does not differ significantly from healthy controls,
copper levels were found to be substantially lower (45%) in substantia nigra of PD
patients [187,188]. This reduction in the copper content of the substantia nigra in
PD has been discussed to result in an impairment of copper-dependent pathways,
thereby contributing to the pathogenesis of PD [189]. In support of this view, copper
supplementation has been shown to prevent the increase in lipid peroxidation, stria-
tal dopamine depletion, and the reduction in the activity of tyrosine hydroxylase in
an animal model for PD [190], while copper chelation was reported not to be protec-
tive in PD animal models [191]. Recently, the hypoxia imaging agent, Cu(II)(atsm)
has been found to be neuroprotective in several animal models of PD possibly by
inhibiting the nitration of α-synuclein [192].

5.3 Huntington Disease

Huntington disease is a rare autosomal-dominant, progressive neurodegenerative


disease that results in motor, cognitive, and psychiatric abnormalities [193]. The
genetic defect underlying HD is an expansion of a CAG repeat in exon 1 of
the huntingtin gene that is translated into an expanded polyglutamine domain at the
N-terminus of the huntingtin protein [194]. The exact pathogenic mechanism in HD
remains to be elucidated. Accumulation of copper in the HD brain has been hypoth-
esized to promote disease progression by promoting the aggregation of the hunting-
tin protein [195]. In addition treatment with the copper chelator tetrathiomolybdate
delayed the decline in motor function in mouse models for HD, further supporting
a potential role of copper in disease progression [196].

5.4 Motor Neuron Disorders

Motor neuron disorders (MNDs) involve both the central and peripheral nervous
systems and are relatively common diseases. The anterior horn of the spinal cord
contains the cell bodies of the motor neurons whose axons can extend for consider-
able distances (up to 1 m) to distal limb muscles. The degeneration of these neurons
leads to peripheral motor neuropathies. Inherited peripheral neuropathies can affect
sensory and autonomic nerve cells as well as motor neurons [197]. The best known
MND is amyotrophic lateral sclerosis (ALS).
ALS is a progressive neurodegenerative disease preferentially but not exclu-
sively affecting motor neurons in the spinal cord, brainstem, and brain [198]. About
10% of cases are genetic, and the most common genetic form is due to mutations
11 Copper: Effects of Deficiency and Overload 379

in the copper/zinc SOD1. This finding and the demonstration of abnormalities of


copper homeostasis in ALS were the first indications that copper was playing a role
in MNDs [199]. At least 10 genes of diverse function are known to cause hereditary
peripheral neuropathies [200] but there is no obvious common pathological mecha-
nism. The recent finding that the two specific mutations in ATP7A (T994I and
P1386S) causing distal hereditary neuropathy, result in mislocalization and defec-
tive intracellular trafficking of ATP7A in patient’s fibroblasts suggest that abnormal
copper homeostasis may lead to the degeneration of the distal motor neurons [158].
Given the role of SOD1 in ALS and ATP7A in peripheral neuropathy, together with
many other studies linking copper more broadly with other neurological diseases,
further studies to clarify the role of copper homeostasis in neurological health and
disease are warranted.

5.5 Prion Diseases

Refolding of the normal prion protein (PrPc) into an abnormal conformation (PrPsc)
has been associated with transmissible neurodegenerative diseases, such as
Creutzfeld-Jacob disease, Kuru and fatal familial insomnia in humans, bovine spon-
giform encephalopathy in cattle and scrapie in sheep, which are summarized as
prion diseases or transmissible spongiform encephalopathies [201]. Most attention
has been paid to the role of the abnormal prion protein in disease, and there is much
less data relating to the normal physiological role of the cellular prion protein (PrPc),
but evidence is growing that its role involves copper in some capacity. The PrPc is
ubiquitously expressed but most abundantly in neurons [202]. The N-terminal
region of the protein contains four to five octapeptide repeats, which have copper
binding sites of various affinities and binding of copper induces a conformation
change in the protein [203].
Cells expressing PrPc are much more resistant to copper-treatment than PrPc-
deficient cells [204]. Copper has been shown to stimulate endocytosis of PrPc [205].
Based on this observation the PrPc has been suggested to serve as a receptor for
cellular uptake or efflux of copper [205]. The copper contents of synaptosomes of
PrP-deficient mice were found to be lower compared to that of wild-type mice,
which has led to the proposal that the PrPc may play a role in regulating copper
release at the synapse [206]. Evidence for a role of the prion protein in copper
homeostasis remains controversial, however, recently it was found that PrPc
increased when cells were copper deficient and this resulted in an increase in copper
uptake [207]. Relevant to the role of copper in neurological diseases, the prion pro-
tein has been found to regulate the activity of the N-methyl-D-aspartate (NMDA)
receptor and indeed it has been proposed that copper modulation of the NMDA
receptor possibly by PrPc could be a unifying theme in many neurological disorders
[208]. Intriguingly, it has recently been found that mice with a mutation in ATP7A
have a delayed onset of prion disease [209].
380 Scheiber, Dringen, and Mercer

6 Overview and Future Developments

Copper has been known to be an essential element since the 1920s but despite many
decades of research, the full biological roles of this element have yet to be clarified.
With the advent of molecular tools, the intricate nature of copper homeostasis has
become clear. The isolation of the copper efflux genes ATP7A and ATP7B in the
early 1990s was a turning point for the field and presaged the heady decades since,
when so many of the molecular players that regulated copper status have been
identified. The interactions between many trace elements and copper are being
identified at a molecular level, particularly with iron and zinc.
We have briefly touched on some areas, where the importance of adequate dietary
copper is underappreciated, such as cardiovascular diseases and immune system
function. This is an area that promises to develop over the next few years, leading
to wider appreciation among health professionals of the importance of adequate
copper intake. Over the last 10 years the neurological importance of copper has
begun to be appreciated.
It is very exciting that abnormalities of copper homeostasis are apparently
playing an important role in neurological disorders such as AD, PD, and motor
neuron disorders. We anticipate that many of the hints of copper involvements in
these diseases will become solid evidence in the next decade, and it is even possible that
therapies based on modification of copper metabolism will lead to cures of these
devastating conditions.

Abbreviations

Aβ amyloid β
AD Alzheimer disease
ALS amyotrophic lateral sclerosis
ATOX 1 human antioxidant protein 1 (also known as HAH1)
ATP7A a copper transporting ATPase
ATP7B a copper transporting ATPase
atsm diacetylbis(N(4)methylthiosemicarbazonato)
Atx1 antioxidant protein1
CCS copper chaperone for superoxide dismutase
Cp ceruloplasmin
Ctr1 copper transporter 1
DOPA 3,4-dihydroxyphenylalanine
DβM dopamine-β-monoxygenase
EPR electron paramagnetic resonance
GPI glycosylphosphatidylinositol
GSH glutathione
gtsm glyoxalbis(N(4)-methylthiosemicarbazonato)
hCTR human copper transporter
11 Copper: Effects of Deficiency and Overload 381

HD Huntington disease
IMS intermembrane space
LOX lysyl oxidase
MBD metal binding domain
MD Menkes disease
MDS myelodysplastic syndrome
MNDs motor neuron disorders
MT metallothionein
NMDA N-methyl-D-aspartate
OHS occipital horn syndrome
PAM peptidylglycine α-amidating monoxygenase
PD Parkinson disease
PrPc normal prion protein
PrPSC pathogenic prion protein
ROS reactive oxygen species
SOD superoxide dismutase
SOD1 Cu/Zn superoxide dismutase
SOD2 Mn superoxide dismutase
SOD3 extracellular superoxide dismutase = EC-SOD
TGN trans-Golgi network
TM transmembrane helices
WD Wilson disease

Acknowledgment J.M. is grateful for funding support from the National Health and Medical
Research Council of Australia grant APP1003903.

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Chapter 12
Zinc and Human Disease

Wolfgang Maret

Contents
ABSTRACT ............................................................................................................................. 390
1 INTRODUCTION ............................................................................................................. 390
2 ZINC BIOCHEMISTRY ................................................................................................... 391
2.1 Zinc in Enzymes and Proteins ................................................................................... 392
2.1.1 Catalytic Zinc ................................................................................................ 392
2.1.2 Structural Zinc ............................................................................................... 392
2.1.3 Regulatory Zinc ............................................................................................. 393
2.2 Zinc in Vesicles: Intracellular and Intercellular Signaling
with Zinc(II) Ions ...................................................................................................... 393
2.3 Cellular Zinc Homeostasis ........................................................................................ 394
2.4 Zinc and Oxidoreduction (Redox) ............................................................................ 395
2.5 Global Functions of Zinc .......................................................................................... 396
3 ZINC IN ORGAN PATHOPHYSIOLOGY ....................................................................... 398
3.1 Liver and Gastrointestinal System ............................................................................ 398
3.2 Cardiovascular and Pulmonary System..................................................................... 399
3.3 Immune System......................................................................................................... 400
3.4 Central and Peripheral Nervous System ................................................................... 401
3.5 Reproductive System................................................................................................. 402
3.6 Sensory Systems ....................................................................................................... 403
3.7 Other Systems ........................................................................................................... 403
4 ZINC IN DISEASE............................................................................................................ 404
4.1 Genetic Disease ......................................................................................................... 404
4.2 Metabolic and Chronic Disease ................................................................................ 404
4.2.1 Diabetes ......................................................................................................... 404
4.2.2 Cancer ........................................................................................................... 406
4.2.3 Neurodegeneration ........................................................................................ 406
4.3 Infectious Disease ..................................................................................................... 407
5 GENERAL CONCLUSIONS ............................................................................................ 407

W. Maret (*)
King’s College London, School of Medicine, Diabetes and Nutritional Sciences Division,
Metal Metabolism Group, London, SE1 9NH, UK
e-mail: wolfgang.maret@kcl.ac.uk

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 389
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_12, © Springer Science+Business Media Dordrecht 2013
390 Maret

ABBREVIATIONS .................................................................................................................. 409


ACKNOWLEDGMENT .......................................................................................................... 409
REFERENCES ........................................................................................................................ 409

Abstract The vast knowledge of the physiologic functions of zinc in at least 3000
proteins and the recent recognition of fundamental regulatory functions of zinc(II)
ions released from cells or within cells links this nutritionally essential metal ion to
numerous diseases. However, this knowledge so far has had remarkably limited
impact on diagnosing, preventing, and treating human diseases. One major road-
block is a lack of suitable biomarkers that would detect changes in cellular zinc
metabolism and relate them to specific disease outcomes. It is not only the right
amount of zinc in the diet that maintains health. At least as important is the proper
functioning of the dozens of proteins that control cellular zinc homeostasis, regulate
intracellular traffic of zinc between the cytosol and vesicles/organelles, and deter-
mine the fluctuations of signaling zinc(II) ions. Cellular zinc deficiencies or over-
loads, a term referring to zinc concentrations exceeding the cellular zinc buffering
capacity, compromise the redox balance. Zinc supplementation may not readily
remedy zinc deficiency if other factors limit the capability of a cell to control zinc.
The role of zinc in human diseases requires a general understanding of the wide
spectrum of functions of zinc, how zinc is controlled, how it interacts with the
metabolism of other metal ions, in particular copper and iron, and how perturbation
of specific zinc-dependent molecular processes causes disease and influences the
progression of disease.

Keywords human diseases • zinc • zinc homeostasis • zinc metalloproteins • zinc


signaling

Please cite as: Met. Ions Life Sci. 13 (2013) 389–414

1 Introduction

The biochemistry of zinc deserves much more attention than it generally receives in
textbooks in the biomedical sciences and in some parts of the scientific literature.
There are historic reasons for this lack of coverage. Unlike iron, which was easily
detected and analyzed in blood and tissues due to the color of its complexes, zinc
compounds are colorless. Therefore the field of zinc biology developed much later
and only with the advent of new methods to analyze zinc in biological material.
Also, unlike iron, where a large amount is found in heme, zinc is not predominantly
part of a single substance but instead serves as a cofactor of at least 3000 human
proteins. This distribution among so many proteins dilutes zinc and requires sensi-
tive methods for the speciation and characterization of proteins. Zinc is involved in
a much wider variety of processes and molecular mechanisms than vitamins and
other cofactors with more specific chemical functions. The notion of zinc being a
12 Zinc and Human Disease 391

trace metal also somewhat confuscates the issues at hand. While the total amount of
zinc in a human (70 kg) is 2–3 g and about as much as that of iron, cellular zinc
ion concentrations are rather high, almost as high as those of major metabolites
such as ATP.
A multi-authored text summarizes the knowledge accumulated since the field was
last reviewed two decades ago in terms of the biochemical basis of zinc physiology
as it relates to the numerous functions of zinc in metalloenzymes and transcription
factors [1,2]. What has changed in the time between these accounts is the increase of
the number of zinc proteins by one order of magnitude, demonstrating a much more
general role of zinc in protein structure and in protein-protein interactions, an under-
standing of the biological and chemical aspects of how cellular zinc is controlled
(zinc homeostasis), and the discovery that zinc(II) ions function in cellular regulation
and information transfer. All these advances demonstrate that the present knowledge
has by far surpassed the already impressive zinc biochemistry, which in 1993 was
deemed to be, based on functions of zinc, “too numerous to cite” [2].
The implications of zinc biology for human health are enormous as about half of
the world’s population is believed to be at risk for zinc deficiency [3]. The World
Health Organization (WHO) has identified zinc deficiency as the fifth most impor-
tant risk factor for morbidity and mortality in developing countries (11th world-
wide) [4]. The figure translates into 3.2% of all lost disability-adjusted life years
(DALYs). These estimates are derived primarily from the incidence of infectious
and parasitic diseases due to compromised immune functions in zinc deficiency.
Clearly, this measured outcome is far from inclusive. It does not take into account
the functions of zinc in human memory acquisition and storage, behavior, growth
retardation and development, delayed wound healing, the effects of environmental
exposures to substances interfering with zinc metabolism, or the role of zinc in
aging and in chronic diseases, such as cancer, diabetes, and neurodegeneration.
Rather than trying to summarize all the functions of zinc in physiology, which
would be an immense task well beyond the scope of this chapter, the subject matter
is approached by discussing the general significance of zinc in biochemistry for
health and the specific involvement of zinc in pathophysiology and diseases at the
molecular level. This approach will necessitate a certain degree of selectivity when
referencing from the immense literature.

2 Zinc Biochemistry

Almost all our knowledge about the molecular roles of zinc is based on the interac-
tion of zinc with proteins. Whether any interactions with other biomolecules are
important is not known. Zinc has a role in enzymatic catalysis and in the structure
and regulation of proteins. The coordination chemistry of zinc in proteins has
been discussed in detail [5]. A major aspect of the cellular biology of zinc includes
the storage of zinc(II) ions in cellular vesicles/organelles, in which relatively high
concentrations can be reached and from which zinc(II) ions are released in a
392 Maret

controlled way. In this regard, zinc resembles calcium and is quite different from
iron, which uses redox chemistry of the central atom and a protein (ferritin) for
storage and release.

2.1 Zinc in Enzymes and Proteins

Discoveries of zinc in numerous proteins demonstrated the key role of zinc for life.
They occurred in the order of (i) zinc as a catalytic ion in enzymes, (ii) zinc in the
structure of proteins, and (iii) zinc in the regulation of proteins [6]. By analyzing
sequences of proteins from databases and detecting signatures for metal-binding
sites with characteristically spaced amino acids providing the ligands, it became
possible to estimate the number of zinc proteins in genomes, the so-called zinc pro-
teome [7]. The estimate is about 3000 human zinc proteins [8].

2.1.1 Catalytic Zinc

The field of zinc metalloproteins began with the discovery of carbonic anhydrase as
a zinc enzyme in 1939 [9]. Gradually, it became known that every enzyme class
contains zinc enzymes. By far the largest number of zinc enzymes is in the class of
hydrolases in the form of hundreds of zinc proteinases. Many proteinases are called
metalloproteinases, e.g., matrix metalloproteinases (MMP), when in fact “metallo”
stands for zinc only. For the most part, zinc enzymes seem to be absent in the major
biochemical pathways of intermediary metabolism. This absence does not mean,
however, that zinc has no roles in metabolism. It appears that rather than being a
permanent constituent of metalloenzymes in these pathways, zinc has a role in con-
trolling some of them.

2.1.2 Structural Zinc

Only a few structural zinc sites in enzymes were known before zinc finger proteins
were discovered in 1986 [10]. The discovery revealed a new principle, namely the
widespread use of zinc in proteins – which for the most part are not enzymes – to
form domains for interacting with and recognizing DNA (transcription factors)/
RNA, other proteins, or lipids. Many additional “zinc finger motifs” were found,
thus establishing zinc as an important metal ion in the tertiary structure of proteins
and for the interaction of proteins, the interactome. Also, zinc participates in the
interaction between peptide chains and determines the quaternary structure of some
proteins. How many of these zinc-binding sites at the interface between subunits
exist is presently unknown, leaving a final count open with regard to the already
remarkably high number of zinc-requiring proteins.
12 Zinc and Human Disease 393

2.1.3 Regulatory Zinc

A regulatory role of zinc in protein structure – as opposed to a role of zinc as a


permanent cofactor in regulatory proteins – is not as firmly defined as the literature
indicates. In contrast to catalytic and structural zinc in proteins, where sites are
thought to be always fully occupied with zinc, regulation requires zinc association
and dissociation. With the exception of metallothionein, variable zinc contents of
proteins as a function of physiological changes have not been established
unequivocally.
Since there is now evidence for zinc(II) ions being signaling ions in the cell, this
issue is receiving renewed scrutiny. Some zinc-binding sites satisfy criteria for serv-
ing in regulation, which occurs at remarkably low zinc(II) ion concentrations and
with controlled release of zinc(II) ions. The proteins targeted and their coordination
sites are the subject of recent investigations and include many proteins that were not
known to be zinc proteins but bind zinc in their active sites very tightly and need to
be activated by removal of the inhibitory zinc [11,12].

2.2 Zinc in Vesicles: Intracellular and Intercellular Signaling


with Zinc(II) Ions

Mainly by employing histological staining techniques, it was recognized that many


cells contain zinc(II) ions that apparently are not protein-bound [13]. These zinc(II)
ions are found predominantly in intracellular compartments. From vesicular/organ-
ellar stores, tightly controlled processes release zinc(II) ions into the cytosol or into
the extracellular space. Examples of zinc secretion from cells are the exocytosis of
vesicles loaded with zinc in specialized neurons in the hippocampus, in pancreatic
β-cells of the islets of Langerhans, and in epithelial cells of mammary glands [14].
Other cells secreting zinc(II) ions include somatotrophic cells in the pituitary gland,
pancreatic acinar cells, Paneth cells in the crypts of Lieberkühn, cells of the tubulo-
acinar glands of the prostate, epithelial cells of the epididymal ducts, and osteo-
blasts [13]. Zinc(II) ions are also released intracellularly from the endoplasmatic
reticulum [15]. Together, these and additional findings led to the concept that
zinc(II) ions are messengers in cellular control and in intra- and intercellular com-
munication [16,17]. The targets of these regulatory zinc(II) ions seem to be primar-
ily proteins, again linking zinc to the functions of proteins. The types of functions
of zinc(II) ions secreted from cells are the subject of present investigations. They
include the modulation of postsynaptic receptors (neurons), supplying the milk with
zinc (mammary gland epithelial cells), and, more speculative, keeping secreted
enzymes inhibited (pancreatic acinar cells and prostate epithelial cells), preventing
proteins from forming amyloids (pancreatic β-cells) or simply being bactericidal.
Within cells, the released zinc(II) ions affect many signaling pathways [18]. Altered
signal transduction activity associated with changed phosphorylations of proteins
and the very tight inhibition of several protein tyrosine phosphatases indicate a role
394 Maret

of zinc in phosphorylation signaling and suggests even more abundant interactions


of zinc with proteins than based on the estimate of 3000 human proteins [19–24].
The regulatory functions of zinc depend on how cellular zinc homeostasis is controlled
and at which amplitudes and frequencies zinc(II) ion transients occur [25].

2.3 Cellular Zinc Homeostasis

Proper control of cellular zinc is critical for the balance between health and disease.
How this control is achieved at the molecular level has been the subject of the latest
phase of biological zinc research. Very tight control of cellular zinc is necessary to
make the right amount of zinc available for protein structure and function, folding,
and aggregation and to prevent zinc from interfering with the metabolism of other
metal ions. The number of proteins involved in controlling cellular zinc and the fact
that proteins control zinc subcellularly is remarkable and attests to the importance
of this transition metal in biology. In humans, ten proteins of the ZnT family
(SLC30A) export zinc from the cytosol, either out of the cell or into vesicles/organ-
elles, fourteen proteins of the Zip family (SLC38A) import zinc into the cytsol from
the extracellular space or from vesicles/organelles, and at least a dozen metallothio-
neins (MTs) buffer and translocate zinc [26–28]. Another major factor in the control
of cellular zinc is the role of metal response element-binding transcription factor-1
(MTF-1) [29], which is a sensor of elevated zinc(II) ion concentrations and regu-
lates zinc-dependent gene expression. In addition to this direct role of zinc in gene
expression, there are multiple effects on signal transduction pathways. Accordingly,
many investigations using transcriptomics demonstrated that variation of zinc con-
centrations affects a myriad of gene products.
The zinc homeostatic proteins have dynamic coordination environments with
specific mechanisms for handling transition metal ions [30,31]. MTs, for instance,
have different binding constants for the seven zinc(II) ions and carry, release, and
accept zinc ions dependent on cellular conditions [32,33]. In addition, they are
redox-active zinc proteins. Zinc itself, on the contrary, is redox-inert. In MTs, the
oxidation of the sulfur donors in the cysteine ligands of zinc causes zinc dissocia-
tion while the reduction of the oxidized cysteines generates zinc-binding capacity
[34]. This property establishes redox cycles that link redox changes and the avail-
ability of cellular zinc [35,36].
The zinc homeostatic proteins are integrated into metabolism and exquisitely con-
trolled by major signal transduction pathways. Thus, they do not work in isolation
and are not only involved in housekeeping of zinc but control zinc(II) ion fluxes for
specific cellular functions. Aside from compiling the “parts list” of proteins involved
in cellular zinc homeostasis, significant advances have been made in understanding
the concentrations at which cellular zinc is controlled. In contrast to other metal ions
such as magnesium and calcium, most of the zinc is protein-bound with high affinity.
The consequence is that only picomolar concentrations are in the form of non-pro-
tein-bound “free” zinc(II) ions [37]. But this pool of zinc is not negligible and under-
12 Zinc and Human Disease 395

goes controlled fluctuations [38]. Minute increases of cytosolic free zinc(II) ion
concentrations have potent biological effects, which led to the concept of free zinc(II)
ions being cellular signaling ions at much lower concentrations than signaling
calcium(II) ions. In fact, the two ions complement each other in signaling and their
different coordination chemistries allow signaling with metal ions over a wide range
of concentrations [39]. Zinc buffering determines the amplitudes of the zinc(II) ion
transients, and ultimately cellular zinc deficiencies and overloads [37].
Owing to the fact that many proteins bind zinc, the overall cellular zinc buffering
capacity is high but only the cellular zinc buffering capacity in the range of physiologi-
cal zinc(II) ion concentrations is important. This buffering capacity is rather limited.
Compromising it, e.g., by decreasing the concentrations of critical sulfhydryls involved
in binding zinc, results in higher free zinc(II) ion concentrations with functional conse-
quences. About a third of the cellular zinc buffering capacity relies on sulfhydryl donors
(thiols) as zinc-binding ligands [21]. Some environmental agents and therapeutic drugs
react with thiols and make fewer thiols available for zinc buffering. Such reactions
change the zinc buffering capacity and increase the availability of free zinc(II) ions,
which then bind to and change the functions of proteins that are not targeted under
physiological conditions. This issue can hardly be over-emphasized because it demon-
strates that factors other than zinc itself affect zinc-dependent functions. The concept of
metal buffering in biology includes dynamic changes in buffering capacity. A unique
feature in the cellular control of zinc and calcium is muffling, which refers to the trans-
port of zinc(II) ions into vesicles/organelles and out of the cell. Muffling also contrib-
utes to buffering because the activity of transporters increases or decreases the cellular
metal ion concentrations [28]. Thus, the capacities of the transport systems and the
vesicular stores also contribute to zinc buffering.
A central regulatory hormone of zinc metabolism akin to hepcidin in iron metab-
olism is not known. Knowledge about systemic control of zinc is lacking except for
the acute phase response where adrenocorticotropic hormone (corticotropin, ACTH)
decreases zinc in the blood [40].

2.4 Zinc and Oxidoreduction (Redox)

Zinc occurs as the redox-inert zinc(II) ion in biology. Because of this, the often
quoted antioxidant properties of zinc can be indirect only, i.e., pro-antioxidant [41].
Zinc has this property only in a certain range of concentrations. Outside this range,
it is a pro-oxidant, also by indirect mechanisms [41]. Cellular zinc deficiency and
zinc overload cause oxidative stress. These opposing effects demonstrate a major
function of zinc in redox metabolism and how important it is to control cellular
zinc(II) ion concentrations in the correct range. The pro-antioxidant effects of zinc
are due to (i) binding to and protecting free sulfhydryls against oxidation, (ii) com-
peting with redox-active transition metal ions and suppressing the production of
damaging free radicals, and (iii) inducing the synthesis of antioxidants, such as the
expression of genes coding for antioxidant enzymes through MTF-1 and Nrf-2
396 Maret

(NF-E2-related factor) dependent gene transcription. Zinc deficiency compromises


these three functions and therefore constitutes a pro-oxidant condition that can trig-
ger a cascading effect: oxidative stress releases zinc from zinc/thiolate coordination
environments in proteins, such as metallothioneins and increases the oxidative
stress through the actions of the released zinc if the buffering capacity is too weak.
Pro-oxidant effects of zinc(II) ions at higher than normal concentrations have been
linked to zinc inhibition of antioxidant enzymes, the mitochondrial respiratory
chain with concomitant increased formation of reactive species, and other proteins,
for example the cellular iron exporter ferriportin [42].
Biomarkers of oxidative stress or inflammation decreased when normal healthy,
middle aged or elderly humans were supplemented with zinc [43,44]. Sufficient
zinc may need to be present in cells before an oxidative insult occurs in order to
support an antioxidant effect, e.g., liver protection against alcohol, cardiac protec-
tion (infarct size), vascular protection (postischemic injury), and protection of tis-
sues against the oxidative stress of diabetic complications. In isolated cells and in
mice, zinc deficiency exacerbates endothelial cell dysfunction through pro-
inflammatory pathways that involve NF-κB and peroxisome proliferator-activated
receptor (PPAR) signaling, while prior zinc supplementation reduces fatty acid or
tumor necrosis factor α-induced oxidative stress [45,46].

2.5 Global Functions of Zinc

Globally, the pro-oxidant effects express themselves as cytotoxic, pro-inflammatory,


and pro-apoptotic functions while the pro-antioxidant effects translate into cytopro-
tective, anti-inflammatory, and anti-apoptotic functions [41]. With molecular func-
tions in so many proteins in metabolism and signal transduction, it is understandable
that zinc is involved in proliferation, differentiation, and apoptosis of all cells with
profound implications for healthy growth, renewal, and repair of cells. Zinc defi-
ciency retards growth, compromises the immune and nervous systems, and affects
virtually any other organ system. The most cited clinical manifestations in humans
are skin lesions, depressed mental functions, impaired night vision, anorexia, hypo-
gonadism, depressed wound healing, and changed immune functions. In animals,
reduced growth, decreased food intake, alopecia, skin lesions (parakeratosis and
hyperkeratinization), impaired skeletal development and abnormal gait and stance
are observed. Zinc is involved in normal development through many specific zinc
finger transcription factors, in genetics and epigenetics through zinc enzymes that
control methylation of DNA and methylation and acetylation of histones, and in
maintaining the integrity of DNA through its role in DNA repair enzymes.
The involvement of zinc in the NF-κB pathway of inflammation serves to illustrate
the widespread role of zinc in many signaling pathways. In this pathway, no less
than 24 zinc(II) ions are involved in various aspects of protein structure (Figure 1).
Human IKKβ has a zinc-binding site in its kinase domain, thus increasing the number
12 Zinc and Human Disease 397

TNFα TNFα
TNFα
TNF Receptor

E1, E2 ITCH RNF11


Cell membrane
TAX1BP1 RIP 1
TRADD TRADD

Cytoplasm TRAF2 A20 K48


TRAF2
K48
RIP 1 K48
RIP 1
DeUbiquitinates RIP1
K63 Re-Ubiquitinates with K48 linked Lys

K63
Tab2 or 3 TAK1
Targets mRNA for degradation K63

Zn
TNFα Zn
Nup475/TTP/Tis11

o
em
o

Degradation via the


em

IKKα

γ/N
IKKα P
/N

26S Proteasome

K

IK
IKKβ
K63
IK

IKKβ
K63 Activated IKK Complex
IKK Complex

NFkB starts transcription Ser


P Ser
of genes for many pro- Ser P
P Ser Ser
inflammatory proteins IkB E1, E2, E3 P
including TNFα, A20, IkB Ser
Nup475/TTP/Tis11. P65 P50
P65 P50 NLS IkB
mRNA P65 P50
NFkB K48
Phosphorylated NFkB
Cytoplasm K48
K48

NLS
Nucleus P65 P50

Figure 1 Zinc proteins involved in turning off the NF-κB pathway of inflammation. A balance
sheet of the number of zinc-containing proteins is given. The figures were kindly provided by
Barbara Amann, Department of Chemistry, Goucher College, Towson, MD, USA.

of zinc(II) ions in this pathway to 25 [47]. This complexity and the pleiotropic functions
of zinc demonstrate the inherent difficulties that investigators face in defining single
modes of action for zinc or finding suitable biomarkers for either the cellular zinc
status or specific zinc-dependent events.
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One of the major issues in overcoming this difficulty is the lack of knowledge
about the hierarchy of cellular zinc distribution under zinc-limiting conditions. Do
some proteins hold on to their zinc whereas others yield their zinc to support more
crucial functions? Or are all zinc-dependent proteins affected to the same extent?
Are vesicular pools of stored zinc(II) ions and signaling functions affected first
before the functions of zinc metalloproteins are compromised? Of course, such
questions need to be asked for systemic zinc homeostasis as well: Which organs/
tissues yield their zinc first and are therefore primarily affected by zinc deficiency?
Compartmental models have described re-distribution of zinc from the bone/skele-
ton to the liver, but answers lie in the affinities of zinc for cellular proteins and the
kinetics of cellular proteins that re-distribute zinc.
With this knowledge, it is clear that the subject of zinc in organ pathophysiology
and disease remains largely phenomenological as it has rarely been possible to
relate pathology to single or specific zinc-dependent molecular events. It would be,
however, a severe mistake to conclude that the zinc status is not a major determinant
in the etiology and the progression of diseases. Unfortunately, such a conclusion
seems to be the prevailing assessment of a large part of the medical community due
to the absence of any suitable clinical marker of cellular zinc status.

3 Zinc in Organ Pathophysiology

The following short summaries are merely snapshots of examples where the field
has matured to indicate specific roles of zinc in organ pathophysiology and diseases.
In most instances, pathways common to all cells are discussed in the literature of
specific disciplines, but the knowledge applies across disciplines and will become
the focus of zinc biology in the years to come. Challenges remain to tease apart the
specific roles of zinc in zinc-dependent molecular pathways from the myriad of cel-
lular functions of zinc. The involvement of zinc in the NF-κB pathway serves to
illustrate this point (see Figure 1). The following summaries remain limited in scope
as they focus on recently emerging general principles involving zinc(II) ion fluxes
rather than focusing on zinc metalloproteins or the many interactions of zinc with
membrane ion channels and other membrane proteins. Some topics will be treated
only cursorily in order to keep a focus on molecular events and to avoid too much
phenomenology. Comprehensive reviews on most of the topics will be cited.

3.1 Liver and Gastrointestinal System

The liver is a central organ in zinc distribution. Therefore, liver disease can affect
zinc-dependent functions of many other organs and can lead to zinc deficiency [48].
In zinc deficiency caused by factors other than liver disease, the liver is less pro-
12 Zinc and Human Disease 399

tected against various insults. Early observations indicated marked abnormalities of


zinc metabolism in post-alcoholic (Laennec’s) cirrhosis with low serum and liver
zinc and zincuria that correlated with the severity of the disease. A considerable
improvement of liver function was noted when patients were supplemented with
zinc and it was suggested that alcoholic liver disease is a conditioned zinc deficiency
[49,50]. These observations have been confirmed repeatedly and demonstrate the
therapeutic potential of zinc [51]. Alcohol consumption is also a risk factor for the
severity of hepatitis C. The hepatitis C virus increases hepatic mitochondrial oxida-
tive stress, thereby sensitizing hepatocytes to further oxidative insults from exces-
sive alcohol consumption [52]. Nutritional and conditioned zinc deficiencies, which
are pro-oxidative conditions, are therefore cumulative risk factors for liver disease.
Zinc is effective in treating alcoholic and viral liver disease. In liver regeneration,
the zinc transporter Zip14, which also transports non-transferrin bound iron, is
upregulated. The additional cellular influx of zinc affects the phosphorylation of the
hepatocyte growth factor receptor c-Met via inhibiting its dephosphorylation by
protein tyrosine phosphatase-1B (PTP-1B) [53].
The gastrointestinal tract is equally important in the systemic control of zinc because
it is the main organ for zinc uptake, which increases under zinc deficiency, as well as
zinc excretion. Body zinc is a rather closed system where only a few milligrams
(one thousandth of the total of 2–3 g) need to be replenished every day. Additional
conservation of body zinc is afforded through re-uptake of the zinc secreted from the
salivary glands, stomach, bile, and pancreas. Paneth cells located in the crypts of
Lieberkühn in the small intestine secrete zinc(II) ions that are thought to be adjuvants
to the microbicidal properties of secreted defensin peptides (cryptidins) [54].

3.2 Cardiovascular and Pulmonary System

The cellular role of zinc in protecting the vasculature and the extracellular role of
zinc in hemostasis have implications for atherosclerosis and thrombosis. Zinc defi-
ciency is associated with bleeding and clotting abnormalities through the involve-
ment of zinc in platelet aggregation, coagulation, anticoagulation and fibrinolysis
[55]. Beyond these roles with significance for cardiovascular disease, zinc has been
implicated in congestive heart failure, myocardial infarction, arrhythmias, and in
diabetic cardiomyopathy through its involvement in diabetes [56]. Numerous inves-
tigations support a role of zinc in the pathways leading to heart disease. Some
human zinc supplementation trials have shown positive effects on clinical parame-
ters related to heart disease although the lack of suitable biomarkers of zinc status
remains a serious issue in interpreting the findings [57]. Experiments with rats dem-
onstrated that suboptimal dietary intake of zinc promotes vascular inflammation and
atherogenesis by affecting lipoprotein levels and by enhancing proliferation of vas-
cular smooth muscle cells [58,59]. A proteomics analysis of the rats has already
provided significant insights into the pathways involved [60].
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The lung epithelium and endothelium have been the subject of extensive investigations
with regard to zinc [61,62]. As is the case in many other tissues, zinc chelation and
zinc deficiency render the lung endothelium susceptible to injury, whereas zinc(II)
ions released in the cell have a protective effect, though this effect is clearly a matter
of the amount of zinc released because amounts that exceed the cellular zinc buffer-
ing capacity cause injury. The investigations have demonstrated that the zinc/thio-
late clusters of metallothionein are oxidized in vivo and serve as a source of zinc(II)
ions by converting redox signals into zinc signals [63,64]. Hormones and agents
that stimulate the cellular production of nitric oxide (NO), hydrogen peroxide, or
other reactive species release cellular zinc(II) ions. The pathway with oxidative
mobilization of zinc from metallothionein and subsequent zinc inhibition of enzymes
operates in many tissues [11].
Hormone → NO (reactive species) → Zn/S (metallothionein) → Zn2+
→ inhibition of enzymes
The central role of metallothionein in this pathway draws attention to the many
factors involved when it serves as a source of zinc released in cells. The differential
gene expression of the about twelve human metallothioneins in tissues, their
amounts, metal loads, and genetics, and their different reactivites towards thiol-
reactive agents all determine the balance between cytoprotective and cytotoxic
functions of zinc [65,66]. The roles of metallothioneins in diseases cover most of
the areas where zinc is involved and are not discussed here as they have been the
subject of a recent monograph [67].

3.3 Immune System

Zinc has extensive roles in both the adaptive (specific) and the innate (non-specific)
immune response at multiple levels, including the development of immune cells and
gene expression in these cells that either affects the cells themselves or other cells
through secreted cytokines [68]. Investigations with rodents demonstrated that
immune responses decline significantly (>50%) in zinc deficiency [69]. Human zinc
deficiency leads to atrophy of the thymus with apoptotic cell death of precursor
lymphocytes as well as to deficits in erythropoiesis resulting in anemia [70]. T- and
B-cells are the basis of the adaptive immune system. Zinc deficiency affects T-cell
function more readily than B-cell function, but there are fewer B-cells formed
because of the pro-apoptotic effect of zinc deficiency in lymphopoiesis. In human
zinc deficiency, there is a shift in T-cell populations (Th1/Th2 balance) with the
result of less interferon γ, interleukin-2, and TNFα being produced [71]. T-cell
receptor-mediated T-cell activation also causes influx of extracellular zinc via the
zinc transporter Zip6. The zinc inhibits subsynaptically the recruitment of the pro-
tein tyrosine phosphatase SHP-1 to the T-cell receptor [72]. Zinc is also needed to
link the T-cell receptor CD4/CD8 and the tyrosine kinase Lck at protein interface
sites between the two proteins and to induce the subsequent dimerization of this
protein complex [73,74].
12 Zinc and Human Disease 401

The innate immune response and inflammation constitutes the first line of
defense of the host. It includes granulocytes, monocytes/macrophages, dendritic
cells, and natural killer cells. Zinc is involved in the development, maturation, and
function of all these cells. Zip6 is downregulated and cellular zinc decreases when
toll-like receptor-4 of dendritic cells is stimulated [75]. Zip6 is needed to initiate
zinc-dependent expression of major histocompatibility class II molecules. Zinc sig-
naling in the immune system recently gained additional attention when a role of a
protein kinase C-induced zinc(II) ion signal in the formation of neutrophil extracel-
lular traps (NET) was discovered [76]. NETosis is a process, in which components
such as DNA, chromatin, and proteins are released from cells to capture bacteria.
At the molecular level, a re-distribution of zinc is important for the immune
response. The acute phase response to injury or infection decreases plasma zinc and
increases cellular zinc [40]. In the liver, the pro-inflammatory cytokine interleukin-
6 induces the expression of Zip14 and metallothionein, resulting in zinc influx
and binding of zinc [77]. Subsequent to restriction of zinc in the blood, induced
cellular zinc(II) ion fluxes are critical for functions of immune cells. FcεR1 receptor
stimulation of mast cells increases cellular zinc(II) ions and so does stimulation of
Jurkat T cells and monocytes [78,79]. The increase in mast cell zinc is important for
allergic and autoimmune reactions (anaphylaxis, asthma, atopic dermatitis). Under
conditions of zinc deficiency, the cellular re-distribution of zinc cannot take place,
compromising the function of immune cells and increasing morbidity and mortality,
especially in the critically ill with sepsis.
Another important observation is that the zinc transporter Zip8 is a transcrip-
tional target of NF-κB [47]. Zinc transport through Zip8 suppresses pro-inflammatory
conditions by zinc-dependent down-regulation of IκB (IKK) kinase activity. Zinc
deficiency, in contrast, results in increased inflammation. Zip8 transports iron in
addition to zinc [80].
Control of zinc homeostasis must be intact for proper immune functions. Zinc
supplementation must be chosen carefully. Too much zinc causes copper deficiency,
leukopenia, inhibits immune functions, and counteracts the acute phase response,
which removes zinc from the circulation so that an invading pathogen does not have
access to the zinc it needs. Zinc deficiency, on the other hand, increases bacterial
invasion, in particular through an inflammatory response and the damaging effects
on mucosal functions in the gastrointestinal and respiratory tracts [81]. These effects
of zinc on the immune system are important for autoimmune diseases and neoplas-
tic growth, and the efficacy of vaccinations in zinc-deficient individuals. Since zinc
affects B-cells indirectly through its effect on T-cells, T-cell function should be opti-
mal prior to vaccination.

3.4 Central and Peripheral Nervous System

In addition to the functions of zinc in every nerve cell, specialized neurons in the
cerebral cortex store zinc in synaptic vesicles. Upon neuronal stimulation, zinc(II)
ions are released from these vesicles and have multiple effects on the postsynaptic
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neurons. Therefore, control of synaptic zinc homeostasis is critical [82]. ZnT3 and
MT3 (growth inhibitory factor) participate in the loading of the synaptic vesicles
with zinc. The role of zinc(II) ions in synaptic neurotransmission and in excitotoxic-
ity has been investigated extensively [83,84]. In a provocative article entitled “Do
we need zinc to think?” the role of synaptic zinc was discussed [85]. It is becoming
clear that synaptic zinc is involved in cortical plasticity affecting learning and mem-
ory and thus critical to the function of the hippocampus [86–88].
Stroke leads to ischemic neuronal injury and neurodegeneration [89]. Release of
vesicular zinc associated with a stroke either affects the receptors at the postsynap-
tic neuron, such as NMDA channels, acid-sensing channels or GABAA receptors,
or enters the postsynaptic neuron through the AMPA receptor and other calcium
channels and acts intracellularly. In the neuron, zinc is also released from proteins by
oxidative/nitrosative stress and acidosis, both of which are consequences of ischemia.
The zinc(II) ions then enter mitochondria and inhibit the respiratory chain and anti-
oxidant enzymes in the matrix with the result of mitochondria churning out more
reactive oxygen species. Reperfusion following ischemia may augment the injury.
The fine balance between zinc being released for protective functions and zinc
being neurotoxic and the timing of the events make it very difficult to intervene
therapeutically with either chelating agents or with zinc supplementation. The pro-
tective functions of zinc are evident in preconditioning that lowers the damage of an
ensuing ischemic insult.
Zinc(II) ions released from vesicles through exocytosis and from cellular pro-
teins by oxidative and nitrosative stress also contribute to neurotoxicity in traumatic
brain injury and seizures [90–92].
For human brain health and for public health in general, it is important to realize
that subclinical zinc deficiency impairs brain function [93].

3.5 Reproductive System

The observation of hypogonadism in zinc deficiency underscores the role of zinc in


the reproductive system. The prostate, seminal fluid, and sperm are all very rich in
zinc. Testicular zinc is required for spermatogenesis. The high amount of zinc in the
prostate has been linked to the inhibition of mitochondrial aconitase for the produc-
tion of high concentrations of citrate, which together with secrected zinc(II) ions is
important for the physiology of the seminal fluid [94]. The high concentrations of
zinc in prostate epithelial cells has been suggested to have antiproliferative and
antitumor effects [94].
Significant advances have been made recently in elucidating the role of zinc in
oocytes. At the end of maturation, mouse oocytes accumulate zinc and arrest at
metaphase II after the first meiotic division. The fertilized oocytes then secrete zinc(II)
ions with characteristic “zinc sparks” into the environment in order to lower intracellular
12 Zinc and Human Disease 403

zinc as a requirement for resuming the meiotic cell cycle [95]. Zinc is also involved in
prophase I arrest through its effect on the MOS-MAPK pathway [96]. The authors
comment on these remarkable findings in the following way: “These results establish
zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation,
including the maintenance of and release from the first and second meiotic arrest
points.” Oocytes interact with cumulus cells and their cellular zinc content is intri-
cately linked to the control of zinc homeostasis in cumulus cells [97].
These recent discoveries will impact significantly our knowledge about fertility,
reproductive health, and embryonic development.

3.6 Sensory Systems

Among the sensory systems, most work has focused on the eye, where the retina and
the retinal pigment epithelium/choroid complex are particularly rich in zinc [98,99].
Drusen, deposits in the retina, accumulate metal ions, suggesting a pathology
similar to that of perturbation of metal homeostasis in the deposits of Alzheimer’s
disease (see below). Dietary supplementation with zinc has become a method of
treatment for age-related macular degeneration.
Loss of taste acuity is a clinical sign of zinc deficiency; hearing and smelling
may also be affected. The olfactory bulb has very high zinc concentrations. Zinc is
stored in vesicles of olfactory sensory neurons and can be released by electrical
stimulation [100].

3.7 Other Systems

Other organ pathologies are also linked to zinc. There is a relative extensive litera-
ture on the role of zinc in skin diseases, wound healing, and bone health.
One additional general subject is the role of zinc in growth. Stunting is a conse-
quence of zinc deficiency. Hypopituitarism was observed when human zinc defi-
ciency was first described [101]. Therefore, research has addressed the role of zinc
in the growth hormone (somatostatin) – insulin-like growth factor-1 (IGF-1) axis.
Neuroendocrine cells store secreted proteins in dense cores of secretory granules.
Zinc has a role in the secretory pathway of growth hormone [102]. It binds to growth
hormone in a stoichiometry close to 1:1 in the secretory granules of the pituitary
glands and induces the dimerization of the hormone [103,104]. And anterior pitu-
itary cells, which release growth hormone, secrete zinc(II) ions [13]. Growth
hormone then stimulates the synthesis of IGF-1 in the liver. The function of IGF-1
in tissues (muscle, bone) is zinc-dependent in pathways that also involve Zip7,
Zip13, and Zip14 [19,105–107].
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4 Zinc in Disease

4.1 Genetic Disease

In addition to the many changes in mRNA levels of zinc transporters observed in


diseases, a number of mutations in zinc transporters are associated with human
diseases (Table 1). A polymorphism of MT1A, an Asn27Thr substitution, changes the
zinc-binding properties of the protein and is associated with type 2 diabetes and coro-
nary heart disease [108]. Therefore, not only the availability of zinc in the diet but also
the proteins that control cellular zinc homeostasis are important for health. Very little is
known, however, about genetically determined differences in requirements for zinc or
sensitivities towards an excess of zinc, nor have the genetics of the about 3000 human
zinc proteins been much explored with regard to disease-causing mutations. In addition
to the metabolic effects of mutations in zinc homeostatic proteins, the effect of such
mutations on mental health are quite remarkable (Table 1). There are individuals with
hyperzincemia [109]. Their high zinc in the blood is associated with calprotectinemia.
Calprotectin is a protein of the S100 family that binds zinc. It is released from leuko-
cytes in the acute phase response, and is involved in the innate immune response by
binding manganese to deprive invading bacteria of this metal ion [110]. Hyperzincemic
individuals present with the same symptoms as those with zinc deficiency.

4.2 Metabolic and Chronic Disease

4.2.1 Diabetes

A role of zinc in diabetes was considered for a long time but did not receive much
attention because insights into molecular mechanisms were lacking, the many key
functions of zinc in proteins and in the control of metabolism were not yet known,

Table 1 Polymorphisms/mutations in human zinc transporters causing, or being associated with,


human diseases.
ZnT2 Transient neonatal zinc deficiency [111]
ZnT8 Risk for diabetes type 2 [112]
ZnT10 Broad phenotype with neurologic, hepatic, and hematologic disturbances,
hypermanganesemia, obesity [113,114]
Zip2 Carotid artery disease [115]
Zip3 Bipolar disorder [116]
Zip4 Acrodermatitis enteropathica [117,118]
Zip8 Schizophrenia, obesity (dyslipidemia), coronary artery disease [119,120]
Zip11 Major depressive disorder [121]
Zip12 Schizophrenia [122]
Zip13 Spondylocheiro dysplastic form of Ehlers–Danlos syndrome (SCD-EDS) [123,124]
12 Zinc and Human Disease 405

and cause versus effect could not be addressed [125–127]. Already in the 1930s, it
was reported that the pancreas of cadavers from diabetics has only about half the
amount of zinc compared to a healthy pancreas. Also, diabetics have significant
hyperzincuria. However, a zinc deficiency is not readily established as reliable clini-
cal markers of cellular zinc status are not available. Molecular studies in three areas
have now significantly strengthened the link between zinc and diabetes. Zinc
enhances the insulin action in peripheral tissues; it has a role in insulin storage and
secretion in pancreatic β-cells; and it has pro-antioxidant functions in protecting the
endocrine pancreas and peripheral tissues. The following summaries are based on
recent reviews that cite the original work [112,128,129].

4.2.1.1 Zinc in Pancreatic β-Cell Physiology

In the granules of human pancreatic β-cells, human insulin is stored as a crystalline


hexamer with two bound zinc ions. The zinc(II) ions that are co-secreted with
insulin may affect glucagon secretion from α-cells, channel proteins, and/or prevent
amyloidogenesis of co-secreted proteins. For instance, zinc inhibits the fibrillation
of monomeric insulin and the formation of the dimer of the human islet amyloid
polypeptide (IAPP, amylin).
The zinc transporter, ZnT8 provides zinc to the insulin-storing granules. A strong
association between a polymorphism in the SLC30A8 gene (coding for ZnT8) and
type 2 diabetes exists in different populations. ZnT8 with an Arg instead of a Trp
at position 325 in its cytoplasmic domain increases the risk for diabetes by 53%.
The risk allele is the most prevalent in populations, 55% in Asians, 75% in
Europeans, and 95% in Africans. There is another variant with Gln at position 325.
ZnT8 is also a significant autoantigen in type 1 diabetes and the single nucleotide
polymorphism modulates the ZnT8 autoantibody specificities, indicating that the
amino acid substitution has probably a critical role. Mice with a β-cell specific
knock-out of ZnT8 have decreased zinc in the granules, altered insulin secretion,
and mild glucose intolerance. Other zinc transporters participate in zinc metabolism
of β-cells and some of them are associated with diabetes type 2.

4.2.1.2 Zinc in the Physiology of Cells Targeted by Insulin

Already in the 1960s it was shown that zinc has an insulin-sparing effect and that zinc-
deficient rats are less sensitive to insulin. Zinc stimulates lipogenesis in isolated adi-
pose tissue and affects glucose uptake in target tissues. Remarkably, for the culture of
some mammalian cells, zinc can replace insulin in serum-free media. These insulin-
mimetic effects of zinc are intracellular. Zinc increases the phosphorylation of the
insulin/IGF-1 receptor and hence protein phosphorylations downstream in insulin sig-
nal transduction. One molecular target of zinc is the protein tyrosine phosphatase
PTP-1B, a major regulator of the phosphorylation state of the insulin receptor. Zinc
inhibits this enzyme with an apparent zinc binding constant of 15 nM [19,130].
406 Maret

4.2.1.3 Zinc and Oxidative and Carbonyl Stress

Diabetic hyperglycemia causes the glycation of proteins. The resulting advanced


glycation end products (AGEs) increase reactive carbonyls, resulting in so-called
carbonyl stress, which modifies, among other targets, sulfhydryl groups, lowers the
zinc buffering/binding capacity, and mobilizes zinc from proteins [131]. Oxidative
stress is a cause of insulin resistance [132]. The role of zinc in mediating oxidative
stress thus establishes a causal link between zinc and insulin resistance.

4.2.2 Cancer

The roles of zinc in the immune system and thus immune surveillance and in main-
taining genome stability are all relevant to cancer [133]. In addition, metastasis and
angiogenesis require zinc. Matrix metalloproteinases are involved in metastasis.
Cancer is often associated with low serum zinc, and with increased or decreased
zinc in the malignant tissue. Zinc deficiency is a major risk factor for esophageal
and oral small cell carcinoma and is involved in the development of chemically
induced esophageal cancer [134,135]. It causes a pro-inflammatory environment
through the expression of onco-micro RNAs [136]. The diminished cytoprotection
in zinc deficiency constitutes a risk factor for reactions of biomolecules with envi-
ronmental carcinogens and therefore has general implications for carcinogenesis.
Zinc chelation arrests cell growth. Zinc is required for cells to pass through the
G1 and G2 restriction points of the cell cycle and for differentiation. In addition,
mRNAs of specific cyclins decrease under zinc deficiency [137]. Fluctuations of
free cellular zinc(II) ions have been observed at the two restriction points [38].
Changes in zinc and in zinc homeostatic proteins (zinc transporters and metallothio-
neins) have also been observed in many cancers. Zinc transporters have a role in cancer
signaling through the apparent activation of protein tyrosine kinases [138]. Very tight
zinc inhibition of protein tyrosine phosphatases supports such activation [22].
Zip6 (LIV-1) is an estrogen-induced protein in breast cancer cells [139]. Zip6 is
regulated by the transcription factor STAT3 and has a role in the EMT (epithelial to
mesenchymal transition) in metastasis [140]. The increase of cellular zinc(II) ions
caused by Zip6 induction inhibits downstream events, in particular the expression of
E-cadherin, which is involved directly in cell detachment and hence metastasis [141].

4.2.3 Neurodegeneration

Among the neurodegenerative diseases, Alzheimer’s disease (AD) received the


most attention with regard to zinc’s involvement in both the Aβ and tau protein
pathologies. The reason is that zinc and other metals (Fe, Cu) were found in
the amyloid plaques of AD at relatively high concentrations [142]. This finding led
to the hypothesis that Aβ disrupts the control of neuronal zinc homeostasis by
sequestering metals and that inappropriately localized zinc leads to a loss of zinc’s
12 Zinc and Human Disease 407

function in neurotransmission. Zinc induces the aggregation of Aβ into an insoluble


form, the processing of amyloid precursor protein (APP), the metabolism of iron
and copper, and interferes with microtubule-associated tau, the protein forming
neurofibrillary tangles, the second hallmark of AD pathology [143]. Elevated levels
of zinc inhibit the iron exporter ferroportin, possibly leading to iron overload and
associated oxidative stress in neurons [42].
A quite remarkable observation is the shared phylogenetic origin of some Zip
proteins and the prion proteins [144]. A role of the prion protein in uptake of zinc
into neuronal cells indicates functional significance for neurodegeneration [145].

4.3 Infectious Disease

With compromised immunity of the host in zinc deficiency, infection with parasites
is a major health concern, in particular for those at risk: children, pregnant women,
and the elderly. Treating and preventing diarrhea in children under the age of
five with zinc is robust and saves a high number of lives in the developing world.
There is correlative evidence between zinc deficiency and malaria, measles, HIV,
tuberculosis, and respiratory tract infections such as pneumonia. However, evidence
for efficacy of the treatment and prevention of these diseases with zinc is less
clear [146].

5 General Conclusions

The importance of proper control of cellular zinc is being recognized in several


specialized disciplines of academic medicine. Zinc is involved in pathways com-
mon to all cells and in virtually all aspects of molecular and cellular biology. While
changed levels of zinc in diseases were mostly considered consequences of the dis-
eases, causation is now indicated strongly in many cases. Zinc biology, therefore,
will have a major impact on the practice of medicine in many disciplines, including
those where the roles of zinc are not considered at present.
Translation of the knowledge into practice has been, and continues to be, ham-
pered by the lack of suitable clinical markers, especially markers for milder forms
of zinc deficiency [147]. Biomarker discovery can be better pursued now because
many additional and wider functions of zinc are known. In particular the knowledge
about proteins involved in zinc homeostasis raise expectations that biomarkers
reflecting the zinc status akin to transferrin and ferritin in iron metabolism will be
discovered. The majority of clinical cases of zinc deficiency present themselves
with overt signs. Mild zinc deficiency, though known to affect the nervous and
immune system, is generally not addressed in the clinical setting.
Blood zinc represents only about 0.1% of total body zinc and corresponds to
about the amount of zinc replenished every day, but the requirements are different
408 Maret

Table 2 Causes for zinc deficiency [149].


Nutritional
General malnutrition but also wrong diets, including diets high in phytate; total parenteral nutrition
Conditioned
Diseases: Liver disease (cirrhosis, chronic active hepatitis, fatty liver disease) and intestinal disease
(Crohn’s disease, colitis ulcerosa, celiac disease), pancreas disease (chronic pancreatitis), terminal
kidney insufficiency, acute myocardial infarct, infections, tumors, diabetes mellitus, collagenoses
Iatrogen: Treatment with penicillamine, contraceptives, corticoids, and certain antibiotics
Genetic
Sickle cell disease and inherited diseases of zinc metabolism

for children, pregnant and lactating women, the elderly, healthy and sick humans,
and populations that are at risk for zinc deficiency. As a systemic marker, blood
zinc it is not a sensitive marker for the cellular zinc status, in particular since acute
infections can lower serum zinc and increase cellular zinc, cells differ in their zinc
requirements and kinetics, and major functions of zinc are within cells. Zinc
therapy is already proven to be effective in diseases, such as intestinal diseases,
acrodermatitis enteropathica, transient neonatal zinc deficiency, and Wilson’s
disease.
Although many deficits have been linked unequivocally to zinc deficiency and
zinc appears to be a panaceum for a large number of ailments, zinc supplementation
is often ineffective in reversing deficits [148]. This failure has perplexed some and
is sometimes used to refute the hypothesis that zinc deficiency is the cause of a
condition. However, there are multiple reasons for the occasional failure of revers-
ing symptoms with zinc supplementation. The control of zinc metabolism is remark-
ably complex and encompasses competition with and interactions among other
metal ions; zinc supplementation depends on the dose as too much zinc may have the
opposite effects; and last but not least, restoration of function may require additional
factors because other micronutrient deficiencies often accompany zinc deficiency.
Supplementing only zinc may not restore the control of cellular zinc homeostasis.
Additional zinc may be a pro-oxidant and harmful under conditions of sustained
oxidative stress when free zinc(II) ion concentrations are already higher than normal
and the zinc buffering capacity is reduced. Under conditions such as oxidative
stress, the binding capacity of cellular thiols is reduced and supplemental zinc may
not be retained in the cells or bind to non-physiological targets with adverse side
effects. Restoring control of cellular, not necessarily systemic, zinc homeostasis,
restoring the cellular redox state, and addressing the factors that condition zinc defi-
ciency (Table 2) seem to be a prerequisites to successful zinc therapy in the many
conditions that may require zinc.
With the discovery of the many proteins controlling cellular zinc homeostasis
and the genetics of these proteins, the focus of attention shifted from the presence
or absence of zinc to the functions of the proteins that determine the cellular alloca-
tion of zinc to zinc proteins, re-distribute zinc under changing conditions, and,
importantly, regulate the functions of zinc in intra- and intercellular communication.
Specific roles of zinc in cellular signaling pathways are being identified and
12 Zinc and Human Disease 409

examined. Changed functions of zinc homeostatic proteins affect the redox state,
inflammation, genetic stability, and cellular signaling and metabolism through
altered cellular zinc metabolism.

Abbreviations

ACTH adrenocorticotrophic hormone, corticotropin


AD Alzheimer’s disease
AGE advanced glycation end product
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
APP amyloid precursor protein
DALY disability-adjusted life years
EMT epithelial-to-mesenchymal transition
GABAA γ-aminobutyric acid A
HIV human immunodeficiency virus
IAPP islet amyloid polypeptide, amylin
IGF-1 insulin-like growth factor 1
IKKβ IκB kinase β
MMP matrix metalloproteinase
MOS-MAPK mos (a protein serine/threonine kinase)-mitogen-activated protein kinase
MRE metal response element
MT metallothionein
MTF-1 metal response element (MRE)-binding transcription factor-1
NET neutrophil extracellular trap
NF-κB nuclear factor κ light-chain-enhancer of activated B cells
NMDA N-methyl D-aspartate
PPAR peroxisome proliferator-activated acceptor
PTB-1B protein tyrosine phosphatase-1B
TNFα tumor necrosis factor α
WHO World Health Organization
Zip Zrt-, Irt-like proteins
ZnT zinc transporter

Acknowledgment I thank Dr. Barbara Amann, Department of Chemistry, Goucher College,


Towson, MD, for providing the figures.

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Chapter 13
Molybdenum in Human Health and Disease

Guenter Schwarz and Abdel A. Belaidi

Contents
ABSTRACT ............................................................................................................................. 416
1 INTRODUCTION ............................................................................................................. 417
1.1 Chemistry and Biology of Molybdenum .................................................................. 417
1.2 Molybdenum Uptake................................................................................................. 417
1.3 Molybdenum Toxicity ............................................................................................... 418
1.4 Molybdenum Enzymes.............................................................................................. 418
2 DEFICIENCIES IN MOLYBDENUM ENZYMES .......................................................... 419
2.1 Xanthine Dehydrogenase and Oxidase ..................................................................... 420
2.1.1 Structure and Function .................................................................................. 420
2.1.2 Xanthinuria Type I ........................................................................................ 420
2.1.3 Xanthinuria Type II ....................................................................................... 421
2.1.4 Hyperuricemia ............................................................................................... 421
2.2 Aldehyde Oxidase ..................................................................................................... 422
2.3 Sulfite Oxidase and Sulfite Toxicity .......................................................................... 423
2.3.1 Cysteine Catabolism...................................................................................... 423
2.3.2 Sulfite Oxidase Localization, Structure, and Function.................................. 425
2.3.3 Sulfite Toxicity and Sulfite Oxidase Deficiency............................................ 425
2.4 Mitochondrial Amidoxime-Reducing Component ................................................... 426
3 MOLYBDENUM COFACTOR DEFICIENCIES ............................................................. 426
3.1 Biochemistry of Molybdenum Cofactor Biosynthesis .............................................. 427
3.1.1 Pterin Synthesis ............................................................................................. 427
3.1.2 Dithiolene Synthesis...................................................................................... 428
3.1.3 Molybdate Insertion ...................................................................................... 429
3.1.4 Maturation of Moco ...................................................................................... 430
3.2 Genetics of Human Molybdenum Cofactor Synthesis .............................................. 430
3.2.1 Structure and Organization of Moco Synthesis Genes.................................. 430
3.2.2 Mutations in Molybdenum Cofactor-Deficient Patients ............................... 432
3.2.3 Biochemical Classification of Molybdenum Cofactor Deficiency ................ 434

G. Schwarz (*) • A.A. Belaidi


Institute of Biochemistry, Department of Chemistry, Center for Molecular Medicine,
University of Cologne, Zülpicher Strasse 47, D-50674 Köln, Germany
e-mail: gschwarz@uni-koeln.de

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 415
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_13, © Springer Science+Business Media Dordrecht 2013
416 Schwarz and Belaidi

3.3 Pathophysiology of Molybdenum Cofactor Deficiency ............................................ 434


3.3.1 Clinical Presentation of Patients with Molybdenum
Cofactor Deficiency....................................................................................... 434
3.3.2 Molecular Basis of Neurodegeneration ......................................................... 435
3.4 Animal Models .......................................................................................................... 436
3.4.1 Mocs1-Deficient Mice ................................................................................... 436
3.4.2 Gephyrin-Deficient Mice............................................................................... 437
3.5 Treatment of Molybdenum Cofactor Deficiency ...................................................... 437
3.5.1 Treatment of Mocs1-Deficient Mice with cPMP .......................................... 437
3.5.2 Treatment of MoCD Type A Patients with cPMP ......................................... 438
3.5.3 Treatment of MoCD Type B and C Patients ................................................. 439
3.5.4 Dietary Restriction and Treatment of Sulfite
Oxidase Deficiency ....................................................................................... 439
4 ASSOCIATION OF MOLYBDENUM WITH OTHER DISORDERS ............................. 440
4.1 Copper Homeostasis Disorders ................................................................................. 440
4.2 Epilepsy and Neuropsychiatric Disorders ................................................................. 441
4.3 Ethylmalonic Encephalopathy .................................................................................. 441
5 CONCLUDING REMARKS AND FUTURE DEVELOPMENTS .................................. 442
ABBREVIATIONS AND DEFINITIONS .............................................................................. 443
ACKNOWLEDGMENTS........................................................................................................ 444
REFERENCES ........................................................................................................................ 444

Abstract Molybdenum is an essential trace element and crucial for the survival of
animals. Four mammalian Mo-dependent enzymes are known, all of them harbor-
ing a pterin-based molybdenum cofactor (Moco) in their active site. In these
enzymes, molybdenum catalyzes oxygen transfer reactions from or to substrates
using water as oxygen donor or acceptor. Molybdenum shuttles between two oxida-
tion states, MoIV and MoVI. Following substrate reduction or oxidation, electrons are
subsequently shuttled by either inter- or intra-molecular electron transfer chains
involving prosthetic groups such as heme or iron-sulfur clusters. In all organisms
studied so far, Moco is synthesized by a highly conserved multi-step biosynthetic
pathway. A deficiency in the biosynthesis of Moco results in a pleitropic loss of all
four human Mo-enzyme activities and in most cases in early childhood death. In this
review we first introduce general aspects of molybdenum biochemistry before we
focus on the functions and deficiencies of two Mo-enzymes, xanthine dehydroge-
nase and sulfite oxidase, caused either by deficiency of the apo-protein or a pleiotro-
pic loss of Moco due to a genetic defect in its biosynthesis. The underlying molecular
basis of Moco deficiency, possible treatment options and links to other diseases,
such as neuropsychiatric disorders, will be discussed.

Keywords cyclic pyranopterin monophosphate • mitochondria • molybdenum


cofactor • neurodegeneration • sulfite oxidase • S-sulfocysteine • substitution
therapy

Please cite as: Met. Ions Life Sci. 13 (2013) 415–450


13 Molybdenum in Human Health and Disease 417

1 Introduction

1.1 Chemistry and Biology of Molybdenum

Molybdenum is the only metal of the 2nd row (4d) of the periodic table with biological
activity. It is found in nature in different oxidation states ranging from zero to six
and forms complexes with organic and inorganic ligands with coordination numbers
between four and eight. Molybdenum is the 25th most abundant element in seawater
at an average concentration of 100 nM; whereas its concentration in continental water
is much lower (5 nM). In living organisms, molybdenum is present at low concen-
trations. In humans, highest Mo levels are found in kidney, liver, small intestine,
and adrenals [1]. In serum, the concentration is about 0.6 ng mL–1 [2], but depends
on dietary intake [3]. The oxyanion molybdate [MoVIO4]2– is the only known form
that cells can take up from the environment. In solution, molybdate can be chemi-
cally adsorbed onto positively charged iron, aluminium or manganese oxides [4].
Its availability increases as the pH increases, mainly due to decreased association
with metal oxides.
The discovery of molybdenum in enzymes such as nitrogenase, nitrate reduc-
tase (NR), and xanthine oxidase (XO) demonstrated the biological importance of
molybdenum as catalyst in the active site of those enzymes. Up to now, more than
50 different Mo-dependent enzymes have been found in all kingdoms of life [5].
Most of them are of bacterial origin and, except for nitrogenase, they all bind
molybdenum via a pterin-based prosthetic group forming the so-called molybde-
num cofactor (Moco). In eukaryotes, Moco (Figure 1a) is composed of a fully
reduced pterin backbone with a C6-substituted four-carbon side chain forming a
third pyran ring that hosts a terminal phosphate and the unique dithiolene group,
which binds molybdenum [5].

1.2 Molybdenum Uptake

All organisms, that depend on molybdenum, take up the oxyanion molybdate as


only source of molybdenum. In prokaryotes, molybdate enters the cell through
the action of proteins belonging to the ATP-binding cassette (ABC) transporters
family [6]. In contrast to bacteria, molybdate transport in eukaryotes is poorly
understood and the first eukaryotic molybdate transporters were identified only
recently [7,8]. Genome-wide sequence analysis revealed that orthologues of
bacterial ABC-type molybdate transporters are absent in eukaryotes. Given the
known interference between molybdate, sulfate, and phosphate transport in
plant cells, molybdate transporters were identified within the large and hetero-
geneous family of sulfate transporters [ 9 ]. The transporter MOT1 ( MO lybdate
Transporter, type 1 ) was simultaneously identified in the alga Chlamydomonas
418 Schwarz and Belaidi

reinhardtii and the plant Arabidopsis thaliana [7,8]. MOT1 exhibits both a
high-specificity and high-affinity transport for molybdate.
Physiological studies in Chlamydomonas suggested the presence of a second
transporter, which was recently identified as first member of the MOT2 family of
molybdate transporters. MOT2 belongs to the ubiquitous major facilitator super-
family (MFS) of transporters with orthologues in plants and animals. Saccharomyces
expressing the human member of the MOT2 family (HsMOT2) shows molybdate
uptake activity comparable to cells expressing MOT1 or MOT2 from Chlamydomonas
(Km = 546 nM) [10]. Therefore, HsMOT2 represents the first animal protein able
to facilitate molybdate transport. Future studies are required to characterize the
cellular localization and functional relevance of MOT2 proteins within the ssMo
homeostasis in higher eukaryotes.

1.3 Molybdenum Toxicity

Severe molybdenum toxicity has only been reported in ruminants. Under conditions
of high molybdate uptake, the reducing sulfide-rich gastrointestinal track promotes the
formation of tetrathiomolybdate (MoS42 −), which in turn readily reacts with CuI or
CuII to form insoluble copper-thiolate-molybdenum complexes [11]. Therefore, high
molybdenum intake causes a secondary copper deficiency and is called molybdenosis
or hypocuprosis [12]. Molybdenum toxicity in ruminants is characterized by severe
diarrhea, anorexia, greying of hair, anemia, and leg stiffness accompanied with
infertility or sterility. These symptoms are readily reversed by copper supplementa-
tion. The ability of tetrathiomolybdates to interact with copper has been used in
the treatment of copper-dependent disorders such as Wilson’s disease, which in the
absence of treatment results in hepatic and neurological dysfunctions due to intra-
cellular copper accumulation [13].
Monogastric animals are less sensitive to molybdenum toxicity. In humans, cases
of molybdenum toxicity are extremely rare and confined to geographic areas with
high amounts of molybdenum in drinking water or soils [14]. In some regions of
Armenia the population is exposed to high dietary molybdenum intake (10–15
mg/day as compared to 1–2 mg/day under normal conditions). In those individuals
aching joints and symptoms resembling gout have been reported, probably due to
increased XDH/XO activity causing elevated uric acid production, the major gout
(see Section 2.1.4). The tolerable intake for molybdenum has been estimated to be
9 μg Mo kg–1 day–1 [15].

1.4 Molybdenum Enzymes

Five Mo-enzymes are found in eukaryotes that belong to two families (Figure 1).
Nitrate reductase, sulfite oxidase (SO), and the amidoxime-reducing component
13 Molybdenum in Human Health and Disease 419

Figure 1 Structure of Moco in the two families of Mo-enzymes and the domain organization of
the four mammalian Mo-enzymes. (a) Chemical structure of Moco found in sulfite oxidase (SO),
with cysteine ligation, and Moco found in xanthine oxidase (XO), with a terminal sulfido ligand.
(b) Domain structure of mammalian SO, mitochondrial amidoxime-reducing component (mARC),
xanthine oxidoreductase (XOR), and aldehyde oxidase (AOX). Domains are depicted according
to their relative size. Moco and dimerization domains with similar structures are identified with
the same grade of gray shade. Other domains binding prosthetic groups are shown as white boxes.
D: dimerization domain, b5: cytochrome b5 domain, Fe-S cluster domain, FAD domain.

(mARC) are members of the SO family. In this family, Moco is covalently bound to
the enzyme via an invariant cysteine residue forming the third S-ligand in the
molybdenum coordination sphere.
The other so-called XO family is formed by two very homologous members,
xanthine dehydrogenase (XDH) or oxidase (XO), and aldehyde oxidase (AOX)
[16]. Eukaryotic Mo-enzymes are involved in key processes in the global carbon,
nitrogen, and sulfur cycles, such as nitrate reduction, sulfite detoxification, and
purine catabolism. Except nitrate reductase, which is only found in autotroph organ-
isms such as plants, fungi, and algae, all other four Mo-enzymes are expressed in
humans and will be discussed in the following sections (Figure 1).

2 Deficiencies in Molybdenum Enzymes

Deficiencies in one or more Mo-enzymes have been described in humans with


different pathophysiologies ranging from normal life to neonatal death. All defi-
ciencies belong to the large family of inborn errors in metabolism; however, the
numbers of known cases for the individual disorders are very low. Therefore, reliable
projections on incidence and prevalence cannot be drawn for deficiencies affecting
420 Schwarz and Belaidi

Mo-enzymes. In addition, considerable heterogeneity in symptoms and disease


progression further contribute to misdiagnoses and limited understanding of the
underlying pathophysiology. Only recently, novel therapeutic approaches have
fostered systematic analyses of patient cohorts. In the following sections we will
introduce the four mammalian Mo-enzymes, focus on the individual pathways
they are involved in, and discuss the clinical and cellular consequences of their
dysfunction.

2.1 Xanthine Dehydrogenase and Oxidase

2.1.1 Structure and Function

Eukaryotic XDH and XO (EC 1.17.1.4) are key enzymes in the degradation of
purines, catalyzing the two terminal steps in purine catabolism, the oxidation of
hypoxanthine to xanthine and xanthine to uric acid. The enzyme can function either
as a dehydrogenase using NAD+ as electron acceptor or, upon reversible oxidation
of two conserved cysteines, as an oxidase using dioxygen as terminal electron
acceptor. In contrast, proteolytic cleavage of XDH converts the enzyme irreversibly
into the XO form [17,18]. The protease, responsible for this cleavage remained
unknown until today, but it is thought to be localized to the mitochondrial inner
membrane space and might be released upon apoptotic permeabilization of mito-
chondria [19]. In the following we will use the term xanthine oxidoreductase (XOR)
for both forms of the enzyme.
XORs are homodimeric enzymes harboring three cofactor-binding domains
(Fe-S clusters, FAD, and Moco) and an additional domain important for dimeriza-
tion [20] (Figure 1b). Hydroxylation of purine substrates causes two-electron
reduction of Moco and the electrons are subsequently transferred singly via two
[2Fe-2S] clusters to the FAD cofactor where either NAD+ or dioxygen is reduced
in XDH or XO, respectively. The latter co-substrate produces superoxide anions,
which renders XO an important target for cellular stress responses. For more details
on structure-function relations in XOR see the latest review of Hille, Nishino, and
Bittner [21].

2.1.2 Xanthinuria Type I

Deficiency of XOR results in the accumulation of xanthine in urine (xanthinuria)


[22] with hypoxanthine being also elevated. Hereditary type I xanthinuria lacks
only XOR activity, while type II and molybdenum cofactor deficiency (MoCD) lack
two or more Mo enzyme activities, respectively. Type I xanthinuria patients carry
mutations in the XDH gene [23]; up to date only a handful cases have been geneti-
cally classified. The combined incidence for classical type I and type II xanthinuria
has been estimated to 1/69,000 [24].
13 Molybdenum in Human Health and Disease 421

The affected individuals by xanthinuria type I may develop urinary tract calculi,
acute renal failure, or myositis due to tissue deposition of xanthine, while some
subjects with homozygous xanthinuria remain asymptomatic [25]. The fact that
xanthine accumulation is much higher than the one of hypoxanthine suggests an
increased salvage of hypoxanthine, which was experimentally proven by feeding
studies using radio-labeled purines [26]. Given the relatively broad substrate speci-
ficity, XOR can hydroxylate a number of exogenous substrates such as thiopurines,
which are chemotherapeutics used for the treatment of acute lymphoblastic leuke-
mia and autoimmune diseases [27]. Therefore polymorphisms in the XDH gene may
increase the toxicity of drugs such as 6-mercaptopurine.

2.1.3 Xanthinuria Type II

Xanthinuria type II is characterized by the simultaneous loss of two Mo-enzyme


activities, XOR and AOX [23]. The molecular basis of this dual loss of Mo-enzyme
function is due to a mutation in the MCSU gene, encoding for a two-domain protein
catalyzing the sulfuration of Moco in enzymes of the XO family [28] (for details see
Section 3.1.4). Both types of hereditary xanthinuria are clinically similar, but
patients with type I retain their ability to metabolize allopurinol (via the activity of
AOX, see Section 2.1.4), those with type II xanthinuria cannot.
Both types result in plasma uric acid levels below 5 mmol/L and elevated plasma
xanthine concentrations (>10 mmol/L). Less than half of the affected people develop
symptoms, which are caused by deposition of xanthine in the urinary tract. This
often results in haematuria or renal colic, and rarely, in acute renal failure or chronic
complications related to urolithiasis [23]. In very few cases, muscle pains caused by
xanthine deposition have been reported [29]. In one case with hereditary xanthin-
uria type II the association with mental delay, autism, cortical renal cysts, osteopenia,
hair and teeth defects, and various behavioral symptoms was observed [30].
Although the underlying disease-causing mechanism remains unclear, xanthinuria
joins the growing list of metabolic disorders, such as phenylketonuria, histidinaemia,
dihydropyrimidine dehydrogenase deficiency, and 5’-nucleotidase superactivity,
contributing to the development of complex neuropsychiatric disorders [31].

2.1.4 Hyperuricemia

Hyperuricemia is characterized by an accumulation of uric acid resulting in an


increased urinary excretion. The major cause of the disease is seen in an imbalance
between the rates of production and excretion of uric acid. Primary hyperuricemia
is due to a decreased renal excretion of uric acid, while causes of secondary hyper-
uricemia are more complex and include increased XOR activity, increased purine
release due to chronic inflammation, increased purine synthesis and high dietary intake
of purines. Longstanding hyperuricemia leads to gout, which is characterized by the
deposition of urate crystals in the joints and periarticular structures. Hyperuricemia
422 Schwarz and Belaidi

and gout have been associated with the development of cardiovascular disease.
Epidemiological studies have shown that certain foods increase the risk of develop-
ing gout and hyperuricemia. Low-fat dairy products, purine-rich vegetables, whole
grains, nuts and legumes, and less sugary fruits, coffee, and vitamin C supplements
decrease the risk, whereas intake of red meat, fructose-containing beverages, and
alcohol increase the risk of gout [32]. Since more than 50 years, gout is effectively
treated with allopurinol, a suicide inhibitor of XOR, but recently, new drugs, developed
by structure-based drug design, have entered the clinics [33].
Recent data indicate that XOR also plays an important role in various forms of
ischemic and other types of tissue and vascular injuries, inflammatory diseases, as
well as chronic heart failure [34]. Therefore, uric acid and other purine-related bio-
markers gain increased interest in epidemiological studies of such diseases.
Furthermore, XOR has also been implicated in nitrite-dependent synthesis of nitric
oxide (NO) [35], which in turn would counteract hyperuricemia-related cardiovas-
cular disorders. However, pharmacological studies do not support such findings
[36]. Future studies are needed to increase our understanding of the roles of foods,
urate transporters and other molecular mechanisms on the risk of developing gout
as well as hyperuricemia and their relation to cardiovascular disorders.

2.2 Aldehyde Oxidase

AOX enzymes (EC 1.2.3.1) originate from a duplication of the xdh gene in eukary-
otes before the origin of multi-cellularity [37]. Therefore, both enzymes contain the
same cofactor-binding domains (Fe-S clusters, FAD, and Moco) as well as a dimer-
ization domain (Figure 1b). The human genome harbors a single AOX gene together
with two pseudogenes clustered on chromosome 2q. However, mouse and rat
genomes express four aox genes giving rise to four iso-enzymes [38]. The crystal
structure of AOX3 from mouse has been recently determined showing high struc-
tural similarity to XOR [39].
Similar to XOR, mammalian AOX produces superoxide and hydrogen peroxide
as secondary products. However, the major difference between the two enzymes
relies on substrate specificity. In fact, AOX exhibits a broad substrate spectrum
including heterocycles, aldehydes, purines, and pteridines [40]. AOX substrates are
often involved in the metabolism of drugs and xenobiotics and the presence of high
levels of human AOX in the liver renders this class of enzymes highly interesting in
the field of drug discovery [41]. Up to now, the physiological function and endog-
enous substrates of AOXs are unknown, although results from AOX-knockout mice
revealed a significant role in the synthesis and biodisposition of endogenous reti-
noids in the Hardarian glands and skin [42].
In addition, AOX may participate in the oxidation of endogenous products involved
in various metabolic pathways such as neurotransmitters (i.e., serotonin), the conversion
of the hydroquinone-precursor gentisate aldehyde into gentisate, the catabolism of
valine, leucine or isoleucine as well as vitamins (nicotinamide and pyridoxal) [40].
Besides acting as oxidase, a secondary function as reductase was also attributed to
13 Molybdenum in Human Health and Disease 423

AOX as it was found to catalyze the reduction of N-oxides, sulfoxides, nitro compounds,
and heterocycles under certain conditions [43–45]. However, the reductase activity of
AOX was only observed in vitro and its physiological relevance remains unclear. This
ample substrate specificity and diverse functionality of either the oxidase or reductase
rendered AOX very attractive in medical chemistry and toxicology. Thus, its function
as a drug-metabolizing enzyme has been confirmed for different xenobiotics such as
the antitumor agents, methotrexate [46], and 6-mercaptopurine [47], the antidepres-
sant, citalopram [48], and other compounds of medical relevance [41].

2.3 Sulfite Oxidase and Sulfite Toxicity

2.3.1 Cysteine Catabolism

The intracellular pool of cysteine is relatively small as compared to the much larger
pool of glutathione (GSH) [49]. Under oxidizing extracellular conditions, cysteine
is oxidized to cystine. Thus, the plasma cysteine concentration is low (10–25 μM),
compared with that of cystine (50–150 μM) [50]. Cysteine and cystine are trans-
ported by different membrane carriers, and cells have typically different transport
affinities for one or the other compound [51]. Hepatocytes have low or no capacity
for import of cystine. However, GSH is abundant in the liver and reduces cystine
extracellularly to cysteine, which is then imported into hepatocytes [50].
Cysteine undergoes catabolism via two pathways: an oxidative pathway
involving cysteine dioxygenase (CDO; EC 1.13.11.20) and a non-oxidative path-
way involving several enzymes (Figure 2). In mammals, the major route of cys-
teine catabolism follows the oxidation to cysteine sulfinic acid (CSA) catalyzed
by CDO [52]. This irreversible reaction involves the transfer of two oxygen
atoms to the sulfhydryl group of cysteine. CSA can be either decarboxylated in
the cytosol to form hypotaurine, which is further oxidized to taurine (Figure 2).
Taurine is the most abundant non-proteinogenic amino acid in the body and is
mainly produced in the liver [53]. Alternatively, CSA can be deaminated yield-
ing the putative compound β-sulfinylpyruvate, which spontaneously decomposes
into pyruvate and sulfite [52]. The latter undergoes oxidation to sulfate catalyzed
by SO (see Section 3.3.2).
The non-oxidative degradation pathway of cysteine involves the contribution of
one of the three enzymes, cystathionine γ-lyase (CSE; EC 4.4.1.1), cystathionine
β-synthase (CBS; EC 4.2.1.22), and 3-mercaptopyruvate sulfurtransferase (MSPT;
EC 2.8.1.2) (Figure 2). Given that all three enzymes are physiologically active with
other substrates, high Km values were found for cysteine, which do not correlate
with the physiological concentration of cysteine [54]. However, all three enzymes
have been shown to be involved in the cysteine-dependent production of hydrogen
sulfide [55], which was considered to be toxic as reported in several poisoning
cases [56]. However, the fact that significant levels of hydrogen sulfide have been
found in the brain of rats, humans, and cattle [57–59] suggested a functional role
as neural messenger [60].
424 Schwarz and Belaidi

Figure 2 Cysteine catabolism and altered metabolites in sulfite oxidase deficiency. Components
of the transsulfuration pathway (methionine to cysteine; light orange box), cysteine oxidative
(CDO-dependent; gray box), and non-oxidative catabolism (green box) are summarized with all
involved enzymes and metabolites. Changes in MoCD are highlighted in blue with corresponding
arrows indicating an increase or decrease in concentration in comparison to healthy controls.
Enzyme abbreviations are: MAT, methionine-S-adenosyl transferase; MT, methyl transferase;
SAHH; S-adenosylhomocysteine hydrolase; BHMT, betaine-homocysteine methyl transferase;
MS, methionine synthase; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase (cystathionase)
GCS, γ-glutamylcysteine synthetase; GS, glutathione synthetase CDO, cysteine dioxygenase;
CSD, cysteinesulfinate decarboxylase; AAT, aspartate aminotransferase; SO, sulfite oxidase;
MPST, 3-mercaptopyruvate sulfurtransferase; SQR, quinone oxidoreductase; SDO, sulfur dioxy-
genase; ST, sulfur transferase; KG, α-ketoglutarate.

Hydrogen sulfide is further oxidized to thiosulfate in mitochondria by three


sequential enzymatic reactions (Figure 2) [61]. First, the mitochondrial membrane
flavoprotein quinone oxidoreductase (SQR) converts sulfide to a protein-bound per-
sulfide and transfers two electrons to the ubiquinone pool [62]. Next, the persulfide
is handed over to a sulfur dioxygenase, which converts the persulfide molecule to
sulfite using molecular oxygen. Sulfite, generated by sulfur dioxygenase may also
13 Molybdenum in Human Health and Disease 425

be converted to sulfate by the action of SO in mitochondria [62]. Finally, a sulfur


transferase adds a second persulfide molecule from SQR to sulfite yielding the final
product thiosulfate [61].
The relative contribution of the non-oxidative pathway in cysteine catabolism is
usually low and insensitive to cysteine dietary intake [63]. In contrast, CDO concen-
tration and activity increases significantly in response to a higher dietary sulfur-
containing amino acid intake. Thus, it has been assumed that cysteine levels in the
body are predominantly controlled via CDO [64]. However, non-oxidative cysteine
catabolism is increased if CDO activity is lost as observed in CDO−/− mice, which in
addition to increased plasma cysteine and sulfate levels, revealed high concentra-
tions of hydrogen sulfide [65].

2.3.2 Sulfite Oxidase Localization, Structure, and Function

In vertebrates, SO catalyzes the two-electron oxidation of sulfite to sulfate coupled to


the reduction of two molecules of cytochrome c [66]. Sulfite oxidation represents the
terminal step in the oxidative degradation of cysteine. Vertebrate SO forms homodi-
mers and each monomer harbors an N-terminal cytochrome b5-type heme domain, a
catalytic Moco domain, and a C-terminal dimerization domain [67]. In vertebrates,
SO is localized in the mitochondrial intermembrane space where electrons derived
from sulfite oxidation are directly passed to the physiological electron acceptor cyto-
chrome c [68]. Recently, the maturation of mammalian SO has been clarified showing
that it combines a conventional leader sequence-based translocation mechanism with
the folding trap mechanism for which the presence of Moco is strictly required [69].
The catalytic mechanism of SO involves transfer of two electrons from sulfite to
Moco (MoVI → MoIV) followed by two one-electron transfer steps via the heme
domain to cytochrome c. Within the crystal structure of chicken SO a large distance
of 30 Å has been found between the heme and Moco domain [67], which would not
support the high electron transfer rate observed between the two domains by elec-
trochemical methods [70]. Based on the inhibition of internal electron transfer by
solution viscosity [71] and the use of deletions of the solvent-exposed tether con-
necting the heme and Moco domain, it was shown that during catalysis both domains
undergo conformational changes in order to enable efficient electron transfer [72].
SO is structurally very similar to nitrate reductase [73], which is only found in
plants, fungi, and algae. Regardless of the high structural similarity, SO is unable to
reduce nitrate to nitrite unless key residues are replaced by site-directed mutagene-
sis [74]. Interestingly, we recently found that SO is able to reduce nitrite to nitrate
under anaerobic conditions [75], providing a first and novel hint towards a second
function of SO in nitrite-dependent NO synthesis [76].

2.3.3 Sulfite Toxicity and Sulfite Oxidase Deficiency

Sulfite toxicity is characterized by an accumulation of sulfite, which can be detected


in urine of affected patients using commercial sulfite strip tests. Sulfite accumulation
and toxicity is observed either in isolated SO deficiency (SOD) caused by mutations
426 Schwarz and Belaidi

in the SUOX gene resulting in a loss of SO activity or in MoCD caused by mutations in


any of the genes within the Moco biosynthetic pathway resulting in loss of activity
of all Mo-enzymes [5,77]. SOD is less prevalent than MoCD and approximately
30 cases are reported up to now [78]. SUOX mutations affecting substrate binding,
specificity or catalytic activity were biochemically characterized [67,79,80].
SOD and MoCD are clinically nearly indistinguishable and patients of both
groups are characterized by a severe neurodegenerative phenotype manifested in the
early infancy by intractable seizures, hyper- and hypotonus, mental retardation,
developmental delay, and lens dislocation and patients usually die in early child-
hood (more details below) [77]. Given the overlapping clinical phenotype between
SOD and MoCD one can assume that SO is the most critical Mo-enzyme to ensure
survival during the neonatal period of life.

2.4 Mitochondrial Amidoxime-Reducing Component

Recently, two isoforms of the mitochondrial amidoxime-reducing component have


been identified in mammals, thus constituting a novel type of Mo-containing
enzymes [81]. Mouse mARC1 localizes to the outer mitochondrial membrane [82].
It is targeted by a bipartite N-terminal signal peptide leading to its tail-anchored
integration with cytosolic exposure of the protein. Besides a role of mARC2 in the
regulation of NO synthesis [83], no primary physiological substrates are known for
mARC proteins [84]. Human mARC proteins seem to be involved in pro-drug
metabolism given their ability to reduce several N-hydroxylated substances com-
monly used as pro-drugs.
All mARC proteins contain a C-terminal Moco-binding domain, which has been
proposed to form a novel class of molybdo-enzymes in plants [85]. In contrast,
studies in C. reinhardtii have demonstrated that a highly conserved cysteine residue
present in all mARC proteins is essential for catalytic activity, which is a typical
feature present in proteins of the SO family (Figure 1b) [86]. So far, all character-
ized mARC proteins require the formation of complexes with cytochrome b5 and
with a NADH/cytochrome b5 reductase for catalytic activity suggesting the forma-
tion of an intermolecular electron transfer chain [87].

3 Molybdenum Cofactor Deficiencies

MoCD is characterized by the simultaneous loss of all Mo-enzyme activities due to


a mutational block in the biosynthesis of Moco. Human MoCD is a rare autosomal
recessive disorder, which mostly affects neonates and is characterized by progressive
brain injury leading to early childhood death [88]. In the following sections we first
introduce the biochemistry of Moco and the genetics underlying MoCD before the
pathophysiology of the disease, models and treatment options will be discussed.
13 Molybdenum in Human Health and Disease 427

3.1 Biochemistry of Molybdenum Cofactor Biosynthesis

The structure and function of Moco is universal in all eukaryotes (Figure 1a) [5].
First genetic and later biochemical studies in fungi, bacteria, plants, and finally
animals (including humans) demonstrated that also the biosynthesis of Moco is
highly conserved in all kingdoms of life [89]. Eukaryotic Moco biosynthesis can
be divided into three major steps based on the two first identified intermediates
cyclic pyranopterin monophosphate (cPMP) [90], previously named Precursor Z
[91] and the metal-binding pterin (MPT) [92]. Each of the steps involve the action
of one or more proteins producing additional reaction intermediates (pyranopterin
triphosphate [93], thio-pyranopterin phosphate [94], and adenylated MPT [95])
(Figure 3).

3.1.1 Pterin Synthesis

Similar to the biosynthesis of other pterins and flavins, Moco synthesis starts with
GTP [96]. Labeling studies in E. coli proposed a complex and unique rearrange-
ment mechanism where the C8 atom of the purine base is removed as a formyl
group and subsequently inserted between the 2’ and 3’ ribose carbon atoms result-
ing in the formation of the four-carbon pyran ring [97,98] present in both, cPMP
and Moco. This reaction leads to the formation of the pterin backbone of the cofac-
tor (Figure 3). cPMP is the most stable biosynthetic intermediate with a half-life of
several hours, depending on the environment [99]. Its chemical structure has been
resolved first by high-resolution mass spectrometry and 1H NMR spectroscopy and
confirmed by 13C NMR showing its pyranopterin nature with an unusual geminal
diol [90,99].
The proteins MoaA and MoaC, catalyzing cPMP synthesis, have been best
studied in bacteria. Human proteins are encoded by the MOCS1 gene and will be
discussed later (Section 3.2.1). MoaA and homologous proteins are members of
S-adenosylmethionine (SAM)-dependent radical enzymes containing one or two
[4Fe-4S] clusters [100,101]. The N-terminal [4Fe-4S] cluster in MoaA promotes
SAM cleavage to generate a 5’-deoxyadenosyl radical, which initiates the transfor-
mation of 5’-GTP (bound to the C-terminal [4Fe-4S] cluster) by abstracting the
3’ proton from the ribose resulting in the formation of pyranopterin triphosphate
(Figure 3) [93]. This mechanism is believed to be conserved, given that plant and
human orthologues are able to complement E. coli moaA mutants [102,103]. The
second protein essential for cPMP synthesis is MoaC and it is involved in pyrophos-
phate release and formation of the geminol diol [96]. Plant proteins catalyzing
cPMP synthesis have been found to localize to mitochondria [104], which is consis-
tent with a recent finding for human MOCS1 proteins (see Section 3.2.1). The bio-
genesis of Fe-S clusters and high abundance of GTP within mitochondria might
have been the driving force for the subcellular localization of these proteins in
eukaryotes [105].
428 Schwarz and Belaidi

Figure 3 Biosynthesis of the molybdenum cofactor. Major and transient intermediates of the
three steps are shown. Co-substrates for each reaction step are depicted at the arrows.

3.1.2 Dithiolene Synthesis

In the second step of Moco biosynthesis two sulfur atoms are incorporated into
cPMP to form the dithiolene function in MPT. The reaction is catalyzed by MPT
synthase, a heterotetrameric complex built of two small and two large subunits that
13 Molybdenum in Human Health and Disease 429

stoichiometrically converts cPMP into MPT (Figure 3). The reaction mechanism of
MPT synthesis involves a stepwise transfer of sulfur, which is accompanied by the
formation of a mono-sulfurated intermediate (Figure 3) and hydrolysis of the cyclic
phosphate (thio-pyranopterin phosphate) [94]. Each small subunit of MPT synthase
carries a sulfur atom as thiocarboxylate [106], which is transferred by an ATP-
dependent reaction involving an adenylyltransferase [107]. While in bacteria sepa-
rate cysteine desulfurases can provide sulfide for the subsequent sulfuration reaction
following the hydrolysis of the protein-adenylate, in eukaryotes a rhodanese-like
domain (MOCS3) binds sulfur as persulfide at a conserved cysteine, which is
believed to mediate the subsequent sulfuration of the small subunit [108].

3.1.3 Molybdate Insertion

Once MPT is formed, it is ready to coordinate molybdenum via the dithiolene func-
tion. However, prior to molybdenum coordination, MPT is activated by adenyl-
ylation. While in bacteria two different proteins (MogA and MoeA) catalyze MPT
adenylylation [109] and molybdenum insertion [110], respectively, eukaryotic
organisms mostly use two-domain proteins (Figure 3) in which both activities have
been fused and in case of mammals, including humans, the resulting gephyrin rep-
resents a multi-functional protein [111]. Gephyrin’s G-domain binds MPT with
high affinity and forms adenylylated MPT as demonstrated by functional and struc-
tural studies [95,112]. Furthermore, in the complex structure of the gephyrin-
homologous Cnx1 G-domain with either MPT or MPT-AMP, a bound copper was
found at the dithiolene [95]. A possible role of this copper can be seen either in
protecting MPT from oxidation or in facilitating molybdenum insertion by provid-
ing a suitable leaving group. Following its synthesis by the G-domain, MPT-AMP
is transferred to the gephyrin E-domain where MPT-AMP is subsequently hydro-
lyzed in a reaction that is molybdate- and Zn2+- or Mg2+-dependent (Figure 3) [113].
Adenylylated molybdate has been proposed as reaction intermediate in analogy to
the adenylylated sulfate in sulfate assimilation [113].
Based on the fusion of two catalytic domains in gephyrin, intramolecular
product-substrate-channeling has been proposed. Recently, we developed a fully
defined in vitro system for Moco biosynthesis allowing the direct comparison of
the reaction rates of the individual gephyrin domains with holo-gephyrin [114]. In
contrast to the isolated domains, holo-gephyrin exhibited a 300-fold increased
ATP-dependent Moco synthetic activity with a Km for molybdate being close to the
intracellular concentration of molybdate as well as the Km of the respective molyb-
date transporter [114]. As side effect of the orientation of the fused domains in
gephyrin, novel functions have evolved, one of which rendering gephyrin an
instructive and essential protein in synaptogenesis [111]. Therefore, in the central
nervous system, gephyrin functions in addition to Moco synthesis, as scaffolding
protein at inhibitory synapses where it binds to glycine and γ-amino butyric acid
type A receptors [115].
430 Schwarz and Belaidi

3.1.4 Maturation of Moco

Following the release of Moco from the biosynthetic machinery, the cofactor either
binds directly to enzymes of the SO family or it undergoes additional modification,
in case of enzymes of the XO family. In those cases, one of three oxo ligands of
Moco is replaced by a terminal sulfido ligand. In contrast, in enzymes of the SO
family the third S-ligand is derived from the apo-enzyme by non-enzymatic ligation
to a conserved cysteine residue. In all cases, the third sulfur ligand occupies one of
the four corners of the square pyramidal coordination of molybdenum ligands
(Figure 1).
In humans, the sulfuration of Moco is catalyzed by a Moco sulfurase (MCSU)
[116]. In vitro studies using the plant orthologue (ABA3) demonstrated its ability to
sulfurate Moco in holo-enzymes of the XO family [117], while in vivo it remains
unclear whether the transfer of sulfur to Moco takes place before or after cofactor
insertion into apo-enzymes. Human MCSU and orthologues present two-domain
proteins with an N-terminal cysteine desulfurase domain and a Moco sulfurase
C-terminal domain (MOSC) that binds Moco and is believed to mediate protein-
protein interactions with apo-enzymes [118,119]. Following the desulfuration of
L-cysteine, a protein-bound persulfide is formed on a conserved cysteine and subse-
quently transferred to bound Moco [120].

3.2 Genetics of Human Molybdenum Cofactor Synthesis

Human genes and gene products involved in Moco synthesis are named according
to the MOCS nomenclature (Mo Cofactor Synthesis) [121] with the exception of
GEPHYRIN, which has been identified earlier as neuronal scaffolding protein
[122]. Due to its “bridging” function between inhibitory neuroreceptors and the
subcellular cytoskeleton, the greek word for bridge “gephos” has been chosen and
kept, regardless of the identification of GEPHYRIN’s primary function in Moco
synthesis.

3.2.1 Structure and Organization of Moco Synthesis Genes

Shortly after the identification of the first human Moco synthesis gene MOCS1
[121], the other three genes in the pathway (MOCS2, MOCS3, and GEPHYRIN)
have been identified based on high homologies of their gene products to plant and
bacterial Moco synthetic proteins [107,111,123]. Remarkably, MOCS1 as well as
MOCS2 show a bicistronic gene structure suggesting a highly coordinated expres-
sion of the encoded gene products. In contrast to MOCS2, the predicted expression
of two open reading frames from the MOCS1 mRNA, MOCS1A and MOCS1B,
encoding for MoaA- and MoaC-homologous proteins could not be verified [103].
Instead, alternative splicing of the MOCS1A transcript was found to result in the
13 Molybdenum in Human Health and Disease 431

Figure 4 Types of molybdenum cofactor and Mo-enzyme deficiencies. (a) Three major steps of
Moco synthesis, the involved genes and their translation products. Note, GEPH-G and GEPH-E
represent the two functional domains of GEPHYRIN catalyzing two separate steps in Moco
synthesis, respectively. MCSU catalyzes the sulfuration of Moco, which is essential for XOR and
AOX activities. Patients with mARC and AOX deficiencies have not been reported yet. (b) MRI
scans of a MoCD type A patient recorded at an age of 12 and 27 days showing the rapidly progressing
brain damage resulting in brain atrophy and cystic changes in the cerebral cortex.

expression of either MOCS1A alone (splice type I) or MOCS1AB fusion proteins


(splice type II and III, Figures 3 and 4) [124]. The latter only harbors MOCS1B
activity due to the truncation of functionally important C-terminal residues within the
C-terminus of MOCS1A [103]. Besides alternative splicing of exon 9 (producing
splice types II and III) [124], additional splice variants resulting from alternative
splicing of exon 1 have been found [125,126]. Very recently, we could demonstrate
that MOCS1 proteins are localized to mitochondria (similar to their plant ortho-
logues, Roeper and Schwarz, unpublished results). Mitochondrial translocation of
MOCS1A is dependent on exon 1a, while MOCS1AB fusion proteins are translo-
cated also in the absence of exon 1a using another internal targeting signal encoded
by exon 10, which drives MOCS1AB into the inner mitochondrial membrane.
Human MPT synthase is encoded by the bicistronic MOCS2 gene. The two over-
lapping open reading frames (MOCS2A and MOCS2B) are translated by a ribo-
somal leaky scanning mechanism producing both subunits in an approximately
equimolar ratio (Figure 4) [123]. Similar to MOCS1, a fusion of both proteins would
432 Schwarz and Belaidi

abolish the function of the first translation product (small subunit of MPT synthase),
due to the functional requirement of the conserved C-terminal double glycine motif
in thiocarboxylation and subsequent sulfur transfer. MOCS3 encodes for the Moco
sulfurase and has been identified based on sequence homology to plant and E. coli
proteins [107]. Both, MPT synthase (MOCS2A-MOCS2B) as well as the sulfurase
(MOCS3) are localized in the cytosol.
The GEPH gene encodes for the multi-domain cytosolic protein GEPHYRIN
composed of an N-terminal G-domain (GEPH-G), central C-domain, and a
C-terminal E-domain (GEPH-E, Figure 4), which is membrane-associated in the
central nervous system due to its interaction with glycine and GABA type A recep-
tors [115]. In addition to brain and spinal cord, high levels of GEPHYRIN expres-
sion have been identified in liver and kidney [127]. The GEPH gene is highly mosaic
with 27 exons distributed over 760 kb of genomic DNA on chromosome 14q32. At
least six of these exons are subject to alternative splicing producing more than 10
different splice variants [128]. Functional diversity of GEPHYRIN is believed to
rely on a tissue-specific expression of alternatively spliced transcripts [115]. For
example, variants containing the C3 cassette are highly abundant in liver, the organ
with highest Moco synthesis, while variants containing different forms of the C4
cassette were found in brain and spinal cord, where gephyrin is mainly functioning
as receptor scaffold [128,129]. This finding is supported by recent studies showing
that different gephyrin splice variants, which were expressed in Sf9 insect cells,
bind to the glycine receptor with different affinities [130].

3.2.2 Mutations in Molybdenum Cofactor-Deficient Patients

Following the identification of MOCS1 and MOCS2 genes, mutations in patients


with MoCD have been identified [121,131] as well as prenatal diagnosis in carrier
families has been successfully established [132]. Today more than 64 disease-
causing mutations have been identified in MOCS1, MOCS2 and GEPHYRIN, repre-
senting over 100 reported cases worldwide [133]. In nearly all cases, disease-causing
mutations result in a complete loss of enzyme function due to frame shifts, splice
site and nonsense mutations, or missense mutation affecting highly conserved or
invariant residues (Figure 5).
The majority of MoCD patients carry mutations in the MOCS1 gene affecting
either MOCS1A function or the MOCS1B domain in splice type II and III transla-
tion products (Figure 4) [134], representing approximately two thirds of all MOCD
patients identified so far. Up to date at least 40 disease-causing MOCS1 mutations
have been found. In most cases homozygous mutations were found due to the high
consanguinity of heterozygous parents, while compound heterozygous cases are the
minority.
Approximately one third of MOCD patients are affected in the second step of
Moco synthesis due to mutations in the MOCS2 gene. 23 disease-causing mutations
have been found in both open reading frames affecting either the small (MOCS2A)
or large subunit (MOCS2B) of MPT synthase. Interestingly, up to date not a single
13 Molybdenum in Human Health and Disease 433

Figure 5 Multiple sequence alignment of MOCS1A and MOCS1B translation products with
plant Cnx2 and Cnx3 as well as E. coli MoaA and MoaC proteins, respectively. Identified mutations
resulting in amino acid substitutions, frame shifts (fs) or deletions are shown above the aligned
sequences. Note, missense mutations were only found at invariant positions.

MoCD patient has been identified with a MOCS3 mutation. Given the dual function
of MOCS3 in Moco synthesis as well as tRNA thiolation [135], a combination of
both might not be vital.
Mutations in GEPH are very rare. Only two cases have been described so far.
The first patient was the last of three affected infants born to a Danish mother and
father who were cousins. All three infants died in the neonatal period (day 12, 29, and
3, respectively), with typical symptoms of MoCD [136]. Genetic analysis revealed
an early stop codon resulting in a total loss of GEPHYRIN expression. As a result,
434 Schwarz and Belaidi

both functions of GEPHYRIN, Moco synthesis as well as glycine and GABA receptor
clustering are impaired. In light of the neurological phenotype of both disorders
(MoCD and impaired synaptic inhibition) [77,137], a worsening of each of the two
isolated conditions can be assumed (see also next section). A second case with mis-
sense mutation in GEPH affects an invariant aspartate residue (Asp580) within the
active site of the GEPHYRIN E-domain. This patient presented typical symptoms
of MoCD, was two years old at the time of report [138] with severe axial hypotonia,
peripheral hypertonia, and lack of head control and visual contact. Based on the
clinical presentation as well as the underlying genetic defect, one can assume that in
this patient Moco synthesis, namely the hydrolysis of MPT-AMP and molybdenum
insertion is prohibited while GEPHYRIN’s function in receptor clustering is
retained given that the binding site of both, glycine [139] and GABA receptors
[140] are distinct from Moco synthesis [113].
The vast majority of MoCD patients present a very severe neurological pheno-
type. Only a handful cases have been reported with mild forms of the disease. To our
knowledge, only one mild presentation was reported for MoCD type A deficiency
affecting the splicing of MOCS1 exon 9 probably affecting the mitochondrial matu-
ration and/or targeting of MOCS1AB translation products, thus leading to a reduced
but not completely absent cPMP synthetic activity. All other mild cases carry muta-
tions either in MOCS2, thus showing only partially impaired MPT synthesis [141]
(G. Schwarz, unpublished results) or mutations in the SUOX gene [142–144].

3.2.3 Biochemical Classification of Molybdenum Cofactor Deficiency

MoCD can be grouped into three types according to the underlying genetic defect
(Figure 4a). Type A deficiency affects two-thirds of all patients and is caused by
mutations in the MOCS1 gene [133]. While type A patients lack the first Moco
intermediate cPMP, type B patients accumulate cPMP due to defects in the MOCS2
gene, which encodes the MPT-synthase [132]. Biochemical analysis of urine sam-
ples using HPLC and reverse phase chromatography can discriminate between type
A and type B patients by quantifying the cPMP oxidation product compound Z,
which accumulates in urine of type B patients and is completely absent in type A
patients. The two GEPHYRIN-deficient cases belong to type C MoCD deficiency
[136,138].

3.3 Pathophysiology of Molybdenum Cofactor Deficiency

3.3.1 Clinical Presentation of Patients with Molybdenum


Cofactor Deficiency

Duran et al. described the first case of a human patient with MoCD in 1978 [145].
The patient presented in his neonatal period initial feeding difficulties, therapy-
resistant seizures, high pitch crying followed by severe neurological abnormalities,
13 Molybdenum in Human Health and Disease 435

lens dislocation of the eyes, and major dysmorphic features of the head. At the time
of identification, the chemical nature of Moco was not known, neither its biosynthe-
sis. Based on the identified alterations in the biomarkers of two Mo-dependent
enzymes, XO and SO, a defect in either molybdenum metabolism or transport
has been proposed [145]. Since then more than 100 cases with MoCD have
been reported [77,134], which nearly all share a predominant deterioration of the
central nervous system as main disease feature mimicking hypoxic ischemic
encephalopathy during the first days of presentation [146].
In general, first symptoms are observed within the first days of life, which are
initially presented by feeding difficulties accompanied with intractable seizures
with a predominant opisthotonus and an exaggerated startle reaction [138]. Disease
progression is accompanied by psychomotor retardation due to progressive cerebral
atrophy and ventricular dilatation, which are typical in brain MRI of patients
(Figure 4b). In addition, major radiological features of the disease include global
cerebral edema, cystic encephalomalacia, cortical and white matter atrophy, focal or
bilateral changes within the globi pallidi and subthalamic regions, dysgenesis of the
corpus callosum, and ventriculomegaly [146–148]. Patients that survive the neona-
tal period show essentially no neuronal development, are unable in any coordinated
movements, require tube feeding and show no signs of communication with their
environment and usually die within their first years of life [77].

3.3.2 Molecular Basis of Neurodegeneration

In humans, MoCD is clinically nearly indistinguishable from the less prevalent iso-
lated SOD implicating sulfite toxicity as a major underlying cause triggering neuro-
toxicity in MoCD patients. SO, which is localized in the mitochondrial intermembrane
space oxidizes sulfite to sulfate, thus no accumulation of sulfite in the cytosol or
extracellular compartments is seen under wild-type conditions [67]. In MoCD and
SOD, sulfite accumulates first in mitochondria where it has been shown to increase
reactive oxygen species [149]. Sulfite also decreases ATP synthesis in mitochondria
when respiring on glutamate (which is the case in the brain), while using other
respiratory substrates such as malate, α-ketoglutarate, and succinate did not show
any sulfite vulnerability [149]. The mechanism underlying the inhibition of ATP
synthesis has been related to glutamate dehydrogenase inhibition by sulfite, which
in brain, due to the fact that glutamate dehydrogenase operates in the direction of
oxidative deamination, will lead to a decrease in the availability of α-ketoglutarate
and other tricarboxylic acids resulting in an overall decrease in ATP synthesis [149].
Knowing that glutamate itself is neuroexcitotoxic and is the precursor of the inhibi-
tory neurotransmitter GABA, inhibition of glutamate dehydrogenase may also
affect the metabolism of those neurotransmitters, which would be one explanation
for the accelerated injury in neuronal rather than non-neuronal tissue in MoCD.
In the absence of SO activity sulfite crosses the outer mitochondrial membrane
and accumulates in cytosol and later in plasma. Sulfite is a strong reductant and
will therefore reduce disulfide bridges, primarily in membrane and extracellular
proteins, thus affecting their folding, stability, and activity. Probably first, sulfite
436 Schwarz and Belaidi

reacts with cystine leading to the formation of the secondary metabolite


S-sulfocysteine (SSC) [5]. SSC is very abundant in MoCD patients and its excretion
in urine is detectable shortly after birth and increases with age [150], which sup-
ports the view that sulfite is cleared during maternal gestation [151]. SSC is struc-
turally similar to glutamate, is able to bind to NMDA receptors and therefore
postulated as main agent responsible for seizure development and subsequent brain
damage in MoCD [152]. In fact, early studies in rats demonstrated that subcutane-
ous administration of SSC induces the same type of brain damage that glutamate
and other excitatory amino acids are known to cause [152]. In contrast to the neu-
rotransmitter glutamate, whose release in the extracellular compartment is highly
controlled by vesicular fusion and cellular re-uptake, SSC is continuously produced
by sulfite accumulation. In the absence of a specific transporter, SSC is assumed to
persist in the extracellular compartment thus potentiating its excitotoxic effect
leading to neuronal death. Besides SSC, taurine is also elevated in MoCD [153] and
known to be neuroprotective by playing an important role in glutamate and GABA
signaling [154], however, this positive effect seems to be erased by SSC toxicity.
SSC formation goes hand in hand with cystine depletion [77]. Cystine is the
major transport form of cysteine in plasma. In the brain, cystine is taken up into glial
cells, where it is reduced and incorporated into glutathione, the major antioxidant
in neuronal tissue and most abundant low-molecular-weight thiol in animal cells
(0.5–10 mM) [50]. Most of the GSH (85–90%) is present in the cytosol, while
relatively low GSH concentrations are found in plasma (2–20 μM) [155,156].
An increase in cysteine supply via oral or intravenous administration enhances GSH
synthesis and prevents GSH deficiency in humans [157]. Thus, cysteine is generally
considered to be the limiting amino acid for GSH synthesis in humans, rats, and
pigs [49,158,159] and its depletion will have a major impact on cell viability.
Despite the dramatically reduced levels of plasma cystine in MoCD, no information
is available regarding GSH concentrations in affected patients, which together with
SSC, cystine, and glutamate levels in cerebrospinal fluid may further contribute to
the understanding of the mechanism of neurodegeneration in MoCD.

3.4 Animal Models

3.4.1 Mocs1-Deficient Mice

Due to the high prevalence for type A MoCD, a mocs1-knockout mouse was gener-
ated by homologous recombination with a targeting vector [160]. Homozygous
mocs1−/− mice displayed a severe phenotype characterized by a retarded growth,
abnormal behavior, lack of feeding and they died within the first 11 days of life with
an average life span of 7.5 days [160]. As observed in humans, biochemical charac-
terization of mocs1−/− mice revealed markedly elevated urinary sulfite and xanthine
levels while uric acid was undetectable. Furthermore, neither MPT nor Moco could
be detected in homozygous mice and as a result, Mo-enzyme activities were absent.
13 Molybdenum in Human Health and Disease 437

In contrast to humans, no brain atrophy or any other kind of histological damage


could be observed, which could be due to a different developmental stage of mice in
comparison to humans, presenting the last trimester of human brain development
in comparison to neonatal mice. However, in terms of biomarkers and disease
progression (survival), mocs1−/− mice present a suitable animal model to study the
molecular basis and treatment of human MoCD.

3.4.2 Gephyrin-Deficient Mice

Gephyrin-deficient mice present a severe phenotype resembling that of humans


with hereditary MoCD and hyperekplexia, a failure of inhibitory neurotransmission
[161]. This animal model was instrumental in demonstrating the dual role of gephy-
rin in Moco synthesis [111] and receptor clustering. Geph−/− neonates appeared
externally normal, and failed to suckle. In response to mild tactile stimuli, they
retained rigid with a hyperextended posture and exhibited apnea (difficulty in
breathing) by 12 hours after birth being consistent with impairment of inhibitory
glycinergic inputs to motoneurons. Given the hyperekplectic presentation and a
maximal survival of 1 day after birth renders the gephyrin deletion clearly more
severe than MoCD as seen for mocs1−/− mice. On the other hand, motor defects seen
in geph−/− mice occurred earlier with more severe presentation than those observed
in mutant mice that lack the glycine receptor α1 subunit or have reduced levels of
the GlyR β subunit [162]. In light of both observations and the underlying
pathomechanism of MoCD, we propose that the excitotoxic action of accumulating
SSC due to gephyrin-dependent MoCD is worsened by the lack of appropriate syn-
aptic inhibition, caused by the loss of gephyrin-dependent receptor immobilization
in the central nervous system.

3.5 Treatment of Molybdenum Cofactor Deficiency

3.5.1 Treatment of Mocs1-Deficient Mice with cPMP

Following the establishment of an animal model (mocs1−/− mice) for human MoCD
type A, a cPMP fermentation and purification procedure from E. coli has been
developed [90]. To probe that mocs1−/− mice are still able to convert cPMP into MPT
and to determine the required dose, purified cPMP was titrated to crude liver extracts
of mocs1−/− mice and in vitro Moco synthesis was quantified as a function of cPMP
added. Based on those results, an initial dose of 1 μg cPMP per animal was deter-
mined and injected in the liver of Mocs1-deficient mice. cPMP-treated Mocs1-
deficient mice developed normally, gained weight and reached adulthood, were
fertile and not distinguishable from their wild-type littermates [163]. Furthermore,
improvement of the treated animals was directly correlated with cPMP injections as
withdrawal of cPMP caused death within 10–14 days following the final injection.
438 Schwarz and Belaidi

Consequently, a dose and treatment interval-dependent restoration of Moco synthesis


as well as Mo-enzyme activity was observed. In conclusion, a first experimental and
causal treatment approach using cPMP substitution has been established for type A
MoCD [163].

3.5.2 Treatment of MoCD Type A Patients with cPMP

Based on the promising results of cPMP substitution therapy in Mocs1-deficient


mice, cPMP fermentation and purification has been upscaled allowing a first
human exposure following intensive discussion with the Regulatory Authorities
[150]. Prior to treatment, patient’s urine and plasma levels revealed markedly
elevated levels of SSC, thiosulfate, and xanthine, low uric acid, and elevated
urine sulfite and undetectable cPMP in urine, being indicative for MoCD type A,
which has been confirmed by genetic analysis [150]. The devastating character
of the disease was manifested by a rapid and continued increase of urinary sul-
fite, thiosulfate, and SSC levels during the first 36 days of life before starting the
treatment. Furthermore, magnetic resonance imaging showed diffuse cerebral
edema with an elevated lactate peak in the magnetic resonance spectroscopy at
day six and the infant had a markedly abnormal electro encephalogram at seven
days of age.
Treatment of the patient started on day 36 of life and sulfite, SSC, xanthine, and
uric acid were used as biomarkers to monitor treatment efficacy. As starting dose,
80 μg cPMP per kg body weight were chosen based on previous studies in Mocs1-
deficient mice [164] and applying the conversion factor from “mouse to men” of
12.3. Within days after treatment started, the patient showed a remarkable normal-
ization of MoCD biomarkers with sulfite disappearing after 3 days from urine and
SSC dropping continuously during the first week of treatment and reaching near
normal levels following a stepwise dose adjustments to 240 μg/kg body weight.
Xanthine and uric acid normalization was observed within two weeks of treatment.
Clinically, the patient became more alert 48 hours after treatment started; convul-
sions and twitching disappeared within the first two weeks and epileptic discharges
were markedly reduced [150]. Today, the patient is more than five years of age,
shows a delayed neurological development with profound cerebral palsy due to the
preexisting brain damage before treatment was initiated. The patient is alert,
interacts with family members, can sit and stand with support and shows normal
sleeping pattern (Veldman et al., unpublished results).
Based on the first index case, a treatment plan has been developed for cPMP
therapy of MoCD patients and currently six MoCD patients receive cPMP treat-
ment, all showing a similar biochemical and clinical improvement as the index case
(Schwahn et al., unpublished results). Depending on the time of treatment initiation,
patients can reach an almost normal neurodevelopmental outcome [165]. Very
recently, the chemical synthesis of cPMP has been reported, providing a key
milestone in the clinical development of cPMP therapy [166].
13 Molybdenum in Human Health and Disease 439

3.5.3 Treatment of MoCD Type B and C Patients

Treatment of the most frequent MoCD type A deficiency was fostered by the chemi-
cal nature of cPMP, which presents a fully reduced pterin with a relatively long
half-life [99] as compared to other reduced and clinically relevant pterins such as
tetrahydrobiopterin [167]. In contrast, MoCD type B patients are unable to synthe-
size MPT and therefore a substitution therapy with either MPT or mature Moco
would be required. Neither MPT nor Moco have been stably isolated in a protein-
free form yet and are therefore not available for substitution therapies [5].
MoCD type C has only been reported in two cases [134,136]. Given the overall
increased severity of a gephyrin loss of function in both, an animal model [161] as
well as a patient [136], we assume that most cases remain undiagnosed due to their
early neonatal death. Biochemical studies with gephyrin-deficient fibroblasts from
either mouse (L929 cells) [168] or human [136] suggest that in the complete absence
of gephyrin, molybdate excess results in the formation of Moco by chemical liga-
tion of molybdenum to MPT. However, this reaction is only possible, when MPT is
present in high quantities, while MPT-AMP, the reaction product of the G domain
of gephyrin cannot be activated by molybdate. Therefore, the second gephyrin
patient carrying a point mutation within the E-domain [138] would not have bene-
fited from molybdate substitution therapy. In conclusion, we propose that gephyrin
patients with missense mutation within the G-domain that do not impair the synap-
tic function of gephyrin, should be considered for molybdate supplementation.

3.5.4 Dietary Restriction and Treatment of Sulfite Oxidase Deficiency

Knowing that sulfite is the main toxic compound accumulating in MoCD and SOD,
early attempts to reduce its formation by reducing the dietary intake of sulfur amino
acids in patients have been conducted. Boles and colleagues reported a short-term
rapid decrease in urinary sulfite following methionine restriction and cysteine sup-
plementation in a single MoCD patient [169]. However, this improvement could not
be reproduced in a SOD patient [170]. In contrast, increase in urinary sulfite was
observed upon cysteine supplementation and treatment was stopped after four
weeks [170]. Investigation of cysteine catabolism in mammals clearly demonstrated
that increase in dietary cysteine levels are directly correlated with an increased
activity of CDO as the first and rate-limiting cysteine-catabolizing enzyme [171].
Following CDO reaction, either sulfite or taurine are formed when cysteine is sup-
plemented [171]. Thus, it is not surprising that on a long-term therapy, a continuous
cysteine supplementation will lead to an increase in the formation of sulfite and
S-sulfocysteine.
The short-term response to an increased cysteine supplementation reported in a
SOD patient is probably due to the sulfite-chelating capacity of cysteine, which
initially leads to a rapid decrease in sulfite concentration. In contrast, a low protein
diet with reduced intake of both sulfur amino acids methionine and cysteine were
440 Schwarz and Belaidi

effective in reducing sulfite, thiosulfate and S-sulfocysteine in two patients with a


mild form of SOD [143]. Furthermore, both patients grew normally without any
sign for neurological deterioration and show evidence of progress in psychomotor
development. Thus, a control of plasma cysteine and methionine levels should be
considered in both MoCD and SOD. As reduction of methionine and cysteine also
lead to sulfate and taurine decrease, a supplementation with those compounds may
reduce possible side effects resulting from their deficiencies [143]. A patient with
SO deficiency presenting a mild phenotype was recently reported, who showed a
beneficial development after a restricted diet in sulfur amino acids, suggesting
a residual activity of SO in that case [144].

4 Association of Molybdenum with Other Disorders

4.1 Copper Homeostasis Disorders

Copper has been found to bind to MPT and MPT-AMP [95]. It is known that molyb-
denum can act antagonistically to copper. The shortage of molybdate in Australian
farmland triggered excessive fertilization, resulting in molybdate overload of the
soil that caused pathologic symptoms of molybdenosis in animals, which in particu-
lar in ruminants triggered secondary copper deficiency [172]. Later, these
Mo-induced conditions of copper deficiency revealed the pathology of two human
Cu homeostasis disorders: Menkes (Cu deficiency) and Wilson’s (Cu overload) dis-
eases [173]. Consequently, potent Cu chelators such as tetrathiomolybdates were
used to treat Wilson’s disease and a number of other disorders that are linked to Cu
homeostasis, such as neurodegeneration, cancer, and inflammation [174]. For exam-
ple, tetrathiomolybdates are used to inhibit metastatic cancer progression, however,
the molecular mechanism is poorly understood [175]. The underlying chemistry of
the chelation of either copper or Mo to dithiolates most likely explains their antago-
nistic function towards each other.
The function of copper in Moco synthesis has not been clarified yet. Therefore,
future studies are needed to probe the impact of copper homeostasis on Moco syn-
thesis and Mo-enzyme activities. Our own in vitro synthesis studies using protein-
bound MPT-AMP showed an inhibition of Moco synthesis in the presence of 1 μM
CuCl2, providing a link between Mo and copper metabolism [95]. Copper inhibition
of Moco synthesis can be explained by inhibition of the Mg-dependent molybdate
insertion reaction. This finding might suggest that Moco biosynthesis might be
affected under conditions when cellular copper concentrations are increased, as
seen in patients with Wilson’s disease [173], where copper accumulates in liver and
brain, resulting in severe damage of both organs. In contrast, copper deficiency
might also impact Moco biosynthesis. Patients with impaired copper uptake
(Menkes disease) are characterized by hypotonia, seizures, mental retardation,
13 Molybdenum in Human Health and Disease 441

developmental delay and other neurological features. It remains to be elucidated to


which extend an underlying deficiency in Moco synthesis might contribute to the
disease pathology.

4.2 Epilepsy and Neuropsychiatric Disorders

Given the dual functions of gephyrin in Moco synthesis and receptor clustering, the
spectrum of altered gephyrin function is very complex. While deletion or missense
mutations in the GEPH gene result in a very severe MoCD phenotype with loss of
synaptic inhibition, there are also subtle cases of altered GEPHYRIN expression
resulting in various neuropsychiatric disorders.
Mutations in the GEPH gene that do not affect its catalytic function in MoCD
result in hyperekplexia, an impairment in inhibitory synaptic transmission [176].
The GEPH gene has also been associated with cancer development, given that a
fusion with the mixed lineage leukemia gene results in leukemia by transforming
hematopoietic progenitor cells [177]. We have found that stress-induced mis-splicing
of GEPH transcript results in the exclusion of exons 4 to 8 producing truncated
GEPHYRINS, which are able to curtail the function of wild-type GEPHYRIN by
dominant negative interactions [178]. As a result, individuals expressing such
GEPHYRIN variants develop temporal lobe epilepsy. In another study, rare genomic
deletions of GEPH have been found in patients with autism, schizophrenia, and
seizures [179]. Therefore, GEPHYRIN represents a novel contributor to the develop-
ment of complex neuropsychiatric disorders. The investigation of GEPHYRIN’s
Moco biosynthetic activity in such diseases might help to develop new biomarkers
for the diagnosis and progression of neuropsychiatric disorders.

4.3 Ethylmalonic Encephalopathy

Ethylmalonic encephalopathy (EE) is similarly to MoCD an autosomal recessive


inborn error of metabolism caused by defects in the ETHE1 gene, which encodes a
β-lactamase-like, iron-coordinating metalloprotein localized within the mitochon-
drial matrix [180]. As observed in MoCD, EE symptoms develop shortly after birth
and include typical neurological features such as delayed development, encephalopa-
thy, seizures, as well as microangipathy, hypotonia, and chronic diarrhea. ETHE1 is
a mitochondrial sulfur dioxygenase involved in hydrogen sulfide metabolism and
loss of its function leads to an accumulation of hydrogen sulfide and inhibition of
cytochrome c oxidase and short-chain fatty acid oxidation [180]. Besides increased
hydrogen sulfide levels, ETHE-knockout mice and human patients are characterized
by a massive excretion of thiosulfate in urine, a biochemical feature, which is shared
with MoCD. However, hydrogen sulfide and thiosulfate increase was not accompa-
nied by sulfite increase in ETHE1-knockout mice [62].
442 Schwarz and Belaidi

5 Concluding Remarks and Future Developments

Molybdenum is crucial for the survival of all mammals. A deficiency in one of the
four Moco-dependent enzymes can either be asymptomatic in some cases (XDH
deficiency) or lethal in other cases (SOD). MoCD, however, is in nearly all cases a
severe inborn error in metabolism and characterized by a rapidly progressing neuro-
degeneration. Although we begin to understand the underlying mechanism causing
the catastrophic brain damage, future studies are needed to identify key players in
metabolism that initiate neuronal cell death. Here, mitochondrial dysfunction is
very likely to present a crucial entry point of signals contributing to cell death.
Furthermore, the pathogenesis of multi-factorial neurodegenerative disorders such
as Huntington’s disease might be dependent on an altered basic metabolism that
involves Mo-dependent enzymes. In addition, cysteine homeostasis, which is
dependent on a tight control of numerous S-containing intermediates might contrib-
ute to the pathogenesis of such disorders too. In this context, longevity has been
show to be dependent on dietary restriction in methionine intake [181]. This in turn
suggests that high sulfur turn over, probably via the oxidative catabolism of cyste-
ine, is a negative predictor of life span, which is supported by the finding that lon-
gevity is increased when the trans-sulfuration pathway is increased. In conclusion,
SO-dependent sulfite detoxification might be a key regulator of cell survival in
health and disease.
Besides purine catabolism, XO has been implicated in the generation of reactive
oxygen species. Studies using animal models of mild hyperuricemia provided evi-
dence for a pathogenic role of uric acid in the development of hypertension, vascu-
lar disease, and renal disease [182]. The physiological role of other Mo-enzymes
such as AOX and mARC are still poorly understood and novel animal models are
required to identify their primary function in metabolism [183]. SO is the most
important Mo-enzyme in humans. Recently, we found nitrite reductase activity of
SO under hypoxic conditions. In the future it will be important to learn more about
SO’s role in nitrite-dependent NO signaling using novel animal-based approaches.
Finally, Moco biosynthesis provided the evolutionary origin for numerous
eukaryote-specific functions and pathways, such as radical SAM-dependent reac-
tions, the ubiquitin-proteasome pathway including other thiolation reactions, and
synaptic clustering of receptors. While the biochemistry of Moco biogenesis is well
understood, the underlying cell biology, the organization and regulation of interme-
diate fluxes and the assembly and turn over of different Mo-enzymes are still poorly
understood. Not addressed at all is the catabolism of Moco, resulting in the excretion
of a thiolated oxidized pterin, called urothione, whose identity has been uncovered
more than 40 years ago [184]. In future studies, the catabolic machinery, including
enzymes catalyzing the methylation and dephosphorylation of the cofactor need to
be identified.
13 Molybdenum in Human Health and Disease 443

Abbreviations and Definitions

Proteins and genes written in capital letters refer to humans.


AAT aspartate aminotransferase
ABA3 plant orthologue of human Moco sulfurase
ABC ATP-binding cassette
AMP adenosine 5′-monophosphate
AOX aldehyde oxidase
ATP adenosine 5′-triphosphate
BHMT betaine-homocysteine methyl transferase
CBS cystathionine β-synthase
CDO cysteine dioxygenase
cPMP cyclic pyranopterin monophosphate
CSA cysteine sulfinic acid
CSD cysteinesulfinate decarboxylase
CSE cystathionine γ-lyase (cystathionase)
EE ethylmalonic encephalopathy
FAD flavin adenine dinucleotide
GABA γ-amino butyric acid
GCS γ-glutamylcysteine synthetase
Glu glutamic acid
Gly glycine
GS glutathione synthetase
GSH glutathione
GTP guanosine 5′-triphosphate
HPLC high performance liquid chromatography
KG α-ketoglutarate
mARC mitochondrial amidoxime reducing component
MAT methionine-S-adenosyl transferase
MCSU Moco sulfurase
MFS major facilitator superfamily
MoCD molybdenum cofactor deficiency
Moco molybdenum cofactor
MOCS molybdenum cofactor synthesis
MOSC Moco sulfurase C-terminal domain
MOT1 molybdate transporter type 1
MPST 3-mercaptopyruvate sulfurtransferase
MPT metal-binding pterin (or molybdopterin)
MRI magnetic resonance imaging
MS methionine synthase
NAD+ nicotinamide adenine dinucleotide
NADH nicotinamide adenine dinucleotide (reduced)
NO nitric oxide
NR nitrate reductase
444 Schwarz and Belaidi

PPi inorganic diphosphate (= pyrophosphate)


SAHH S-adenosylhomocysteine hydrolase
SAM S-adenosyl methionine
SDO sulfur dioxygenase
Ser serine
SO sulfite oxidase
SOD sulfite oxidase deficiency
SQR quinone oxidoreductase
SSC S-sulfocysteine
ST sulfur transferase
XDH xanthine dehydrogenase
XO xanthine oxidase
XOR xanthine oxidoreductase

Acknowledgments The great work of all current and past postdocs, graduate, master, and bachelor
students as well as technicians is gratefully acknowledged. This work would not have been
possible without many collaborators that helped to build bridges of true interdisciplinarity.
Research funding by the German Science Foundation (DGF), the Federal Ministry of Education
and Research (BMBF), the FP7 EU funding (Marie Curie Program), the Fonds der Chemischen
Industrie (FCI), and the Center for Molecular Medicine Cologne (CMMC) is gratefully acknowledged.

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Chapter 14
Silicon: The Health Benefits of a Metalloid

Keith R. Martin

Contents
ABSTRACT ............................................................................................................................. 452
1 INTRODUCTION ............................................................................................................. 452
2 SILICON BIOCHEMISTRY ............................................................................................. 453
2.1 Silicon Distribution and Prevalence in Nature .......................................................... 453
2.1.1 Dietary Sources ............................................................................................. 454
2.1.2 Non-dietary Sources ...................................................................................... 456
2.2 Silicon Chemical Speciation as Silicates .................................................................. 456
2.3 Silicon Chemistry and Effects on Bioavailability ..................................................... 456
3 SILICON AND ITS POTENTIAL HEALTH BENEFITS ................................................ 457
3.1 Bone Health and Skeletal Development.................................................................... 458
3.2 Vascular Disease and Atherosclerosis ....................................................................... 459
3.3 Neurodegenerative Disease (Alzheimer’s Disease) .................................................. 460
3.4 Diabetes ..................................................................................................................... 462
3.5 Wound Healing.......................................................................................................... 462
4 TOXICOLOGY OF SILICON AND SILICA ................................................................... 463
4.1 Chemical Forms Contributing to Toxicity................................................................. 463
4.2 Routes of Exposure and Safety ................................................................................. 464
4.2.1 Inhalation and Asbestosis .............................................................................. 464
4.2.2 Inhalation and Silicosis ................................................................................. 464
4.3 Mechanisms of Toxicity ............................................................................................ 465
4.3.1 Oxidative Stress............................................................................................. 465
4.3.2 Inflammation ................................................................................................. 466
5 POTENTIAL MEDICINAL USES OF SILICON AND SILICATES............................... 467
6 SUMMARY AND FUTURE DIRECTIONS .................................................................... 468
ABBREVIATIONS .................................................................................................................. 469
REFERENCES ........................................................................................................................ 469

K.R. Martin (*)


School of Nutrition and Health Promotion, Healthy Lifestyles Research Center,
Arizona State University, 500 North 3rd Street, Phoenix, AZ 85004, USA
e-mail: keith.r.martin13@gmail.com

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 451
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_14, © Springer Science+Business Media Dordrecht 2013
452 Martin

Abstract Silicon is the second most abundant element in nature behind oxygen.
As a metalloid, silicon has been used in many industrial applications including use
as an additive in the food and beverage industry. As a result, humans come into
contact with silicon through both environmental exposures but also as a dietary
component. Moreover, many forms of silicon, that is, Si bound to oxygen, are water-
soluble, absorbable, and potentially bioavailable to humans presumably with bio-
logical activity. However, the specific biochemical or physiological functions of
silicon, if any, are largely unknown although generally thought to exist. As a result,
there is growing interest in the potential therapeutic effects of water-soluble silica
on human health. For example, silicon has been suggested to exhibit roles in the
structural integrity of nails, hair, and skin, overall collagen synthesis, bone mineral-
ization, and bone health and reduced metal accumulation in Alzheimer’s disease,
immune system health, and reduction of the risk for atherosclerosis. Although
emerging research is promising, much additional, corroborative research is needed
particularly regarding speciation of health-promoting forms of silicon and its rela-
tive bioavailability. Orthosilicic acid is the major form of bioavailable silicon
whereas thin fibrous crystalline asbestos is a health hazard promoting asbestosis and
significant impairment of lung function and increased cancer risk. It has been pro-
posed that relatively insoluble forms of silica can also release small but meaningful
quantities of silicon into biological compartments. For example, colloidal silicic
acid, silica gel, and zeolites, although relatively insoluble in water, can increase
concentrations of water-soluble silica and are thought to rely on specific structural
physicochemical characteristics. Collectively, the food supply contributes enough
silicon in the forms aforementioned that could be absorbed and significantly improve
overall human health despite the negative perception of silica as a health hazard.
This review discusses the possible biological potential of the metalloid silicon as
bioavailable orthosilicic acid and the potential beneficial effects on human health.

Keywords asbestos • dietary silica • medicine • orthosilicic acid • silicon • therapy

Please cite as: Met. Ions Life Sci. 13 (2013) 451–473

1 Introduction

Silicon is the second most prevalent element in the earth’s crust existing primarily
as oxygen-containing silica and silicates and accounting for around 27% of elemen-
tal mass with oxygen comprising approximately 45% [1–3]. Silica is omnipotent
being present in almost all of the earth’s minerals, rocks, sands, and clays and exists
in myriad chemical forms expressed as quartz, emerald, feldspar, serpentine, mica,
talc, clay, asbestos, and glass all of which have different uses [4,5]. Overall, quartz
and aluminosilicates are the two most predominant silicates [6]. As an element, sili-
con has found widespread use in industrial applications often as a component of
fabricated steel, a component of abrasives (silicon carbide), a building block of
transistors (along with boron, gallium, arsenic, etc.), solar cells, rectifiers, and other
14 Silicon: The Health Benefits of a Metalloid 453

electronic solid-state devices [7]. Industrial applications also include synthesis of


glass when derived from sand-based silica, production of computer chips, and as a
filler for paint and rubber ceramics, in lubricants, concrete and bricks, as well as
being used for medical devices such as silicone implants [5]. Although silicon is
used frequently for technical applications, its exposure to humans is fairly limited
and largely in chemical forms that are not readily absorbed nor bioavailable.
Silica is used widely in the food and beverage industry as a food additive, i.e.,
anti-caking agent in foods, clarifying agent in beverages, viscosity controlling
agent, as an anti-foaming agent, dough modifier, and as an excipient in drugs and
vitamins [5]. Thus, silicon as silica is a dietary component although largely assumed
to be inert when provided in forms typically used in the aforementioned applica-
tions. Nonetheless, humans are exposed to diet-derived forms of silica suggesting
potential capacity for absorption and ultimate bioavailability, which raises the ques-
tion of whether silicon as a molecular component can exert beneficial, biological
effects in humans. Currently, silicon is not recognized as a nutrient in humans
although emerging research suggests benefit from consumption of water-soluble
forms. To that end, there is renewed growing interest in the potential beneficial
effects of silica on human health.
Regarding inadvertent environmental exposure, previous research, in large part,
has explored the toxic effects of inhaled crystalline silica and silica-derived asbestos.
In fact, silicon has long been recognized as a pulmonary carcinogen with resultant
silicosis or asbestosis developing upon prolonged and/or heavy exposure to airborne
material [8]. Silicosis is a disease of the lungs caused by continued inhalation of the
dust of minerals that contain silica and is characterized by progressive fibrosis and
a chronic shortness of breath [9]. Asbestosis is similar in etiology and pathology but
distinct as an exposure. While there are intrinsic dangers associated with inhalation
of crystalline silica, there are multiple forms of silica in nature that are not toxic.
Although non-toxic, the question remains as to the relative water-solubility of dif-
ferent compounds, relative amounts ingested, efficiency of absorption and overall
bioavailability. Low-molecular-weight silica can dissolve in water as silicic acid
rendering it bioavailable and potentially a beneficial component in humans.
Collectively, the lack of understanding of the relative dependence of the physico-
chemical structure of silica and silicates on water-solubility for absorption has lim-
ited overall research interest in aqueous silica. As a result, a clearer understanding
of the chemistry of silica, specifically of aqueous orthosilicic acid, is critical to
fostering much needed research on potential health benefits.

2 Silicon Biochemistry

2.1 Silicon Distribution and Prevalence in Nature

Chemically, silica is an oxide of silicon and represented by silicon dioxide (SiO2).


Silicon itself is a tetravalent metalloid with chemical properties somewhere in
between that of a metal and non-metal element. Its presence is second only to oxygen
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in its abundance on earth comprising almost a third of the earth’s crust. In its pure
form, silicon typically does not exist in a natural elemental state due to its extreme
propensity to undergo reactions with ambient oxygen and water. For example, silica,
SiO2, and other oxides, are ubiquitously found in polymerized combinations with
metals and embedded in geologic rock formations. Given its omnipotence, overall
prevalence and reactivity with other elements, it clearly exists in myriad forms with
differing physicochemical properties with some that are toxic and others that are
seemingly critical for health.
Silica is largely present in geographical formations and not readily released from
these substrates except through natural, but significant, weathering of these structures.
Overall, the forms and resultant molecular sizes of polymers and aggregates are
dependent on pH and concentrations in aqueous matrices [10]. For example, at low
concentrations (<2 mM) silicon exists in a monomeric acidic form (pKa 9.6) as ortho-
silicic acid, which imparts a fair degree of water solubility and certainly more than
the higher-molecular-weight forms. As concentrations increase, polymerization
will occur to form oligomers and eventually colloids, then aggregates and solid
amorphous precipitates with a clear concentration dependence on solubility. As one
might surmise, the increasing molecular weight and structural complexity restricts
water solubility and, as a result, limits potential absorption by humans and animals.

2.1.1 Dietary Sources

Silica exists in the food chain with concentrations tending to be much higher in
plant-based foods, i.e., phytolithic, than animal foods [11]. Beverages, however, are
the major contributor to dietary silica, or silicon, and include water, coffee, and beer
(due to barley, hops, etc.) where fluid ingestion alone can account for ≥20% of
intake [12–14]. Beer is the major source of bioavailable silicon for males with con-
centrations of 9–39 mg/L [14–16]. Silica is also prevalent in municipal water sup-
plies but is particularly high in bottled spring and artesian waters depending on the
respective geological source [17]. In fact, beverages alone contribute up to 55% of
total dietary intake of silicon as water-soluble silica. Dietary grains and grain prod-
ucts including cereals, oats, barley, wheat flour, pasta, pastries, and polished rice
contribute 14% of ingested silicon and vegetables contribute 8% [18]. In the Western
diet, major sources of silicon are cereals (30%) followed by fruits, beverages, and
vegetables, which together make up 75% of total silicon intake [19]. Processing and
refinement of grains remove silicon during the processes but silica-derived food
additives can replace the stripped silicon and increase the content although the rela-
tive absorptivity of added silicon is questionable. Overall, estimation of dietary
intake from all sources is approximately 20–50 mg silicon/day for Western popula-
tions but up to ~200 mg/day for populations consuming a more plant-based diet
such as populations from India and China [12,18,20–22].
The presence of large amounts of silica in geological formations contributes
greatly to the silica content of water. For example, in the United Kingdom, silicon
concentrations are ≤2.5 mg/L in north and west Britain but up to 14 mg/L in south
14 Silicon: The Health Benefits of a Metalloid 455

and east Britain [23–25]. Silica is found in fresh water at concentrations of 1–100 mg/L
depending on the geographical location, e.g., soil content. Typical municipal water
supplies can provide 4–11 mg/L of aqueous silica as noted in a study of the large
cities of France. Levels of around 18–20 mg/L occur in the water of large cities of
the United States. Bottled waters also contain modest concentrations of silica rang-
ing from 8 to 36 mg/L as noted for the French brands Badoit, Vichy Celestian, and
Volvic [26]. Interestingly, bottled water from Malaysia contains 30–40 mg/L silica
and from the Fiji Islands contains 85 mg/L silica, more than four times the levels
found in fresh water and municipal supplies and over twice that of other bottled
waters, presumably due to the leaching of water-soluble silica from volcanic rock.
Collectively, aqueous sources provide a wide range of concentrations of water-
soluble, bioavailable silica.
There are other dietary sources of silicon including primarily food additives and
dietary supplements. For foods, silicon may be added to processed, manufactured,
and distributed foods as anticaking agents, thickeners, stabilizers, and clarifying
agents, significantly increasing the overall silicon concentration [27]. However, sili-
cates are generally considered to be inert and, as a result, not absorbed to any great
extent by humans. In particular, polymeric silicic acids and amorphous silicon diox-
ide are poorly absorbed. Dietary supplements are an alternative silicon source con-
taining orthosilicic acid or other forms that are presumably modified to a form that
is water-soluble, absorbed, and bioavailable although this does not universally apply
[28,29]. The estimated overall bioavailability of silicon from supplements ranges
from <1 to >50%, a remarkably wide range, and depends on the formulation and
concentration [6].
Silica is prevalent in the typical human diet at around 10–25 mg/day and gener-
ally considered safe, even if indigestible and non-absorbable. Although a bio-
marker of silicon status has yet to be developed, approximately 41% of ingested
silicon is excreted in urine, which is significantly correlated with dietary consump-
tion of silicon [20,30]. The lack of clear understanding of the myriad of chemical
forms of silica and significant, widely communicated likelihood of increased risk
of cancer has unduly overshadowed the study of the potential protective effects of
silica on human health. It is the intent of this review to provide insight into the
chemical properties of silica that may render it bioavailable and beneficial to
human health.
Although there are dietary sources of silicon which are thought to exert benefi-
cial effects in humans, there is no recommended dietary allowance (RDA) for sili-
con and, in fact many do not recognize silicon as a micronutrient essential for life,
although 1–2 g is present in the human body [31,32]. However, if one considers the
risk assessment of amorphous silicon dioxide as a common silicon source, although
non-absorbed, the safe tolerable upper intake level (TUL), a component of the
Dietary Reference Intakes (DRIs), is estimated to be 700 mg/day for adults, which
is equivalent to 12 mg silicon/kg body weight/day for a 60 kg adult [33]. However,
only minimal amounts of silicon become water-soluble and ultimately absorbed,
thus the systemic plasma concentration does not increase significantly. The mean
dietary silicon intake reported for a Finnish population was 29 mg silicon/day and
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for a typical British diet 20–50 mg/day corresponding to 0.3–0.8 mg silicon/kg


body weight/day [14,18,34,35]. The estimated dietary intake in the US is 24–33 mg
silicon/day with males generally consuming more [20].

2.1.2 Non-dietary Sources

Given the relative prevalence and widespread use of silica, it seems reasonable that
there are myriad, diverse sources of and exposures to non-dietary silica/silicon.
These occur primarily from exposure to dust, pharmaceuticals, cosmetics, medical
implants, and medical devices. Often the forms of silicon occur as silicates or “sili-
cones,” synthetic organosilicon compounds that, for the most part, are sparse in the
human diet and contribute little silicon overall. Moreover, the forms that do result in
exposure are not readily absorbed or biologically useful. For example, some phar-
maceuticals can increase exposure of silicon to >1 g/d but the molecular species are
largely inert and not absorbed to any significant extent. Examples of silicates include
talc, kaolin, and magnesium, calcium, and sodium salts. This seems to be the case
with other non-dietary sources such as toiletries, e.g., toothpaste, lipstick, etc., and
detergents, tissue implants, etc. [6].

2.2 Silicon Chemical Speciation as Silicates

Silicon is the second most abundant element on earth with properties that are a mix-
ture of both metals and non-metals resulting in classification as an elemental metal-
loid. As stated previously, silicon is rarely found in its elemental form but rather
complexed with oxygen and/or other elements forming silica and silicates. Silicon
dioxide, SiO2, is the oxide of silicon most commonly found in nature as sand or
quartz. Generally, a silicate is any compound containing silicon and oxygen as an
anion, SiO42 −, with most in nature existing as oxides although the non-oxygen con-
taining hexafluorosilicate anion, [SiF6]2−, is also often included as a silicate.
Chemically, silicate anions can form compounds with numerous, diverse cations,
thus this chemical class of compounds is large with formation of aluminosilicates
being the most prevalent in nature [36]. Aluminum is the third most prevalent ele-
ment in the earth’s crust and exists in combination with >270 other minerals.

2.3 Silicon Chemistry and Effects on Bioavailability

Silica, SiO2, is a silicic acid anhydride of monomeric orthosilicic acid (H4SiO4)


which is water-soluble and stable in aqueous solutions when relatively dilute.
Several other low-molecular-weight, but hydrated forms, of silicic acid exist in
14 Silicon: The Health Benefits of a Metalloid 457

aqueous solutions and include metasilicic acid (H2SiO3), lower-molecular-weight


oligomers such as disilicic acid (H2Si2O5) and trisilicic acid (H2Si3O7), as well as
their hydrated forms pentahydro- and pyrosilicic acids [1]. Depending on the envi-
ronmental conditions (temperature, pH, and presence of other ions), concentrations,
and exposure time, formation of numerous potential polymerized silicic acids is
possible through chemical condensation and cross-linking resulting in colloids and
gels [37]. It is the lower molecular weight forms, especially the orthosilicic acid that
is of the greatest research interest in exerting beneficial effects since this form is
preferentially absorbed [38]. In fact, a small human study showed that ingestion of
polymeric forms of silicic acid did not increase urinary levels suggesting little
absorption, but 53% of ingested orthosilicic acid did increase urinary output indicat-
ing absorption [39]. Interestingly, most aqueous silica, i.e., seawater, freshwater,
soil water, etc., occurs as orthosilicic acid (H4SiO4) making it an important environ-
mental exposure in the context of biological systems due both to its water-solubility
and bioavailability [4,40].
As previously noted, orthosilicic acid is water-soluble at relatively low concen-
trations but polymerizes readily at higher concentrations in excess of 100–200 ppm
to form colloids and gels, which are less bioavailable. However, more concentrated
solutions of orthosilicic acid can be stabilized to avoid polymerization. In fact,
choline-stabilized orthosilicic acid, a liquid formulation, has been developed and
approved for human consumption. It is considered non-toxic at high doses with a
lethal dose exceeding 5,000 mg/kg body weight in humans and 6,640 mg/kg body
weight in animals [41,42]. For a 70 kg human, this translates to a safe level of con-
sumption of 350 g. This stabilized form currently represents the most bioavailable
source of supplemental silicon.

3 Silicon and Its Potential Health Benefits

Silicon is the third most abundant trace element in the human body [20,43]. It is
present at 1–10 ppm in hair, nails, the epidermis, and epicuticle of hair [44–46].
Considering the natural abundance, presence of bioavailable chemical forms, expo-
sure to humans through diet, it seems more than plausible that there could, and
likely is, potential benefit to humans. Whether silicon is an essential micronutrient
continues to be debated. It has, however, been reported in the peer-reviewed litera-
ture that silicon is actively involved, and perhaps integral, in bone mineralization
and prevention of osteoporosis, collagen synthesis, and prevention of the aging of
skin, overall condition of hair and nails, reduced risk of atherosclerosis and
Alzheimer’s disease, as well as other biological effects [47–51].
Interestingly, serum levels are similar to other trace elements and appear to be
dependent on life stage, age, and sex with levels of 11–31 μg/dL depending on
population assessed and means of analysis [23,52]. A recent study by Jugdaohsingh
et al. evaluated host factors potentially influencing the absorption and excretion
of dietary silicon. Serum and urine samples were collected from 26 participants
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followed by a single ingestion of 17 mg orthosilicic acid. Analyses of samples over


the subsequent 6 hours indicated that participant age, sex, and estrogen status did
not influence absorption or excretion suggesting more research is needed to better
understand the effects of host factors on disposition of dietary silica [53].

3.1 Bone Health and Skeletal Development

Osteoporosis is a leading cause of morbidity and mortality in the elderly and mark-
edly affects overall quality of life, as well as life expectancy. As a result, there is
considerable interest in elucidation and use of specific nutrients, non-nutritive
dietary components, and/or bioactive compounds of natural origin singly or in com-
bination as a means of mitigating or preventing disease, as well as for maintenance
of bone health. Calcium and vitamin D have largely been the primary focus of nutri-
tional prevention of osteoporosis, however, supplementation with other vitamins
including B, C, and K has been an area of increased research as well as the use of
silicon for maintenance of bone health [54,55].
Osteoporosis is defined as a progressive, debilitating skeletal disorder
characterized by low bone mass and deterioration of the microarchitecture of bone
[56,57]. Indeed, several key animal studies dating back four decades clearly showed
that dietary silicon deficiency caused abnormalities and dysfunction in connective
tissues and bone function [58–62]. Numerous human studies have supported a role
for dietary silicon in bone health including reduction of the risk for osteoporosis.
In a retrospective, clinical study by Eisinger and Clairet, dietary silicon administra-
tion induced significant increases in bone mass and bone mineral density of the
femur in human females [47]. Moukarzel et al. have also shown a direct relationship
between silicon intake and bone mineral density [63]. In osteoporotic participants,
supplementation with silicon increased trabecular bone volume and femoral bone
mineral density [47,64]. Spector et al. showed in osteopenic and osteoporotic study
participants an increase in bone formation markers, i.e., collagen synthesis, and
significant increases in femoral bone mineral density [65]. Maehira et al. [66] have
shown in mice fed five different calcium sources with differing silicon concentra-
tions that soluble silicate and coral sand, with the highest silicon content, signifi-
cantly improved bone biochemical and mechanical properties through induced gene
expression encouraging correction of the imbalance between bone-forming osteo-
blastogenesis and suppression of bone-resorbing osteoclastogenesis [66–68]. Others
have shown in human osteoblasts that orthosilicic acid-releasing zeolites could
induce osteoblastogenesis, formation of extracellular matrix, induced synthesis of
ostecalcin and activity of alkaline phosphatase both produced by osteoblasts and
reflecting biosynthetic activity of bone formation [69–71]. It has also been shown
that silicon supplementation increased hip bone mineral density in men and
pre-menopausal, but not post-menopausal, women although a subsequent study
showed increased bone mineral density in the spine and femur of both pre- and
post-menopausal women currently taking hormone replacement therapy [15,72].
14 Silicon: The Health Benefits of a Metalloid 459

Compelling evidence demonstrates that silicon localizes to bone and that dietary
silicon can strengthen bones and, as a result, reduce the risk of osteoporosis [73].
As mentioned before, stabilized preparations of silicic acid have been developed,
e.g., choline-stabilized orthosilicic acid, permitting water-soluble preparations with
higher concentrations and also markedly enhanced bioavailability. In a randomized
controlled animal study, long-term treatment with choline-stabilized orthosilicic
acid prevented partial femoral bone loss and exerted a positive, beneficial effect on
bone turnover and ultimately bone mineral density [74]. In this study, ovariecto-
mized aged rodents were used suggesting a potential interrelationship between
estrogen and bone health and silicon metabolism. A subsequent study by Macdonald
et al. found that dietary silicon interacts with estrogen to beneficially affect bone
health [72]. Silicon has previously been shown to significantly enhance the rate of
bone mineralization and calcification much like vitamin D, although functioning
independently [75]. There are potentially conflicting reports since Jugdaohsingh
et al. found that silicon supplementation in drinking water did not significantly alter
silicon concentrations in the bones of rodents suggesting an additional nutritional
cofactor might be absent such as vitamin K in rodents fed a low silicon diet [76].

3.2 Vascular Disease and Atherosclerosis

It has been reported that there are higher incidences of sudden death, cerebrovascu-
lar diseases, arterial hypertension, and coronary heart disease in soft water areas of
the United States suggesting, in part, that the absence of components presence in
hard water, i.e., minerals, may be contributors. As a result, a major research effort
has been devoted to identifying potential protective factors in hard water including
calcium, magnesium, manganese, and silicon, as examples, all of which are consid-
ered potentially beneficial [77].
Silicon is recognized by epidemiologic and biochemical studies as a protective
trace element in atherosclerosis. Moreover, the observed decrease in silicon concen-
trations with increasing age has been suggested to contribute to chronic diseases
such as atherosclerosis. The highest concentrations of silica in the human occur in
connective and elastic tissues and especially the normal human aorta where it
appears to function as a crosslinking agent that stabilizes collagen and presumably
strengthens the vasculature [49,78]. Atherosclerosis significantly decreases silicon
levels in arterial walls. Moreover, silicon levels decrease just prior to plaque devel-
opment, which may indicate that silicon deficiencies cause inherent weaknesses in
blood vessel walls.
In a study by Trinca et al., the antiatheromatous effect of sodium silicate was tested
in rabbits given a standard control diet, an atherogenic diet, and a sodium silicate-
supplemented atherogenic diet. Levels of total lipids, cholesterol, triglycerides, free
fatty acids, and phospholipids remained unchanged in sodium silicate supplemented
rabbits fed an atherogenic diet [79]. In a subsequent study, silicon administered orally
or intravenously in rabbits inhibited experimental atheromas normally induced by an
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atheromatous diet, decreasing the number of atheromatous plaques and lipid deposits.
It was proposed that the preservation of elastic fiber architecture, as well as of ground
substance and the lack of free fatty acid accumulation in the aortic intima decreased
plaque formation [80]. In a study by Maehira et al. using soluble silica and coral sand,
as a natural silicon-containing material, the effect on hypertension, a contributing fac-
tor to atherosclerosis, was evaluated in spontaneously hypertensive rats. In rats fed 50
mg/kg dietary silicon for 8 weeks, systolic blood pressure was significantly lowered
by 18 mmHg. Provision of soluble dietary silica also suppressed the aortic gene
expression of angiotensinogen and growth factors related to vascular remodeling.
Silicon also stimulated the expression of peroxisome proliferator-activated receptor-γ,
which has antiinflammatory and antihypertensive effects on vascular cells [81]. In a
study by Oner et al., dietary silica modified the characteristics of endothelial dilation
in aortic rings from rats with modulation of endothelial relaxants and attenuation of
smooth muscle cell responsiveness to nitric oxide [82].
Silicon has also been suggested to exert a protective role in atherosclerosis
through its effects on blood vessel-associated glycosaminoglycans and collagen
integrity and function via its crosslinking capacity [19]. Glycosaminoglycans are
long unbranched (linear) polysaccharides consisting of repeating disaccharide units
including hyaluronan, chondroitin, dermatan, heparan, and keratan. Silicon is also a
constituent of the enzyme prolyl hydroxylase, which synthesizes collagen and gly-
cosaminoglycans. Dietary silicon may facilitate the formation of glycosaminogly-
cans and collagen and/or serve a structural role as a component of glycosaminoglycans
where it crosslinks, and strengthens, polysaccharide chains. Nakashima et al. have
noted that the glycosaminoglycan content of the aorta was inversely correlated with
the severity of atherosclerosis. Interestingly, they showed that the silicon content in
fatty streaks and/or atheroma was significantly higher than in normal human aortic
intimal regions suggesting that the increase of silicon in the aortic intima is related
to the occurrence and/progression of atherosclerosis [83].

3.3 Neurodegenerative Disease (Alzheimer’s Disease)

Metals that can cross the blood brain barrier and generate directly or indirectly oxi-
dative stress can cause significant damage to the neuronal structure of the brain.
Aluminum is abundant in the environment but is not a micronutrient. However,
ingestion and/or exposures can cause deposition and accumulation in the body, e.g.,
brain, where it can cause considerable damage. Aluminum, a nonredox-active metal,
is a well-known toxicant and its salts can accelerate oxidative damage of neurons.
Oxidative stress is one of the critical features in the pathogenesis of Alzheimer’s
disease and has been demonstrated in brain tissue from Alzheimer’s patients.
Aluminum is a contributing factor to oxidative stress, as it generates reactive oxy-
gen species (ROS) shown to cause oxidative damage to neurons through interaction
with iron, a redox-active metal, and promotion of free radical-generating Fenton
reactions, which can increase hallmark aggregation and accumulation of β-amyloid.
14 Silicon: The Health Benefits of a Metalloid 461

Collectively, studies clearly indicate that aluminum promotes oxidative stress capa-
ble of damaging neuronal cell death [84].
The molecular pathogenesis of Alzheimer’s disease includes many risk factors
including extracellular deposition of β-amyloid, accumulation of intracellular neu-
rofibrillary tangles, oxidative neuronal damage and activation of inflammatory cas-
cades [85]. Although the subject of continuing scientific debate, aluminum has been
detected in neurofibrillary tangles in the brains of both Alzheimer’s and Parkinson’s
disease patients with dementia and is proposed to play crucial roles as a crosslinker
in β-amyloid oligomerization [86–88].
Although the neurotoxicity of aluminum is well-documented, the association
with neurodegenerative disorders is the subject of debate as is the potential benefit
of consuming silica [89]. Some epidemiological studies, but not all, suggest that
silica could be protective against aluminum damage, because silica reduces oral
absorption of aluminum and/or enhances its excretion [90–92]. Studies have sug-
gested that oligomeric but not monomeric, viz., orthosilicic acid, silica can prevent
aluminum absorption through the gastrointestinal (GI) tract reinforcing the impor-
tance of chemical speciation [39]. Silicon readily complexes with aluminum and,
in fact, aluminosilicates are the most prevalent silicates in nature. A silicate is any
of numerous compounds containing silicon, oxygen, and one or more metals form-
ing essentially a salt of silicic acid. Aluminum silicates are water-insoluble and
although the processes involved in aluminum bioavailability are unclear regarding
its transport into the central nervous system, numerous reports show that silicic
acid can, in fact, reduce aluminum absorption and ultimately deposition and accu-
mulation within the brain. In an epidemiological study, Rondeau et al. examined
associations between exposure to aluminum or silica from drinking water and risk
of cognitive decline, dementia, and Alzheimer’s disease among 1,925 elderly sub-
jects followed for 15 years. The authors concluded that cognitive decline with time
was greater in subjects with a higher daily intake or geographic exposure to alumi-
num from drinking water. An increase of 10 mg/day in silica intake was signifi-
cantly associated with a reduced risk of dementia [93]. Thus, it appears that the
relative concentration of both aluminum and silica in drinking water are important
in determining benefit or detriment regarding the risk and/or exacerbation of
Alzheimer’s disease [94]. Interestingly, soft water contains less silica acid and
more aluminum while the converse is true for hard water [25]. In a study by Exley
et al. introduction of hard water rich in silica significantly reduced overall alumi-
num levels in the body presumably through reduced absorption of aluminum as
supported by reduced urinary concentrations [95]. A subsequent study showed that
drinking up to 1 L of a silicon-rich mineral water daily for 12 weeks fostered uri-
nary removal of aluminum in both control and Alzheimer patient groups without
increasing urinary excretion of the micronutrients iron and copper [96]. Moreover,
there were clinically relevant increases in cognitive performance in 20% of partici-
pants. Gonzalez-Munoz et al. have shown that beer consumption, a rich bioavail-
able source of silicic acid, can reduce cerebral oxidation caused by aluminum
toxicity by, interestingly, modulating gene expression of pro-inflammatory cyto-
kines and antioxidative enzymes [51].
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3.4 Diabetes

Type 2 diabetes is a disorder of glycemia based largely on the development of


insulin resistance. It has been noted that micronutrients can regulate metabolism
and gene expression associated with glycemia thereby potentially influencing the
development and progression of diabetes [97]. In a report by Oschilewski et al.,
administration of silica to BB-rats, prone to spontaneous diabetic syndrome, com-
pletely prevented the development of diabetes [98]. Rats were treated with 100 mg
silica/kg body weight via intraperitoneal and intravenous routes and observed for
weight changes, glycosuria, and ketonuria. The authors showed nearly complete
inhibition of the development of diabetes (1 of 31 in treated group versus 9 of 31 for
control group) and attribute the protection of silica to reduced infiltration of pancre-
atic islets by macrophages. Kahn and Zinman showed in a previous study exploring
bone health that dietary silicon suppressed bone marrow-derived peroxisome-
proliferator receptor-γ, which regulates bone metabolism, but also regulates glucose
metabolism where it is a ligand-activated transcription factor and a molecular target
of a class of insulin-sensitizing drugs referred to as thiazolidinediones [99].
In the subsequent study, the antidiabetic effects of silicon were investigated in
obese diabetic KKAy mice prone to hyperleptinemia, hyperinsulinemia, and
hyperlipidemia (50 ppm silicon for 8 weeks). Interestingly, silicon and coral sand,
a rich source of silicon, displayed antidiabetic effects through blood glucose
reductions and increases in insulin responsiveness, as well as improvement in the
responses to the adipokines leptin and adiponectin [100]. The authors report this
as a novel function of anti-osteoporotic silicon and suggest use of silicon as a
potential antidiabetic agent capable of reducing plasma glucose and reducing the
risk of diabetic glomerulonephropathy. There is clearly a need for research into
the potential novel therapeutic applications of silicon, as silica, for prevention and
management of diabetes.

3.5 Wound Healing

Silica already finds widespread use in medical and surgical applications including
tissue engineering for regeneration of tissues, e.g., wound repair and organs. This
typically is in the form of collagen scaffolds, which are used as sponges, thin sheets
or gels. Collagen, as a long fibrous structural protein, possesses the appropriate
properties for tissue regeneration including optimal pore structure, permeability,
hydrophilicity and stability in vivo. As a result, collagen scaffolds permit deposition
and growth of cells, e.g., osteoblasts and fibroblasts, promoting normal tissue
growth and restoration [101]. There are studies that suggest that dietary silicon can
also exert beneficial effects on wound repair.
The successful healing of wounds requires local synthesis of significant amounts
of collagen with its high hydroxyproline content drawing upon amino acid precursors
14 Silicon: The Health Benefits of a Metalloid 463

such as proline and ornithine [102]. In animal studies, silica-deficient diets result in
poor formation of connective tissues including collagen and ultimate structural
damage. Silica maintains the health of connective tissues due, in part, to its interac-
tion with the formation of glycosaminoglycans where silicon is consistently found
and presumed to have an active role. As a result, a deficiency in silica could result
in reduced skin elasticity and wound healing due to its role in collagen and glycos-
aminoglycan formation. Seaborn and Nielsen have reported that silicon deprivation
decreases collagen formation in wounds and bone, and decreases ornithine
transaminase enzyme activity in liver [103]. In a rodent study, silicon deprivation
affected collagen formation at several different stages of bone development, the
activities of collagen-forming enzymes, and consequent collagen deposition on
other tissues. This has major implications suggesting that silicon is important in
wound healing and supports that dietary silicon, as silicic acid, can exert therapeutic
effects for this use.

4 Toxicology of Silicon and Silica

4.1 Chemical Forms Contributing to Toxicity

As previously discussed, elemental silicon exists primarily as an oxide largely in


the form of silicon dioxide. Silica, SiO2, is a silicic acid anhydride of monomeric
orthosilicic acid (H4SiO4), which is water-soluble and stable in aqueous solutions
when relatively dilute but can polymerize and complex with numerous minerals to
form silicates with aluminum silicate being the most prevalent. Several other low-
molecular-weight, but hydrated forms, of silicic acid exist in aqueous solutions
and are non-toxic. Forms of silicon that are toxic include long fibrous crystalline
forms such as asbestos. Asbestos is a group of crystalline 1:1 layer hydrated sili-
cate fibers that are classified into six types based on different physicochemical
features [104]. These include chrysotile [Mg6Si4O10(OH)8], the most common and
economically important asbestos in the Northern Hemisphere, and the amphi-
boles: crocidolite [Na2(Fe3+)2(Fe2+)3Si8O22(OH)2], amosite [(Fe,Mg)7Si8O22(OH)2],
anthophyllite [(Mg,Fe)7Si8O22(OH)2], tremolite [Ca2Mg5Si8O22(OH)2], and actino-
lite [Ca2(Mg,Fe)5Si8O22(OH)2].
Silica occurs in both non-crystalline and crystalline forms where crystalline
silica is a basic component of soil, sand, granite, and many other minerals.
Crystalline forms technically are physical states in which the silicon dioxide mole-
cules are arranged in a repetitive pattern with unique spacing, lattice structure and
angular relationship of the atoms. Crystalline silica forms, viz., polymorphs, include
quartz, cristobalite, tridymite, keatite, coesite, stishovite, and moganite. Silicosis
largely occurs due to inhalation of one of the forms of crystalline silica, most com-
monly quartz. All three forms may become respirable size particles when workers
chip, cut, drill, or grind objects that contain crystalline silica.
464 Martin

4.2 Routes of Exposure and Safety

The most noted toxicity associated with silica and asbestos are silicosis and asbes-
tosis, respectively. The key route of exposure leading to toxicity is respiratory with
progressive, debilitating damage from lengthy and/or heavy inhalation of the dust of
silica. In fact, the International Agency for Research on Cancer (IARC) classifies
silica as a “known human carcinogen” based on inhalation as a route of exposure.
Regarding dietary exposure, there is no evidence of carcinogenesis when silica was
fed to rodents for ~2 years (effectively the whole life span) supporting that the route
of exposure is more critical than the chemical form. There are reports that magne-
sium trisilicate (6.5 mg elemental silicon) when used as an antacid in large amounts
for years may be associated with the development of urolithiasis due to formation,
in vivo, of silicon-containing stones although fewer than 30 cases have been reported
in the last 80 years [105].
There are other reports of toxicity from oral ingestion of crystalline and amorphous
silicates. For example, nephropathy can result from finely ground silicates and
nephritis from long-term use of high dose, silica-containing medications as well as
kidney damage and kidney stones [106]. There are some reports of increased risk of
cancer (esophagus and skin) from silica-rich materials such as millet and seeds
[14,107,108]. It is proposed that the overall limitation in absorption of silicon, regard-
less of level of dietary intake, coupled with efficient elimination significantly limits
the potential toxicity of silica. Circumventing this defense mechanism via peritoneal
injections of silicon as shown in animals can easily exceed expected urinary output
beyond that associated with presumed silicon adequacy [30,109].

4.2.1 Inhalation and Asbestosis

When asbestos fibers are inhaled, most fibers are expelled, but some can become
lodged in the lungs and remain there throughout life increasing the risk of asbesto-
sis. Asbestosis is a chronic inflammatory and fibrotic disease affecting the paren-
chymal tissue of the lungs, referred to as interstitial fibrosis, caused by the inhalation
and deposition of fibrous asbestos. Manifestation of the disease occurs typically
after high intensity and/or long-term exposure to asbestos as a specific group of
airborne crystalline silicate fibers. Asbestos fibers are invisible without magnifica-
tion because their size is approximately 3–20 μm wide but as small as 0.01 μm. For
reference, human hair has a width of ~20–180 μm. Given the omnipotence of asbes-
tosis in technical applications, it is considered an occupational lung disease.

4.2.2 Inhalation and Silicosis

Silicosis is also a form of irreversible occupational lung disease, technically a type


of pneumoconiosis that is caused by inhalation of small particles of crystalline
silica dust. Inhaling finely divided crystalline silica dust even in small quantities
14 Silicon: The Health Benefits of a Metalloid 465

(the Occupational Safety and Health Administration (OSHA) allows 0.1 mg/m3)
over time can lead to silicosis, bronchitis, or cancer, as the dust becomes lodged
in the lungs causing chronic irritation with reduced lung capacity. It is marked by
inflammation, pulmonary edema, scarring of the lungs, and formation of nodular
lesions in the upper lobes of the lungs with resultant difficulty in breathing. There
are several different clinical and pathologic varieties of silicosis, including simple
(nodular) silicosis, acute silicosis (silicoproteinosis), complicated pneumoconiosis
(progressive massive fibrosis), and true diffuse interstitial fibrosis [110].

4.3 Mechanisms of Toxicity

The molecular mechanism of silica and asbestos-induced carcinogenesis is complex


and unclear. Clearly, inhalation is the primary route of exposure leading to toxicity
and depends on the shape and size of silica fibers, duration of exposure, and relative
dose, as well as lung clearance capacity and individual genetics [111,112]. Several
mechanisms have been proposed including the adsorption, chromosome tangling,
and oxidative stress hypotheses.
The adsorption theory posits that the surface of asbestos has a high natural affin-
ity for proteins and other biomolecules and presumably disrupts cell function. The
chromosome tangling hypothesis argues that asbestos can interact with chromo-
somes and “tangle” them during cellular division causing clastogenic damage.
Probably the most compelling mechanism at this time is the oxidative stress theory,
which purports that iron associated with asbestos fibers, once internalized, can con-
tribute to Fenton chemistry with generation of reactive, damaging free radicals and
reactive oxygen species. Moreover, deposition of asbestos and silica particles in the
lungs can initiate chronic inflammation via involvement of phagocytic macro-
phages, which also produces copious ROS. Although discussed separately, oxida-
tive stress and inflammation are intimately linked and often occur concurrently, thus
both occur concomitantly in lung disease.

4.3.1 Oxidative Stress

Cumulative supporting evidence suggests a role for ROS and reactive nitrogen
species in the pathogenesis of asbestos- and silica-induced diseases [110,113].
Oxidative damage to the lungs can occur directly through highly reactive hydroxyl
radical formation via the Fenton and Haber-Weiss reactions with fiber surface iron,
and indirectly through inflammation [114–116]. This route involves the recruitment
and activation of ROS-producing inflammatory cells, such as macrophages. Other
cell types also participate in the process including mesothelial cells and lung
fibroblasts, which also produce ROS species in response to silica and/or asbestos.
Numerous in vitro studies have shown the involvement of oxidative stress in damage
caused by silica. For example, Liu et al. tested the effects of silica nanoparticles on
466 Martin

endothelial cells by measuring ROS generation, apoptosis and necrosis, proinflammatory


and prothrombic properties and the levels of the apoptotic signaling proteins and
the transcription factors after exposure to silica nanoparticles (25–200 μg/mL) for
24h [117]. Silica nanoparticles markedly induced ROS production, mitochondrial
depolarization and apoptosis in endothelial cells. Others have shown similar results
with primary endothelial cells exposed to silica nanoparticles with activation and
dysfunction of endothelial cells shown by release of von Willebrand factor and
necrotic cell death [118]. In a study of mesothelial cells, exposure to crocidolite
asbestos induced oxidative stress, caused DNA damage and induced apoptosis
demonstrating that phagocytosis was important for asbestos-induced injury to
mesothelial cells [119].
Several human studies have been conducted to determine if oxidative stress
results from asbestos exposure using a relatively new biomarker of exposure.
Measurement of exhaled breath condensate for markers of oxidative stress is one of
the most promising methods available for determining pulmonary damage from
environmental exposures [120]. An increase in the exhaled breath condensate con-
centrations of 8-isoprostane, an oxidative stress marker, has been observed in
patients with idiopathic pulmonary fibrosis and in a limited study with asbestos-
exposed subjects. Pelclova et al. measured 8-isoprostane, in 92 former asbestos
workers with an average exposure of 24 years [114]. The results indicated higher
levels of 8-isoprostane in exposed subjects compared to control subjects (69.5 versus
47.0 pg/mL) supporting asbestos-induced oxidative stress. In a study involving 83
patients (45 with asbestosis and hyalinosis and 37 with silicosis), concentrations of
8-isoprostane and hydroxynonenal, an oxidative degradation product, were mea-
sured in urine and exhaled breath condensate. The results indicated that most mark-
ers correlated positively and significantly with lung function impairment [121].
These markers as well as others have been effectively developed to detect and con-
firm oxidative stress in patients with asbestosis and silicosis [122,123].

4.3.2 Inflammation

There is growing evidence that amorphous silica can cause an inflammatory response
in the lung. These crystalline silicates are phagocytozed by macrophages that then
release cytokines that attract and stimulate other immune cells including fibroblasts,
which are responsible for the excessive production of collagen (fibrotic tissue) that is
characteristic of silicosis [10]. In a study by McCarthy et al., exposure of human lung
submucosal cells to SiO2 nanoparticles (10–500 nm) for up to 24 hours increased
cyotoxicity and cell death, induced pro-inflammatory gene expression and release of
pro-inflammatory IL-6 and IL-8, and upregulation of pro-apoptotic genes indicating
oxidative stress-associated injury [124]. Bauer et al. also showed that silica nanopar-
ticles caused dysfunction and cytoxicity through exocytosis of von Willebrand factor
and necrotic cell death in primary human endothelial cells [118]. In the study by Liu
et al., incubation of endothelial cells with 200 μg/mL silica caused increased cell
death and the release of numerous, diverse pro-inflammatory mediators (TNF, IL-6,
IL-8, and MCP-1) by remaining viable cells [117].
14 Silicon: The Health Benefits of a Metalloid 467

Silica nanoparticles also activated pro-inflammatory gene expression, e.g.,


NF-κB, and suppressed antiinflammatory gene expression, e.g., Bcl-2. The study
collectively showed that silica nanoparticles damaged endothelial cells through oxi-
dative stress via changes in gene expression associated with inflammation. Others
have shown the role of IFN-γ in the development of murine bronchus-associated
lymphoid tissues induced by silica and activation of NF-κB in silica-induced IL-8
production by bronchial epitehelial cells [125]. Clearly, silicosis is characterized by
mononuclear cell aggregation and lymphocytes are abundant in these lesions [126].
Ironically, short-term studies in rodents exposed to crystalline quartz suggested
that silicon exposure stimulated the immune system and respiratory defense through
activation of neutrophils, T lymphocytes, and NK cells with subsequent increased
production of ROS [127–129]. This is thought to enhance the pulmonary clearance
of microbes. Intriguingly, silica was shown, at least in rats, to activate and increase
proliferation of CD8+ and CD4+ T cells suggesting potential therapeutic use in the
future as an immunostimulant for pulmonary disorders. Recently a supplemental
anionic alkali mineral complex containing sodium silicate (60% of mass) has been
developed and is currently used as immunostimulant in animals including horses,
pigs, etc. [130]. The mechanism of action is not known, however, it has been sug-
gested that orthosilicic acid-generating sodium silicate is the bioactive agent respon-
sible for the immunostimulation. Sodium metasilicate has also been shown to be
immunostimulatory [131]. The seemingly dichotomous actions of silica represent a
conundrum with excessive immunostimulation in silicosis and asbestosis clearly
being detrimental but an apparent capacity of silica to also beneficially boost the
immune system with consequent ROS production.

5 Potential Medicinal Uses of Silicon and Silicates

The potential medicinal uses of silicon in the form of silica have only recently been
recognized particularly with respect to bone health and prevention of neurodegen-
erative diseases. Data are preliminary yet supportive of potential roles in reducing
the occurrence of type 2 diabetes and preserving and producing collagen, e.g.,
wound repair. Silicon is environmentally prevalent representing the second most
abundant element yet the biological availability of silica is limited and distributed
unevenly based largely on geographic location and source. As discussed previ-
ously, it is the orthosilicic acid that is water-soluble and bioavailable yet overall
intake and absorption could be improved. Thus, orthosilicic acid will likely be a
prominent therapeutic medicinal agent and, in fact, many potential therapeutic
applications have already been presented. For example, silicon appears to play a
significant role in maintaining bone health through increased bone formation and
increased bone mineral density and maintenance of connective tissues. Silicon, as
dietary silica, also inhibits absorption of toxic aluminum, which may contribute to
the development of Alzheimer’s disease. This occurs at a time when there is
increased prevalence of osteoporosis and Alzheimer’s disease as populations
worldwide become older. Other potential uses include enhancement of immune
468 Martin

function, preservation and health of skin, hair, and nails, and use as potential
antidiabetic and anticancer agents.
The development of new formulations of orthosilicic acid or orthosilicic acid-
releasing compounds is a promising means of delivering increased concentrations
of bioavailable and safe silicon. Choline-stabilized orthosilicic acid is a newly
developed, concentrated solution of orthosilicic acid in a choline and glycerol
matrix and is promoted as biologically active and the most bioavailable form of
silicon. Moreover, choline-stabilized orthosilicic acid has been approved for human
consumption and is considered relatively non-toxic with a tolerable upper limit
exceeding 5 g/kg body weight [28,41]. There are many other silicon supplements
available including extracts of horsetail, which contains 12 mg silicon per tablet of
which 85% is suggested to be bioavailable [28,29,65,74,132]. Overall, results of the
NHANES III study indicate a median intake of silicon from supplements to be 2
mg/d, but with preparations such as the aforementioned could markedly increase.
A particularly interesting area of research and development has been the emer-
gence and/or use of orthosilicic acid-releasing compounds. Specifically, certain
types of zeolites, a class of aluminosilicates with well-described ion (cation)-
exchange properties have been shown to release orthosilicic acid [1]. Overall, 191
unique zeolites have been described with over 40 naturally occurring zeolites identi-
fied. These are already widely employed in chemical and food industries, agricul-
ture, and environmental technologies but could find much greater use as medicinal
and/or nutritional agents. In fact, the biomedical applications of zeolites include, in
part, modulation of enzyme kinetics, use in hemodialysis, prevention of diabetes,
increased bone formation, function as an antidiarrheal and antibacterial agent and as
vaccine and tumor adjuvants [1]. The numerous biological activities of some types
of zeolites documented so far is thought to be due, in large part, to the orthosilicic
acid-releasing property.

6 Summary and Future Directions

In conclusion, silicon, as silica and silicates, represents a very large family of mol-
ecules with potential health benefits but also with potential toxic effects depending
on the form, water-solubility, route of exposure, and amount consumed. For exam-
ple, inhaled particulate fibrous crystalline silica can be toxic and depends heavily on
route of exposure and chemical form. Silica can also dissolve in water to form non-
toxic bioavailable silicic acids and specifically orthosilicic acid. This form of
absorbable silica found in foods and water sources, is readily absorbed, reaches key
tissue and organ target sites of action, and is efficiently excreted. The lack of appar-
ent toxicity of water-soluble forms that are consumed, as opposed to inhaled, and
the ongoing debate regarding essentiality as a micronutrient have obscured the rela-
tive importance of chemical speciation and potential contributions of silica.
Even though water-soluble to some degree, there are limitations to absorption
dictated largely by chemical instability, e.g., propensity to polymerize, and maximum
14 Silicon: The Health Benefits of a Metalloid 469

allowable concentrations of water-soluble orthosilicic acids. However, there has


been development of acid forms with markedly increased stability and, as a result,
significant increased concentrations and bioavailability of silicon. Choline chloride-
stabilized orthosilicic acid is a pharmaceutical formulation that is particularly prom-
ising but other forms exist including sodium or potassium silicates, and orthosilicic
acid-releasing forms such as zeolites.
Further research on silicon is critically needed particularly focusing on the
physiological roles of silicon and how this relates to human health, as well as the
dependence on chemical speciation. Specifically, ample data exist to support a pos-
sible role of silicon in wound repair, atherosclerosis and hypertension, diabetes,
several bone and connective tissue disorders, neurodegenerative diseases, e.g.,
Alzheimer’s and Parkinson‘s disease, and other conditions that occur particularly in
the aging population. It is also important to further elucidate biochemical mecha-
nisms of action of silicon-containing molecules, as silicic acids, and to extend test-
ing more into whole body systems. Specifically, larger studies with humans are
needed to explore the medicinal and nutritional potential of silicon.

Abbreviations

DRI dietary reference intake


IARC International Agency for Research on Cancer
IFN-γ interferon-γ
IL interleukin
MCP-1 monocyte chemoattractant protein-1
NF-κB nuclear factor B
NHANES National Health and Nutrition Examination Survey
NK cells natural killer cells
NTF tumor necrosis factor
OSA orthosilicic acid
RDA recommended dietary allowance
ROS reactive oxygen species
TUL tolerable upper limit

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Chapter 15
Arsenic. Can This Toxic Metalloid Sustain Life?

Dean E. Wilcox

Contents
ABSTRACT ............................................................................................................................. 476
1 INTRODUCTION ............................................................................................................. 476
1.1 Overview ................................................................................................................... 476
1.2 Chemical Properties of Arsenic ................................................................................ 477
1.3 Environmental Properties of Arsenic ........................................................................ 479
1.4 Biological Properties of Arsenic ............................................................................... 480
2 TOXICITY ......................................................................................................................... 481
2.1 Acute Toxicity ........................................................................................................... 481
2.2 Chronic Toxicity........................................................................................................ 481
3 SUSTAINING ROLES ...................................................................................................... 484
3.1 Nutritional Need for Arsenic? ................................................................................... 484
3.2 Hormesis and Arsenic ............................................................................................... 485
3.3 Surviving High Levels of Arsenic ............................................................................. 486
3.3.1 Microorganisms ............................................................................................. 486
3.3.2 Humans ......................................................................................................... 490
4 BENEFICIAL USES ......................................................................................................... 490
4.1 Pesticides ................................................................................................................... 490
4.2 Pharmaceuticals......................................................................................................... 491
4.2.1 Antibiotics ..................................................................................................... 491
4.2.2 Chemotherapeutics ........................................................................................ 492
5 SUMMARY ....................................................................................................................... 492
ABBREVIATIONS .................................................................................................................. 494
ACKNOWLEDGMENTS........................................................................................................ 494
REFERENCES ........................................................................................................................ 494

D.E. Wilcox (*)


Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA
e-mail: dwilcox@dartmouth.edu

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 475
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_15, © Springer Science+Business Media Dordrecht 2013
476 Wilcox

Abstract It was recently reported that a bacterium, Halomonas species GFAJ-1,


isolated from arsenic-rich Mono Lake and further selected for growth under condi-
tions of high arsenate and low phosphate, is able to grow using arsenic instead of
phosphorus. This claim, and subsequent studies to evaluate GFAJ-1, has brought
new attention to the question of whether arsenic can play an essential or sustaining
role for living organisms. If true, this would be in stark contrast to the well known
toxicity of this element and its ability to cause a number of diseases, including
cancer of the skin, lung, bladder, liver, and kidney. However, while deadly at high
doses, arsenic oxide is also an approved and effective chemotherapeutic drug for
the treatment of acute promyelocytic leukemia (APL). This review examines the
evidence that arsenic may be a beneficial nutrient at trace levels below the back-
ground to which living organisms are normally exposed. It also examines whether
arsenic can be used to sustain organisms growing under high arsenic conditions,
specifically the results from recent studies of arsenic biochemistry motivated by the
report of GFAJ-1. Both of these topics are considered in the context of the toxicity
of this element and its ability to cause cancer and other diseases, yet its Janus-faced
ability to effectively treat APL.

Keywords acute promyelocytic leukemia • arsenic • beneficial nutrient • GFAJ-1


• toxicity

Please cite as: Met. Ions Life Sci. 13 (2013) 475–498

1 Introduction

1.1 Overview

The biochemical and physiological properties of arsenic (As) are invariably linked
with the toxicity of this element. From earliest human use of arsenic as a constituent
of bronze and decorative pigments, there has been a collateral risk of toxicity for all
who worked with it. Even modern beneficial uses of arsenic are predominantly asso-
ciated with its lethality for organisms that affect crops, livestock, and human health.
Recent efforts to understand the correlation between chronic exposure to arsenic,
primarily through drinking water and food, and various diseases, including cancer
and diabetes, reinforces the detrimental side of this element. So, what is a chapter
on arsenic doing in a volume entitled, “Interrelations between Essential Metals Ions
and Human Diseases”?
In contrast to well-known essential trace elements, such as Cu, Mn, I, Mo, and
Se, a number of elements that are commonly found in living organisms at ultra-trace
levels, such as B, Si, V, Ni, and As, have not been recognized as essential for humans.
It has been difficult to establish whether or not there is a requirement for arsenic at
ultra-trace levels because of its prevalence in the environment from natural and
anthropomorphic sources, its ubiquity in living organisms, and the onset of its toxic
15 Arsenic. Can This Toxic Metalloid Sustain Life? 477

effects. Because arsenic has some chemical properties that are similar to those of
essential phosphorus, a relevant question is the interrelationship between these two
elements for living organisms, including the possibility that arsenate may substitute
for phosphate under certain conditions.
Arsenic is certainly associated with human disease, both as a causative agent
and as a therapeutic agent. Consumption of sub-lethal doses leads to a variety of
symptoms that are given the broad medical designation arsenicosis, but a clear
correlation has now been established between chronic exposure to arsenic and cancer
of the skin, lung, bladder, liver, and kidney, type II diabetes, depressed cardiovascular
function, and peripheral neuropathy. However, arsenic has been used to treat human
diseases since early times, and As-based Salvarsan®, which Ehrlich developed for
the treatment of syphilis, was one of the first modern pharmaceuticals. Although
As-based drugs have been largely replaced due to their toxicity, recently arsenic
oxide (As2O3), which was used in traditional Chinese medicine, has been approved
for treatment of acute promyelocytic leukemia (APL).
Although perhaps not a particularly good fit for this volume, the aim of this contri-
bution is to (i) summarize the evidence for beneficial or sustaining roles for arsenic in
living organisms, including its substitution for phosphorus, and (ii) summarize its
Janus-faced role in both causing and treating human disease. Tying these together is the
unifying theme of the toxicity of this element. After a brief introduction to the relevant
chemical properties of arsenic, its availability to living organisms, and its uptake,
metabolism and excretion, I begin with an overview of arsenic toxicity and its associa-
tion with a number of diseases. This is followed by the limited evidence for a beneficial
role for arsenic, including the phenomenon known as hormesis. Microorganisms living
in environments with high levels of arsenic have adapted to use its chemical properties
for their energy-generating pathway, but it appears not, as reported recently [1], by
substituting arsenate for phosphate in DNA and other biological molecules. I finish
with the beneficial roles of arsenic in modern society, and its use in treating human
disease, including its remarkable therapeutic role in treating APL.

1.2 Chemical Properties of Arsenic

Two oxidation states of arsenic, As3+ (r = 0.58 Å) and As5+ (r = 0.46 Å), are found
under environmental and biological conditions. Arsenic(III) is normally three-
coordinate with pyramidal structures due to a stereochemical lone pair of electrons
(Figure 1). Because of its size and polarizability, As3+ has a relatively high stability
with softer S- and Se-donating species. In aqueous solution it is found as the
neutral tris-hydroxo arsenite, As(OH)3 whose lowest pKa is 9.2. Arsenic(V) is nor-
mally four-coordinate with tetrahedral structures. Its greater charge density leads
to a higher stability with harder O-donating species and its prevalence in aqueous
solution as arsenate, AsO43 − in a pH-dependent protonation state (pKa’s 2.3, 7.0, and
11.5). Comparison of arsenic and phosphorus is instructive, due to the similarity
between arsenate and phosphate, PO43 − (pKa’s 2.1, 7.2, 12.7), which have a similar
Figure 1 Structural representations of environmentally, biologically and medically important
arsenic molecules: 1. arsenite (arsenous acid) (monomethyl- and dimethylarsenite have one and
two –CH3’s replacing –OH’s), 2. arsenate (arsenic acid) (monomethyl- and dimethylarsenate have
one and two –CH3’s replacing –OH’s), 3. arsenobetain (AsB), 4. arsenocholine (AsC), 5. arsenosugars,
6. arsenolipids, 7. roxarsone, 8. Salvarsan® (mixture of trimer and pentamer), 9. melarsoprol.
15 Arsenic. Can This Toxic Metalloid Sustain Life? 479

structure and size. However, the arsenate 2-electron reduction potential, ε°’ = +140
mV (pH 7.0, 25°C, versus NHE) is significantly higher than that of phosphate
(ε°’ = –690 mV), resulting in the prevalence of both arsenite and arsenate under
environmental and biological conditions. Since arsenic is larger and has longer
(weaker) bonds than those of phosphorus, substitution reactions of arsenate species
are faster than those of the corresponding phosphate species. Arsenic in both
oxidation states forms stable bonds with carbon, most prevalent of which are
those with one to three methyl groups (Figure 1). Thus, the environmental and
biological chemistry of arsenic can be divided into that of inorganic (arsenite and
arsenate) and various organic species, which lack and contain stable As-C bonds,
respectively.

1.3 Environmental Properties of Arsenic

Although more than 20 years old, the review by Cullen and Reimer [2], recently
augmented by their review of organoarsenic species in this series [3], provides a
thorough overview of arsenic in the environment. Thus, only the prevalence, distribution,
and availability of arsenic will be mentioned here. While not an insignificant compo-
nent of the earth’s crust at 1.8 ppm, arsenic is unevenly distributed. Although found
in the sulfide ores orpiment, As2S3 (yellow arsenic) and realgar, AsS (red arsenic),
arsenic is more commonly a constituent of the mineral arsenopyrite, FeAsS, and
associated with various iron oxides. The release of arsenic from these minerals and
geological formations is governed typically by redox processes.
The concentration of arsenic in the ocean is 1–3 µg/L (ppb), but in typical fresh-
water and aquifers it has a wide range, 0.1–80 µg/L, depending on the local geology
and geological processes. However, some conditions result in even higher local con-
centrations, such as 700 µg/L in certain aquifers in Taiwan and 15 mg/L (ppm) in
Mono Lake in California due to evaporative concentration. The predominant arsenic
species in natural waters depends on the redox potential, which is normally deter-
mined by the amount of dissolved oxygen, and the pH. Arsenate will predominate
under aerobic conditions (e.g., surface water), while arsenite will predominate
under anoxic conditions (e.g., subsurface aquifers). Although the relative amount of
each species can be predicted from thermodynamic considerations, kinetic barriers
and biological redox processes can alter the arsenate-arsenite ratio. Organoarsenic
species can contribute to the total arsenic when biological activity (predominantly
methylation) is prevalent, particularly in marine environments. Human activities
can alter local and regional levels and the distribution of inorganic arsenic (e.g.,
mining and processing As-containing ores, burning As-rich coal) and organic arse-
nic (e.g., agricultural application of organoarsenic pesticides). Arsenic is injected
into the atmosphere by volcanoes and combustion and by microbial activity that
generates volatile arsine (AsH3) and methylated arsenic species. However, the atmo-
spheric residence time of arsenic species is relatively short and the arsenic concen-
tration is generally low (0.02 µg/m3), except in the vicinity of these sources.
480 Wilcox

1.4 Biological Properties of Arsenic

Arsenate and arsenite are taken up by microorganisms, plants (root cells), and ani-
mals (intestinal cells) with different mechanisms. Arsenate is actively imported by
two pathways used for the essential and structurally similar phosphate, as shown in
competition uptake studies [4,5]. The low affinity phosphate inorganic transport
(Pit) pathway uses energy from the trans-membrane proton gradient, while the high
affinity phosphate specific transport (Pst) pathway confers some selectivity for
phosphate over arsenate with a periplasmic phosphate binding protein (PBP) and an
ATP-hydrolyzing membrane transporter [6]. Neutral arsenite, however, diffuses
through membrane-spanning channels created by aquaglyceroporin proteins, which
allow the diffusion of water, glycerol, and other neutral species [7]. The properties
of organoarsenic species are modulated by their organic substituent(s), and this
affects their uptake by these or other pathways.
Due to the prevalence of arsenic there is selective pressure for resistance to this
toxic element, and microorganisms have genomic and plasmid-encoded mecha-
nisms for exporting arsenic upon its inevitable import. The best studied of these
involves proteins encoded by the ars operon, and include the minimal set of ArsR,
a DNA-binding repressor protein that suppresses ars expression until arsenite binds
and it is released from the operator DNA, ArsC, which is a cytoplasmic glutathione-
dependent arsenate reductase, and ArsB , which is a membrane-spanning protein
that exports arsenite using energy from the trans-membrane proton gradient [8].
Some ars operons also code for a membrane-associated ArsA, which couples ATP
hydrolysis to arsenite export by ArsB for more efficient removal of arsenic.
Certain bacteria, as well as higher organisms from fungi to humans, are capable
of methylating arsenic by a mechanism originally outlined by Challenger [9], based
on oxidative addition from the activated methyl donor S-adenosyl methionine.
Enzymes that catalyze this coupling reaction have been isolated [10] and this
appears to be part of a detoxification mechanism, as glutathione complexes of these
methylated species are exported by multi-drug resistance protein (MRP) transport-
ers [11]. Certain microorganisms are capable of demethylating organoarsenic spe-
cies by a mechanism that may involve the reverse process of reductive elimination
[12]. Some plants are capable of growing on soils that have high levels of arsenic,
and these species often hyperaccumulate arsenic, which is transported to the leaves
where it is sequestered in vacuoles [13]. Certain marine plants have pathways to
synthesize various organoarsenic species, such as arsenocholine (AsC) and arseno-
betaine (AsB) (Figure 1), which is found in seaweed and may not only be a product
of arsenic detoxification but also help to maintain osmotic balance. These and other
organoarsenic species (e.g., arsenosugars, arsenolipids) (Figure 1) become widely
distributed in marine food webs [14].
Normal human blood levels of arsenic are 0.3–2 µg/L but can be 1–2 orders of
magnitude higher when elevated levels of arsenic are being consumed in drinking
water or the diet. The overall half-life of arsenic in humans is about 10 hours, though
elimination appears to be triphasic; while some may be retained for longer periods,
there is no biological sink or pool where arsenic accumulates. The urine of individuals
15 Arsenic. Can This Toxic Metalloid Sustain Life? 481

consuming elevated levels of inorganic arsenic contains monomethyl (MMAs) and


predominantly dimethyl (DMAs) arsenic species [15]. These are enzymatically
formed primarily in liver cells by arsenic methyltransferases, As3MT [16]. Although
readily absorbed from the diet, organoarsenic species are rapidly excreted in the
urine, as are the intra-cellularly methylated species MMAs and DMAs after their
active export by MRPs or diffusion out through aquaglyceroporins [17].

2 Toxicity

The toxicity of arsenate is due to its competition with isostructural phosphate, yet
hydrolytic instability of arsenoester bonds results in unstable species, such as the
ATP analog ADP-arsenate [18,19]. Arsenate lowers ATP levels by uncoupling its
synthesis through a general mechanism known as arsenolysis. However, the toxicity
of arsenite, which predominates under reducing intracellular conditions, is due to its
affinity for functionally and structurally important thiols. This makes it an effective
inhibitor of enzymes such as pyruvate dehydrogenase, which is required for the
citric acid cycle [20]. Alternatively, redox generation of reactive oxygen species
(ROS) has been suggested as a mechanism for this inhibition [21]. Methylation of
arsenic modulates its properties and thus its toxicity that, depending on the organ-
ism (e.g., tissue culture, animal model) and the mode of exposure (e.g., oral, injec-
tion), can be higher or lower [22]. The toxicity of organoarsenic species is dictated
to a large extent by the organic substituent(s). AsB and AsC, like various arseno-
sugars and arsenolipids, lack exchangeable valence sites and have low toxicity, par-
ticularly AsB, which is abundant in seafood. A Provisional Tolerable Daily Intake
(PTDI) for inorganic arsenic has been set at 2.1 μg/kg/day [23], though this has
recently been withdrawn [114].

2.1 Acute Toxicity

Ingestion of overtly toxic amounts of inorganic arsenic species (60–120 mg is the


estimated lethal dose of As2O3) leads to gastroenteritis, characterized by vomiting,
abdominal pain, and bloody diarrhea, which lead to dehydration, shock, con-
vulsions, coma, and death. Doses that overwhelm cellular and tissue mechanisms
that remove arsenic also result in circulatory collapse and depression of the central
nervous system.

2.2 Chronic Toxicity

Chronic arsenic exposure leads to symptoms given the general designation arsenic-
osis, which affects millions worldwide and arises primarily through contaminated
drinking water [24,25]. Since arsenic occurs at elevated concentrations in many
482 Wilcox

natural waters and is colorless and odorless, people were unaware when their water
had high levels of arsenic and the consequences of chronic arsenic exposure were
largely unknown. Water testing is now common and the consequences of exposure
are well documented. Nevertheless millions remain exposed because of significant
socio-economic barriers to providing them with clean water.
One of the first correlations between human disease and arsenic exposure was
the large-scale incidence of black foot disease (BFD) in southwestern Taiwan [26].
This condition, which is due to peripheral vascular disease, is manifest as a discol-
oration and blackening of the extremities, especially the feet. Subsequently it was
shown that exposure to elevated arsenic in drinking water from deep artesian wells
of the BFD area of Taiwan correlates with increased incidence of cancer of the skin
[27], diabetes [28] and cardiovascular disease [29,30]. Epidemiological studies
from Taiwan formed the basis for the reduction of the US drinking water limit from
50 μg/L to 10 μg/L, which finally came into effect in 2006. Similar relationships
between cancer and arsenic exposure through drinking water have been docu-
mented in Argentina [31,32], Chile [33,34], and, most recently, Bangladesh [35].
Epidemiological results from the Antofagasta region of Chile show that in utero
and early life exposure to arsenic predisposes individuals to increased incidence of
lung cancer and bronchiectasis in later life [36]. For the well-studied cases in
Taiwan and Chile, exposure occurred in the early to mid 20th Century and drinking
water has since been switched to lower arsenic sources. In contrast, many millions
in Bangladesh and West Bengal are still exposed to high levels of arsenic from
drinking water obtained from deep tube wells [37,38]. The Bangladesh situation
has been widely described as the worst mass poisoning in human history, and came
about through the installation of an extensive series of tube wells in the 1970’s
sponsored by UNICEF in an effort to reduce childhood disease and mortality from
drinking microbially-contaminated surface water [39].
The mechanism(s) by which arsenic causes these diseases from chronic exposure
is not yet well understood. With regard to cancer, while it is genotoxic, arsenic also
affects cell proliferation, cell signaling, DNA structure (methylation), epigenetic
regulation, DNA repair, and apoptosis [40]. With so many effects, some of which
may originate from the non-specific generation of ROS [41], it is difficult to identify
molecular targets for arsenic, and the mechanism by which arsenic causes cancer is
still poorly understood [42].
One case where a specific arsenic target has been identified is its effect on
hormone-regulated pathways. Arsenite is known to disrupt steroid hormone func-
tion at the non-cytotoxic level of 1–5 μM, and in vitro and intracellular studies of the
glucocorticoid receptor (GR) have traced arsenic effects to its DNA-binding domain
(DBD) [43]. This ~90 residue domain has 10 cysteines that bind two Zn2+ ions to
stabilize a folded structure that is competent to bind to its target DNA, the glucocor-
ticoid response element (GRE) (Figure 2 [44]).
The hypothesis that arsenite displaces Zn2+ from the thiols and disrupts the active
structure of the domain was tested and shown to be valid for MMAsIII, but not
arsenite [45], consistent with the higher MMAsIII stabilities with small thiols [46].
Further, based on the measured stability constants and cellular conditions, it was
15 Arsenic. Can This Toxic Metalloid Sustain Life? 483

450
C
G H
S Y I
K R
A G
D R
E V
I K 490
D L
I N
S T 480
C C N DC C
V G P
Zn R Zn
L S A
G A
C 440 C 460 C C
K 470 L R 500 510
V F F K R A V E G QH N Y Y R KC L QA GMN L E A R

445
486 N-term
482
448
457
492
476

C-term

508
469
471

Figure 2 Amino acid sequence (top) and the NMR-determined 2° and 3° structure (bottom) of the
rat GR-DBD (residues 440-510) with the two Zn2+ ions indicated by spheres. Reprinted with
permission from [44]; copyright 1993 American Chemical Society.

estimated that approximately 0.5 μM MMAsIII would displace an essential Zn2+ and
inhibit (IC50) GR binding to GRE. While arsenite may have other effects on the
steroid hormone pathway [47], GR-DBD and homologous DBDs of other steroid
hormone receptors appear to be a target for the monomethylated metabolite of arsenite
[48]. Similarly, MMAsIII is able to compete with an essential Zn2+ for Cys thiols of
the nucleotide excision repair protein Xeroderma pigmentosum group A [49], thereby
identifying a potential target for arsenic in its known role as a co-carcinogen.
With multiple potential mechanisms of action for arsenic-induced disease, health
risks from exposure to arsenic are currently based on a “linear, no threshold” (LNT)
dose-response model from epidemiology data for populations exposed to elevated
levels in Taiwan and Chile [50]. However, some of these data have been re-analyzed
484 Wilcox

and this model called into question [51]. Extrapolation to the low level of exposure
that is widely encountered leads to uncertainty in risk for those populations with
low background levels of arsenic in their drinking water and diet [52].

3 Sustaining Roles

3.1 Nutritional Need for Arsenic?

The currently recognized essential nutrients for humans, besides glucose and the
essential amino acids and fatty acids, includes 13 vitamins (one of which is
Co-containing vitamin B12, required in ultra-trace amounts), six elements (Ca, P,
Mg, Na, K, Cl) required in macroscopic amounts, five elements (Fe, Zn, Cu, Mn, F)
required at trace levels (~1–10 mg/day), and four elements (I, Se, Mo, Cr) required
at ultra-trace levels (<1 mg/day). In addition, there is some evidence that certain
other elements found in humans at ultra-trace levels, including B, Si, Ni, V, and As,
may play an essential biochemical role, but the evidence in each case is not compel-
ling enough for a human nutritional requirement. Some of these, such as Ni, B, and
Si, are required by plants and/or microorganisms.
What is the evidence for any nutritional need for arsenic? Early studies with rats
investigated arsenic deficiency but these tended to be compromised by high back-
ground levels and elevated (toxic?) control (sufficiency) levels. Careful studies of
arsenic deprivation (<50 ng As/g ration) with goats, minipigs, chicken, rats, and
hamsters have shown depressed growth and abnormal reproduction [53,54]. The
latter includes low fertility, elevated perinatal mortality (spontaneous abortions),
maternal death during lactation, and higher offspring mortality. Post-mortem analy-
sis of arsenic-deficient goats found compromised mitochondrial membranes in their
myocardial tissue [53].
Extrapolation of results from these animal studies leads to 12–25 μg/day as an
estimated human dietary requirement [53], which is well below the previous PTDI
(150 μg/day for a 70 kg adult). Since market basket analysis shows that typical
dietary arsenic is <10% of the previous PTDI [55] and typical arsenic intake in water
and food is estimated to be 12–60 μg/day [56], diets not meeting such a need would
be very rare. However, two caveats should be noted. First is a caution about extrapo-
lating results from animal studies to humans. It is known that arsenic metabolism is
different in common animal models: arsenic is uniquely sequestered in the erythrocytes
of rats [57], and certain primates do not methylate ingested arsenic [58]. Second is
the absence of evidence for an essential arsenic role in patients undergoing long-
term total parenteral nutrition (TPN) therapy (intravenous delivery of nutrients).
Such an individual provided the first human evidence that chromium plays a role in
regulating blood sugar levels [59]. Nevertheless, a need for trace amounts of arsenic
might be missed in TPN therapy due to trace impurities of arsenic in the nutrients
for these individuals [60]. Further, animal studies show that arsenic deficiency
15 Arsenic. Can This Toxic Metalloid Sustain Life? 485

affects growth, development, and reproduction, and most TPN patients are adults
where these effects could be missed.
Studies by Nielsen, Uthus and coworkers at the Grand Forks Human Nutrition
Research Center have sought the metabolic or biochemical origin of the effects of
arsenic deprivation. Lower levels of taurine (H2NCH2CH2SO3H), S-adenosyl methi-
onine and polyamines, and elevated S-adenosyl homocysteine are detected in rats
fed a low arsenic (<50 ng/g) diet, suggesting that arsenic may play a role in the
metabolism of methionine or modulation of methylation capacity [54]. Arsenic
deprivation also affects enzymes involved in the biosynthesis of phosphatidylcho-
line [61] that, along with lower levels of taurine, could affect mitochondrial mem-
branes, as found in the myocardial tissue of As-deprived goats [53].
Whether or not there is any beneficial role for arsenic in human physiology and
biochemistry is probably a moot point because it is ubiquitous and prevalent at the
very low levels that might be required for roles in growth, development, and repro-
duction, as found in animals. No well-defined biochemical role has been found for
arsenic that would explain these effects of its deprivation. Ironically, several of the
adverse outcomes on reproduction found with As-deprived animals (spontaneous
abortion, offspring mortality) are also found in human populations exposed to toxic
levels of arsenic [40].

3.2 Hormesis and Arsenic

It has been noted that low levels of some toxic species have a stimulatory effect on
certain biochemical processes and the growth of some organisms, prior to the onset
of toxic effects at higher levels. Known as hormesis, and characterized by an upside
down U response curve, this phenomenon has been found with arsenic in studies
involving microorganisms, plants, invertebrates, and human cell cultures.
Several early studies of the effect of arsenate, and in some cases arsenite, on the
growth of crop plants (pea, wheat, potato, bean, oats) show enhanced growth at low
arsenic concentrations, followed by depressed growth at higher concentrations [62].
More recently this has been found for salt marsh grass (Spartina alterniflora), where
enhanced growth at low arsenate or arsenite (0.2 mg/L) is associated with higher lev-
els of phosphorus in the roots [63]. Similar results were found with an As-accumulating
plant, the Chinese brake fern (Pteris vittata L), but not its non-hyperaccumulating
cousin (Pteris ensiformis L) [64]. Again, the As-stimulated growth was associated
with higher phosphorus in the roots, as well as elevated arsenic transported to the
fronds, as is common for hyperaccumulating plants. Finally, in a model of
As-contaminated Taiwanese aquaculture ponds, the growth of algae (Microcystis
aeruginosa) showed stimulated growth at 0.1 μM arsenate but suppressed growth at
higher concentrations [65]. The connection with phosphorus metabolism in some of these
studies is noteworthy. While arsenic could be mobilizing growth-limiting phosphorus
from soil, the salt marsh grass study was conducted with hydroponically grown plants.
Thus, arsenic stimulation of growth may be due to increased phosphate uptake. It has
486 Wilcox

been suggested that arsenate competition and uncoupling of phosphate-requiring


energy-generating pathways (arsenolysis) is biochemically equivalent to a phosphate
deficiency and stimulates phosphate uptake [66].
A stimulated response at low arsenic levels has also been found in cell culture
and animal studies. This has been reported for: DNA synthesis in human lympho-
cyte cell cultures [67]; growth of various cell types, where the As-enhanced growth
correlated with lower levels of some cytogenetic effects [68]; base excision repair in
human lung fibroblasts and keratinocytes, where low levels of arsenic (<1 μM) cor-
relate with elevated levels of DNA polymerase-β [69]; and estrogen receptor-alpha
expression and activity in breast cancer cells [70]. Arsenite stimulation at low doses
(<1 μM) has also been reported for the intracellular activity of GR (Figure 3) and
other steroid hormone receptors [48]. The freshwater amphipod (Hyalella azteca),
which accumulates arsenic and metals, shows a maximum stimulation of its growth
under conditions where it has accumulated arsenic to a level of ~5 ppm [71]. Finally,
a modest suppression of DNA damage in liver and lung tissue was found in rats
exposed to low levels of arsenite [72].
Enhanced DNA synthesis and repair upon exposure to low levels of arsenic, which
are then overwhelmed at higher levels, may explain some of these results. Thus, there
could be a common basis for certain cases of arsenic hormesis, which may originate
from a general response to toxic species, such as stimulated production of ROS [41].
However, it should be noted that hormesis is not a scientifically well-defined
phenomenon based on known mechanisms and is controversial [73].

3.3 Surviving High Levels of Arsenic

3.3.1 Microorganisms

Certain ecological conditions, such as those found in or near volcanoes, highly


saline lakes, and gold mines that are rich in arsenopyrite, contain high levels of
arsenic, yet some microorganisms survive under these conditions. A major challenge
for these organisms is dealing with the toxicity of arsenate imported by their essential
phosphate pathways, the low affinity Pit pathway and the high affinity Pst pathway,
though the latter provides some selectivity for phosphate over arsenate.
Microorganisms use various strategies to survive under these conditions, including
more efficient arsenic export pathways, such as that encoded by the ars operon, and
oxidizing arsenite in the immediate vicinity to the less toxic arsenate.

3.3.1.1 Redox

Certain microorganisms found under conditions of high arsenic levels are able to
exploit its redox properties for their energy-generating pathways. All living organ-
isms require the energy from hydrolysis of ATP, which is regenerated by ATP
synthase using the trans-membrane proton gradient created by the electron transport
15 Arsenic. Can This Toxic Metalloid Sustain Life? 487

Figure 3 Effect of low a 2.00


concentrations of arsenite on Human WT
human (a) and rat (b) GR 1.75
activation of transcription
of a GRE- and luciferase- 1.50
containing reporter gene

Relative Activity
construct transfected into 1.25
EDR3 hepatoma cells.
Reprinted with permission 1.00
from [43]; copyright 2004
American Chemical Society. 0.75

0.50

0.25

0.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0
As (μM)
b
1.75
Rat WT
1.50

1.25
Relative Activity

1.00

0.75

0.50

0.25

0.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0
As (μM)

pathway. This pathway starts with the oxidation of low potential electron donor
species (i.e., reduced carbon molecules) and ends with the reduction of a high
potential electron acceptor (i.e., oxygen). Since the arsenate reduction potential lies
near the middle of the biologically useful range, depending on the availability of
reductants (e.g., reduced carbon species) and oxidants (e.g., O2), arsenite (As3+)
could serve as the electron donor or arsenate (As5+) could serve as the electron
acceptor for the electron transport pathway.
The first of these two roles was found for chemolithoautotrophs that can use arsenite
oxidation as a source of electrons and CO2 as a carbon source [74]. These were
organisms found initially in cattle dipping solutions, where arsenic was used to kill
ticks on livestock, and later in gold mine tailings. The second of these two roles was
found initially for Eubacteria species [75], and later other bacteria and archaea, that
488 Wilcox

use arsenate as a terminal electron acceptor to support dissimilatory respiratory


[76]. However, the mechanism by which arsenate reduction is coupled to the gen-
eration of a trans-membrane potential gradient is not known. In each of these cases,
abundant arsenic can be used to sustain the organism by its contribution to the
essential energy-generating pathway.

3.3.1.2 Phosphate Substitute?

Since arsenate and phosphate have similar structural properties, it may be possible
for the former to substitute for one or more of the essential roles that the latter plays
in biology. Westheimer considered this notion over 25 years ago [77] and dismissed
it for sound chemical reasons, specifically the rapid hydrolysis of arsenate ester
bonds in aqueous solution. Thus, there was considerable surprise a few years ago
when it was reported that a microorganism, Halomonas strain GFAJ-1, isolated
from As-rich Mono Lake and selected for growth on high arsenic and low phospho-
rus, grew in the presence of arsenate and absence of phosphate [1]. Further, evi-
dence was provided that arsenate was associated with the DNA of this organism,
suggesting that arsenate replaced at least some of the phosphate.
Several critiques of the experimental conditions, data analysis, and conclusions,
as well as alternate explanations and contradictory chemical properties (e.g., reduc-
tion of arsenate to arsenite under intracellular conditions), and the authors’ response
to these criticisms, accompanied publication of the original report [78]. Additional
critiques that elaborated these and other points followed [79–82]. Three types of
studies were motivated by the original report, those aimed to refute the original
results, those aimed to provide alternate explanations, and those aimed to address
the possibility that arsenate could substitute for phosphate.
The first type focused on the unique organism, GFAJ-1, which was made avail-
able to the scientific community. One of these publications showed that its DNA
was hydrolytically stable, contrary to the expectation with arsenate diester linkages,
and mass spectral data indicated the absence of arsenic in the DNA [83]. Another
showed only small amounts of arsenic in various phosphorus metabolites and pro-
vided elemental analysis indicating the absence of arsenic in the GFAJ-1 DNA [84].
In addition, the genome of this organism was mapped and no unusual operons or
genetic properties were found [85].
Two recent studies suggest alternate explanations for results in the original
report. The first provides evidence for arsenic destruction of bacterial ribosomes,
which would liberate enough phosphorus for a small population of As-resistant
organisms to grow slowly after a lag period [86], as reported for the growth of
GFAJ-1. This toxic effect of As had not been reported previously, and may relate to
arsenic effects on known metabolic factors (e.g., starvation) that lead to ribosome
destruction [87]. The second addressed the ability of GFAJ-1 to grow, albeit slowly,
under such high As:P ratios, and focused on the induction and activity of its
periplasmic PBP’s, finding one from GFAJ-1 with a 10-fold higher phosphate-arsenate
discrimination than PBP’s from other organisms [88]. In addition, this study reported
the high-resolution phosphate- and arsenate-bound structures of the Pseudomonas
15 Arsenic. Can This Toxic Metalloid Sustain Life? 489

Figure 4 High resolution a Oδ1


X-ray crystal structures of
phosphate (top) and arsenate
D62
(bottom) bound to the P.

fluorescens PBP, indicating
their hydrogen bonding 122.0°
interaction with Asp-62 2.51 Å
of the protein. Reprinted Oδ2
with permission from [88]; O2
copyright 2012 Nature 179.1°
Publishing Group. 108.7°

P
O1
O3
O4

b Oδ1

D62

127°
162°
Oδ2
O2
2.50 Å
95.4°

As

O1
O3 O4

fluorescens PBP, which identified a unique low-energy hydrogen bond that appears to
provide the selectivity for phosphate (Figure 4). However, the molecular origin of the
unusually high phosphate selectivity of the GFAJ-1 PBP has yet to be determined.
While little support remains for the original claim that GFAJ-1 substitutes arse-
nate for phosphate in its DNA, this report motivated several efforts to examine this
possibility, which may be relevant to alternate biochemistry of extra-terrestrial
organisms. In particular, computational studies have examined the consequences of
a P → As swap in DNA. While the structural differences are modest [89–91], the
arsenate diester bonds are weaker [92] and their hydrolysis would be significantly
faster than that of native DNA [93], as predicted [77] based on data for small
arsenate complexes [94]. DNA, with an estimated hydrolytic half-life of 30 million
years [95], and the information encoded therein would not be sustainable for life
forms that use arsenic instead of phosphorus in an aqueous environment.
Thus, certain microorganisms can not only survive exposure to high levels of
arsenic, but some take advantage of the unique reduction potential of this generally
toxic element and can use abundant arsenite as an electron donor (chemolithoautotrophs)
or arsenate as a dissimilatory electron acceptor under anaerobic conditions for their
electron transport pathway. To survive exposure to this toxic element, organisms
490 Wilcox

employ one or more mechanisms, including a higher phosphate selectivity in their


phosphate import pathway, more efficient or active arsenic export pathways, seques-
tration of arsenic in specialized organelles, and increased repair of damage to DNA
and other biomolecules caused by arsenic.

3.3.2 Humans

Despite the well-known toxicity of arsenic, there are instances, mostly from the 18th
and 19th Centuries, of arsenic being used therapeutically as a general tonic, in some
cases at levels higher than believed to be fatal. The best example of these so-called
‘arsenic eaters’ is the inhabitants of Styria, now part of Austria. Their story was reported
in the scientific literature at the time, formed the basis for a 1939 Ph.D. thesis, and has
been summarized recently [96]. Certain individuals from regions in the Alps began
eating arsenic in the belief that it increased the ability to breathe easily, improved the
complexion, aided digestion, and protected against infectious disease. These individu-
als began by consuming small amounts of arsenic oxide (As2O3) and gradually
increased the amount consumed, such that they were eventually consuming doses that
would otherwise be considered fatal. Stories of these arsenic eaters were greeted with
skepticism at the time, and it was claimed that the consumed substances contained only
minor amounts of arsenic or the arsenic solids were poorly adsorbed during digestion.
However, a relatively compelling case has been made that there was likely some truth
about the arsenic eaters of Styria. Indeed it has recently been suggested that arsenic
exposure might lead to epigenetic changes that favor increased tolerance to altitude
sickness [97]. Survival of supra-toxic doses of arsenic suggests a conditioned enhance-
ment of detoxification mechanisms, possibly overexpression of the arsenic methylation
enzyme, As3MT, and/or MRP that exports arsenic-glutathione conjugates.
A contemporary example of deliberate arsenic consumption is associated with
the Chinese Dragon Boat Festival [98]. Part of the festivities involves consumption
of wine with high levels of realgar (AsS) and face-painting with realgar-based
paints. While this arsenic sulfide is particularly insoluble and, therefore, presum-
ably less bioavailable, elevated arsenic levels in the urine of children after face
painting and adults after drinking the realgar wine are reported. These elevated
urinary levels were reported to persist for quite some time, suggesting that at least
some of the arsenic is bioavailable and absorbed dermally.

4 Beneficial Uses

4.1 Pesticides

Arsenic compounds have a long history of use as pesticides in the US and indeed
worldwide. In the early 20th Century lead arsenate and calcium arsenate were
commonly used in agriculture, most notably against insects in apple orchards [99].
Use of these pesticides decreased after the introduction of DDT but they were not
15 Arsenic. Can This Toxic Metalloid Sustain Life? 491

officially banned in the US until the 1990’s. The use of these arsenical pesticides has
created a problem of legacy arsenic, and lead, in old orchard soils, which has human
health implications when this land is used for urban development [100]. These
contaminated soils are also potential point sources for wider distribution of arsenic
in the environment [101,102]. Copper chrome arsenate (CCA) is a wood preserva-
tive compound that has been used extensively to suppress fungal rot in exterior
lumber for residential and non-residential use. CCA is no longer registered for resi-
dential use in the US but can still be used to treat other lumber products.
Various organoarsenicals have been developed and used for their beneficial prop-
erties. MMAs and DMAs were used extensively as herbicides in cotton production
and on golf courses. In the environment these organoarsenic species are less toxic
than inorganic arsenic, but the possibility for their microbial demethylation into
inorganic species [12] has prompted environmental concerns. Consequently, these
two organoarsenic compounds were not re-registered in the US, effectively banning
them from future use. Various phenyl-arsenic compounds, such as roxarsone,
3-nitro-4-hydroxyphenylarsonic acid (Figure 1), have been used as feed additives in
poultry and swine production, as they control the parasitic protozoan disease coc-
cidiosis in poultry and increase growth, presumably by lowering enteric microflora
levels and thereby increasing nutrient absorption. There has been a concern about
the potential for arsenic contamination of edible chicken meat. However, the US
Food and Drug Administration (FDA) has a requirement for the withdrawal of arse-
nic growth promoters from chicken feed five days before slaughter, and regulations
for the maximum arsenic content in edible muscle and liver. Because poultry manure
contains high levels of unabsorbed roxarsone and is mostly applied back to agricul-
tural land as a soil amendment, there is concern about the fate of this organoarsenic
species, including its microbial degradation to inorganic arsenic [103], and local-
ized arsenic loading of soil. In 2011 roxarsone was voluntarily withdrawn from use
as a feed additive in an agreement between the US FDA and the poultry industry.
Hence, the widespread use of arsenic in agriculture has now largely been discontin-
ued, at least in the US. While none of these widespread beneficial uses of arsenic
have a specific relationship to human disease, they do indicate the anthropomorphic
distribution of arsenic that contributes to its background level and, depending on the
extent and duration of exposure, its potential for toxic disease-causing impact.

4.2 Pharmaceuticals

4.2.1 Antibiotics

The toxicity of arsenic was the basis for its earlier use in formulations found to
be effective against various microbial-based diseases, beginning with Ehrlich’s
development of Salvarsan®, arsphenamine (Figure 1), to treat syphilis [104].
Although largely replaced by therapeutic agents that target specific microbial pathways
and lack arsenic’s known toxicity, the organoarsenical melarsoprol (Figure 1) is still
used to treat trypanosome infections in Africa.
492 Wilcox

4.2.2 Chemotherapeutics

Arsenic compounds have been a component of traditional Chinese medicines,


including those for the treatment of cancer, and Fowler’s solution (1% potassium
arsenite) was in use by the mid-18th Century to treat a number of diseases and con-
ditions, including leukemia. Largely replaced by other chemotherapeutic agents in
the mid-20th Century, recently arsenic was found to be remarkably effective against
one type of leukemia, acute promyelocytic leukemia (APL), leading to high rates of
remission and survival [105]. Arsenic is known to affect a number of cellular pro-
cesses, many of which are associated with elevated ROS levels, that result in apop-
tosis [106]. However, the efficacy of As2O3 in treating APL at relatively low doses
suggests there is a specific molecular target.
This form of leukemia arises from a chromosomal translocation that results in a
fusion protein that is generally formed between the promyelocytic leukemia protein
(PML) and the retinoic acid receptor α (RARα). It was shown that arsenic decreases
the level of this oncogenic PML-RARα fusion protein by inducing its degradation,
which begins with the attachment of ubiquitin or the small ubiquitin-like protein
(SUMO) [107]. This targeting for degradation is associated with the PML protein,
which contains three cysteine-rich Zn-binding sequences, one of which binds two
Zn2+ in a structure known as a RING domain. Both in vitro and in vivo results [108]
suggest that As3+ binding to this domain affects the SUMOylation and/or ubiquitina-
tion of the protein, which labels it for degradation, consistent with an X-ray crystal
structure showing the interaction between a RING domain and a ubiquitin conjugase
[109] (Figure 5). The level of arsenic required for this therapy is not overtly toxic, in
contrast to levels that are required to affect the progression of other cancers by alter-
nate pathways, including those that induce apoptosis of cancer cells. Recently it has
been shown that arsenic oxide is also effective against chronic myeloid leukemia
(CML) and that a target protein containing a RING domain appears to be involved
[111]. A chromosome translocation associated with this cancer also leads to an onco-
genic fusion protein, but here the putative RING-containing target protein is a ligase
that can ubiquitinate the CML fusion protein and whose activity is altered by arsenite.
Thus, for APL, and possibly CML, there appears to be a specific molecular target for
arsenic that is based on the thiophilic nature of arsenite and its competition with Zn2+.

5 Summary

It has been claimed that arsenic has killed more human beings than any other substance
[112]. Whether or not this is true, the hallmark of this element is its toxicity, which
is still a problem. Efforts to provide clean water for large populations in Bangladesh
and nearby Indian provinces have exposed millions to elevated levels of arsenic
and its toxic effects, including higher risk for several types of cancer, diabetes, and
cardiovascular and neurological problems. This has created one of the largest, if not
the largest, public health incident of modern times.
15 Arsenic. Can This Toxic Metalloid Sustain Life? 493

Figure 5 X-ray crystal structure of the ternary complex of the RING-containing ligase c-Cbl, the
ubiquitin-conjugating UbcH7, and a peptide from ZAP-70 tyrosine kinase [109], which serves as
a model for the interaction between the RING domain of PML and the conjugase protein Ubc9
[110]. Reprinted with permission from [109]; copyright 2000 Elsevier.

This review has considered the opposite side of arsenic and reviewed the
literature for evidence of any beneficial roles of this element. Animal studies
indicate that very low levels of arsenic may promote normal growth and develop-
ment, and its absence is detrimental to both the offspring and the mother during
reproduction. However, the levels of arsenic that may be required are below the
levels to which humans are normally exposed in their drinking water and diet,
and below the level that has been directly associated with disease. In fact, a study of
a Danish population suggests that there may be a reduced risk for non-melanoma
skin cancer from low background levels of arsenic in drinking water [113]. Toxic
effects of this element are clearly correlated with exposure levels above this
background, and cause a variety of diseases by mechanisms that are still not
well understood but in some cases can be correlated with the unique chemical
properties of this element.
Can arsenic sustain life? Under certain unique situations, microorganisms can
use arsenite or arsenate for their essential energy-generating pathway. However, the
report of a microorganism, GFAJ-1, that can grow using arsenic instead of phosphorus
has not been supported by subsequent studies. Thus, there is still no evidence that
life under terrestrial conditions, including those with very high levels of arsenic,
can be sustained by substituting arsenate for phosphate. Nevertheless, for humans
suffering from APL, and possibly CML, arsenic can send their disease into remis-
sion, thereby extending and sustaining their life.
494 Wilcox

Abbreviations

APL acute promyelocytic leukemia


AsB arsenobetaine
AsC arsenocholine
BFD black foot disease
CCA copper chrome arsenate
CML chronic myeloid leukemia
DBD DNA-binding domain
DDT 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane
DMAs dimethyl arsenic (As3+ or As5+)
FDA Food and Drug Administration
GR glucocorticoid receptor
GRE glucocorticoid response element
LNT linear, no threshold
MMAs monomethyl arsenic (As3+ or As5+)
MRP multi-drug resistance protein
NHE normal hydrogen electrode
PBP phosphate binding protein
Pit phosphate inorganic transport
PML promyelocytic leukemia protein
Pst phosphate specific transport
PTDI provisional tolerable daily intake
RARα retinoic acid receptor α
RING really interesting new gene
ROS reactive oxygen species
SUMO small ubiquitin-like protein
TPN total parenteral nutrition
UNICEF United Nations Children’s Fund

Acknowledgments I thank Brian Jackson for his contributions to the sections on chronic toxicity
(2.2), deliberate human exposure (3.3.2), and pesticides (4.1). I am grateful for previous support
from the Dartmouth Superfund Research Program, which is supported by the NIH (P42 ES07373).
This contribution is dedicated to the late Paul Saltman.

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Chapter 16
Selenium. Role of the Essential Metalloid
in Health

Suguru Kurokawa and Marla J. Berry

Contents
ABSTRACT.............................................................................................................................. 500
1 Introduction.............................................................................................................. 501
1.1 History of Selenium................................................................................................... 501
1.2 Identification of Selenium as an Essential Micronutrient.......................................... 501
2 Selenium in Biomolecules................................................................................... 502
2.1 Selenocysteine, the 21st Amino Acid in Proteins...................................................... 502
2.2 Selenocysteine tRNA and Biosynthesis of Selenocysteine........................................ 503
2.3 Selenoprotein mRNAs............................................................................................... 506
2.4 Selenocysteine Incorporation into Proteins................................................................ 507
3 Function of Selenoproteins............................................................................... 509
3.1 Human Selenoproteins............................................................................................... 509
  3.1.1 Glutathione Peroxidases (Gpx1, Gpx2, Gpx3, and Gpx6)........................... 509
  3.1.2 Thyroid Hormone Deiodinases (DI1, DI2, and DI3)................................... 511
  3.1.3 Thioredoxin Reductases (TR1, TR2, and TR3)........................................... 512
  3.1.4 Methionine-R-Sulfoxide Reductase (MsrB1).............................................. 513
  3.1.5 15kDa Selenoprotein (Sep15)...................................................................... 513
  3.1.6 Selenophosphate Synthetase 2 (SPS2)......................................................... 513
  3.1.7 Selenoprotein P (Sepp1)............................................................................... 514
  3.1.8 Selenoprotein W (SelW).............................................................................. 514
  3.1.9 Selenoprotein V (SelV)................................................................................ 514
3.1.10 Selenoprotein T (SelT)................................................................................. 515
3.1.11 Selenoprotein M (SelM)............................................................................... 515
3.1.12 Selenoprotein H (SelH)................................................................................ 515

S. Kurokawa • M.J. Berry (*)


Department of Cell & Molecular Biology, John A. Burns School of Medicine,
University of Hawaii at Manoa, Honolulu, HI 96813, USA
e-mail: kurokawasuguru@gmail.com; mberry@hawaii.edu

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 499
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8_16, © Springer Science+Business Media Dordrecht 2013
500 Kurokawa and Berry

3.1.13 Selenoprotein O and I (SelO and SelI)......................................................... 515


3.1.14 Selenoprotein S (SelS)................................................................................. 515
3.1.15 Selenoprotein K (SelK)................................................................................ 516
3.1.16 Selenoprotein N (SelN)................................................................................ 516
4 Selenium and Disease............................................................................................. 516
4.1 Overview of Selenium-Related Diseases................................................................... 516
  4.1.1 Selenium Deficiency.................................................................................... 517
  4.1.2 Selenium Toxicity (Selenosis)...................................................................... 517
4.2 Selenium in Brain Function....................................................................................... 518
4.3 Selenium in Diabetics................................................................................................ 518
4.4 Selenium in Reproduction.......................................................................................... 519
5 Health Benefits of Selenium in Humans...................................................... 520
5.1 Molecular Forms of Selenium in Diet........................................................................ 520
5.2 Selenium Transport in Mammals............................................................................... 521
  5.2.1 Tissue-Oriented Selenium Transport............................................................ 521
  5.2.2 Selenium Excretion...................................................................................... 523
5.3 Human Dietary Standards for Selenium.................................................................... 524
6 General Conclusions............................................................................................. 525
Abbreviations and Definitions............................................................................... 525
Acknowledgment........................................................................................................... 527
References......................................................................................................................... 527

Abstract  Selenium is an essential micronutrient in mammals, but is also recognized


as toxic in excess. It is a non-metal with properties that are intermediate between the
chalcogen elements sulfur and tellurium. Selenium exerts its biological functions
through selenoproteins. Selenoproteins contain selenium in the form of the 21st
amino acid, selenocysteine (Sec), which is an analog of cysteine with the sulfur-
containing side chain replaced by a Se-containing side chain. Sec is encoded by the
codon UGA, which is one of three termination codons for mRNA translation in
non-selenoprotein genes. Recognition of the UGA codon as a Sec insertion site
instead of stop requires a Sec insertion sequence (SECIS) element in selenoprotein
mRNAs and a unique selenocysteyl-tRNA, both of which are recognized by special-
ized protein factors. Unlike the 20 standard amino acids, Sec is biosynthesized from
serine on its tRNA. Twenty-five selenoproteins are encoded in the human genome.
Most of the selenoprotein genes were discovered by bioinformatics approaches,
searching for SECIS elements downstream of in-frame UGA codons. Sec has been
described as having stronger nucleophilic and electrophilic properties than cysteine,
and Sec is present in the catalytic site of all selenoenzymes. Most selenoproteins,
whose functions are known, are involved in redox systems and signaling pathways.
However, several selenoproteins are not well characterized in terms of their
function. The selenium field has grown dramatically in the last few decades, and
research on selenium biology is providing extensive new information regarding its
importance for human health.

Keywords  essential nutrient • selenium • selenocysteine • selenoprotein

Please cite as: Met. Ions Life Sci. 13 (2013) 499–534


16  Selenium. Role of the Essential Metalloid in Health 501

1  Introduction

1.1  History of Selenium

Selenium is a non-metal element, but sometimes considered as a metalloid, with the


symbol Se and atomic number 34. It was first described in 1818 by the Swedish
chemist Jöns Jacob Berzelius (1779–1848). He named this element selenium (Greek
σελήνη selene meaning Moon) after the Greek moon goddess Selene (reviewed in
[1]). Berzelius was investigating the cause of illness among workers at a sulfuric acid
manufacturing plant when he found the element in the bottom sludge of a sulfuric acid
preparation. He reported that selenium had similarities with the previously known
element tellurium (named for the Earth). He also reported close similarities between
selenium and sulfur. At that time, selenium was thought to be a toxic element.

1.2  Identification of Selenium as an Essential Micronutrient

Most of the early research on selenium was done with the goal of addressing sele-
nium toxicity. In the 1930s, selenium was found to cause poisoning of livestock in
areas with high selenium content in the soil. In the mid 20th century, selenium was
recognized as a micronutrient and its biological function was studied with regard to
its importance in human nutrition. In 1957, Klaus Schwarz, a German scientist
working at the National Institutes of Health in Bethesda first reported on the health
benefits of selenium. Schwarz had studied yeast as a protein source in Germany
during World War II and continued the study in the United States, eventually discov-
ering that feeding torula yeast instead of brewer’s yeast as a protein source to vita-
min E-deficient rats led to necrotic liver formation. Schwarz and Foltz announced
that the selenium contained in the fractionated brewer’s yeast was responsible for
preventing liver necrosis [2]. Selenium deficiency was also recognized in studies in
Oregon [3], which showed a myopathy known as ‘white muscle disease’ in calves
and lambs to be associated with selenium-depleted soil. Selenium supplementation
to livestock has subsequently had great economic impacts in several countries,
including New Zealand and Finland.
In 1979 in China, a congestive cardiac myopathy termed Keshan disease was
the first reported human disease associated with selenium deficiency [4]. Keshan
County in northeastern China, for which the disease was named, is a predomi-
nantly rural region where the diet consisted almost entirely of food produced
locally on selenium-deficient land. The disease was also reported in New Zealand
and Finland where the level of selenium in the soil is low [5]. Further details are
discussed in Section 4.1.
In the 1970s selenium was found to be present in glutathione peroxidase as
the amino acid selenocysteine (Sec) [6], and the focus on selenium studies shifted
to the field of molecular biology. As a micronutrient, a recommended dietary allowance
502 Kurokawa and Berry

(RDA) for selenium was established in 1989 (70 μg/d for men and 55 μg/d for
women) [7] and revised in 2000 (55 μg/d) [8]. In 1996, dietary recommendations
from the World Health Organization (WHO) were issued (34 μg/d for men and 26
μg/d for women) [9].

2  Selenium in Biomolecules

2.1  Selenocysteine, the 21st Amino Acid in Proteins

Selenoproteins are defined as proteins containing the 21st amino acid, Sec [10]. The
discovery of selenoproteins occurred in 1973, when Hoekstra and coworkers at the
University of Wisconsin identified the presence of selenium in glutathione peroxi-
dase as the first animal selenoprotein [6]. Research then focused on the catalytic
role of the amino acid in the active site of selenoproteins. In 1976, Thressa Stadtman
et al. identified glycine reductase as a selenoprotein [11] and in the 1980s, Böck and
colleagues identified additional selenoproteins in bacteria [12]. The application of
bioinformatics and SECIS specific algorithms allowed for identification of seleno-
protein genes from the expressed sequence tags (ESTs) database [13,14]. All of the
25 selenoprotein genes in humans were thus identified in 2003 [15].
Sec (Figure 1, left) contains a selenol group which is highly reactive at physio-
logical pH. The reactivity of the thiol group (pKa 8.3) of cysteine is modulated by
microenvironmental conditions, and when deprotonated, is nucleophilic and easily
oxidized. Since the selenol group of Sec has a lower pKa (5.47), it is fully deproton-
ated. Due to the higher reduction potential of Sec, it is more efficient in participat-
ing in redox reactions, and is specifically used as a catalytic amino acid in most
selenoproteins.
The other selenium-containing amino acid is selenomethionine (Figure 1, right).
Like methionine, selenomethionine is not synthesized de novo in humans, but is
supplied from plants. Selenium is misincorporated at random in place of sulfur in
methionine biosynthesis, followed by the ribosome failing to distinguish between

Figure 1  Selenium-containing amino acids in mammals. Selenocysteine is found in selenoproteins.


At the physiologic pH, its selenol is mostly deprotonated. Sec can be synthesized by mammals.
Selenomethionine is classified as non-polar amino acid. Like the essential amino acid methionine,
selenomethionine is also not synthesized de novo in mammals. Selenomethionine appears to be
distributed nonspecifically in the proteins in place of methionine residues.
16  Selenium. Role of the Essential Metalloid in Health 503

selenomethionine- and methionine-loaded tRNA during translation [16]. Since its


selenium is covalently bound between two carbon atoms, selenomethionine is
considerably less reactive than Sec.

2.2  Selenocysteine tRNA and Biosynthesis of Selenocysteine

Sec is a naturally occurring amino acid in eukaryotes, archaea, and eubacteria. Sec
is cotranslationally incorporated into selenoproteins by Sec tRNA decoding of
UGA, which is normally a termination codon [10,17,18]. In 1970, a seryl-tRNA
was identified that specifically decoded the stop codon, UGA [19]. In assessing
whether nonsense suppressor tRNAs occurred in mammalian cells, the minor
seryl-tRNA was identified as a possible candidate. Extensive characterization of
this tRNA subsequently revealed it to be Sec tRNA[Ser] (reviewed in [20]). Unlike
the other 20 amino acids, the biosynthesis of Sec occurs on its transfer RNA
(tRNA[Ser]Sec) [21]. The biosynthesis of Sec was first characterized in bacteria [22]
by Böck and coworkers in the late 1980s to early 1990s [23,24], and subsequently
in mammalian cells [21]. In the first step, tRNA[Ser]Sec is aminoacylated with serine,
providing the carbon backbone for Sec, thus the tRNA has been designated as
tRNA[Ser]Sec [21,25]. The pathway for mammalian Sec biosynthesis has been eluci-
dated more recently, and is discussed below.
Sec tRNA[Ser]Sec has been isolated and sequenced from bovine liver [19,26,27], rat
liver [28], mouse liver, and HeLa cells [29]. tRNA[Ser]Sec is the longest tRNA in
mammals with a length of 90 nucleotides [26,27,30], and contains several modified
bases. It was the first mammalian tRNA shown to contain mcm5U34 and mcm5Um34
[28] (Figure 2). Full expression of selenoproteins requires modification of tRNA[Ser]Sec
[31]. Interestingly, two major isoforms of tRNA[Ser]Sec differ by a single methyl
group at the wobble position (Um34) and synthesize different subclasses
of selenoproteins [20]. The non-Um34 isoform supports the synthesis of a subclass
of selenoproteins, designated housekeeping, while the Um34 isoform supports
the expression of another subclass, designated stress-related selenoproteins [32].
The modification of i6A37 is also required for stress-related selenoproteins [33].
Um34 methylation of tRNA[Ser]Sec requires aminoacylated tRNA[Ser]Sec, most likely
with Sec [34]. Recently, it was shown that N6-isopentenylation of base A37 is
catalyzed by Trit1, a dimethylallyl:tRNA[Ser]Sec-transferase [35].
In the first step of Sec biosynthesis, seryl-tRNA synthetase (SerRS) attaches
serine to Sec tRNA in the presence of ATP and Mg2+ as follows [36–38]:

tRNA[ ] + serine + ATP ←→ seryl-tRNA[ ] + AMP + PPi


Ser Sec Ser Sec

In 1970, a kinase that phosphorylated what was presumed to be a minor species
of seryl-tRNA to form O-phosphoseryl-tRNA was reported. Because it recognized
the stop codon UGA, it was initially thought to be a suppressor tRNA [19,39]. This
seryl-tRNAUGA was eventually identified as Sec tRNA[Ser]Sec, and the kinase origi-
nally found by Mäenpää and Bernfield was shown to be O-phosphoseryl-tRNA[Ser]Sec
504 Kurokawa and Berry

Figure 2  Cloverleaf model of bovine liver Sec tRNA[Ser]Sec and its modifications. Sec tRNA[Ser]Sec
sequences in mammals are 90 nucleotides long. The tRNA contains base modifications at position 34
(methyl carboxymethyl-5’-uridine; mcm5U), 37 (isopentenyladenosine; i6A), 55 (pseudouridine; Ψ)
and 58 (N1-methyladenosine; m1A). The two isoforms differ from each other by a single methyl
group on the position 34 (mcm5U or mcm5Um).

kinase (PSTK) [40]. PSTK was identified using a comparative genomics approach
that searched completely sequenced genomes of archaea for a kinase-like protein
that was present in those organisms (Methanococcus jannaschii and Methanopyrus
kandleri) that utilized the selenoprotein synthesizing machinery and was absent in
those that did not. Two kinase-like genes were identified in the two archaea that
synthesize selenoproteins. However, they were not found in the other twelve archaea
which lack that ability. Further comparison was carried out in eukaryotic genomes
that synthesize selenoproteins (nematodes, Drosophila, and mice) and those that do
not (yeasts) for homologous sequences to the two candidate genes. A single candidate
was detected and the putative pstk was cloned from mouse genomic DNA [40].
The protein’s biochemical properties showed it to be phosphoseryl-tRNA kinase
(PSTK). The reaction is as follows:

seryl-tRNA[ ] + ATP ←→ O-phosphoseryl-tRNA[ ] + ADP


Ser Sec Ser Sec

16  Selenium. Role of the Essential Metalloid in Health 505

Figure 3  Biosynthesis of Sec and de novo synthesis of cysteine. Sec is synthesized on tRNA[Ser]Sec
by generation of selenophosphate from selenide and ATP (upper portion of the figure for the final
steps in Sec biosynthesis). The de novo synthesis of cysteine on Sec tRNA[Ser]Sec occurs when
sulfide replaces selenide (lower portion of the figure for the final steps in cysteine biosynthesis).

The selenium donor is monoselenophosphate, which is formed from selenite and


ATP by selenophosphate synthetase (SPS). SPS has two homologs, SPS1 and SPS2
[41,42]. The gene product of SPS2 is a selenoprotein [42]. Biochemical analysis
demonstrated that mouse SPS2 generates selenophosphate in the presence of sele-
nide and ATP [43]. The reaction is as follows:
selenide + ATP → selenophosphate + AMP + Pi

Sec synthetase (SecS) then mediates the generation of selenocysteyl-tRNA[Ser]Sec
and inorganic phosphate. Hatfield’s group identified SecS using a computational
and comparative genomic strategy, similar to that used to identify pstk, in searching
for a SecS gene in eukaryotes [44]. The sequence of the purported mammalian SecS
matched a 48 kDa soluble liver antigen (SLA) [45], which was previously reported
as an antigen in patients with autoimmune chronic hepatitis and co-precipitated
with Sec-tRNA[Ser]Sec in extracts from such patients. SecS binds tightly with
phosphoseryl-­tRNA[Ser]Sec[46]. The reaction is as follows (Figure 3):

O-phosphoseryl-tRNA[ ] + selenophosphate → selenocysteyl-tRNA[ ] + PPi


Ser Sec Ser Sec

Recently, it was shown that cysteine can be synthesized on the tRNA[Ser]Sec by


replacing selenide with sulfide in the Sec biosynthetic pathway [47] (de novo cysteine
biosynthesis is discussed in Section 3.1.6).
506 Kurokawa and Berry

2.3  Selenoprotein mRNAs

The specific, cotranslational incorporation of Sec into eukaryotic proteins requires


the presence of a SECIS element within the selenoprotein mRNA. The SECIS ele-
ment was identified through bioinformatic and functional studies of the selenopro-
tein genes encoding cytosolic glutathione peroxidase (Gpx1) and type 1 deiodinase
(DI1) [48,49]. Deletion analyses of the respective 3’UTRs resulted in production of
truncated selenoproteins where the Sec UGA codon was recognized as a stop codon.
Analysis of the DI1 and Gpx1 cDNAs from multiple species revealed the consensus
SECIS element to be a conserved stem-loop structure containing three short, highly
conserved nucleotide sequences [48].
Typical SECIS elements are depicted in Figure 4 and are based on an experi-
mental secondary structure model [50]. Alignment of selenoprotein 3’-UTRs
revealed the consensus sequences of AUGA, AAA, and UGAU as the SECIS core
[48]. The core (quartet non-Watson-Crick base pairs; two shared tandem G•A,
A•G motif) is a conserved feature of SECIS elements which introduces a kink-
turn like structure [50]. This structure is recognized by SECIS binding protein 2
(SBP2) and plays a key role for SECIS function [51]. For example, mutation of
the SECIS core of selenoprotein N (AUGA to ACGA) causes myopathy [52].
The secondary structure of the SECIS element shows conserved AA residues in
an apical loop or internal bulge [53]. This motif is essential and is recognized
by the RNA binding protein nucleolin [54] (SECIS binding proteins are further
discussed in Section 2.4).
The sequences flanking the SECIS element need to be open and non-base paired.
The stem length of the SECIS is between 9 to 15 Watson-Crick base pairs, a dis-
tance which approximates a single turn of an A-form double helix. Interestingly,
human selenoprotein P (Sepp1), which has 10 UGA Sec codons, has two distinct
SECIS elements, both necessary for synthesis of the full-length protein. The 5’ ele-
ment has an extended apical loop (SECIS1, form 2) and the 3’ element has the basic
stem-loop structure (SECIS2, form 1) [55]. These Sepp1-SECISs have distinct
functions, SECIS1 being more efficient than SECIS2 [56]. SECIS elements have
been utilized to develop software-based search algorithms in an attempt to identify
potential selenoprotein cDNAs in the GenBank [13]. The algorithms search data-
bases for the primary sequence and secondary structure features of SECIS elements,
calculating the free energy of the secondary structures. This strategy was initially
used to identify several selenoproteins (SelT and SelR [13]) (SelX, SelN, and SelZ
[14]) and the SECIS elements activity was validated by incorporation of Sec into
Gpx1 protein [57]. A more sophisticated approach was subsequently developed,
searching for SECIS elements, conservation of open reading frames downstream of
UGA codons between species (which identified SelH, SelI, SelK, SelO, SelS, and
SelV), and conservation between Sec and Cys-containing orthologs (Gpx6) [15].
These novel proteins indeed contain selenium, which was verified by subsequent
radioactive 75Se labeling analysis.
16  Selenium. Role of the Essential Metalloid in Health 507

Figure 4  Diagram of two classes of eukaryotic SECIS elements. Secondary structure models of
Form 1 and 2 SECIS. The critical structural features are labeled. The eukaryotic SECIS element is
located in 3’ UTR. N, any nucleotide; A/G and A/C indicate that A is the prevalent base.

2.4  Selenocysteine Incorporation into Proteins

SECIS element and RNA binding protein interaction is required for decoding of
UGA codons as Sec. SECIS binding protein-2 was identified by Copeland et al. as
a 94 kDa protein that specifically recognizes the AUGA core of SECIS elements
[51,58]. SBP2 works as a factor for the recoding of an in-frame UGA as Sec, activity
uncovered by an in vitro translation system in rabbit reticulocyte lysate. SBP2 contains
an RNA binding domain found in several ribosomal proteins and eukaryotic transla-
tion termination release factor 1.
Sec incorporation requires the eukaryotic selenocysteyl-tRNA-specific elongation
factor (eEFSec). eEFSec was identified using searches of the murine and human EST
databases for homology to a putative archaeal alternative translational elongation
factor SELB sequence [59,60]. eEFSec reveals a high degree of conservation in the
amino-terminal elongation factor domain and purified recombinant eEFSec protein
specifically binds to selenocysteyl-tRNA but not seryl-tRNA [59]. This discrimination
508 Kurokawa and Berry

between correctly charged selenocysteyl-tRNA and seryl-tRNA reveals the mechanism


for preventing misincorporation of serine at UGA codons instead of Sec.
eEFSec has been shown to interact with SBP2 in an RNA-dependent manner
[59]. Electrophoretic mobility shift assays (EMSA) with in vitro transcribed
32
P-labeled SECIS elements and recombinant purified proteins showed SBP2 and
eEFSec bind the SECIS element independently. In contrast, co-­immunoprecipitation
studies of the mutant SECIS elements with eEFSec and SBP2 revealed a lack of
ability to bind the SECIS element. This result suggests eEFSec has the ability to
bind SECIS elements and SBP2 enhances its binding specificity.
L30 is a component of the large ribosomal subunit that binds to a specific
sequence in 28S rRNA. It is a ubiquitously expressed abundant protein in mamma-
lian tissues, and it has been demonstrated to overlap with SBP2 in binding to SECIS
elements [61]. In vitro binding studies demonstrated that L30 binds to the SECIS
element and forms a kinked conformer to facilitate SBP2 binding since SBP2 only
binds to the kinked SECIS form.
Eukaryotic initiation factor 4A-III (eIF4a3) has been identified as a selenium
status sensitive RNA-binding protein [62]. eIF4a3 is ubiquitously expressed and
belongs to the DEAD box family of RNA-dependent ATPases [63].
Nucleolin, a protein that facilitates ribosome biogenesis in the nucleolus, has
been demonstrated to exhibit SECIS element binding [64]. This protein alters
mRNA stability or translation in the cytoplasm of specific selenoprotein mRNAs. It
binds the upper part of the basal stem of SECIS elements but the consensus sequence
for nucleolin binding in SECIS elements has not been identified [54]. siRNA knock-
down of nucleolin demonstrated synthesis of essential selenoproteins was reduced
but without effect on non-essential selenoprotein synthesis [54]. It is postulated that
nucleolin may bind to the essential selenoproteins’ SECIS element and facilitate
SECIS element-protein interactions with SBP2 or other factors in the Sec incorpo-
ration pathway (Figure 5).
Supramolecular protein-protein and protein-RNA interactions are implicated in
both the biosynthesis of selenocysteyl-tRNA and the incorporation of Sec [65].
Co-immunoprecipitation studies described the following interactions that have been
characterized to date.
Protein:RNA interactions
SecS-selenocysteyl-tRNA
Secp43- selenocysteyl-tRNA
eEFsec- selenocysteyl-tRNA
SBP2-SECIS mRNA and rRNA
L30- SECIS mRNA and rRNA
Protein:protein interactions
SBP2-eEFSec
SecS-SPS1
SecS-Secp43
As several selenoprotein biosynthesis factors have both nuclear localization signals
and nuclear export signals, this suggests that these factors may shuttle between
16  Selenium. Role of the Essential Metalloid in Health 509

Figure 5  A model of Sec biosynthesis and incorporation. Aminoacylation of tRNA[Ser]Sec and its
conversion to Sec-tRNA[Ser]Sec is depicted along the top. Recruitment of transcription factors
(SBP2, eEFSec) are depicted top right. Shuttling of the complex of Sec-tRNA[Ser]Sec into the nucleus
and the association with eEFSec, SBP2, and SECIS elements are depicted along the right bottom.
Cytoplasmic export and translation are shown along the left bottom.

the cytoplasm and the nucleus. SPS1, Secp43, and eEFSec were observed in both the
cytoplasmic and nuclear fractions [65]. In summary, the co-­immunoprecipitation
studies suggest that SPS1, SecS, Secp43, and selenocysteyl-tRNA may form a complex
in the cytoplasm that subsequently enters the nucleus. SecS may then leave the
complex, replaced by eEFSec and SBP2 and subsequent shuttling to the cytoplasm.

3  Function of Selenoproteins

Twenty-five selenoproteins have been identified in human genome databases [15].


Comparative genomic analyses of selenoproteins provide insights into the biologi-
cal functions of selenium. Figure 6 summarizes the human selenoprotein families.

3.1  Human Selenoproteins

3.1.1  Glutathione Peroxidases (Gpx1, Gpx2, Gpx3, and Gpx6)

In the early 1970s, glutathione peroxidase was identified as the first true selenopro-
tein [6]. The Gpx family has 8 known homologous proteins (Gpx1–Gpx8) and in
humans, Gpx1, Gpx2, Gpx3, Gpx4, and Gpx6 are Sec-containing. Gpxs break down
hydroperoxides in a reduced glutathione (GSH)-dependent reaction. The selenolate
510 Kurokawa and Berry

Figure 6  Selenoproteins found in humans. SECIS type and Sec residues belong to the thioredoxin
motif as shown (C; cysteine, x: any amino acid, U; Sec residue). On the right, relative size of
selenoproteins are shown (relative to a 100 amino acid scale). The location of Sec within the
protein sequence is shown by a black line.

(R-Se-) in Gpx is oxidized by hydroperoxide and forms seleninic acid (R-SeOH).


The seleninic acid reacts with a GSH to form the GS-Se-R. A second GSH reduces
the GS-Se-R intermediate back to R-SeH and releases GSSG and water as the
by-­product. The reaction is a ping-pong mechanism:
Gpx-Se − + H 2 O2 → Gpx-SeOH + OH −
Gpx-SeO − + H + + GSH → Gpx-Se-SG + H 2 O
Gpx-Se-SG + GSH → Gpx-Se + H + + GSSG

Gpx kinetics are identical for Gpx1, Gpx3, and Gpx4. The oxidizing substrates
for Gpx2 have not been identified. Gpx1 is expressed in all cells and is more abundant
in liver and kidney. It is a homotetramer and localizes to cytosol and mitochondria,
reacting with hydrogen peroxide and soluble low molecular mass hydroperoxides
such as t-butyl hydroperoxide, cumene hydroperoxide, hydroperoxy fatty acids and
16  Selenium. Role of the Essential Metalloid in Health 511

hydroperoxy lysophosphatides. In tissues, which do not express GSH synthetase,


Gpx1 can use γ-glutamylcysteine as a reductant for H2O2. Gpx1 knockout mice
show increased susceptibility of liver and lung to ROS [66,67].
Gpx2 is expressed in the gastrointestinal epithelium [68]. It is a homotetramer
and localizes to the cytosol. Gpx2 is upregulated in epithelium-derived tumors
which include colon adenocarcinoma [69–72], Barrett’s esophagus [73], squamous
cell carcinoma [74], and lung adenocarcinomas of smokers [75]. It is suggested to
act as a barrier against absorption of food-born hydroperoxides. Gpx2-knockout
mice display an increased rate of spontaneous apoptosis at crypt bases, an effect that
is stimulated under restricted selenium supply [68,76]. Since selenium supplemen-
tation partially prevented the spontaneous apoptosis in crypt bases, Gpx1 might be
compensating the Gpx2 depletion. Gpx1/Gpx2 double-knockout mice spontane-
ously develop colitis and intestinal cancer, a model now used to study spontaneous
inflammatory bowel disease and predisposition to intestinal cancer [77].
Gpx3 is a tetramer, primarily synthesized in kidney proximal tubule cells and
secreted into plasma [78,79]. Gpx3 produced by the kidney binds to renal proximal
tubules, basement membranes of epithelial cells throughout the intestine, the epi-
didymis, bronchi, and lung type II pneumocytes [80,81]. The epididymis synthe-
sizes Gpx3 and releases it into its lumen [81]. Gpx3 knockout mice did not show an
obvious phenotype. Since the apparent Km of Gpx3 for GSH (5.3 mM) [82] is sig-
nificantly higher than plasma GSH concentrations (<0.5 μM) [83], Gpx3’s enzy-
matic function is unknown. Down-regulation of Gpx3 is observed in many types of
cancer and hypermethylation of the Gpx3 promoter is detected in patients with
Barrett’s esophagus cancer [84] and prostate cancer [85]. Gpx3 has been suggested
to be a novel tumor suppressor.
Gpx4 has 3 isoforms (cytosolic, sperm nuclear, and mitochondrial forms), which
catalyze reduction of lipid peroxides and cholesterol ester hydroperoxides inside
cellular membranes. Gpx4 is a monomer protein [86] that reacts with GSH and can
use protein thiols instead of GSH when GSH is limiting. This has been shown for
Gpx4 reactivity with sperm mitochondria-associated cysteine rich protein and also
for Gpx4 against itself [87]. The mitochondrial form of Gpx4 has vital function in
the sperm midpiece serving a structural role during spermatogenesis [87]. While
total Gpx4 knockout is lethal [88,89], mitochondrial Gpx4-knockout mice devel-
oped normally [90]. Male mitochondrial Gpx4 mice are infertile since Gpx4 is nec-
essary for sperm as an inactive structural protein. Mice in which the nuclear form of
Gpx4 was knocked out were not only viable but also fully fertile [91].
Gpx6 is closely related to Gpx3, and is a homodimer selenoprotein only in
humans [15].

3.1.2  Thyroid Hormone Deiodinases (DI1, DI2, and DI3)

The iodothyronine deiodinase family has three homologues (DI1, DI2, and DI3) in
mammals. DIs catalyze reductive deiodination of thyroid hormones, regulating
their activity. T3 enters the nucleus and binds to thyroid hormone receptors which
512 Kurokawa and Berry

bind T3-responsive genes and regulate their transcription [92]. Thyroxin (T4) is
predominantly produced in the thyroid gland but its affinity for the thyroid hormone
receptors is about tenfold lower than T3 [93]. DI1 and DI2 are involved in activation
of the thyroid hormone by outer ring deiodination of T4 to produce T3 [94]. DIs are
homodimeric thioredoxin-like fold membrane-spanning proteins. DI1 and DI3 are
found in the plasma membrane, while DI2 is present in endoplasmic reticulum (ER)
[95–97]. DI1 is expressed at highest levels in liver and kidney, and produces most
of the circulating T3 [98] while DI2 is most abundant in thyroid, heart, skeletal
muscle, brown adipose tissue, and the central nervous system. DI2 on the ER may
preferentially supply T3 to the nucleus.
Interestingly, human DI2 has an active site Sec and a second Sec 7 amino acids
from the C-terminus. The second Sec and the remaining seven C-terminal amino
acids are not critical for DI2 enzyme activity and the function of the C-terminal
region is unknown [99]. DI2 is expressed in the brown adipose tissue (BAT) and its
activity increases in BAT during cold stress, resulting in increased intracellular T3
levels [100–102]. DI2-knockout mice exhibit a mild phenotype; they are unable to
control normal body temperature following cold exposure and also show bone
development defects [103,104]. Further analysis of DI2-knockout mice on a high fat
diet showed a tendency of weight gain and development of insulin resistance [105].
On the other hand, DI3 inactivates T3 and T4 to biologically inactive T2 or reverse
T3 (rT3) by preferentially removing the iodine from the inner ring of the molecule.
DI3 is found in fetal tissue and placenta and is thought to function in protecting tis-
sues from premature exposure to T3 [106].

3.1.3  Thioredoxin Reductases (TR1, TR2, and TR3)

The thioredoxin reductase (TR) family has three selenoprotein homologues (TR1,
TR2, and TR3). TRs reduce oxidized thioredoxin (thioredoxin-S2) with NADPH as
a cofactor. A C-terminal conserved motif (-GCUG) contains Sec, which is crucial
for the enzyme activity. Reduced thioredoxin is reoxidized by disulfides in proteins
generating thiols.
thioredoxin-S2 + NADPH + H + → thioredoxin- ( SH )2 + NADP + ( catalyzed by TR )
thioredoxin- ( SH )2 + protein-S2 → thioredoxin-S2 + protein- ( SH )2

TRs also have broad substrate specificity, being able to use hydrogen peroxide,
selenite, lipoic acid, ascorbate, and ubiquinone [107] as substrate. TR1 is localized
in the cytosol. TR2, known as thioredoxin/glutathione reductase (TGR) [108], has
the function of formation/isomerization of disulfide bonds during sperm maturation
[109]. TR3 is a mitochondrial protein. Knockout of either TR1 or TR3 in mice is
fatal [110,111]. Knockout of the TR1 gene results in early embryonic death at day
6.5 (E6.5). TR3-knockout mice develop exencephaly and die during midgestation
(E10.5). Thus both genes are indispensable for mouse embryo development.
16  Selenium. Role of the Essential Metalloid in Health 513

3.1.4  Methionine-R-Sulfoxide Reductase (MsrB1)

Methionine, a sulfur containing amino acid, is highly susceptible to accumulated


reactive oxygen species. Since sulfur of methionine is a prochiral atom, oxidized
methionine generates two diastereomers, methionine-S-sulfoxide and methionine-
R-­sulfoxide. Methionine sulfoxide reductases (Msrs) reduce oxidized methionine
residues in proteins and free methionine sulfoxides. Msrs have two stereospecific
families, MsrA (reduces S-form of methionine sulfoxide) and MsrB (reduces
R-form of methionine sulfoxide) [112]. Oxidized methionine may lead to confor-
mational changes of proteins, e.g., ribosomal protein L12 and calmodulin are
impaired by oxidation of methionines and restored by MsrA. On the other hand,
methionine oxidation of calcium/calmodulin-dependent protein kinase II in the
absence of calcium activates the protein. MsrB1 was identified through bioinfor-
matics and initially designated as selenoprotein X but was subsequently renamed
selenoprotein R [13,14]. MsrB1 efficiently acts on methionine sulfoxide in protein
but it has very low activity on the free form of methionine sulfoxide. MsrB1 is a
zinc-containing protein, primarily synthesized in liver and kidney, and localizes in
the cytosol and nucleus. Its activity is the highest of the Msrs because of the pres-
ence of Sec in its active site. MsrB1 expression and activity is highly dependent on
selenium status, and selenium deficiency decreases MsrB1 activity. Although
MsrB1 affects redox regulation in liver and kidney, knockout of the gene in mice
showed that MsrB1 is not an essential selenoprotein for development.

3.1.5  15kDa Selenoprotein (Sep15)

Sep15 has an ER signal peptide and localizes in the ER [113]. The C-terminal
domain has a thioredoxin-like fold [114,115]. Sep15 has been suggested to take part
in the process of rearrangement of disulfide bonds or reduction of incorrectly formed
disulfide bonds in misfolded glycoproteins bound to UDP-glucose:glycoprotein
glucosyltransferase [114,116,117]. There are several studies suggesting an associa-
tion of Sep15 with cancer, but reports are contradictory as to whether it promotes or
restricts cancer growth. Earlier studies had shown that Sep15 expression was
reduced substantially in a malignant prostate cell line and in hepatocarcinoma [115].
An increase of Sep15 expression in colon cancer has been found [118], and targeted
down-regulation of Sep15 inhibited growth of colon cancer cells [119]. Additionally,
Sep15 knockout mice form significantly fewer carcinogen-induced aberrant crypt
foci in colonic epithelia in vivo compared to controls [120]. Thus, the tumor-­
suppressor activity or oncogenic activity of Sep15 may be tissue-dependent.

3.1.6  Selenophosphate Synthetase 2 (SPS2)

SPS2 is a factor for selenoprotein biosynthesis, with an enzymatic activity to


synthesize selenophosphate from ATP and selenite [44]. SPS2 could serve as an
514 Kurokawa and Berry

autoregulator of selenoprotein synthesis because it is a selenoprotein [42]. Sodium


sulfide is also a substrate for SPS2 and generates thiophosphate that would be used
as an active sulfur donor in making Cys attached to tRNA[Ser]Sec. Mass spectrometry
(MS) analysis of purified TR1 and TR3 from livers of mice that had been fed
selenium-­deficient (0 ppm selenium), or selenium-adequate (0.1 ppm selenium)
diets showed Cys in place of Sec [47]. Cys was not detected in these selenoproteins
on a selenium-enriched (2.0 ppm selenium) diet. Cysteine insertion instead of
Sec occurs by sulfide competing with selenide in generating the active donor
catalyzed by SPS2.

3.1.7  Selenoprotein P (Sepp1)

Sepp1 is synthesized primarily in the liver and secreted into the plasma. Sepp1 is the
only selenoprotein with multiple Sec residues. Human Sepp1 has 10 Sec residues
[55]. The N-terminal domain contains a Sec residue in a thioredoxin-like motif and
the C-terminal domain contains nine Sec residues in human Sepp1. Four isoforms
of Sepp1 have been identified; the shortest isoform terminates at the second UGA,
and has been verified as encoding Sec by mass spectrometry of this isoform purified
from rat plasma [121]. Sepp1 is a secreted, heparin-binding glycoprotein. Two
histidine-­rich domains separate the N-terminal and C-terminal regions. Sepp1 deliv-
ers selenium to organs where apolipoprotein E receptor 2 or megalin are expressed
[122–124]. More details on selenium transport via Sepp1 receptor mechanisms are
provided in Section 5.2.

3.1.8  Selenoprotein W (SelW)

SelW was identified in 1993 as a 6 kDa protein, the smallest selenoprotein in mam-
mals. SelW is primarily expressed in muscle, where its absence was notable in
muscle of Se-deficient sheep. The expression level of SelW in vertebrates is highly
sensitive to dietary Se intake. SelW has a thioredoxin-like fold structure and a Sec-­
containing redox center located in an exposed loop [125]. SelW was first identified
in Se-deficient sheep [126]. SelW contains a CXXU redox motif (where C is Cys,
X is any amino acid, and U is Sec) that is conserved among various mammalian
species. SelW may be involved in oxidative metabolic pathways and functions as an
antioxidant protein [127,128]. SelW was the first selenoprotein to be linked to mus-
cular disorders [125].

3.1.9  Selenoprotein V (SelV)

SelV is a homologue of SelW with additional N-terminal sequence and unknown


function. SelV is primarily expressed in testis [15].
16  Selenium. Role of the Essential Metalloid in Health 515

3.1.10  Selenoprotein T (SelT)

SelT is highly expressed in kidney, brain, heart, thymus, and testis [129]. SelT has
a thioredoxin-like fold and belongs to a new redox protein family. SelT is likely an
ER resident protein and the thioredoxin fold domain is exposed to the ER or cytosol.
Recently, the role of SelT in regulation of calcium homeostasis and neuroendocrine
secretion in response to a pituitary adenylate cyclase-activating polypeptide was
demonstrated [130].

3.1.11  Selenoprotein M (SelM)

SelM is a homologue of Sep15 and has an ER signal [15]. SelM contains a thiore-
doxin fold motif and is abundantly expressed in the brain [116]. SelM knockdown
experiments in cell culture revealed a role for SelM in calcium regulation [131].
Overexpression of SelM reduced peroxide-induced calcium influx in a neuronal cell
line [131]. Knockdown of SelM increased cytosolic calcium concentrations and
resulted in apoptotic cell death [131].

3.1.12  Selenoprotein H (SelH)

SelH has a thioredoxin-like fold motif with a small DNA-binding domain (AT-hook
motif) and is localized in the nucleus [132]. SelH is highly responsive to selenium
status and is upregulated under conditions of elevated copper in mouse liver [133].
SelH overexpression was shown to upregulate expression levels of genes involved
in de novo GSH synthesis and phase II detoxification [134]. Chromatin immunopre-
cipitation experiments demonstrated SelH binds to sequences containing heat shock
and/or stress response elements, suggesting SelH may function in a regulatory role
in response to redox status. Overexpression of SelH demonstrates neuroprotection
against UVB-induced cell death in neurons in culture and increases the levels of
mitochondrial biogenesis regulators, mitochondrial cytochrome c content, mito-
chondria mass and enhanced respiration [135]. SelH may transduce oxidant signals
by modulating gene expression.

3.1.13  Selenoprotein O and I (SelO and SelI)

The functions of SelO and SelI are unknown. SelI localizes in the ER.

3.1.14  Selenoprotein S (SelS)

SelS has an ER signal and is induced by ER stress [15,136,137]. SelS is a compo-


nent of the ER-associated protein degradation (ERAD) system [138,139]. ERAD
516 Kurokawa and Berry

protects cells from accumulation of misfolded proteins, transferring these proteins


from the ER to the proteasome [140,141]. SelS interacts with Derlin-1, a ER mem-
brane integral protein [141]. The specific function of SelS in the ERAD system is
unknown. An association of SelS expression and type 2 diabetes has been reported
[142]. Further details are discussed in Section 4.3.

3.1.15  Selenoprotein K (SelK)

SelK has been postulated to function in regulation of endoplasmic reticulum stress


[143] and facilitation of calcium flux in immune cells [144]. SelK mRNA is
expressed in immune cells and lymphoid tissues, spleen, intestine, and testis. SelK
predominantly localizes to the ER but has no ER localization signal [144]. Thus, the
protein may be bound by a chaperone or other ER-localized protein for insertion
into the ER. Studies with SelK-knockout mice suggest that this protein is not
required for growth or development of mice [144]. SelK is cleaved by calpain and
the cleaved form is highly abundant in unactivated macrophages [145]. SelK cleav-
age is inhibited by upregulation of the Toll-like receptor-induced calpain inhibitor,
calpastatin [145].

3.1.16  Selenoprotein N (SelN)

SelN is ubiquitously expressed with highest expression in muscle [146] and is local-
ized in the ER membrane [146]. It has a predicted redox active CUGS motif. Loss
of SelN function is associated with congenital muscular dystrophy [147]. SelN has
been reported to interact with ryanodine receptors, and may affect calcium flux
[148]. Another study demonstrated SelN deficiency was associated with oxidative
stress [149,150].

4  Selenium and Disease

4.1  Overview of Selenium-Related Diseases

Recently, selenium supplementation trials found that moderately higher selenium


intake may influence redox status through selenoprotein synthesis to cause type 2
diabetes. Thus selenium homeostasis needs to be tightly regulated for healthy life.
The range of selenium intake for optimal health in humans and animals is narrow,
such that low selenium intake is associated with developmental defects and disease
states and high selenium results in toxicity. We discuss below the conditions associ-
ated with both cases, specifically myopathies, selenosis, brain degeneration, type 2
diabetes, and male infertility.
16  Selenium. Role of the Essential Metalloid in Health 517

4.1.1  Selenium Deficiency

Three diseases have been reported to be associated with severe selenium deficiency,
due to their occurrence in areas with selenium poor soils and their reversal upon
selenium supplementation. It should be noted that selenium may be a cofactor in
these diseases, with other factors contributing to their incidence or severity.
(1) Keshan disease, was first described as a juvenile cardiomyopathy in the early
1930s in the Chinese medical literature [151]. Women and children were sus-
ceptible to the development of Keshan disease, which had a high mortality rate.
Supplementation of individuals with sodium selenite tablets could prevent the
development of Keshan disease [4]. Since the incidence of Keshan disease fluc-
tuated seasonally and annually, viral infection was also considered as a possible
cofactor [152]. Heart tissues from Keshan disease victims were subsequently
shown to contain coxackie viruses. Further, studies in selenium-deficient mice
demonstrated that coxsackie virus B4 infection induced severe heart pathology
[153]. Selenium-adequate mice showed mild heart pathology when infected
with the virus, which suggests that selenium deficiency in combination with
coxsackie virus infection was required for the development of Keshan disease.
(2) Kashin-Beck disease is a chronic, endemic osteochondropathy, accompanied
by joint necrosis [154]. This syndrome affects individuals in specific regions of
Tibet, northeastern to southwestern China, Siberia, and North Korea. While
individuals with this disease show skeletal pathology, they are not reported to
develop dysfunction of other organs or tissues. Kashin-Beck disease may
require other factors since the disease is clustered within specific regions and/
or families. A polymorphism in the Gpx1 gene was reported as a potential
genetic risk factor for Kashin-Beck disease [155].
(3) Myxedematous endemic cretinism, which is induced by thyroid atrophy, results
in mental retardation [156,157]. Myxedematous endemic cretinism is highly
prevalent in central Africa, where iodine and selenium-poor areas overlap with
thiocyanate-rich areas.

4.1.2  Selenium Toxicity (Selenosis)

Blood selenium levels greater than 100 μg/dL can lead to selenosis. Symptoms of
selenosis include hair loss, white blotchy nails, a garlic breath, gastrointestinal dis-
orders, fatigue, irritability, and neurological damage [158]. Selenosis in humans is a
rare event except in very high selenium areas. Extreme cases of selenosis can be
fatal, due to cirrhosis of the liver [159].
Elemental selenium and metallic selenides have relatively low toxicities because
of their low bioavailability. Selenates and selenites are very toxic. Organic selenium
compounds which occur in metabolic processes, such as Sec, selenomethionine,
and methylated selenium compounds are toxic in large doses. In the 10th edition of
RDAs in 1989, it was pointed out that sensitive biochemical indices of selenium
518 Kurokawa and Berry

overexposure were not available and no attempt was made to establish an upper
limit of selenium intake [7]. In 2000, The Institute of Medicine of the National
Academy of Science provided a DRI, which set the tolerable upper intake levels of
selenium to 400 μg/d [8]. The Lowest Observed Adverse Effect Level is 910 μg/d
[160], and the No Observed Adverse Effect Level is 200 μg/d.

4.2  Selenium in Brain Function

The supply of selenium to the brain is prioritized for normal development and brain
function. There is a “hierarchy” of tissues with respect to selenium supply under
low selenium status, whereby brain tends to maintain its selenium compared to
other tissues [161,162]. Brain expresses almost all selenoproteins, especially within
neurons [163]. Sepp1-knockout mice produce severe brain selenium deficiency
when maintained on a selenium-deficient diet, with neurological impairments that
lead to death within weeks [164,165]. Sepp1-knockout mice fed a 0.10 ppm sele-
nium diet developed spasticity and abnormal movements in addition to poor perfor-
mance on the rotarod test and pole climb. Sepp1-knockout mice fed 0.25 ppm
selenium diet produced no neurological dysfunction.
Recently, syndromes of congenital selenoprotein biosynthetic deficiency have
been discovered. Progressive cerebellar-cerebral atrophy (PCCA) was identified in
several non-consanguineous Jewish Sephardic families of Moroccan or Iraqi ances-
try [166]. The syndrome was mapped to homozygous or compound heterozygous
missense mutations of the SecS gene, with no enzymatic activity. PCCA involves
mental retardation, progressive microcephaly, and spasticity [166]. PCCA repre-
sents the first clinical syndrome related to selenocysteine biosynthesis in humans.
Clinically, cerebral and cerebellar atrophy involves gray and white matter [166].

4.3  Selenium in Diabetics

Body glucose homeostasis is regulated by functional insulin circulation and signal-


ing. Type 2 diabetes is characterized by high blood glucose in the context of insulin
resistance. The Nutrition Prevention Cancer (NPC) trial which is a double-blind,
randomized, placebo-controlled clinical trial to test micronutrients for cancer pre-
vention revealed a more than two-fold increase in type 2 diabetes incidence in the
selenium-supplemented compared to the placebo group [167–169]. The Selenium
and Vitamin E Cancer Prevention Trial (SELECT) revealed a similar trend [170],
however non-significant in a 10-year follow-up, but still concerning enough to lead
to the trial’s termination.
A recent study reported that Sepp1 is associated with development of type 2
diabetes [171]. Sepp1 mRNA levels were increased in people with insulin resis-
tance. Hepatic Sepp1 mRNA is upregulated by glucose and hepatic Sepp1 mRNA
levels correlated with insulin resistance [171]. Furthermore, insulin suppressed
16  Selenium. Role of the Essential Metalloid in Health 519

Sepp1 protein expression in hepatocytes. It is postulated that Sepp1 induces insulin


resistance in liver and muscle, resulting in hyperglycemia [171].
Targeted removal of the tRNA[Ser]Sec gene in hepatocytes revealed increases in
plasma apolipoprotein E and cholesterol levels, up-regulation of cholesterol biosyn-
thesis genes and down-regulation of cholesterol metabolism or transport genes in
the hepatocytes [172]. These results suggest hepatic selenoproteins are responsible
for ApoE and cholesterol metabolism.
Recently, overproduction of the antioxidant selenoprotein, Gpx1 in mice resulted
in a type 2 diabetes-like phenotype [173]. Lei’s group developed Gpx1-overproducing
mice, which became obese at 6 months of age. Gpx1 catalyzes the reduction of
hydrogen peroxide and organic hydroperoxides using GSH as the cofactor. Insulin
production is regulated by pancreatic duodenal homeobox 1, forkhead box A2, and
mitochondrial uncoupling protein 2, expression and function of which are affected
by intracellular ROS [174,175]. Pancreatic β cells express relatively low amounts of
Gpx1, and may be susceptible to oxidative stress [176].
ER stress caused by a disruption in ER homeostasis is associated with type 2 dia-
betes [177]. Up-regulation of SelS mRNA in liver, adipose tissue and skeletal muscle
is associated with type 2 diabetes in Psammomys obesus [142]. Hepatic SelS mRNA
expression and protein content are increased by deprivation of glucose in P. obesus
[137] but not in non-diabetic P. obesus [142]. In addition, a high concentration of
glucose reduces SelS mRNA expression in cultured hepatocytes [142]. A recent
study demonstrated that SelS mRNA expression was increased by insulin stimulation
in human subcutaneous adipocytes from type 2 diabetic patients but not in nondia-
betic subjects [178]. Thus, SelS may be involved in development of type 2 diabetes.
ER stress arrests translation in DI2 synthesis, which leads to a reduction in T3
production [179]. Chemical chaperones (tauroursodeoxycholic acid or 4-­phenylbutyric
acid) can resolve ER stress and restore glucose tolerance in a DI2-­dependent manner
[180]. DI2 is necessary for T3-induced adaptive thermogenesis [103] and DI2-
knockout mice showed obesity and glucose intolerance when placed on thermoneu-
trality and a high-fat diet [180]. The SNP of DI2 (A/G) at codon 92 has been identified
[181] and this SNP leads to the Thr92Ala variant, which strongly associates with
insulin resistance [182] and subsequently type 2 diabetes [181]. DI3-knockout mice
were found to be glucose-intolerant and exhibited a reduction in pancreatic β-cell
mass and insulin content [183]. The absence of DI3 in the β-cells exposes them to T3
and leads to impaired β-cell function [183] and insulin secretion.

4.4  Selenium in Reproduction

For at least five decades, selenium was recognized as an important factor for male
fertility in rats, mice, and livestock. Selenium deficiency in human reproduction
data is contradictory because of the limited number of patients analyzed. Thus, the
role of selenium in human reproduction was largely inferred from studies in labora-
tory animals. Testis uptake and retain selenium even under conditions of substantial
520 Kurokawa and Berry

selenium deficiency. Feeding a selenium-deficient diet for two generations gener-


ated abnormal spermatozoa in rats [184,185]. In severe selenium deficiency, male
rats and mice become sterile as spermatogenesis is arrested, the seminiferous epi-
thelium is degenerated and abnormal sperm morphology is observed. Interestingly,
when 75Se was injected into rats, most of the selenium accumulated in the mid-piece
of the spermatozoon that harbors the helix of mitochondria embedded in a keratin-­
like matrix [186]. Recently, TR2 was shown to be abundant in elongating sperma-
tids at the site of the mitochondrial sheath formation [109].
In 1999 the Ursini and Flohé laboratories identified Gpx4 as the major compo-
nent of the mitochondrial capsule [87]. Gpx4 activity is not detected with the spe-
cific substrate phosphatidylcholine hydroperoxide in spermatozoa but
immunohistochemical staining of Gpx4 was observed. Gpx4 in the mitochondrial
mid-piece of mature spermatozoa is a chemically inactive form, likely due to disul-
fide and selenyl sulfide bridges that form high molecular weight complexes with
other capsular proteins [87]. The Gpx4 protein can be solubilized out of this com-
plex by strong reduction and chaotropic agents and detected by MALDI-TOF mass
spectrometry or Western blotting [87]. Prolonged preincubation with 0.1M DTT or
mercaptoethanol leads to recovery of enzymatic activity. Unlike Gpx1-3, Gpx4 can
accept electrons from protein thiols instead of GSH when GSH is limiting. As the
spermatids mature, GSH and protein thiols decrease, resulting in Gpx4 polymeriza-
tion. The inactive Gpx4 complex contains sperm mitochondrion-associated cysteine-­
rich protein fragments (SMCP), voltage-dependent anion channel and three types of
keratins (type II keratin kb1, keratin k5, and acidic keratin complex I) [187]. SMCP
with its 30% cysteine residues is the most likely candidate to react with Gpx4.
SMCP-knockout mice showed infertility and asthenozoospermia in some mice, but
marginal disturbance of spermatogenesis in others [188].
Reduced Gpx4 production in spermatozoa resulted in bent tails with slight angu-
lations to hairpin structures and abnormal kinking at the mid-pieces [189]. Knockout
of the mitochondrial Gpx4 isoform resulted in viability but male infertility, and the
spermatozoa presented severe morphological abnormalities [90]. Knockout of the
entire Gpx4 gene was embryonic lethal, whereas nuclear Gpx4-knockout mice are
viable and fertile but exhibit transient nuclear instability and delay in chromatin
condensation of spermatozoon [91]. Since cytosolic Gpx4 exists in the nucleus, lack
of nuclear Gpx4 function may be compensated by cytosolic Gpx4 [91,190].

5  Health Benefits of Selenium in Humans

5.1  Molecular Forms of Selenium in Diet

Selenium exists in the environment in several inorganic and organic forms.


Elemental selenium exists stably as selenite and selenate. Organic forms of sele-
nium are found in biological matter, and include the methylated selenium com-
pounds, selenoamino acids, and selenoproteins. However selenium is ubiquitous,
and the amounts can vary widely [191,192].
16  Selenium. Role of the Essential Metalloid in Health 521

Selenium is primarily supplied in diets from grains and animal products [193].
Plants have no true selenoproteins. Selenomethionine is produced in plants due to
indiscriminate substitution of selenium for sulfur in methionine biosynthesis.
Drinking water contains very low amounts of water-soluble inorganic forms of sele-
nium (0.12 to 0.44 μg/L) [194,195] and this contribution to selenium as a dietary
source is very minor. In the United States, the amount of selenium in water is regu-
lated by the Environmental Protection Agency under the Safe Drinking Water Act.
The federal standards allow up to 50 mg/L in drinking water [196].
Grains, wheat, and corn used for breads and other food products contain seleno-
methionine (~55%) as a bioavailable selenium source [197]. Sec and selenite/sele-
nate are also detectable in substantial amounts in wheat (~20% respectively). The
content of selenium in plants can vary widely, as much as 500-fold, depending on the
soil selenium. In regions where soil selenium is low, such as Southwestern Oregon in
the United States, Northeastern China, Finland, and New Zealand, sodium selenite is
added to fertilizers as well as animal feed [198,199]. High-­selenium regions, where
soils exceed 600 mg/kg selenium, are found in parts of North and South America,
China, and Russia [200,201]. In regions with high selenium in the soil, plants may
accumulate up to 3,000 μg selenium per gram and may potentially be toxic [202].
Selenium absorption by plants depends on the pH of the soil, i.e., selenite in acidic
soils and selenate in alkaline soils. Because selenate is a more water-soluble form of
selenium, it is thought to be more available for plant uptake [203,204].
Fruit and vegetables contain only small amounts of selenium. Some vegetables
can grow under selenium-rich soil and accumulate selenium (onions, leeks, garlic,
and broccoli) [205]. These vegetables accumulate selenium up to 50-fold or higher.
Vegetables grown in high-selenium soil contain mostly selenomethionine, meth-
ylselenocysteine, and γ-glutamyl-methylselenocysteine [206,207]. Fungi, such as
mushrooms and yeast, can accumulate selenium and may contain more than 20
selenium-containing compounds (Sec, selenomethionine, methylselenocysteine,
and Se-adenosylselenohomocysteine) [208].
Meats are good sources of selenium, but the selenium content in livestock is
dependent on diet and the region in which the animals feed. Selenium supplementa-
tion of cattle, hogs, and chicken is a common practice [209]. Animal meat contains
mostly selenomethionine (up to 60%) and Sec (up to 50%). The remaining selenium
species are small selenium-containing molecules. These ratios can vary depending
on what form of selenium is consumed. Selenite or selenate in food will be con-
verted into Sec. Animals fed selenomethionine-containing food increase the content
of selenomethionine and Sec in the meat [210].

5.2  Selenium Transport in Mammals

5.2.1  Tissue-Oriented Selenium Transport

Selenium is differentially distributed into different organs. It has been proposed


there is a hierarchy of selenium requirements for selenium in tissues. Brain and
testis tend to preserve selenium for their essential functions under selenium
522 Kurokawa and Berry

­deficiency [161,211]. Dietary selenium is absorbed in intestine and transferred


into liver. The body selenium content is regulated by hepatic production of methylated
selenium compounds and its urinary excretion, not by intestinal absorption of sele-
nium. Selenium is primarily transported in the plasma to the organs via Sepp1
[164,165]. Kidney expresses Sepp1 at 38% of the liver level. Skeletal muscle,
heart, and testis followed with 10, 6, and 6%, respectively, in mouse [212]. Brain
expresses Sepp1 at less than 2% of the liver level. Sepp1 is also expressed in many
tissues at very low levels. The human plasma concentration of Sepp1 is approxi-
mately 5.6 mg/L. Since the liver exports selenium in the form of Sepp1, dietary
selenium deficiency dramatically decreases liver selenium concentrations. As dis-
cussed above, Sepp1-knockout mice exhibited a severe phenotype, including neu-
rological ­dysfunction and male infertility. Testis requires selenium for sperm
maturation, and testis of Sepp1-knockout mice becomes severely selenium-defi-
cient unless a high-­selenium diet is fed. Testis selenium concentrations in Sepp1-
knockout mice decrease to 8% of the wild-type value, whereas brain retains 56%
of the wild-type value when mice were fed 0.25 ppm selenite diet for 4 weeks after
weaning, a diet considered as selenium-adequate. Deletion of Sepp1 causes
increased excretion of selenium in the urine [213]. Selective deletion of Sepp1 in
hepatocytes showed liver selenium is maintained but whole-body selenium con-
centration decreased to 58% of the control value in mice fed 0.25 ppm selenium
diet for 4 weeks beginning at weaning [212]. However. in other tissues selenium
concentrations also decreased to varying degrees, but brain and testis retained sele-
nium better than other tissues. Under conditions of selenium deficiency, selective
deletion of Sepp1 in hepatocytes resulted in elevation of liver selenium concentra-
tion to 500% of the control value, accounting for 53% of whole-body selenium.
These results demonstrate the central role of Sepp1 production by hepatocytes and
the critical role of secretion of Sepp1 from liver in maintaining selenium homeo-
stasis [212].
Sepp1 is observed in testis Sertoli cells in endocytosed vesicles. A lipoprotein
receptor family member, apolipoprotein E receptor-2 (apoER2) was the first identi-
fied Sepp1 receptor in testis Sertoli cells [122]. ApoER2-knockout mice exhibit
very low testis selenium concentrations. Thus, most of the selenium in testis is taken
up in the form of Sepp1 by apoER2. ApoER2 is also highly expressed in the brain
[214], where it was shown to play a crucial role in uptake of Sepp1 and preservation
of selenium when dietary selenium is limiting [123].
Megalin, also a lipoprotein receptor family member, was identified as a Sepp1
receptor in kidney brush border of proximal convoluted tubule (PCT) cells [124].
Several isoforms of Sepp 1 have been identified in rat [121,215]. Shortened iso-
forms may result from termination of Sepp1 translation at UGAs in the open read-
ing frame of Sepp1 mRNA. One of the shortest isoforms of mouse Sepp1Δ240–361 is
small enough to pass through the renal glomerulus. Since megalin-knockout mice
lose Sepp1 in urine, megalin prevents loss of selenoproteins in urine [216]. However,
another plasma selenoprotein, Gpx3, which accounts for 21% of selenium in plasma
[217], is not a selenium source of selenoproteins [218].
16  Selenium. Role of the Essential Metalloid in Health 523

Figure 7  Selenium containing compounds in mammals. Methylselenocysteine is supplied by plants.


Selenosugar is a major urinary selenium compound which is synthesized in liver. Dimethylselenide
is found in breath.

When selenium intake is high, non-Sepp1 selenium forms including low molecu-
lar weight selenium compounds are taken up by kidney. Under selenium deficiency,
low molecular weight selenium compounds are not sufficient to support tissue
selenium requirements [212].
In summary, selenium is transported to tissues primarily via Sepp1 and small
molecules. Plasma Sepp1 is an efficient form of selenium transporter. Gpx3 is
another plasma selenoprotein but does not appear to transport selenium for specific
uptake by cells. Small selenium compounds can transfer selenium but this requires
much higher selenium intake and the pathway appears to be nonspecific. The func-
tions of small molecule selenium compounds need further characterization.

5.2.2  Selenium Excretion

Selenium is excreted in urine, in feces, and by other routes, which include exhala-
tion in breath and loss through hair and skin cells.
Urine. Once selenium is absorbed by the body, it is excreted mostly into the urine.
Urinary selenium excretion increased with increases in dietary selenium intake.
Trimethylselenonium ion was identified as a prominent form of selenium in rat
urine [219], and is the major excreted form when selenium is in excess [220].
Recently, Suzuki’s group identified the major selenium metabolite in urine as
1β-methylseleno-N-acetyl-D-galactosamine (selenosugar) within the required to
low-toxic range [221]. This selenosugar is synthesized in liver [221] (Figure 7).
Breath. Volatilization of selenium into breath is observed only at high selenium
intakes [222]. The volatile compound dimethylselenide was identified as one of the
methylated forms of selenium that account for most selenium excretion in urine and
breath [223].
Feces. Fecal selenium excretion was regulated by dietary selenium intake at defi-
cient to moderately high selenium intakes. Fecal selenium excretion plateaued at
moderately high selenium intake [224]. Characterization of fecal selenium excre-
tion has been relatively minimal.
524 Kurokawa and Berry

5.3  Human Dietary Standards for Selenium

In 1980, the National Research Council (NRC) established an estimated safe and
adequate daily dietary intake for selenium in humans [225]. The recommendation
for adults was set from 50 to 200 μg/d based on extrapolations from animal studies.
In 1989, the Dietary Reference Intake (DRI) was established for selenium, with a
RDA of 70 μg/d for men and 55 μg/d for women in accordance with the World
Health Organization [7].
Selenoproteins are the major form of functional selenium, thus selenium nutri-
tional requirements have been assessed through selenoprotein optimization [226].
Plasma contains two selenoproteins, Gpx3 and Sepp1. Plasma Gpx activity and
Sepp1 concentration decrease to less than 5% of selenium-replete values in animals
with severe selenium deficiency. Thus, plasma levels of these selenoproteins are
used primarily as nutritional biomarkers of selenium.
In 2001, Burk’s group carried out a study in a low-selenium area of China [227].
They concluded that full expression of glutathione peroxidase was achieved with 37
μg Se/d as selenomethionine and with 66 μg/d as selenite. However, full expression
of selenoprotein P was not achieved at the highest doses of either form. There are
several forms of selenium that exist in dietary food and supplements (e.g., high-­
selenium yeast and selenomethionine). Yeast contains selenium mostly as seleno-
methionine but has a significant amount of selenium in other forms. Burk’s group
carried out a study supplementing moderate (approximately 200 μg/d) to high levels
(approximately 600 μg/d) of selenium supplements in three forms (selenized yeast,
selenomethionine, selenite) to selenium-replete individuals in the US [228]. Since
selenomethionine is nonspecifically incorporated to proteins, high-yeast selenium
supplement and selenomethionine raised the plasma selenium concentration in a
dose-dependent manner but plasma selenoproteins did not respond to selenium sup-
plementation in selenium-replete individuals. Selenite intake did not increase
plasma selenium concentration and was excreted into urinary selenium compounds.
In the study, total intakes of over 800 μg/d selenium for 16 weeks showed no signs
of selenium toxicity. The authors concluded that the 800 μg/d can be used safely in
studies of limited duration if the subjects are monitored closely for signs of sele-
nium toxicity.
In 2010, the Burk group further studied the effect of selenium supplementation
in a selenium-deficient human population in China [229]. They studied healthy
Chinese individuals who had a daily dietary selenium intake of 14 μg/d and showed
that supplementation with 35 μg selenium/d for 40 weeks optimized the Sepp1 in
the healthy selenium-deficient Chinese individuals. Gpx activities were optimized
by a total intake of 35 μg selenium/d. The investigators concluded that adjustment
for the difference in weight between the Chinese subjects (58 kg) and US residents
(76 kg) and for variation among individuals would yield a selenium requirement for
US adults of ≈75 μg/d. These studies indicate that once the selenium requirement
has been met, selenoproteins are not increased and plasma Sepp1 concentration is
the best marker of human selenium nutritional status.
16  Selenium. Role of the Essential Metalloid in Health 525

6  General Conclusions

It has been two centuries since the identification of selenium by Berzelius. Selenium
is essential for life processes. Although it is a rare element, many organisms have
evolved to maximize selenium’s properties. It is integrated into the biology of many
life forms, to the extent of being critical for life. The selenium field has been dra-
matically expanding over the last few decades. However, functions of most of the
selenoproteins and selenium containing molecules still remain unclear. Continued
research of the biochemical properties of selenium will hopefully lead to new dis-
coveries to improve human health.

Abbreviations and Definitions

Ψ55 tRNA pseudouridine position at 55


28S 28S ribosomal RNA
ADP adenosine 5′-diphosphate
AMP adenosine 5′-monophosphate
apoER2 apolipoprotein E receptor-2
ATP adenosine 5′-triphosphate
BAT brown adipose tissue
cDNA complementary DNA
Cys cysteine
DI iodothyronine deiodinase
DRI dietary reference intake
DTT dithiothreitol
EF elongation factor
eEFSec eukaryotic selenocysteyl-tRNA-specific elongation factor
eIF4a3 eukaryotic initiation factor 4A-III
EMSA electrophoretic mobility shift assays
ER endoplasmic reticulum
ERAD ER-associated protein degradation
EST expressed sequence tag
γ-GCS γ-glutamylcysteine synthetase
Gpx glutathione peroxidase
GS glutathione synthetase
GSH reduced form of glutathione or γ-glutamylcysteinylglycine
GSSG oxidized form of glutathione, glutathione disulfide
i6A isopentenyladenosine
kDa kilo dalton
L12 large ribosomal subunit protein L12
L30 large ribosomal subunit protein L30
m1A N1-methyladenosine
MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight
526 Kurokawa and Berry

mcm5U methylcarboxymethyl-5’-uridine
mcm5Um methylcarboxymethyl-5’-uridine-2’-O-methylribose
mRNA messenger ribonucleic acid
MS mass spectrometry
Msr methionine sulfoxide reductase
mV millivolts
NADP+ nicotinamide adenine dinucleotide phosphate
NADPH nicotinamide adenine dinucleotide phosphate (reduced)
NPC Nutrition Prevention Cancer
NRC National Research Council
Nrf2 leucine zipper transcription factor NF-E2 factor 2
PCCA progressive cerebellar-cerebral atrophy
PCT proximal convoluted tubule
pKa acid dissociation constant
PPi pyrophosphate (diphosphate)
PSTK O-phosphoseryl-tRNA[Ser]Sec kinase
RDA recommended dietary allowance
ROS reactive oxygen species
rRNA ribosomal ribonucleic acid
rT3 reverse triiodothyronine
SBP2 SECIS binding protein-2
Sec selenocysteine
SECIS Sec insertion sequence
Secp43 Sec tRNA[Ser]Sec associated 43 kDa protein
SecS Sec synthetase
Sel(X) selenoprotein X (X is any selenoprotein)
SELB Sec-specific translation elongation factor
SELECT Selenium and Vitamin E Cancer Prevention Trial
selenosugar1 β-methylseleno-N-acetyl-D-galactosamine
Sep15 15 kDa selenoprotein
Sepp1 human selenoprotein P
siRNA small interfering RNA
SLA soluble liver antigen
SMCP sperm mitochondrion-associated cysteine-rich protein
SPS selenophosphate synthetase
T3 3,3′,5-triiodo-L-thyronine or triiodothyronine
T4 thyroxin
TGR thioredoxin/glutathione reductase
TR thioredoxin reductase
Trit1 tRNA isopentenyltransferase, mitochondrial
tRNA transfer ribonucleic acid
UDP-glucose uridine diphosphate glucose
Um34 single methyl group on the ribosyl moiety at position 34
UTR untranslated region
UVB ultraviolet, 315-280 nm wave length
WHO World Health Organization
16  Selenium. Role of the Essential Metalloid in Health 527

Acknowledgment  This work was supported by National Institutes of Health grants R01DK047320
to MJB.

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Index

A disturbance, 40
Aβ amyloid plaques, 277 imbalance, 43, 44
metabolism, 377 Acid dissociation constants (pKa), 146, 148,
ABC. See ATP-binding cassette 151, 267, 454, 477, 502
superfamilies (ABC) Acidosis, 40–42, 44, 402
Absorption of Acid phosphatase, 86, 150
calcium, 84, 85, 127 Acid-sensing channel, 402
chromium, 176, 177 Acinetobacter baumannii, 11
cobalamin, 296, 298–300, 311, 313 Acireductone dioxygenase, 323, 336–338,
iron, 242, 243, 247, 253–255, 257, 341, 342
258, 348 ACOG. See American College of Obstetrics
potassium, 34 and Gynecology (ACOG)
silicon, 464 Aconitase, 246, 402
sodium, 37 Acrodermatitis enteropathica, 11,
Accumulation of 404, 408
calcium, 70, 89, 97, 119 Acrylamide production, 314
copper, 366, 376, 378 ACTH (adrenocorticotropic hormone), 395
glycogen, 155 Actin, 108, 109, 112, 125
hydrogen sulfide, 441 Actinobacteria, 349
iron, 208, 256, 277–280, 363 Actinolite, 463
magnesium, 51, 54, 55, 63–65 Action of
manganese, 204, 208, 209, 213, 216 potassium on membranes, 32, 33
methyltetrahydrofolate, 310 sodium on membranes, 32, 33
sulfite, 425, 435, 436 Active sites, 106, 150, 151, 159, 217, 314,
uric acid, 421 323, 336–341, 345, 350, 362, 393, 416,
xanthine, 420, 421 417, 434, 502, 512, 513
ACD. See Allergic contact dermatitis (ACD) Acute
Aceruloplasminemia, 363 lymphoblastic leukemia, 421
Acetate, 14, 256, 322, 325 promyelocytic leukemia (APL), 476, 477,
Acetohydroxamate (and acid), 234, 268, 269 492, 493
Acetylation of histones, 328, 396 renal failure, 38, 40, 41, 45, 421
Acetylcholine, 33 toxicity of arsenic, 481
AcetylCoA/CoA ratio, 107 AD. See Alzheimer’s disease (AD)
Acetyl-CoA decarbonylase synthase, 342, 343 Addison disease, 44
Acid-base Adenosine
balance, 31, 37 deoxy-, 303, 427

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 535
Metal Ions and Human Diseases, Metal Ions in Life Sciences 13,
DOI 10.1007/978-94-007-7500-8, © Springer Science+Business Media Dordrecht 2013
536 Index

Adenosine 5′-diphosphate (ADP), 97, 107, Agency for Healthcare Research


111, 306, 481, 504 and Quality, 183
-arsenate, 481 Age-related macular degeneration, 279,
Adenosine 5′-monophosphate (AMP) 280, 403
-activated protein kinase Aging, 64, 254, 255, 391, 457, 469
(AMPK), 115, 187, 189 Agranulocytosis, 274, 276
c-, 53–55, 68, 100, 428, 429, 431, 432, 434 Agricultural laborers, 251
Adenosine 5′-triphosphate (ATP), 33, 51, Akt, 154–156, 159, 161, 186, 187, 189, 190
52, 54, 55, 65, 95, 104, 106–108, Albumin, 3, 10, 15, 18, 19, 21, 53, 73, 145,
111, 114, 124, 206, 209, 215, 217, 155, 203, 206, 207, 256, 274, 323
263, 302, 303, 306, 309, 341, 342, parv-, 52, 119
348, 368, 369, 391, 417, 429, 435, Alcohol, 58, 65, 257, 396, 399, 422
480, 481, 486, 503–505, 513 dehydrogenase, 65
ADP ratio, 107, 111 Alcoholism, 58, 65, 66
-cob(I)alamin adenosyltransferase, 302 Alcohol liver disease (ALD), 65
hydrolysis, 108, 342, 368, 480, 486 Aldehyde oxidase (AOX), 419, 421–423,
production, 106, 108, 215 431, 442
-sensitive K+ channels, 111 deficiency, 431
synthase, 107, 486 -knockout mice, 422
synthesis, 108, 215, 263, 435 Aldosterone, 32, 33, 36, 37, 42–44, 59, 62,
Adenosylcobalamin (Ado-Cbl), 297, 298, 302, 67, 68
303, 305, 311 deficiency, 44
S-Adenosyl-homocysteine, 309 Alga, 160, 161, 165, 417
hydrolase, 309, 424 Alkaline phosphatase (ALP), 87, 88, 201, 458
S-Adenosyl-methionine (AdoMet/SAM), 304, Alkalosis, 39, 41–44, 67
307–310, 312, 427 Allergen, 332–335
-dependent enzymes, 236 Allergic contact dermatitis (ACD),
Adenosyltransferase, 302, 309 332, 334, 335
Adenylate (or adenylyl) cyclase, 66, 515 Allopurinol, 421, 422
Adenylyltransferase, 429 ALS, 118, 121, 122, 212, 213, 362, 378, 379
Adequate intake, 173–175, 202 Aluminosilicates, 452, 456, 461, 468
ADH. See Antidiuretic hormone (ADH) Aluminum(III), 456, 460, 461, 463, 467
Adhesives, 200 from drinking water, 461
Adipocytes, 64, 155, 180, 186–189, 519 oxides, 417
3T3-L1, 187, 189 Alzheimer’s disease (AD), 102, 118, 120–122,
Adiponectin, 462 213, 277–280, 311, 376, 377, 380, 403,
Adipose tissue, 145, 146, 405, 512, 519 406, 407, 452, 457, 460, 461, 467, 469
Ado-Cbl. See Adenosylcobalamin Amanita muscaria, 141–143
AdoMet. See S-Adenosyl-methionine Amavadin, 141–143
ADP. See Adenosine 5′-diphosphate (ADP) American College of Obstetrics and
Adrenal cortex, 37 Gynecology (ACOG), 71
β-Adrenergic stimulation, 33, 36 American Diabetes Association, 184
Adrenocorticotropic hormone (ACTH), 395 American trypanosomiasis, 162
Adults, 3, 10, 17, 18, 20, 21, 30, 34, 41–43, 2-Amidopyridin-4-one, 275
101, 152, 173, 179, 181, 183, 191, Amiloride, 53
202, 204, 214, 247–249, 253, 260, Amino acid(s) (see also individual names)
296, 298, 331, 345, 346, 455, 484, hydroxylation, 237
485, 490, 524 metabolism, 349
Aerobactin, 283 mutations, 68
Aerosols, 143, 325 sequences, 300, 307, 368, 483
Affinity constants (see also Binding constants, γ-Aminobutyric acid (GABA), 432, 434–436
Formation constants, and Stability Aminoglycoside, 58
constants), 233, 234, 245 5-Aminolevulinate (δ-aminolevulinate)
Africa, 13, 253, 491, 517 synthase, 246, 263, 264
Index 537

Ammonia, 205, 303, 336, 338, 347 Anti-epidermal growth factor receptor
Amoebae, 141, 162, 163 antibodies, 71–73
Amoebiasis, 140, 162–165 Antifungal
Amoebocidal drug, 163 properties, 5
Amosite, 463 therapy, 9
AMP. See Adenosine 5′-monophosphate Antigen-specific T-cells, 332, 333
AMP-activated protein kinase (AMPK), Antiinflammatory agents, 71, 217
115, 187, 189 Antimicrobial activity, 161
AMPA receptor, 402 Antioxidant(s), 3, 4, 15–18, 213, 215, 218,
Amphotericin, 58 326, 329, 436
Amylin, 405 enzymes, 16, 21, 201, 395, 396, 402
β-Amyloid or Amyloid-beta (Aβ), 120, 213, supplementation, 17
277, 377, 460, 461 Antiparasitic properties of vanadium, 162
Amyloid precursor protein (APP), 120, 278, Antiretroviral therapy, 16
279, 284, 376, 377, 407 Antituberculosis drugs, 162
Amyotrophic lateral sclerosis (ALS), Antitumor activity, 147, 157
118, 121, 122, 212, 213, 277, Antiviral activity, 159, 161
362, 378, 379 APL. See Acute promyelocytic
Anaphylaxis, 401 leukemia (APL)
Anemia, 7, 8, 18, 64, 65, 231, 345, 348, 371, Apolipoprotein E (ApoE), 514, 519, 522
372, 374, 400, 418 receptor-2 knockout mice, 522
of chronic disease, 8, 254, 255 Apoptosis, 65, 101, 113–116, 121, 123, 157,
Anesthetic drugs, 124 158, 215, 218, 323, 329–331, 334, 347,
Angiogenesis, 70, 72, 159, 330, 331, 360, 406 366, 396, 400, 466, 482, 492, 511, 515
Angiotensin II, 36, 37, 373 Apparent binding constants, 177
Animal(s) (see also individual names) zinc, 405
guts, 338, 350 Aquacobalamin, 298, 307
nutrition, 173 Aquaglyceroporins, 480, 481
studies, 6, 22, 284, 458, 459, 463, 484, Arabidopsis thaliana, 418
486, 493, 524 Archaea, 323, 336, 339, 342, 349, 350, 487,
Animal models, 45, 65, 172, 212, 214, 280, 503, 504
313, 314, 316, 323, 324, 326, 436, 437, Argentina, 482
439, 442, 481, 484 Arginase, 20–22, 205
for Parkinson Disease, 378 Arginine vasopressin (AVP), 39
mouse. See Mice and Mouse Aromatic amines, 233
of iron overload, 264 ArsA, 480
rat. See Rat Arsenate, 477–481, 485–490, 493
rodent, 193, 203, 250 diester bonds, 489
Annexins, 51, 87, 91, 92 lead, 490
Anorexia, 4, 42, 45, 396, 418 reductase, 480
Antacid, 56, 58, 250, 464 Arsenic, 452, 476–493
Anthophyllite, 463 -accumulating plants, 485
Antibacterial acid, 478
activity, 160 -based drugs, 477
agent, 468 chemical properties, 477–479
properties, 5 chronic exposure, 476, 477, 481, 482
Antibiotics, 12, 13, 58, 254, 273, 408, 491 deficiency, 484
Anticoagulation, 399 dimethyl-(DMAs), 481, 491
Antidiabetic eaters, 490
agents, 64, 462 excretion, 477
effect of silicon, 462 half-life in humans, 480
vanadium compounds, 154 in agriculture, 491
Antidiarrheal agent, 468 in drinking water, 482, 493
Antidiuretic hormone (ADH), 36–38, 40 inorganic, 479, 481, 491
538 Index

Arsenic (cont.) H+-, 99


in the environment, 479, 491 Menkes P-type, 18
levels in urine, 490 ATP7B, 363, 365–371, 374, 376, 380
metabolism, 484 ATP-binding cassette superfamilies (ABC),
methyltransferases (As3MT), 481, 490 206, 209, 341, 417
organo-, 479, 491 ABC transporter, 300, 342, 417
stimulation of growth, 485 Atrophic gastritis, 299
toxicity, 477, 481 Australia, 374, 440
uptake, 477 Austria, 490
yellow, 479 Autoimmune
Arsenic(III), 477, 487, 492 diseases, 106, 255, 401, 421
Arsenic(V), 477, 487 gastritis mouse model, 313
Arsenicosis, 477, 481 Autoimmunity, 313
Arsenic oxide (As2O3), 477, 490, 492 Autophagy, 98, 113–116
lethal dose, 481 Autosomal recessive disease, 11
Arsenite, 478–483, 485–489, 492, 493 Autoxidation of
Arsenobetain (AsB), 478, 480, 481 catecholamines, 362
Arsenocholine (AsC), 478, 480, 481 iron(II), 240
Arsenolipids, 478, 480 AVP. See Arginine vasopressin (AVP)
Arsenopyrite, 479, 486 Azotobacter, 141
Arsenosugars, 478, 480
Arsenous acid, 478
Arsine (AsH3), 479 B
Arsphenamine, 491 Bacillus, 282
ArsR, 344, 347, 480 Bacillus pasteurii, 338
AsB. See Arsenobetain (AsB) Bacteria(l), 3, 5, 6, 22, 141, 165, 205, 282,
Asbestos, 452, 453, 463–467 296, 302, 314, 323, 324, 327, 336–340,
-induced carcinogenesis, 465 342, 343, 346, 349, 401, 404, 417, 427,
workers, 466 429, 480, 487, 502, 503
Asbestosis, 453, 464, 466, 467 gram-negative, 8, 161
AsC. See Arsenocholine (AsC) gram-positive, 161
Ascidia, 112 infection, 159–161, 283, 345, 347
Ascophyllum nodosum, 165 methionine synthase, 304, 307, 308
Ascorbate or ascorbic acid, 20, 147, 148, 176, nitrogen-fixing, 141
191, 253, 329, 330, 365, 512 Bacterium bifidum, 349
Asia, 13 Bangladesh, 164, 482, 492
As3MT. See Arsenic methyltransferases Barium(II), 206, 208
(As3MT) Barrett’s esophagus cancer, 511
Aspergillus, 9 Bartter’s syndrome, 39, 41
Asthma, 144, 324, 401 BB-rats, 462
Astrocytes, 96, 122, 158, 201, 205, 210, 215, BCB. See Blood-cerebrospinal
216, 218, 363 fluid barrier (BCB)
Astrocytosis, 204, 212, 213, 215, 216 β-cells, 63, 111, 393, 400, 401,
Ataxia, 116–118 405, 519
Atherosclerosis, 61, 62, 64, 399, 452, 457, B12-deficient rats, 305, 309, 313
459, 460, 469 Beef meat, 298, 299
Atopic dermatitis, 401 Beer, 38, 454, 461
ATP. See Adenosine 5′-triphosphate (ATP) Belgrade rat, 264, 265
ATP7A, 18, 363–371, 373–375, 379 Beta blockers, 37
ATP/ADP ratio, 107, 111 Beta-globulin, 203
ATPases, 82, 93, 109, 300, 347 Betaine, 314
Ca2+/Mn2+, 203 Beverages, 143, 422, 453, 454
efflux pump, 347 BFD. See Black foot disease (BFD)
Index 539

Bifidobacterium longum, 349 formation, 86, 87, 201, 458, 467, 468
Bile, 18, 20, 21, 202, 203, 369, 370, 399 health, 403, 452, 458, 459, 462, 467
excretion of copper, 371, 376 malformation, 371
Bilirubin, 3 marrow, 247, 262, 372, 462
Binding constants (see also Affinity constants, mass, 458
Formation constants, and Stability mineral density, 51, 458, 459, 467
constants), 177, 405 mineralization, 84, 452, 457, 459
Bioavailability (of), 153, 253, 254, 266, 296, morphogenetic protein 6, 280
452, 453, 455–457, 459, 461, 469, 517 Bordatella, 282
B12, 296 Borrelia burgdorferi, 5
selenium, 517 Bovine
silica, 455 liver, 503, 504
Bioinformatics, 502, 506, 513 spongiform encephalopathy, 379
Biomarkers (for), 10, 15, 22, 390, 422, 437, Bradycardia, 56
438, 466 Bradyrhizobium japonicum, 343
chromium, 178, 179 Brain, 21, 85, 101, 105, 120, 146, 158, 180,
folate metabolism, 315 201–208, 210, 213, 214, 216, 217, 251,
inflammation, 396 263, 277, 281, 284, 331, 332, 367, 370,
methionine metabolism, 315 371, 376–378, 423, 432, 435, 436, 440,
molybdenum-dependent enzymes, 435 460, 461, 515, 518, 521, 522
neuropsychiatric disorders, 441 cattle, 423
oxidative stress, 14 degeneration, 516
selenium, 524 human, 215, 402, 437
silicon, 455 injury, 402, 426
zinc, 396, 399, 407 rat, 423
Biomineralization, 86–88 Breast
Biosynthesis of cancer, 281, 406, 486
phosphatidylcholine, 485 tumors, 156
selenocysteine, 503–505, 509 Brewer’s yeast, 174, 183, 186, 501
selenocysteyl-tRNA, 505, 507–509 Brody disease, 124, 125
the molybdenum cofactor, 427–430 Bromoperoxidase, 165
Biotin, 306 Bronchitis, 144, 465
Bipolar disorder, 404 Bronze, 476
Black foot disease (BFD), 482 Brucella suis, 342
Blood Buffers, 31, 93, 394
clotting, 201, 361 HEPES, 191
donation, 249, 250 Bulimia, 42
fat, 173 Bumetanide, 39
manganese, 21, 202, 204, 217 Burns, 3, 4, 16–19, 22, 44
plasma, 15, 83, 84, 143, 145, 146, 323 patients, 13, 15
potassium, 41
pressure, 36, 37, 44, 45, 60, 61, 110,
173, 460 C
-retinal barrier, 280 Cadmium, 5, 21, 207
selenium, 15, 517 cADPR. See Cyclic ADP-ribose (cADPR)
sugar, 31, 184, 185, 484 Caenorhabditis elegans, 218, 314
transfusion, 258, 261–264, 276 CAKI-1 mice, 157
Blood-brain barrier (BBB), 206, 207, 209, Calbindins, 84, 87
210, 281 Calcineurin (Cn), 101, 104–106
Blood-cerebrospinal fluid barrier (BCB), 207 Calcitonin, 84
Bone(s), 50, 56, 83–87, 126, 146, 147, 324, Calcium(II), Ca2+, 33, 39, 45, 81–127,
325, 375, 463, 469 201, 206, 212, 392, 394, 395, 456, 458,
composition, 324 459, 513, 515
540 Index

Calcium(II), Ca2+ (cont.) Cancer (see also Carcinoma and Tumor), 42,
absorption, 84, 85, 127 69, 70, 72, 73, 102, 113, 114, 141,
accumulation, 70, 89, 97, 119 144, 156–159, 163–165, 231, 254,
and bioenergetics, 106–108 255, 331, 344–346, 349, 363, 391,
arsenate, 490 440, 441, 452, 455, 464, 465, 468,
as antagonist of Na+, 88–93 476, 492, 493, 513, 518
as regulator, 100–116 Barrett’s esophagus, 511
as signaling agent, 88–93 bladder, 477
-ATPase, 36, 61, 98 breast, 281, 406, 486
-binding proteins, 96, 98 cervical, 157
carbonates, 83, 85 colon, 69, 70, 73, 157, 513
cytosolic, 88, 89, 95, 96, 100, 108, 111, gastric, 102
115, 119, 120, 125 gastrointestinal, 281
free, 61, 83, 87, 89, 93, 96, 111, 113 intestinal, 511
homeostasis, 86, 102, 113–115, 117, kidney, 157, 477
119–121, 124, 215 liver, 281, 477
hydroxyapatite, 85 lung, 144, 477, 482
in biological fluids, 84, 85 nasal, 325
in blood, 83, 84 ovarian, 72, 157
-induced Ca2+ release (CICR), 95, 99, prostate, 511
109–112, 124 renal, 331
in plasma, 83, 84 respiratory tract, 325
Mn2+-ATPases, 203 skin, 477, 482
overload, 86, 88, 89, 96, 102, 113, 116, testicular, 157, 158
119, 122, 126 Candida, 9
oxalate, 83 albicans, 373
phosphate, 83, 85, 87, 88 Carbohydrate metabolism, 31, 107, 175, 201
picolinate, 83 Carbonates, 83, 85
regulation, 97, 116, 329, 515 Carbon dioxide, 336, 487
release, 33, 94–96, 98, 99, 104, 109–111, Carbonic anhydrase IX, 330
113, 120, 124, 125 Carbon monoxide, 140, 336, 337, 342, 343
secretion, 110–111 dehydrogenase, 342, 343
signaling, 94, 97, 98, 100, 116, Carboxylates, 233, 237, 271, 273, 338
118, 121 Carcinogenicity, 192, 325, 327, 345, 465
sulfate, 83 Carcinoma (see also Cancer and Tumor)
transport, 96–98, 106, 208 colon adeno-, 511
uniporter, 93, 203, 215 esophageal, 406
uptake, 94, 99 gastric, 344
Calcium channel(s), 59, 85, 87, 92, hepato-, 513
95–100, 109, 111, 117–120, 124, liver, 256
206, 209 lung adeno-, 511
L-type, 100, 109, 119 oral small cell, 406
Calmodulin, 52, 91, 105, 106, 513 squamous cell, 511
-dependent kinase (CaMK), 100, 101, Cardiac
104, 105 arrest, 31, 41, 43, 56, 57
Calpains, 101–103, 119, 126 arrhythmias, 42, 59, 60
Calpastatin, 103, 516 diseases, 123, 124, 276
Calprotectin, 5, 404 hypertrophy, 159
Calprotectinemia, 404 iron, 274
Calretinin, 84, 119 muscles, 109, 123
cAMP. See Cyclic adenosine monophosphate myocytes, 63, 95, 98, 105, 109
(cAMP) surgery, 17
Campylobacter jejuni, 339 tissue, 274
Index 541

Cardiomyopathies, 102, 123, 124, 256, 257, 362 Sertoli, 363, 522
Cardio-protective effects of vanadium, 160 T-, 17, 101, 105, 106, 160, 163, 323, 325,
Cardiovascular 332–335, 347, 400, 401, 467
diseases, 60, 161, 184, 185, 311, 312, 315, Cellular
349, 363, 373, 380, 399, 422, 482 calcium, 66, 94, 102, 115
effects, 159–162 homeostasis, 30, 114, 201, 327, 331
β-Carotene, 17 iron transport, 241–244
Carotid artery disease, 404 magnesium, 52, 54, 55, 58–65, 69, 73
Caspases, 113, 334 potassium, 63
Catalases, 3, 4, 15, 235, 329, 362 sodium, 60, 63
Cataract formation, 102 Central core disease, 124, 125
Catecholamine, 55, 58, 59, 210, 211, 214, Central nervous system (CNS), 31, 203, 205,
362, 364 210, 244, 256, 284, 363, 376, 429, 432,
Catechols, 267, 271, 361, 364 435, 437, 461, 481, 512
Cathepsin K, 86 Ceramics, 200, 453
Cattle, 296, 373, 379, 423, 487, 521 Ceramide, 66
Cbl. See Cobalamin (Cbl) Cereals, 10, 249, 252, 253, 454
CblC protein, 299–302 Cerebral
CBS. See Cystathionine β-synthase (CBS) atrophy, 435
C2C12 skeletal muscle cells, 187 edema, 435, 438
cDNA, 69, 506 palsy, 71, 438
CDO. See Cysteine dioxygenase (CDO) Cerebrospinal fluid (CSF), 203, 212, 277, 436
Celiac Ceruloplasmin (Cp), 4, 18, 19, 203, 244, 330,
disease, 250, 408 361–363, 365, 371, 372
sprue, 59 Cervical cancer, 157
Cell(s), 18, 21, 30, 31, 34, 36, 42–44, 54, 55, Chagas’ disease, 162, 163, 165, 337, 346
61, 69, 82–85, 88, 89, 92, 93, 95, 97, Channels
99, 101, 106, 113, 114, 116, 117, acid-sensing, 402
119–121, 126, 176, 186–189, 192, 201, calcium. See Calcium channels
210, 213, 215, 216, 234, 235, 238, 240, ion, 95, 105, 208, 398
247, 248, 274, 283, 323, 324, 327, ionotropic glutamate receptor, 209
329–331, 366, 367, 379, 390, 393, 396, ligand-gated, 95
398, 406–408, 423, 462, 510, 516, 523 magnesium, 51
β, 63, 111, 393, 400, 401, 405, 519 membrane, 93–95, 112
C2C12 skeletal muscle, 187 N-methyl-D-aspartate, 402
cycle, 69, 281, 403, 406 nucleotide-gated, 348
cycle arrest, 158 ORAI1, 97, 98
death, 37, 94, 101, 102, 113–116, 121, potassium, 54, 111
127, 214, 216, 279, 324, 442, 466, 515 ryanodine receptor, 95, 98, 99, 109
EDR3 hepatoma, 487 sodium, 32, 118, 119
endothelial, 61, 70, 206, 208, 247, 333, store-operated, 97, 98, 209
334, 396, 466, 467 transient receptor potential (TRP), 51
erythroid, 241, 244 two-pore (TPC), 94, 95, 99, 118
eukaryotic, 92, 349, 364 voltage-regulated, 97, 206, 208, 209, 520
lysis, 33, 37 Chaperones, 98, 114, 342, 343, 516
membrane, 3, 31–33, 51, 53, 55, 65, 92, CH3-Cbl. See Methylcobalamin (CH3-Cbl)
120, 146, 154, 190, 208, 209, 323 Chelating agents, 266, 268, 270, 282,
natural killer (NK cells), 10, 12, 15, 371, 402
401, 467 Chemokines, 324, 332
nerve, 31, 32, 378, 401 Chemolithoautotrophs, 487, 489
neuroendocrine, 96, 403 Chest syndrome, 262
proliferation, 69, 70, 158, 281, 324, 327, CHF. See Congestive heart failure (CHF)
328, 330, 331, 366, 482 Chicken, 42, 425, 484, 491, 521
542 Index

Childhood hepatitis B, 19
diarrhea, 12 inflammation, 14, 421, 465
early death, 426 kidney disease (CKD), 44, 45, 57
Children, 7, 12, 13, 16, 18–20, 40–42, 153, myeloid leukemia (CML), 492, 493
202, 249, 258, 261, 262, 371, 373, 407, nickel exposure, 323
408, 490, 517 toxicity of arsenic, 481
Chile, 253, 482, 483 Chrysotile, 463
Chinese hamster ovary cells, 187 CICR. See Ca2+-induced Ca2+ release (CICR)
Chlamydomonas, 418 Cirrhosis, 18, 38, 39, 44, 204, 256, 375, 376,
reinhardtii, 417, 418, 426 399, 408, 517
Chloride wasting, 67 Cisplatin, 43, 54, 58, 157, 158, 165
Chlorothiazide diuretics, 38 Citalopram, 423
Cholestasis, 18, 20, 21 Citrate, 53, 83, 148, 209, 232, 256, 277, 402
Cholesterol, 61, 180–182, 189, 190, 193, 201, Citric acid cycle, 106, 107, 481
459, 511, 519 CKD. See Chronic kidney disease (CKD)
metabolism, 193, 519 Clastogenic, 191, 465
Choline transporters, 209 Claudin, 54, 68
Chondrocytes, 87 Clay, 452
Chromate, 191 Cleavage of
Chromatin, 213, 215, 327–329, 401, DNA, 329
515, 520 the Co-C bond, 303
damage, 329 Clinical trials, 273, 377
Chromium, Cr, 19, 20, 171–193, 327, 484 phase I, 273
51
Cr, 176 phase II, 273, 377
absorption, 176, 177 Clioquinol, 280
biomarkers, 178, 179 Clusters, 86, 88, 95, 98, 109, 159, 243, 244,
deficiency, 173–177, 179, 192 264, 278, 336, 338, 400, 419, 420, 422
detoxification, 189 [2Fe-2S], 236, 420
excretion, 177, 178 [3Fe-4S], 340
histidine, 186, 187 4Fe-4S, 236, 246, 332, 340, 427
in infectious diseases, 20 iron-sulfur, 240, 241, 263, 284, 416
picolinate ([Cr(pic)3]), 173, 181, 183–187, CML. See Chronic myeloid leukemia (CML)
189–192 CN-Cbl. See Cyanocobalamin (CN-Cbl)
13
supplementation, 179, 182–184 C NMR, 427
-vanadium-iron alloys, 164 CNS. See Central nervous system (CNS)
Chromium(III), 172–174, 176, 177, 179–181, CoA. See Coenzyme A (CoA)
183, 185, 186, 188, 190–193 Coagulation, 92, 399
in drinking water, 175 factors, 361
intake, 174, 175, 177, 178 Cobalamin (Cbl), 296–310, 312, 313, 315
nicotinate, 183, 192 absorption, 296, 298–300, 311, 313
supplementation, 178, 179, 181–185, 189, adenosyl-(Ado-Cbl), 297, 298, 302, 303,
192, 193 305, 311
Chromium(VI), 188 biochemistry, 297, 298
Chromodulin, 188 biological half-life, 313
Chromosome binding proteins, 298–302, 308, 315
57
aberrations, 191, 192, 327, 329 Co-labeled, 300
translocation, 492 cyano-. See Cyanocobalamin (CN-Cbl)
Chronic -dependent enzymes, 297, 300,
arsenic exposure, 476, 477, 481, 482 303–310, 315
constipation, 350 in food, 296
diseases, 8, 254, 255, 277, 391, metabolism, 300, 301
404–407, 459 neuropathy, 310, 312, 313
heart failure, 422 reductase, 302
hemodialysis, 18 transport, 298, 313
Index 543

Cob(I)alamin, 298, 302, 306, 307 Contraceptives, 408


Cob(II)alamin, 298, 302, 303, 305, 307 Convulsions, 68, 146, 374
Cob(III)alamin, 298 Copper, Cu, 3–5, 10, 13, 15, 17–19, 22,
Cobaloxime, 297 149, 205, 208, 214, 231, 266, 267,
Cobalt, Co2+, 5, 206, 242, 296–316, 332, 341, 271, 327, 329, 332, 348, 360–380,
342, 347, 348 401, 406, 407, 418, 429, 440, 441,
57
Co-labeled cobalamin, 300 461, 476, 484, 491, 515
deficiency, 296, 297 accumulation, 366, 376, 378
essentiality, 296 amine oxidase, 361
overload, 315 biochemistry, 361–371
Cobamide, 297, 298 chaperones, 364, 365, 367, 370
Cobinamide, 297, 308 chelating agents, 371
Coccidiosis, 491 chelation, 376, 378
CO dehydrogenase, 323, 343 chrome arsenate (CCA), 491
Coenzymes deficiency, 4, 18, 363, 367–369, 371–377,
A (CoA), 107, 279, 311 401, 418, 440
B, 350 deficient rats, 373
M, 341, 350 detoxification, 366
Coffee, 252, 422, 454 efflux, 364, 368–371, 379, 380
Colitis, 59, 408, 511 excretion, 371, 376
ulcerosa, 408 homeostasis, 18, 332, 361, 364–371, 376,
Collagen, 86, 88, 361, 363, 373, 457–460, 379, 380, 440, 441
462, 463, 466, 467 -induced toxicity, 367
Colon in infectious diseases, 19
adenocarcinoma, 511 overload, 367, 371, 375, 376, 440
cancer, 69, 70, 73, 157, 513 sequestration, 366–367
Coma, 336, 481 storage, 18, 19, 366–367
Combustion of petroleum, 143 supplementation, 371, 374, 378
Committee on the Scientific trafficking, 18, 365, 367–368
Evaluation of Dietary Reference uptake, 364–366, 370, 373, 379, 440
Intakes of the National Academies zinc ratio, 4, 19
of Science, 180 Copper(I), Cu+, 360, 361, 364, 365, 368,
Common cold, 12, 13, 18 377, 418
Complex I, 119, 215, 278, 279, 520 -GSH, 366
Complexins, 110, 111 Copper(II), Cu2+, 332, 360, 361, 377
Computer chips, 453 Cu2+/Cu+ redox system, 361
Concrete, 453 Copper transporter receptor 1 (Ctr1), 364–366,
Conditional stability constant, 238 369, 370, 374
Conformational changes, 102, 103, 111, 163, Copper/zinc superoxide dismutase (SOD1,
164, 189, 308, 333, 369, 425 Cu/Zn-SOD), 3, 10, 18, 21, 149, 212,
Congenital adrenal hyperplasia, 43 361, 362
Congestive Corallina inaequalis, 160, 161
cardiac myopathy, 501 Coral sand, 458, 460, 462
heart failure (CHF), 38, 39, 43, 59–61, 72, 399 Corn, 521
Connective tissues, 201, 372–375, 458, 463, Coronary diseases, 18, 60, 404, 459
467, 469 Corrinoids, 296, 297, 299
dysfunction, 363 Corrin ring, 297, 298
Conn syndrome, 43 Corticotropin, 395
Contaminated Corynebacterium, 282
drinking water, 481 Cosmetics, 456
environments, 323 Countries. See individual names
soils, 491 Coxsackie virus, 517
surface water, 482 Coxsackievirus B4, 161
Continents. See individual names Cp. See Ceruloplasmin (Cp)
544 Index

cPMP. See Cyclic pyranopterin Cytokines, 3, 4, 12, 17, 65–67, 70, 71, 86,
monophosphate (cPMP) 190, 255, 324, 332, 333, 335, 367,
Creutzfeld-Jacob disease, 379 400, 401, 466
Critically ill patients, 3, 4, 13, 14, 16, 17 Cytoplasm, 53–55, 65, 86, 96, 100, 103, 113,
Crocidolite, 463, 466 114, 122, 208, 244, 283, 341, 342,
Crohn’s disease, 59, 250, 408 347, 508, 509
Crude oil, 142 Cytoslic
Cryptidins, 399 calcium, 88, 89, 95, 96, 100, 108, 111,
Cryptococcus, 9 115, 119, 120, 125
Crystalline silica, 453, 463, 464, 468 glutathione peroxidase (Gpx1), 506
Crystal structures of
AdoMet, 308
Cbl, 308 D
Cbl binding proteins, 299 DAergic
Ctr1. See Copper transporter receptor 1 (Ctr1) cell death, 214
Cunninghamella bertholletiae, 9 neurons, 210, 211, 214, 217, 218
Cushing syndrome, 43 DAG. See Diacylglycerol (DAG)
Cyanide, 340 Daily
Cyanocobalamin (CN-Cbl), 297, 298, 301, 302 intake of manganese, 202
Cyclic iron requirements, 249
adenosine monophosphate (cAMP), 53–55, Dantrolene, 124, 125
68, 100 DAT. See Dopamine transporters (DAT)
ADP-ribose (cADPR), 97, 112 db/db mouse, 181
pyranopterin monophosphate (cPMP), DBI. See Dimethylbenzimidazole (DBI)
427–429, 434, 437–439 DβM. See Dopamine-β-monooxygenase
Cyclosporin, 58, 106 (DβM)
Cynomologous macaques, 213 DDT. See Dichlorodiphenyltrichloroethane
Cyprus, 264 (DDT)
Cystathionine Deafness, 117, 123
β-synthase (CBS), 309, 311, 423, 424 Deferasirox, 9, 258, 273, 274, 276, 277, 284
γ-lyase (cystathionase) (CSE), 423, 424 Deferiprone, 9, 269, 274–277, 280, 284
Cysteine Deferitrin, 273
biosynthesis, 505 Deferoxamine, 9
catabolism, 423–425, 439 Deficiency (of)
dietary intake, 425 aldehyde oxidase, 431
dioxygenase (CDO), 423–425, 439 aldosterone, 44
homo-, 311 arsenic, 484
methylseleno-, 521, 523 B12, 296, 297, 300, 310–314, 371, 372
seleno-. See Selenocysteine chromium, 173, 175, 179
sulfinic acid (CSA), 423 cobalt, 296, 297
S-sulfo-(SSC), 436–440 copper, 4, 18, 363, 367–369, 371–377,
supplementation, 439 401, 418, 440
Cystic folate, 312
encephalomalacia, 435 frataxin, 263
fibrosis, 11–12, 17, 18, 22 glutaredoxin, 264
Cytochrome(s) iron, 7, 18–21, 203, 248–255, 264,
a, 235 265, 284
b, 235 magnesium, 56–58
b5, 419, 425, 426 manganese, 202, 205
deficiency, 362, 368 mARC, 431
P450, 235 methylmalonyl-CoA mutase, 302, 314
Cytochrome c, 113, 215, 235, 362, 425, 515 molybdenum cofactor, 426, 431, 435, 437
oxidase, 235, 361, 362, 365, 367, oxygen, 324
368, 375, 377, 441 phosphate, 486
Index 545

potassium, 31, 35 Dichlorodiphenyltrichloroethane (DDT),


selenium, 15–17, 501, 513, 517–520, 32, 490
522–524 Dietary intake of
sulfite oxidase, 424–426, 439, 440 copper, 18, 363, 369, 370, 376, 380
thiamine, 42, 58 low protein, 439
xanthine oxidoreductase, 420 magnesium, 51, 52, 54, 57, 58, 62, 63, 66
zinc, 391, 395, 396, 398–408 manganese, 20, 22, 201–203
Dehydratases, 236 nickel, 325, 349
Dehydration, 39, 44, 481 potassium, 30
Dehydrogenases reference intake (DRI), 455, 518, 524
alcohol, 65 selenium, 14, 514, 522–524
Delirium tremens, 43, 58, 65 silica, 454, 458, 460, 467
Dementia, 120, 278, 313, 363, 461 silicon, 454, 455, 457–460, 462, 463
Demethylating organoarsenic species, 480 sodium, 30
Demethylation of histones, 237 vanadium, 140, 143, 144, 146
Dendritic cells, 334, 401 zinc, 10, 371, 399
Dengue fever, 159, 161 Digitalis, 44, 58, 60
Dentin, 85–87 Dihydrofolate reductase, 309
Deoxyadenosine, 303 Dihydrogen, 336, 350
5′-Deoxyadenosyl radical, 427 DI2-knockout mice, 512, 519
Depression, 41, 42, 56, 58, 71, 92, 404, 481 DI3-knockout mice, 519
Dermal absorption of Dimethylarsenic (DMAs), 481, 491
manganese, 201 Dimethylbenzimidazole (DBI), 297,
nickel, 349 298, 308
Dermatitis, 11, 201, 323, 334, 335, 404, 408 N,N-Dimethyl-2,3-dihydroxybenzamide
atopic, 401 (DMB), 267–269
Desferrioxamine (DFO), 261–263, 266, Dimethylselenide, 523
270–274, 276, 282, 284 Diol dehydratase, 303
-B, 233–235, 266, 269 Dioxygen. See Oxygen
Desferrithiocin (DF), 268, 269, 273, 282 Dioxygenases
Detoxification (of), 147, 189, 239, 323, 331, acireductone, 323, 336–338, 341, 342
365, 366, 419, 442, 480, 490, 515 cysteine, 423–425, 439
arsenic, 480 Diphosphate (pyrophosphate) (PPi), 87,
cadmium, 366 88, 427
methylglyoxal, 336 Disease(s) (see also individual names), 3–22,
DF. See Desferrithiocin (DF) 29–45, 50–73, 83–127, 141, 147, 153,
DFO. See Desferrioxamine (DFO) 159–165, 173, 180, 181, 184, 185,
DI1. See Type 1 deiodinase (DI1) 200–219, 250, 253–255, 257, 258,
Diabetes, 8, 18, 38, 40, 43, 62–64, 72, 172, 260–265, 276–280, 284, 296–315, 323,
173, 179, 186, 256, 391, 399, 404–406, 324, 336–349, 361–363, 367, 372–380,
462, 468, 469, 476, 482, 492 390–409, 417–442, 453, 457–461, 464,
mellitus, 11, 39, 44, 152–156, 363, 408 465, 467, 469, 476, 477, 482, 483,
type 1, 11, 63, 152, 154, 181, 405 490–493, 501, 511, 516–520
type 2, 153, 165, 177, 182–185, 190, 193, 519 Addison, 44
Diabetic(s), 8, 9, 11, 20, 37, 41, 44, 59, 63, 64, alcohol liver (ALD), 65
152, 165, 178, 182–185, 193, 396, 399, Alzheimer’s disease (AD), 102, 118,
405, 406, 462, 518, 519 120–122, 213, 277–280, 311, 376, 377,
animals, 63, 165 380, 403, 406, 407, 452, 457, 460, 461,
rats, 153, 177 467, 469
Diacylglycerol (DAG), 104, 112 autoimmune, 106, 255, 401, 421
Dialysis patients, 7 autosomal recessive, 11
Diarrhea, 11, 34, 38–41, 58, 59, 163, 335, 375, black foot, 482
407, 418, 441, 481 Brody, 124, 125
childhood, 12 cardiovascular. See Cardiovascular diseases
546 Index

Disease(s) (cont.) Diuresis, 36, 37, 40, 41, 43


cardiac, 123, 124, 276 Divalent
carotid artery, 404 cation transporter, 208, 323, 325
celiac, 250, 408 metal transporter 1 (DMT1), 5, 20, 203,
central core, 124, 125 241, 242, 246, 250, 254, 265, 365
Chagas, 162, 163, 165, 337, 346 Dj-1-knockout mice, 217
chronic, 8, 44, 45, 57, 254–255, 277, 391, DMAs. See Dimethylarsenic (DMAs)
404–407, 459 DMB. See N,N-dimethyl-2,3-
coronary, 18, 60, 404, 459 dihydroxybenzamide (DMB)
Creutzfeld-Jacob, 379 DMD. See Duchenne muscular dystrophy
Crohn’s, 59, 250, 408 (DMD)
falling, 373 DMT1. See Divalent metal transporter 1
ferroportin, 257, 258 (DMT1)
heart, 61, 173, 261, 399 DN. See Dopaminergic neurons (DN)
Huntington’s (HD), 118, 120, 121, 213, DNA, 3, 11, 100, 101, 106, 121, 141, 149,
376, 378 151, 158, 159, 163–165, 215, 264, 308,
infectious, 1–23, 322, 324, 407, 409 310, 315, 326–330, 336, 343, 344, 392,
inflammatory bowel, 254, 511 396, 401, 432, 477, 480, 482, 488, 489,
Kashin-Beck, 517 504
Keshan, 501, 517 c, 69, 506
kidney, 44, 185, 323 cleavage, 329
liver, 10, 58, 65, 257, 376, 398, damage, 3, 146, 147, 327, 329, 466, 486, 490
399, 408 fragmentation, 215, 329
lyme, 5 methylation, 328, 330, 396, 482
metabolic, 349, 373, 404–407 polymerase, 151, 486
motor neuron, 362, 372 repair, 146, 327, 396, 482, 486
Mseleni, 202 strand scission, 329
muscle, 124–126, 501 synthesis, 51, 69, 151, 312, 486
neurodegenerative, 94, 118–122, 280, 376, Dogs, 273
379, 406, 467 Dopamine, 211, 214, 218, 251, 363, 364, 378
neurological, 39, 117, 201, 361, 372, -β-monooxygenase (DβM), 361, 364
376, 379 transporters (DAT), 209
Niemann-Pick, 118 Dopaminergic neurons (DN), 119, 279, 377
pancreatic, 10 Down’s syndrome, 202
Parkinson’s (PD), 118, 119, 121, 122, 206, DRI. See Dietary reference intake (DRI)
208, 211, 212, 214, 216–218, 277–280, Drinking water, 142–144, 175, 418, 459, 461,
284, 376–378, 380, 461 476, 480–482, 484, 493, 521
Perthest, 202 Drosophila, 191, 192, 504
Picks, 118, 277 Drugs (see also individual names), 16, 36, 39,
prion, 376, 379 44, 53, 54, 56, 58, 64, 106, 124, 125,
pulmonary, 39 141, 158, 160, 162–165, 172, 217, 270,
renal, 44, 45, 57, 67, 255, 442 283, 395, 421, 422, 453, 462, 477
selenium-related, 516–518 absorption, 270
sickle cell, 14, 22, 258, 262, 408 amoebocidal, 163
skeletal muscle, 124–126 anesthetic, 124
vascular, 459, 460, 482 antituberculosis, 162
Whipple’s, 59 arsenic-based, 477
Distribution of immunosuppressive, 106
magnesium, 50, 51 -induced disorders, 38
potassium, 34, 41 metabolism, 422
sodium, 34 toxicity, 421
vanadium, 141–147 Dry cell batteries, 200, 201
Disulfide bonds, 350, 512, 513 dTMP. See Thymidine 5′-monophosphate
Dithiolene synthesis, 428, 429 (dTMP)
Index 547

Duchenne muscular dystrophy (DMD), Endocytosis, 146, 176, 177, 186, 208, 300,
124–126 366, 379
Dust, 143, 322, 325, 453, 456, 464, 465 Endonucleases, 119
Dysarthria, 363 Endoplasmic reticulum (ER), 54, 65, 93–99,
Dysentery, 163 104, 106, 111, 113–117, 120, 121, 512,
Dysfunction of 513, 515, 516, 519
cystathione β synthase, 311 Endothelial cells, 61, 70, 206, 208, 247, 333,
methionine synthase, 302, 310, 312, 313 334, 396, 466, 467
methylenetetrahydrofolate reductase, 311 Energy metabolism, 31, 51, 108, 201, 324,
Dystonia, 212, 217, 363 330, 361
Dystrophin, 125, 126 England, 164
Enstatite, 143
Entamoeba histolytica, 163
E Enterobactin, 267–271, 282, 283
Early childhood death, 426 Enterocyte membranes, 203
Earth’s crust, 83, 142, 143, 200, 231, 322, 452, Enteroviruses, 39
454, 456, 479 Environmental manganese exposure, 201
Eating disorders, 42 Environmental Protection Agency, 521
ECF. See Extracellular fluid (ECF) Enzymes (see also individual names), 5, 10,
EC-SOD. See Extracellular superoxide 18, 30, 44, 51, 54, 63, 70, 86, 96, 98,
dismutase (EC-SOD, SOD3) 99, 101–104, 106–108, 119, 120,
Eczematous skin reaction, 332 141, 150, 151, 160, 189, 191, 202,
Edema, 31, 38, 39, 435, 438, 465 204, 205, 214, 217, 231, 236–238,
EDTA. See Ethylenediamine-N,N,N′,N′- 240, 243, 244, 246, 247, 263, 279,
tetraacetic acid (EDTA) 297, 300, 302–311, 314, 315, 323,
EE. See Ethylmalonic encephalopathy (EE) 324, 327, 328, 330, 336–343, 345,
eEFSec. See Eukaryotic selenocysteyl-tRNA- 346, 348–350, 360–365, 367–370,
specific elongation factor (eEFSec) 373, 374, 377, 392, 393, 400, 405,
EF. See Elongation factor (EF) 417, 418–424, 427, 430, 432, 435,
EF-hand proteins, 84, 90, 100, 105 439, 442, 460, 461, 463, 468, 480,
EGF. See Epidermal growth factor (EGF) 481, 485, 490, 512
Ehlers-Danlos syndrome, 404 antioxidant, 16, 21, 201, 395, 396, 402
Ehrlich ascites tumor cells, 156 inhibition, 270
Elastin, 361, 363, 373 Epidermal growth factor (EGF), 54, 69, 72, 73
Electrical potential, 31, 32, 42, 51 signaling, 69
Electrode coating, 200 Epigenetic effects in nickel
Electrolyte(s), 30–32, 56 carcinogenesis, 327
disorder, 38, 43, 44 Epilepsy, 202, 441
disturbance, 40, 42, 57 seizures, 69
Electron paramagnetic resonance (EPR), Epinephrine, 36, 61
188, 361 EPR. See Electron paramagnetic
Electron transport, 30, 235, 278, 486, 487, 489 resonance (EPR)
chain, 106, 215 ER. See Endoplasmic reticulum (ER)
Electrophoretic mobility shift assays Erythrocytes, 6, 15, 21, 60, 146, 250, 254,
(EMSA), 508 255, 261, 283, 284, 484
Elongation factor (EF), 91, 92, 98, 100, Erythroid cells, 241, 244
102–106, 108, 109, 122, 507 Erythrophagocytosis, 244
Embryo development, 112, 512 Erythropoiesis, 247, 250, 255, 258, 261,
Emerald, 452 262, 400
EMSA. See Electrophoretic mobility shift Erythropoietin
assays (EMSA) gene, 315
Enamel, 42, 85, 86 therapy, 250
Encephalopathy, 179, 204, 379, 435 Escherichia coli, 6, 8, 283, 304, 307–309, 340,
Endocrine tissue, 241, 255, 258, 272 342, 343, 427, 432, 433, 437
548 Index

Esomeprazole, 72 F
Esophageal carcinoma, 406 FAD. See Flavin adenine dinucleotide (FAD)
Essential elements, 141, 147, 172, 175–177, Falciparum, 13
179, 180, 189, 192, 201, 324, 348, Falling disease, 373
360, 380 Familial
Essentiality of, 201–206, 324, 468 fatal insomnia, 379
chromium, 173 hemiplegic migraine (FHM), 118
nickel, 340, 348–350 hypomagnesemia, 68
(micro)nutrients, 5, 201, 296, 324, 345, Fan worms, 143
455, 457, 484, 501, 502 Farm animals, 173, 185, 373
trace elements, 173, 174, 176, 476 Fatal familial insomnia, 379
vanadium, 165 Fatty acids, 3, 153, 311, 396, 441, 459, 460,
Estimated safe and adequate daily dietary 484, 510
intake of metabolism, 189–190, 306
chromium, 173 Fatty liver disease, 408
selenium, 524 FDA. See Food and Drug Administration (FDA)
Estrogens, 52, 190, 218, 458, 459, 486 Fecal
ESTs. See Expressed sequence tags selenium excretion, 523
database (ESTs) zinc loss, 11
Ethanolamine ammonia-lyase, 303 Federal Trade Commission of the United
Ethanol metabolism, 65 States, 173
Ethylenediamine-N,N,N′,N′-tetraacetic acid Feed additives, 491
(EDTA), 234, 235, 253, 335 Feldspar, 452
Ethylmalonic encephalopathy (EE), 441 Females, 21, 155, 156, 161, 177, 191, 192,
Eubacteria, 336, 487, 503 249, 458
Eukaryotes, 314, 323, 327, 336, 345, 346, Fenton-like reaction, 146, 166
348, 349, 351, 366, 417, 418, 427, Fenton reaction, 233, 460
429, 503, 505 Feroxamine, 9
Eukaryotic Ferredoxins, 236
cells, 92, 349, 364 Ferriportin, 396
pathogens, 336, 346 Ferritin, 6, 9, 18, 146, 158, 202, 237–240, 246,
SECIS elements, 507 247, 277, 283, 392, 407
selenocysteyl-tRNA-specific elongation mRNA, 246
factor (eEFSec), 507–509 Ferrochelatase, 244, 265
Europe, 163, 257, 274, 335 Ferroportin-1 (Fpn), 20, 209, 210
European Food Safety Authority, 192 Ferroportin disease, 257, 258
Excitotoxicity, 102, 117, 118, 122, 205, 212, Ferrovanadin, 144
218, 375 Ferroxidase, 4, 19, 238, 239, 244, 265, 279,
Excretion of 361, 362, 367
calcium, 127 FHM. See Familial hemiplegic migraine
copper, 18, 371, 376 (FHM)
silicon, 457 Fibrinolysis, 92, 399
vanadium, 141, 145 Fibrosis, 62, 256, 453, 465, 466
Exocytosis, 110–112, 115, 393, 402, 466 Fiji Island, 455
Experimental animals (see also Animal Finland, 501, 521
studies), 326 Fireworks, 200
Exposure to nickel, 323, 324, 328, 335 Fish, 95, 112, 247, 296
Expressed sequence tags database (ESTs), Flagellates, 141, 162, 163
502, 507 Flavin adenine dinucleotide (FAD), 148, 308,
Extracellular 419, 420, 422
fluid (ECF), 31, 32, 34, 38–41, 43, 50, Flavin mononucleotide (FMN), 308, 314
85, 87, 122 Fluorophores, 201
superoxide dismutase Fly agaric, 141–143
(EC-SOD, SOD3), 362 FMN. See Flavin mononucleotide (FMN)
Index 549

Folate Gastrointestinal
deficiency, 312 absorption of manganese, 21, 202
metabolism, 308, 310, 315 cancer, 281
Folic acid hemorrhage, 375
fortification, 311, 312 surgery, 371, 372
in blood, 308 tract, 10, 11, 18, 34, 41, 144, 203, 250,
Food, 143, 163, 174, 175, 180, 183, 250, 252, 270, 277, 282, 399, 401, 418, 461
253, 296, 298, 299, 311, 315, 335, 339, Gelatinase, 86
345, 349, 396, 422, 468, 476, 480, 484, Gene
501, 521, 524 expression, 100, 106, 113, 121, 155,
additive, 453–455 324, 327–331, 333, 334, 345, 349,
Food and Drug Administration (FDA), 33, 72, 394, 395, 400, 458,460–462, 466,
184, 185, 192, 202, 274, 491 467, 515
Food and Nutrition Board of the Institute of mutations, 256, 297, 299, 311, 316
Medicine of the National Academy of transcription, 10, 99–101, 105, 115, 121,
Sciences, 174 324, 395
Forkhead transcription factor, 101 Genetic
Formation constants (see also Affinity copper deficiencies, 374, 375
constants, Binding constants, copper overload, 375, 376
and Stability constants), 151, 152 engineering, 313
Forsterite, 143 hearing loss, 122, 123
Fowler’s solution, 492 Genomes, 101, 314, 327, 347, 362, 392, 406,
Fpn. See Ferroportin-1 (Fpn) 422, 488, 504, 509
France, 165, 455 Genotoxic effects of nickel, 327
Frataxin (FXN), 244, 263, 265 Gentamicin, 43, 54, 58
deficiency, 263 Gentisate aldehyde, 422
Free radicals, 4, 14, 201, 214, 217, 244, 270, Gephyrin, 429–434, 441
271, 279, 374, 395, 465 -deficient mice, 437
Friedreich’s ataxia, 263–265, 277, 278, 280 loss, 439
Frogs, 95, 112 GFAJ-1, 488, 489, 493
Fruit Gitelman/Bartter’s syndrome, 59
bats, 305, 313 Gitelman’s syndrome, 67, 68
flies, 191, 264, 327 Global cycles
Fuel additives, 200 carbon, 419
Fumes, 216, 217, 322, 325 nitrogen, 338, 419
Function of selenoproteins, 509–516 sulfur, 419
Fungi (or fungal), 5, 6, 9, 150, 282, 336, Globulins
342, 419, 425, 427, 480, 521 β, 203
infections, 8, 9 β micro-, 257
Fura-2, 201 macro-, 10, 206
Furosemide, 39 Glomerulonephropathy, 462
FXN. See Frataxin (FXN) Glucagon, 55, 405
Glucocorticoid
receptor (GR), 482, 483, 486, 487
G response element (GRE), 482, 483, 487
GABA. See γ-Amino butyric acid (GABA) Glucose, 8, 39, 63, 153–156, 165, 177–184,
Galactomannan, 164 281, 330, 348, 484
Gallium(III), 266 homeostasis, 153, 518
Gambia, 7 intolerance, 11, 20, 174, 179, 405, 519
Gastric metabolism, 20, 31, 153, 174, 175, 179,
cancer, 102 183, 186, 188, 205, 462
carcinomas, 344 tolerance, 173–176, 178, 181, 182, 519
lymphomas, 344 tolerance factor (GTF), 20, 172, 174,
Gastroenteritis, 481 186, 188
550 Index

Glucose (cont.) GPx. See Glutathione peroxidase (GPx)


transporter (GLUT4), 111, 154, 156, 187, GR. See Glucocorticoid receptor (GR)
189, 190 Gram-negative bacteria, 8, 161
uptake, 64, 111, 153–155, 186, 187, 189, 405 Gram-positive bacteria, 161
GLUT4. See Glucose transporter (GLUT4) Granulocytes, 14, 401
Glutamate, 96, 117, 118, 160, 188, 201, 205, Gräsbeck-Imerslund syndrome, 299
215, 216, 218, 347, 375, 435, 436 GRE. See Glucocorticoid response element
-activated ionic channels, 208 (GRE)
aspartate transporter (GLAST), 216 Greece, 264
dehydrogenase, 435 Growth
synthetase, 20 hormone, 403
(excito)toxicity, 117, 118, 122, 218, 375 retardation, 12, 296, 391
trafficking, 205 GS. See Glutamine synthetase (GS)
Glutamatergic signaling, 120, 205 GTF. See Glucose tolerance factor (GTF)
Glutamine, 117, 201 GTP. See Guanosine 5′-triphosphate (GTP)
synthetase (GS), 201, 205, 424 GTPase, 302, 343
transporter, 216 Guanine
Glutaredoxins, 241 8-oxo-, 330
deficiency, 264 Guanosine 5′-triphosphate (GTP),
Glutathione (GSH), 3, 147, 148, 241, 242, 303, 427
278, 365, 366, 423, 436, 480 hydrolysis, 303
reduced, 278, 279, 325, 329, 509 Guatemala, 251
reductase (TGR), 329, 512 Gut’s microflora, 349, 350
synthesis, 436, 515
synthetase, 511
S-transferase, 69 H
Glutathione peroxidase (GPx), 4, 15, 16, 329, Haber-Weiss-type reaction, 214, 465
362, 501, 502, 506, 509–511, 524 Haematuria, 421
Gpx1, 506, 509–511, 519 Hallervorden-Spatz syndrome, 279
Gpx1-knockout mice, 511 Halomonas, 476, 488
Gpx4-knockout mice, 511, 520 Haloperoxidases, 141, 150, 151, 160
GlxI. See Glyoxalase I (GlxI) Hamsters, 326, 484
Glycated hemoglobin, 64, 180, 182–184 Haptocorrin, 299, 300
Glycemia, 63, 462 Haptoglobin, 244
Glycerol Harvard Stop Test, 250
dehydratase, 303 HCP-1 transporter, 252
diacyl-(DAG), 104, 112 HD. See Huntington’s diseases (HD)
Glycine max, 343 Headache, 31, 118, 335, 375
Glycine Heart (see also Cardiac and Cardiovascular),
metabolism, 308, 309 22, 30–32, 63, 66, 96, 108, 109,
reductase, 502 123, 126, 145, 146, 239, 255, 256,
Glycogen accumulation, 155 272, 370, 371, 512, 515, 522
Glycoprotein, 15, 203, 244, 299, 513, 514 damage, 42, 124
Glycosuria, 40, 59, 462 disease, 61, 173, 261, 399
Glycosylphosphatidylinositol (GPI), 363 failure, 38, 43, 44, 124, 126, 163,
Glycosyl transferase, 205 254, 262
Glyoxalase (Glx), 336 muscle, 108
I (GlxI), 323, 336, 337, 341, 346 transplant, 254
II (GlxII), 336, 337 HEK293 T cell model, 209
Goats, 296, 348, 484, 485 HeLa cells, 157, 163, 503
Gold mines, 486, 487 Helicobacter
Golgi system, 89, 93 hepaticus, 342
Gout, 418, 421, 422 pylori, 250, 338, 339, 342–346
GPI. See Glycosylphosphatidylinositol (GPI) Hematopoietic stem cell transplantation
G proteins, 96, 118, 303 (HSCT), 8, 9
Index 551

Hematuria, 45 cellular, 30, 114, 201, 327, 331


Heme, 6, 236, 240, 242, 252, 263, 264, 297, copper, 18, 332, 361, 364–371, 376, 379,
315, 367, 390, 416, 425 380, 440, 441
-containing proteins, 235, 236, 247, 315 glucose, 153, 518
oxidase, 244 iron, 5, 146, 244, 246, 247, 254, 264, 277,
oxygenase, 242 332, 362, 363
Hemochromatosis magnesium, 54–56, 62–64, 73, 206
juvenile, 257, 258 manganese, 202, 203, 206, 210, 213
Hemodialysis, 4, 7, 18, 57, 468 molybdenum, 418
Hemoglobin (Hb), 6, 235, 236, 244, 247, 248, nickel, 324, 340–349, 351
250, 252, 260, 261, 264, 348 potassium, 32–37, 41
disorders, 258, 260 sodium, 34, 36, 37
glycated, 64, 180, 182–184 zinc, 10, 366, 391, 394, 395, 398, 401–404,
production, 260 406–408
Hemoglobinopathies, 258–263 Homocysteine metabolism, 311
Hemojuvelin, 257, 265 Homocysteinemia, 311–312, 315
Hemolysis, 57, 244 Homocystinuria, 300, 302
Hemopexin, 6, 244 Homolytic cleavage of AdoCbl, 305
Hepatic Hormesis, 477, 485, 486
cirrhosis, 38, 204, 217 Hormones, 39, 44, 50, 53, 55, 63, 68, 83, 84,
dysfunction, 418 127, 180, 400, 403, 458
encephalopathy, 204, 211 adrenocorticotropic (ACTH), 395
Hepatitis C, 16, 399 antidiuretic (ADH), 36–38, 40
Hepatocarcinoma, 513 growth, 403
Hepcidin, 9, 247–248, 255–258, 265, 283, 395 parathyroid (PTH), 53, 68, 72, 84
-knockout mice, 280 replacement therapy, 458
HEPES buffer, 191 steroid, 482, 483, 486
Hephaestin, 244, 265, 361, 372 thyroid, 511, 512
Herbicides, 491 HPLC. See High performance liquid
Hereditary chromatography (HPLC)
B12 deficiency, 300 HPO. See Hydroxypyridinones (HPO)
hemochromatosis, 9, 255–258 HRE. See Hypoxia-responsive enhancers (HRE)
molybdenum cofactor deficiency, 437 HSCT. See Hematopoietic stem cell
peripheral neuropaty, 375, 379 transplantation (HSCT)
Herpes simplex, 161 Htt. See Huntingtin (Htt)
HFE hemochromatosis, 257 Human, 2, 3, 5, 6, 15, 18, 21, 22, 57, 62, 84,
Hfe protein, 257, 265 123, 139, 141, 153, 174, 175, 179, 180,
High performance liquid chromatography 185, 201, 202,204, 205, 246, 247, 283,
(HPLC), 434 296, 297, 302, 303, 315, 360, 362, 364,
Histidine, 141, 144, 145, 153, 186, 187, 234, 368, 370–372, 374, 377, 394, 396, 400
237, 238, 323, 331, 333, 334, 338, 362 457, 484, 490, 493, 502, 511, 516
metabolism, 308 blood, 177
Histone(s) blood levels of arsenic, 480
acetylation, 328, 396 brain, 215, 402, 437
methylation, 237, 328 central nervous system, 363
modifications, 328 dietary standards for selenium, 524
Histoplasma, 9 endothelial cells, 333, 466
HIV. See Human immunodeficiency genome, 101, 314, 422, 500, 509
virus (HIV) glucocorticoid receptor, 486, 487
1
H NMR, 427 health, 30, 200, 218, 296, 313, 315,
H2O2. See Hydrogen peroxide (H2O2) 321–351, 361, 391, 415–444, 452,
HOCl. See Hypochlorous acid (HOCl) 453, 455, 469, 476, 494, 500, 525
Homeostasis, 34, 52, 240, 323, 324 immune response, 345
calcium, 86, 102, 113–115, 117, 119–121, kidney cancer cells, 157
124, 215 lymphocyte cells, 486
552 Index

Human (cont.) 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic


mARC, 426 acid (HEPES), 191
MCM, 303–305, 314 Hydroxylation of amino acids, 237
methionine synthase, 307, 308 Hydroxyl radicals, 214, 233, 234, 271, 329,
molybdenum cofactor deficiency, 373, 374, 465
426, 431, 435, 437 Hydroxypyridinones (HPO), 267, 271,
molybdenum cofactor synthesis, 430–434 275, 276
molybdenum levels, 417 8-Hydroxyquinoline, 267–269, 280
MPT synthase, 431 Hyperaccumulation of
serum transferrin, 239 copper, 369
stomach, 345 nickel, 323
testes, 363 Hyperaldosteronism, 44, 58, 62
urine, 177 Hyperalgesia, 64
Human immunodeficiency virus Hypercalcemia, 39, 84
(HIV), 4, 7, 8, 15, 16, 19, 140, 160, Hypercalciuria, 68
165, 254, 264, 407 Hypercholesterolemia, 155
HIV-1, 15, 16, 159–161 Hyperekplexia, 437, 441
HIV-2, 160, 161 Hyperglycemia, 38, 39
Human pharmacokinetics and pharmacology Hyperhomocysteinemia, 311–312
(of), 5, 10, 11, 14, 15, 18–21, 144–147 Hyperinsulinemia, 462
chromium, 19, 20 Hyperkalemia, 30, 31, 43–45, 56
copper, 18, 19 Hyperleptinemia, 155, 462
iron, 5 Hyperlipidemia, 39, 155, 179, 462
manganese, 20, 21 Hypermagnesemia, 55–57
selenium, 14, 15 Hypermagnesuria, 67
zinc, 10, 11 Hypernatremia, 30, 31, 40, 41, 44
Huntingtin (Htt), 120, 121, 213 Hypernucleophilic, 298, 306
Huntington’s disease (HD), 118, 120, 121, Hyperparathyroidism, 45, 57
213, 376, 378 Hypertension, 37, 40, 44, 58–62, 64, 72, 373,
Hut/CCR5 cells, 160 442, 460, 469
Hyalella azteca, 486 Hypertrophy, 124
Hydrogen Hyperuricemia, 421, 422, 442
bonds, 158, 245, 330, 489 Hyperzincemia, 404
metabolism, 336 Hypoalbuminemia, 10, 40, 73
Hydrogenases, 323, 336, 339, 342, 343, 346 Hypoaldosteronism, 44
chaperones, 345 Hypocalcemia, 40, 56, 72, 73
[NiFe], 339, 340, 345 Hypochlorous acid (HOCl), 323
Hydrogen peroxide (H2O2), 4, 20, 152, Hypocuprosis, 418
158, 190, 191, 205, 214, 233, Hypogonadism, 256, 396, 402
235, 271, 323, 329, 362, 400, 422, Hypokalemia, 30, 39–45, 67, 72, 73
510–512, 519 Hypomagnesemia, 39, 42, 52, 55–73
Hydrogen sulfide, 423–425, 441 Hypomorphic mouse model, 314
accumulation, 441 Hyponatremia, 30, 31, 38–41, 44
metabolism, 441 Hypophosphatemia, 42, 58
Hydrolases, 205, 306, 392 Hypopigmentation, 364
Hydrolysis of Hypopigmentation of hair, 374
ATP, 108, 342, 368, 480, 486 Hypopigmentation of skin, 374
cisplatin, 158 Hypotension, 38, 56
triphosphate, 343 Hypotonia, 56, 440, 441
urea, 336, 338 Hypotonicity, 38, 39
Hydrolytic cleavage, 149 Hypoxanthine, 420, 421
Hydroxide, 147, 232, 233, 338 Hypoxia, 315, 330, 331, 360
Hydroxyapatite, 50, 86–88, 146, 147 -responsive enhancers (HRE), 325, 330
3-Hydroxy-1,2-dimethylpyridin-4(1H)-one, 268 Hypoxic ischemic encephalopathy, 435
Index 553

I iNOS. See Inducible nitric oxide synthase


IARC. See International Agency for Research (iNOS)
on Cancer (IARC) Inositol 1,4,5-triphosphate (InsP3), 93–98,
ICP-MS. See Inductively coupled plasma mass 112–114, 121
spectrometry (ICP-MS) Inositol 1,4,5-triphosphate receptor (InsP3R),
ICU. See Intensive care unit (ICU) 94, 95, 97–99, 110, 114, 115, 117, 120,
Idiopathic 121, 124
chronic toxicosis, 375, 376 InsP3. See Inositol 1,4,5-triphosphate (InsP3)
Parkinson’s disease, 211 InsP3R. See Inositol 1,4,5-triphosphate
IF. See Intrinsic factor (IF) receptor (InsP3R)
IFN. See Interferon (IFN) Institute of Medicine of the National Academy
Ig. See Immunoglobulins (Ig) of Science, 174, 518
IGF-1. See Insulin-like growth factor 1 Insulin, 20, 33, 43, 55, 63, 152, 153, 177, 178,
(IGF-1) 181, 183, 519
IL-1. See Interleukin-1 (IL-1) -enhancing action, 141, 164
Imidazoles, 233, 237 -like growth factor 1 (IGF-1), 403, 405
Immune receptor (IR), 152–154, 156, 186, 189,
cascade, 332 190, 405
function, 11, 15, 373, 391, 396, 401 sensitivity, 63, 172, 175, 176, 178,
response, 12, 17, 65, 323, 332, 333, 347, 184–186, 189, 190, 193
400, 401 signaling, 186–189
system, 10, 17, 21, 71, 164, 332, 373, 374, storage, 405
380, 400, 401, 406, 407, 452, 467 Integrins, 69, 205
Immunoglobulins (Ig), 10, 145 Intensive care unit (ICU), 13–16, 19, 58
Immunosuppressive drugs, 106 sepsis, 16–17
Implanted medical devices, 325 Intercellular signaling with zinc(II),
India, 14, 164, 274, 454 393, 394
childhood cirrhosis, 375, 376 Interference between molybdate, sulfate,
Indomethacin, 217 and phosphate transport, 417
Inducible nitric oxide synthase (iNOS), 216 Interferon (IFN), 17
Inductively coupled plasma mass spectrometry -α (IFN-α), 4
(ICP-MS), 279 -γ (IFN-γ), 373, 400, 467
Industry, 183, 322, 452, 453, 468 therapy, 19
workers, 201 Interleukin (IL), 4, 373
Infants, 11, 71, 211, 248, 249, 371, 374, 433, 438 -1 (IL-1), 11, 71, 373
Infections -2 (IL-2), 10, 400
bacterial, 159–161, 283, 345, 347 International Agency for Research on Cancer
Infectious diseases, 1–23, 322, 324, 407, 409 (IARC), 323, 464
Infertility, 418, 516, 520, 522 Interrelationship between arsenic and
Inflammation, 6, 19, 62, 64–67, 70, 126, 190, phosphorus, 477
283, 333, 334, 363, 377, 396, 401, 409, Interstitial fibrosis, 464, 465
440, 465–467 Intestinal
Inflammatory bowel disease, 254, 511 cancer, 511
Influenza, 140, 159, 165 cells, 52, 53, 176, 369, 480
virus, 161 influenza, 34
Inhalation of Intestine, 34, 42, 53, 56, 58, 68, 72, 73, 84, 85,
crystalline silica, 453 104, 109, 127, 144, 163, 348, 349, 366,
manganese, 201 371, 511, 516, 522
nickel, 323, 326, 349 small, 10, 18, 19, 21, 51, 86, 176, 203,
silica dust, 464 250, 369, 370, 399, 417
vanadium, 144 Intracellular
Inner membranes, 94, 107, 108 calcium, 61, 64, 93–99, 112, 114, 116,
Inorganic diphosphate (= pyrophosphate) 120, 206, 329
(PPi), 87, 88, 427 copper trafficking, 365, 367–368
554 Index

Intracellular (cont.) autxidation, 240


distribution of nickel, 324, 325 fumarate, 253, 254
processing of cobalamin, 298, 300–302 gluconate, 254
signaling with zinc(II), 393–394 sulfate, 232, 253, 254, 284
traffic of zinc, 390 Iron(III), Fe3+, 6, 144, 146, 179, 203, 206, 208,
Intrinsic factor (IF), 299, 300 231–235, 237–239, 241, 242, 244,
-cobalamin, 299 252–254, 256, 261,266, 267, 269–276, 372
receptor, 300 chelating agents, 268
Iodine, 512, 517 hydroxide, 254
Ion oxide, 255
channels, 95, 105, 398 polymaltose, 254
gradients, 33, 210 pyrophosphate, 253
Ionotropic glutamate receptor channels, 209 uptake, 206, 208
IR. See Insulin receptor (IR) Iron(IV), 231, 237
IRE. See Iron responsive elements (IRE) Iron deficiency anemia, 248–255, 264, 265, 284
Iron, Fe, 2–9, 15, 18–22, 83, 146, 176, 177, Iron-regulatory protein 1 (IRP-1), 246, 332
179, 186, 200, 202–210, 229–286, 297, Iron responsive elements (IRE), 245, 246
323, 329, 339, 340,348, 380, 390–392, Iron responsive protein (IRP), 241, 245
401, 406, 407, 460, 461, 463, 465, 484 Iron-sulfur cluster(s), 240, 241, 263, 284, 416
absorption, 242, 243, 247, 253–255, 257, biosynthesis, 263
258, 348 Iron-sulfur proteins, 236
accumulation, 208, 256, 277–280, 363 IRP. See Iron responsive protein (IRP)
administration, 6 IRP-1. See Iron-regulatory protein 1 (IRP-1)
chelators or chelation therapy, 230, 231, Irritable bowel syndrome, 350
258, 261, 263, 266, 267, 270–277, 279, Ischemic neuronal injury, 402
281, 282, 284 Isocitrate dehydrogenase, 106, 204
deficiency, 7, 1821, 203, 248–255, 264, 8-Isoprostane, 466
265, 284 Italy, 264
-deficient rats, 08
-dependent enzymes, 237, 238, 240, 284
detoxification, 239 J
efflux, 244, 246, 248 JCR:LA-cp rats, 180, 185
excretion, 272, 275, 276 Juvenile hemochromatosis, 257, 258
export, 240, 244, 247
homeostasis, 5, 146, 244, 246, 247, 254,
264, 277, 332, 362, 363 K
infection, 282–284 Kaolin, 456
in food, 252, 253 Karelianite, 143
in infectious diseases, 6–9 Kashin-Beck disease, 517
in the brain, 277 Kenya, 253
in the cerebrospinal fluid, 277 Keratinocytes, 334, 486
metabolism, 244–246, 257, 264, 345, 361 Keshan disease, 501, 517
overload, 8, 9, 241, 255–257, 261–264, Ketoaciduria, 59
272, 276, 284, 407 α-Ketoglutarate, 106, 424, 435
oxides, 479 Ketonuria, 462
physiology, 246–248 Kidney, 10, 32, 34–37, 39, 40, 42, 50, 52, 58,
production, 283 67, 72, 73, 84, 86, 127, 145, 146, 177,
scavengers, 271 261, 366, 367, 370, 371, 417, 432, 476,
supplementation, 7, 8, 231, 253–255, 257 477, 510–513, 515, 522, 523
supplementation therapy, 231 cancer, 157
toxicity, 270–272 cells, 241
transport, 240–246, 257, 264 disease, 44, 185, 323
Iron(II), Fe2+, 5, 106, 158, 208, 231–235, failure, 56, 375
237–242, 244, 252–254, 266, 267, 270, stones, 336, 464
271, 278, 315, 328,330, 332, 337, 372 transplant, 59
Index 555

Kinases Lipid(s), 146, 175, 177, 183, 201, 274, 323,


AMP-activated protein (AMPK), 366, 392, 459, 460, 481
115, 187, 189 biosynthesis, 98
calcium-dependent (CaMK), 100, 101, metabolism, 172, 181, 186, 349
104, 105 peroxidation, 190, 214, 329, 378
mitogen-activated protein (MAPK), peroxides, 511
66, 187, 190, 333 Lipocalins, 6, 300
myosin light chain (MLCK), 104, 109 Lipoic acid, 512
pantothenate kinase-2 (PANK 2), 279, 280 Lipopolysaccharides (LPS), 14, 66, 334
phosphatidylinositol 3-kinase (PI3K, Akt), Lipoprotein receptor, 244, 522
154, 156, 187 Lipoxygenase, 270
phospho-, 147 Listeria monoctyogenes, 347
phosphorylase (PhK), 104 Listeriolysin, 347
O-phosphoseryl-tRNA[Ser]Sec (PSTK), Lithium, Li+, 114
503, 504 batteries, 165
protein. See Protein kinase Liver, 9–11, 15, 18, 19, 21, 22, 31, 63, 104,
pyruvate dehydrogenase, 328 145, 146, 158, 163, 164, 177, 202–204,
tyrosine, 153, 186, 189, 400, 406, 493 210, 239, 252, 255–258, 297, 298, 305,
Kissing bugs, 163 309, 311, 314, 331, 348, 349, 366, 367,
KK-Ay mice, 155, 462 369–371, 375, 396, 401, 403, 417, 422,
KK/HIJ mice, 187 423, 432, 437, 440, 453, 476, 477, 481,
Klebsiella 486, 491, 501, 503–505, 510–515, 517,
oxytoca, 303 518, 522, 523
pneumoniae, 8 cancer, 281
degeneration, 173
disease, 10, 58, 65, 257, 376, 398, 399, 408
L disorder, 174
Lactate, 144, 148, 336, 438 failure, 38
Lactating women, 408 iron, 274
Lactobacillus, 349 transplantation, 9, 10
fermentum, 349 Livestock, 476, 487, 501, 519, 521
Lactoferrin, 6, 283 Loop of Henle, 34–37, 39, 58
Laennec’s cirrhosis, 399 Low chromium rodent diets, 173–176
Lambs, 374, 501 Low density lipoprotein (LDL), 373
Latin America, 163 LOX. See Lysyl oxidase (LOX)
Laughing gas (N2O), 309, 313 LPS. See Lipopolysaccharides (LPS)
Laxative, 41, 42, 56 Lung
LDL. See Low density lipoprotein (LDL) adenocarcinoma, 511
Lead arsenate, 490 cancer, 144, 482
Legumes, 252, 422 Lyases, 205
Leishmania, 163, 164, 336, 346 Lyme disease, 5
amazonensis, 164 Lymph nodes, 15
Leishmaniasis, 140, 162, 164, 165, 337, 346 Lymphocytes, 10, 14, 65, 332, 333, 400, 467, 486
Lens dislocation, 426, 435 T-, 19, 160, 373, 467
Leptin, 180, 181, 462 Lymphoma
Lethal dose of As2O3, 481 gastric, 344
Leukemia Lysyl oxidase (LOX), 361, 363, 365, 368, 375
acute lymphoblastic, 421
cells, 164
promyelocytic (APL), 476, 477, 492, 493 M
Leukocyte antigen, 251 Macroglobulin, 10
Leukopenia, 372, 401 α2-, 206
Levodopa, 217 Macrophages, 5, 8, 10, 65, 163, 190, 206, 242,
Lewy bodies, 119, 212, 216, 277, 279, 377 244, 247, 248, 255, 279, 283, 334, 346,
Lichen, 150, 154 347, 373, 401, 462, 465, 466, 516
556 Index

Macular degeneration, 279, 280, 284, 403 copper homeostasis, 365


MAC value for V2O5, 144 cytochrome c oxidase, 362, 368
Magnesium, Mg2+, 43, 49–75, 83, 86, diet, 173, 174
88–90, 143, 205, 206, 212, 217, metallothioneins, 367
267, 323, 326, 328, 367, 394, molybdenum-dependent enzymes, 416
456, 459, 503 tissues, 238, 241, 508
absorption, 51–54, 58, 63, 72, 73 Mammary glands, 393
accumulation, 51, 54, 55, 63–65 Manganese, Mn, 20–22, 199–220, 323, 326,
channels, 51 362, 404, 459
54
chloride, 53 Mn, 204, 206
citrate, 53 absorption, 20, 21, 202, 203, 208
deficiency, 56–58 accumulation, 204, 208, 209, 213, 216
-deficient animals, 63, 66 biochemistry, 204–206
dietary intake, 51, 52, 54, 57, 58, 62, blood, 21, 202, 204, 217
63, 66 -citrate, 206, 209
excretion, 58–60 daily intake, 202
free, 52–54, 61, 65 deficiency, 202, 205
gluconate, 53 deposition in the brain, 203
homeostasis, 54–56, 62–64, 73, 206 dietary intake, 20, 22, 201–203
hydroxide, 53 efflux, 209, 210
in disease, 55–73 essentiality, 201, 202
level in blood, 56 excretion, 21, 202, 203
loss, 59, 65 homeostasis, 202, 203, 206, 210, 213
oxalate, 53 in infectious diseases, 21, 22
oxide, 53 neurotoxicity, 214, 215, 218
storage, 56 oxides, 417
sulfate (MgSO4), 53, 66, 70, 71 pharmacokinetics, 202–204
supplementation, 58, 60–62, 64–66, 72, 73 physiology, 204–206
transport, 53–55 toxicity, 206, 212, 216, 218
trisilicate, 464 trafficking, 206, 210
Magnetic resonance imaging (MRI), 200, 204, transport, 20, 206–210
279, 431, 435, 438 uptake, 207–209
Magnetic susceptibility, 188 Manganese(II), Mn2+, 5, 21, 98, 151, 200, 203,
Malabsorption of 206–208, 214, 215, 217
cobalamin, 299, 300 detoxification, 203
zinc, 11 Manganese(III), 200, 203, 208, 214, 215
Malaria, 7, 8, 13, 22, 231, 253, 258, 259, 283, Manganese(IV), 200, 203
284, 407 Manganese(V), 200
resistance, 258 Manganese(VI), 200
Malate, 108, 435 Manganese(VII), 200
Malaysia, 455 Manganese superoxide dismutase (Mn-SOD,
MALDI-TOF mass spectrometry, 520 SOD2), 3, 4, 20, 22, 205, 212, 213,
Males, 21, 34, 155, 175, 177, 192, 252, 253, 362, 367
454, 456, 511, 516, 519, 520, 522 Manganism, 204, 206, 210–212, 216–218
Malignancies, 9, 18, 254 Manganoproteins, 205
Malignant hyperthermia (MH), 124, 125 Manitol diuresis, 40
Malnutrition, 40, 58, 164, 250, 313, 408 Manufacture of glass, 200
Mammal(ian), 84, 112, 114, 172, 175, 192, MAPK. See Mitogen-activated protein kinases
247, 283, 296, 303–310, 314, 324, 363, (MAPK)
374, 423, 426, 429, 439, 442, 502–504, mARC. See Mitochondrial amidoxime-
511, 514, 521–523 reducing component (mARC)
brain, 201 Marine
cells, 50, 51, 53, 99, 186, 191, 240, 241, algae, 141, 150
243, 244, 308, 327, 364, 405, 503 food webs, 480
Index 557

Mass spectrometry (MS), 188, 427, 514, 520 Metabolism (of)


Matrix aβ amyloid plaques, 377
-assisted laser desorption/ionization amino acids, 349
time-of-flight (MALDI-TOF), 520 arsenic, 484
metalloproteinases (MMP), 392, 406 carbohydrates, 31, 107, 175, 201
vesicles (MV), 87 catecholamines, 364
MCM. See Methylmalonyl-CoA mutase cholesterol, 193, 519
(MCM) cobalt, 300, 301
MCU. See Mitochondrial Ca2+ uniporter drugs, 422
(MCU) energy, 31, 51, 108, 201, 324, 330, 361
MDS. See Myelodysplastic syndrome (MDS) ethanol, 65
MeaB, 302, 303, 305 fatty acids, 189, 190, 306
Measles, 407 folate, 308, 310, 315
Meat, 252, 257, 296, 298, 299, 422, 491, 521 glucose, 20, 31, 153, 174, 175, 179, 183,
MECAM, 267–269 186, 188, 205, 462
Mechanisms (of) glycine, 308, 309
neurodegeneration, 436 histidine, 308
ping-pong, 306, 510 homocysteine, 311
reaction, 305 hydrogen sulfide, 441
toxicity, 214–216, 465–467 iron, 244–246, 257, 264, 345, 361
Medicinal use of lipids, 172, 181, 186, 349
silica, 467 metal, 3, 368, 403
silicon, 467, 468 methionine, 315, 485
Megalin, 313, 514, 522 methylmalonyl-CoA, 314
Megaloblastic anemia model, 297, 309, 310, methylmalonyl-CoA mutase, 303–306
312–314 nickel, 324
Meiotic cell cycle, 403 phosphate, 141, 147
Melanin phosphorus, 485
neuro-, 277, 377 propyonyl-CoA, 306
synthesis, 361 proteins, 201
Melanocytes, 363 serine, 308
Melanoma, 331, 493 sodium, 348
Melarsoprol, 478, 491 superoxide, 336
Melastatin, 51 valine, 311
Melissa officinalis, 218 zinc, 11, 391, 395, 399, 405, 408, 409
Membrane Metal-binding pterin (MPT), 215, 216,
channels, 93–95, 112 427–429, 431, 432, 436, 437
potential, 31, 94, 96, 107, 110, 206, 215, -AMP, 429, 434, 439, 440
243, 488 synthase, 428, 429, 431, 432, 434
trafficking, 92 synthesis, 429, 434
vesicles, 63, 92 Metallic selenides, 517
Menkes P-type ATPase, 18 Metallo-chaperones, 341–343
Menstruation, 247, 250 Metalloproteinase 9, 86
Mental retardation, 69, 426, 440, 517, 518 Metallothioneins (MTs), 365–367, 394, 396,
6-Mercaptopurine, 421, 423 400, 406, 424
3-Mercaptopyruvate sulfurtransferase (MSPT), MT-1, 367
423, 424 MT-2, 367
Mercurial diuretics, 38 MT-3, 367
Mesothelial cells, 465, 466 MT-4, 367
Metabolic Metal response element-binding transcription
acidosis, 40, 41, 45, 362 factor-1 (MTF-1), 394, 395
alkalosis, 39, 41, 42, 44, 67 Metastases, 70
bone disease, 373 Meteorites, 143
diseases, 349, 404–407 Metformin, 64
558 Index

MetH, 304, 307–309 Mexico, 164


Methane formation, 336, 350 MH. See Malignant hyperthermia (MH)
Methanobrevibacter smithii, 350 Mica, 452
Methanococcus jannaschii, 504 Mice (see also Mouse), 6, 9, 15, 21, 62, 68, 70,
Methanogenic archaea, 350 117, 118, 122, 123, 153, 155–157, 163,
Methanopyrus kandleri, 504 187, 192, 203, 208, 212, 213, 217, 218,
Methionine 257, 264, 265, 280, 283, 313, 326, 334,
adenosyl-transferase, 309 345, 364, 366, 369, 370, 372–374, 396,
aminopeptidase, 314 405, 422, 425, 436–438, 441, 458, 504,
biosynthesis, 502, 521 511–514, 516–520, 522
-loaded tRNA, 503 CAKI-1, 157
metabolism, 308, 309, 312, 314, 315, 485 DI2-knockout, 512, 519
-R-sulfoxide reductase (MsrB1), 513 DI3-knockout, 519
seleno-, 16, 502, 503, 517, Dj-1-knockout, 217
521, 524 Gpx1-knockout, 511
supplementation, 310, 312, 313 Gpx4-knockout, 511, 520
Methionine synthase (MS), 297, 298, KK-Ay mice, 155, 462
300–304, 306–315, 424, 514 KK/HIJ mice, 187
-deficient mouse, 310 Mtrr-deficient, 314
metabolism, 309 mutant SOD1, 122
reductase (MSR), 301, 302, 307–309, 314 nude, 345
Methotrexate, 423 obese diabetic KKAy, 462
Methylation of histones, 237 parvalbumin null, 52
Methylcobalamin (CH3-Cbl), 297, 298, 302, prion protein-deficient mice, 379
303, 306, 307, 313 SelK-knockout, 516
Methyl-cyclopentadienyl manganese Sep15 knockout, 513
tricarbonyl (MMT), 200 Sepp1-knockout, 518, 522
Methylenetetrahydrofolate (CH2-H4folate), septic, 6
308, 309, 311, 312 SOD1-deficient, 373
reductase (MTHFR), 309–312 transferrin knockout, 265
Methylmalonic acidemia, 306 Micelles, 83, 149, 190
Methylmalonic aciduria, 300, 302, 311, 315 Michaelis-Menten constant, Km, 107, 418, 423,
CblA type, (MMAA), 302, 303, 305 429, 511
CblB type, (MMAB), 302 Microangipathy, 441
CblC type and homocysteinuria, Microcystis aeruginosa, 485
(MMACHC), 299, 300, 302 Microcytic anemia, 264, 265
CblD type and homocysteinuria, Microcytosis, 250
(MMADHC), 302 β2-Microglobulin, 257
Methylmalonic anemia, 306 Micronutrients, 3, 16, 17, 296, 324,
(S)-Methylmalonyl-CoA hydrolase, 306, 311 349, 408, 455, 457, 460–462, 468,
Methylmalonyl-CoA metabolism, 314 501, 502, 518
Methylmalonyl-CoA mutase (MCM), 297, Microorganisms (see also individual names
298, 300–306, 309, 311, 314 and species), 3, 83, 248, 256, 282, 296,
deficiency, 302, 314 336–346, 349, 350, 373, 374, 477, 480,
knock-out mouse model, 314 484–490, 493
metabolism, 303–306 Migraine, 118
reaction mechanism, 305 Milk, 83, 187, 248, 283, 296, 367, 370,
Methylobacterium extorquens, 305 374, 393
Methylselenocysteine, 521, 523 Minasragrite, 143
Methyltetrahydrofolate (CH3-H4folate), Mineralization
306–311 bones, 51, 84, 452, 457–459, 467
accumulation, 310 Mineralized tissues, 85, 88
Metronidazole, 163 Miners, 201, 211
Metvan, 156, 158 Mining, 144, 325, 479
Index 559

Mitochondria(l), 3, 20, 22, 54, 55, 65, 88, 89, Monkeys, 204, 213, 313
94, 97, 98, 101, 106–108, 115, Monoamine oxidase, 281
118–122, 125, 149, 203, 204, 211, 215, Monoclonal antibodies, 69, 70
217, 243, 263, 277, 279, 302, 309, 366, Monocytes, 65, 66, 71, 190, 323, 401
402, 420, 424, 425, 427, 431, 435, 510, Mono Lake, 479, 488
515, 520 Monomethylarsenic (MMAs), 481, 491
amidoxime-reducing component (mARC), MMAsIII, 482, 483
419, 426, 431, 442 Monosiga brevicollis, 314
Ca2+ uniporter (MCU), 97 Morocco, 253
damage, 119 MOT1. See Molybdate transporter type 1
dysfunction, 3, 212, 215, 216, 219, (MOT1)
377, 442 Motor neuron disease, 362, 372
iron transport, 243, 244 Mouse (see also Mice), 9, 88, 117, 155, 181,
membranes, 96, 113, 204, 484, 485 187, 208, 213, 265, 310, 313, 314, 334,
oxidative phosphorylation, 362 345, 347, 367, 373, 374, 377, 378, 402,
Mitogen-activated protein kinases (MAPK), 422, 436, 438, 439, 503–505, 512, 522
66, 187, 190, 333 adipocytes, 155
mk mouse, 264 db/db, 181
MMAs. See Monomethylarsenic (MMAs) genome, 504
MMP. See Matrix metalloproteinases (MMP) liver, 503, 515
Mn-SOD. See Manganese superoxide mARC, 426
dismutase (Mn-SOD, SOD2) methionine synthase-deficient, 310
MoCD. See Molybdenum cofactor deficiency mk, 264
(MoCD) Mocs1–/–, 436, 437
Moco. See Molybdenum cofactor (Moco) Mocs1-knockout, 436
Mocs1-knockout mouse, 436 mottled, 364
Mocs1–/–mice, 436, 437 Mouse models (for/of), 9, 117, 213, 313, 314,
MOCS1 protein, 427, 431 347, 367
Models of Parkinson’s disease, 208 Alzheimer’s disease, 377
Molecular oxygen, 233, 235, 424 diabetes, 181
Molybdate, 417, 418, 429, 439, 440 Huntington’s disease, 378
overload, 440 Wriggle Sagami, 117
supplementation, 439 YAC128Q, 213
Molybdate transporter, 417, 418, 429 MPT. See Metal-binding pterin (MPT)
type 1 (MOT1), 417, 418 MRI. See Magnetic resonance imaging (MRI)
type 2 (MOT2), 418 mRNA
Molybdenosis, 418, 440 synthesis, 344
Molybdenum, Mb, 415–442 translation, 66, 264
enzymes, 418–426, 431, 442 MS. See Methionine synthase (MS)
homeostasis, 418 Mseleni disease, 202
in drinking water, 418 MSR. See Methionine synthase
in soils, 418 reductase (MSR)
toxicity, 418 MTF-1. See Metal response element-binding
uptake, 417–418 transcription factor-1 (MTF-1)
Molybdenum(IV), 425 MTHFR. See Methylenetetrahydrofolate
Molybdenum(VI), 425 reductase (MTHFR)
Molybdenum cofactor (Moco), 417–420, 422, MTs. See Metallothioneins (MTs)
425–442 Mucin, 203
biosynthesis, 427–430 Mucopolysaccharide synthesis, 205
deficiency (MoCD), 420, 424, 426, Mucor, 9
431–442 Mucormycosis, 9
maturation, 430 Multicopper oxidases, 362, 372
structure, 419 Multi-drug resistance protein (MRP), 300,
sulfurase, 430, 432 480, 481, 490
560 Index

Multi-mineral dietary supplements, 53 Natural resistance-associated macrophage


Multiple sclerosis, 277 protein (NRAMP), 5, 206, 208
Multi-vitamin dietary supplements, 53 Nausea, 13, 31, 41, 45, 56, 118, 375
Muscle Necrosis, 37, 123, 466, 501, 517
cells, 33, 37, 62, 65, 66, 70, 94, 109, 110, liver, 173, 501
124, 187, 399, 460 Neonates, 20, 21, 426
contractions, 30, 31, 108–110, 115, 123, 149 death, 374, 419, 439
disease, 124–126, 501 Nepal, 7, 40
fiber, 33, 125 Nephrocalcinosis, 68
stiffness, 125 Nephron, 51, 53, 54, 62, 67
weakness, 31, 56, 58, 125 Nephropathy, 42, 64, 464
Muscular Nerve cells, 31, 32, 378, 401
disorders, 514 Nervous system, 32, 58, 201, 396
dystrophy, 102, 124–126, 516 Neurasthenia, 165
hypotonia, 374 Neurodegeneration, 208, 214, 216, 230, 265,
system, 30, 33 277, 280–281, 284, 362, 375–377, 391,
Mushrooms, 141, 521 402, 406, 407, 435–436, 440, 442
Mutant SOD1 mice, 122 Neurodegenerative diseases, 94, 118–122,
Mutation of 280, 376, 379, 406, 467
DMT1 gene, 250 Neuroendocrine cells, 96, 403
glutaredoxin-5 gene, 250 Neuroexcitotoxicity, 435
MV. See Matrix vesicles (MV) Neurofibrillary tangles, 120, 376, 407, 461
Mycobacterial urease, 347 Neurological diseases, 39, 117, 201, 361, 372,
Mycobacterium, 282, 283 376, 379
bovis, 347 Neuromedin C, 332
tuberculosis, 161, 162, 283, 343, 346, 347 Neuromelanin, 277, 377
Myelodysplastic syndrome (MDS), 8, 258, Neuromodulation, 360
262–263, 276–277, 372 Neuronal
Myeloma cells, 157 death, 212, 436
Myeloneuropathy, 371 diseases, 116–118
Myocardial infarction, 38, 43, 58–61, 159, 399 excitotoxicity, 102
Myoglobin, 235, 250, 252 iron accumulation, 279
Myosin, 51, 104, 108, 109, 123, 149 Neurons, 33, 58, 65, 94–96, 98, 101, 110,
light chain kinase (MLCK), 104, 109 117–122, 146, 205, 209–212, 216–218,
Myositis, 421 277, 376, 379, 393, 401–403, 407, 460,
Myxedemateous endemic cretinism, 517 515, 518
Neuropathy, 64, 179, 372, 376, 378, 379
Neuropsychiatric disorders, 421, 441
N Neurotoxicity, 205, 211–218, 273, 402
NAADP. See Nicotinic acid adenine of aluminum, 461
dinucleotide phosphate (NAADP) Neurotransmitters, 93, 96, 105, 118, 122, 201,
NADH. See Nicotinamide adenine 206, 364, 422, 435, 436
dinucleotide, reduced (NADH) Neurotrophins, 205
Nails, 201, 452, 457, 468, 517 Neutropenia, 9, 18, 371, 372, 374
Nanoparticles, 281 Neutrophils, 10, 323, 334, 373, 467
Nasal cancer, 325 New Zealand, 501, 521
National Academy of Science, 4, 174, 202, NF-κB pathway, 396–398
518 Nickel, Ni, 315, 321–351
National Research Council (NRC), 202, 524 acetate, 322, 325
National Research Council of the National allergy, 332–335
Academies of Science, 173 availability, 345, 349, 350
Natriuresis, 36, 37, 39, 43 carbonyl, 323
Natural killer cells (NK cells), 10, 12, 15, carcinogenicity, 327
401, 467 chloride, 322, 325
Index 561

cobalt/permeases (NiCoT), 342 signaling, 205, 442


-containing active site, 337–340, 350 synthase (NOS), 21, 109, 159, 161, 216,
-containing alloys, 334 218, 330, 425, 426
-containing fuel, 322 synthesis, 425, 426
-dependent infectious diseases, 336–348 Nitrile hydratase, 314
excess, 341 Nitrilotriacetate, 234
exposure, 323, 324, 328, 335 Nitrite, 422, 425, 442
genotoxic effects, 327 Nitrogen-fixing bacteria, 141
homeostasis, 324, 340, 343, 345–348, 351 3-Nitro-4-hydroxyphenylarsonic
homeostasis in pathogenic acid, 491
microorganisms, 340–344 Nitrosative stress, 402
immune reaction, 323 Nitrous oxide (laughing gas) (N2O), 309, 313
-induced carcinogenesis, 324–332 -exposed rat model, 313
-induced neoplastic transformation, 326–332 NK cells. See Natural killer cells (NK cells)
-induced teratogenicity, 323 N-methyl-D-aspartate (NMDA), 101, 105,
membrane transporters, 341, 342 120, 121, 218, 379, 402, 436
metabolism, 324 channel, 402
mining, 325 NMR. See Nuclear magnetic resonance
molecular chaperones, 342, 343 NO. See Nitric oxide (NO)
oxides (NiO), 322 N2O. See Nitrous oxide (laughing gas) (N2O)
-protein interactions, 344 Non-corrinoid cobalt, 314–316
sensors, 331, 341, 343, 344 -containing proteins, 314
silicates, 322 Non-heme iron
smelting, 325 absorption, 253
storage, 343 aggregates, 263
sulfate, 322 enzymes, 237
sulfides, 322, 325, 329 Non-transferrin bound iron (NTBI), 238, 241,
toxicity, 329, 331 255, 256, 258, 272, 276–278, 399
transport, 341 Norepinephrine synthesis, 361
uptake, 323–326, 341 Nori, 296
Nickel(I), 350 Normal hydrogen electrode (NHE), 267, 271,
Nickel(II), Ni2+, 206, 208, 328, 331–335, 337, 274, 479
338, 340–348 North America, 163
accumulators, 345, 346 North Korea, 517
homeostasis, 341, 343, 349 NR. See Nitrate reductase (NR)
Ni2+/Co2+ sensors, 348 NRAMP. See Natural resistance-associated
Ni3+/Ni2+ redox couple, 329 macrophage protein (NRAMP)
permease, 345 NRC. See National Research Council (NRC)
uptake, 344 NTBI. See Non-transferrin bound iron (NTBI)
utilization, 341 Nuclear factor B (NFκB), 71, 217, 218, 325,
Nicotinamide adenine dinucleotide (NAD), 396, 401, 467
107, 148, 420 Nuclear magnetic resonance (NMR), 188
1
Nicotinamide adenine dinucleotide, reduced H, 427
(NADH), 107, 108, 147, 214 Nuclear transcription factor NFκB, 4
Nicotinic acid adenine dinucleotide phosphate Nucleic acids. See DNA and RNA
(NAADP), 97, 99, 112 Nucleolin, 213, 506, 508
Niemann-Pick disease, 118 Nucleotide(s) (see also individual names)
[NiFe]-hydrogenase, 339, 340, 345 -gated channel functions, 348
Nifurtimox, 164 triphosphate hydrolysis, 343
Nigeria, 40 Nutrients, 3–6, 111, 114, 116, 139, 172, 201,
Nitrate (NO3−), 339 202, 204, 296, 298, 313, 315, 316, 345,
Nitrate reductase (NR), 417, 419, 425 346, 349, 353, 458, 484, 491, 501, 502
Nitric oxide (NO), 21, 22, 61, 216, 400, Nutrition, 3, 107, 173, 186, 253, 296, 298, 374
422, 460 copper deficiency, 371–374, 376
562 Index

Nutritional immunity, 4, 5 Oxidases


Nutrition Prevention Cancer (NPC) trial, 518 aldehyde (AOX), 419, 421–423, 431, 442
multicopper, 362, 372
Oxidative stress, 3, 19, 22, 43, 64, 119, 121,
O 190, 205, 208, 211–219, 278, 279,
Obesity, 62, 64, 153, 173, 181, 184, 372, 324, 329–331, 334, 367, 373, 377,
404, 519 395, 396, 399, 406–408, 460, 461,
-related insulin resistance, 181 465–467, 516, 519
Occipital horn syndrome (OHS), biomarker, 14
372, 374–375 Oxidovanadium(IV), 142, 144, 146, 153, 158,
Occupational exposure to 160, 165
manganese, 201 Oxidovanadium(V), 141, 158
nickel, 335 Oxoglutarate dehydrogenase, 107
vanadium, 144 8-Oxoguanine, 330
Occupational Safety and Health Oxygen (O2), 107, 146, 147, 235, 237, 329,
Administration (OSHA), 465 330, 336, 339, 362, 420, 487
Odontoblasts, 86, 87 -containing free radicals, 244
Office of Dietary Supplements deficiency, 324
of the National Institutes singlet, 161
of Health, 182 transfer, 423
OHS. See Occipital horn syndrome (OHS)
Oligodendrocytes, 210
Oliguria, 44, 45 P
Omeprazole, 58 Paints, 200, 453, 490
OMM. See Outer mitochondrial membrane Pancreatic
(OMM) β-cells, 110, 111, 393, 405, 519
Oncogenes, 327, 331 disease, 10
Onco-micro RNA, 406 Pancreatitis, 13, 59, 408
Oocytes, 96, 112, 402, 403 Pancytopenia, 371
ORAI1 channels, 97, 98 Paneth cells, 393, 399
ORAI proteins, 98 Pantothenate kinase-2 (PANK 2), 279, 280
Oral small cell carcinoma, 406 Para-aminosalicilic acid, 217
Organ dysfunction, 3 Paracellin-1, 54, 68
Organoarsenicals, 491 Paracoccidioides, 9
pesticides, 479 Paralysis, 41, 43, 146, 374
Ornithine transaminase, 463 Paraquat, 119
Orpigment, 479 Parasites, 141, 162–165, 248, 283, 284, 336,
Orthosilicic acid (OSA), 453–459, 463, 346, 407
467–469 Parasitic infection, 15, 249
Osmotic gradient, 36 Parathyroid
Osteoblastogenesis, 458 gland, 87
Osteoblasts, 84, 86, 87, 393, 458, 462 hormone (PTH), 53, 68, 72, 84
Osteochondropathy, 517 Parenchymal cells, 255, 256
Osteoclastogenesis, 458 Parenteral nutrition, 4, 19, 21, 211
Osteoclasts, 84, 86–88 PARK9, 20, 209, 218
Osteoporosis, 72, 202, 311, 371, 373, 374, Parkin, 119, 216–218
457–459, 467 Parkinsonian
Outer membrane, 6, 98, 113, 342 disturbances, 201
transporters, 345 -like symptoms, 211, 217
Outer mitochondrial membrane (OMM), Parkinsonism, 209, 211, 377, 378
97, 426, 435 Parkinson’s disease (PD), 118, 119, 121, 122,
Ovarian cancer, 72, 157 206, 208, 211, 212, 214, 216–218,
Oxalate, 53, 83, 176, 234 277–280, 284, 376–378, 380, 461
Oxaloacetate, 205 PARK2 mutations, 216
Index 563

Parvalbumin, 52, 119 ester hydrolysis, 150


null mice, 52 metabolism, 141, 147
Pathogenic bacteria, 282, 343 Phosphatidylcholine
Pathogens, 4, 6, 12, 22, 283, 336, 338–340, biosynthesis, 485
345, 373 Phosphatidylinositol 3-kinase (PI3K, Akt),
Pathology associated with 154, 156, 187
potassium, 41–45 Phosphatidylserine, 87
sodium, 38–41 Phosphocitrate (PC), 88
Patronite, 143 Phosphodiester, 149, 308
PD. See Parkinson’s disease (PD) Phosphoester hydrolysis, 151
Penicillamine, 371, 408 Phosphoester linkage, 158
Penicillin, 43 Phosphokinases, 147
Pentamidine, 58 Phospholipase (PL), 92, 112, 119
Peptic ulcer, 344 Phosphorus metabolism, 485
Peptidylglycine α-amidating enzyme (PAM), Phosphorylase kinase (PhK), 104
361, 364, 368 O-Phosphoseryl-tRNA[Ser]Sec kinase (PSTK),
Periplasmic phosphate binding protein (PBP), 503, 504
480, 488, 489 Phosphotyrosyl phosphatase, 150
Pernicious anemia, 297, 298, 312 Photophobia, 118
Peroxidases Phytates, 10, 176, 252, 408
bromo-165 Phytoremediation, 323
Peroxide, 147, 159, 191 Pick’s
Peroxynitrite, 21, 218 bodies, 277
Perthest disease, 202 disease, 118, 277
Pesticides, 119, 479, 490–491 Pigments, 363, 403, 476
PET. See Positron emission tomography (PET) Pigs, 313, 436, 467
Phagocytes, 8, 326, 347 liver, 314
Phagocytosis, 244, 323, 466 PI3K. See Phosphatidylinositol 3-kinase
Pharmaceuticals, 44, 72, 153, 165, 261, 456, (PI3K, Akt)
469, 477, 491, 492 Ping-pong mechanism, 306, 510
Pharmacodynamics, 141, 144–147 Pioglitazone, 64
Pharmacokinetics, 5, 10–15, 18–20, 141, Pituitary cells, 403
144–147, 158, 204, 273 Placebo, 3, 9, 12–14, 16, 17, 182, 184,
Pharmacological agents, 71–73 185, 518
Pharmacology, 192, 193 Placenta, 7, 370, 371, 374, 512
Phase I clinical trials, 273 Plant(s), 10, 83, 143, 201, 205, 252, 323, 336,
Phase II clinical trials, 273, 377 361, 417–419, 425–427, 430–433, 480,
1,10-Phenanthroline, 156, 159, 164, 234, 266 484, 485, 501, 502, 521, 523
Phenoxide, 233 urease, 323, 338
PhK. See Phosphorylase kinase (PhK) Plaques, 120, 377, 459, 460
Phlebotominae, 164 Plasma, 2, 10, 15, 21, 22, 39, 44, 87, 203, 206,
Phlebotomy, 257, 258 209, 272, 311, 363, 435, 436, 511, 514,
Phonophobia, 118 522, 524
Phosphatases, 88, 104–107, 140, 147, 151, calcium, 83, 84, 87
156, 158, 164, 186, 324, 369 cysteine/cystine, 423, 425, 436, 440
acid, 86, 150 insulin, 175
alkaline (ALP), 87, 88, 201, 458 iron, 255
serine/threonine, 205 lipid, 181
Phosphate, 45, 85–89, 93, 108, 141, 143, manganese, 21, 202, 203
146–152, 165, 233, 256, 417, 427, 429, membrane, 63, 82, 85–87, 93–100, 102,
476, 477, 479–481, 485, 486, 488–490, 104, 105, 108–113, 115, 117, 122, 126,
493, 505 189, 209, 257, 275, 323, 341, 365, 366,
deficiency, 486 369, 375, 512
di-. See Pyrophosphate potassium, 30
564 Index

Plasma (cont.) Pregnancy, 7, 8, 16, 18, 38, 202, 249, 250,


selenium, 15, 16 264, 312, 348, 363, 366, 407
sodium, 30 Prenatal diagnosis, 264, 266, 432
sulfate, 425 Preterm labor, 66, 70, 71
uric acid, 421 Primates, 174, 204, 216, 273, 484
vanadium, 145 Prion diseases, 376, 379
zinc, 10–12 Prion protein (PrPc), 372, 379, 407
Plasmodium falciparum, 284 -deficient mice, 379
Platelets, 60 Production of sulfuric acid, 165
aggregation, 399 Progressive
Platinum(II), 158 cerebellar-cerebral atrophy (PCCA), 518
PMCA pump, 93, 94, 109, 117, 122, 123 microcephaly, 518
Pneumoconiosis, 464, 465 supranuclear palsy, 277
Pneumocystis, 9 Proinflammatory cytokines,
Pneumonia, 12, 13, 15, 17, 19, 144, 407 65, 70, 71, 255
Poisoning of Prokaryotic pathogens, 346–348
livestock, 501 Prolyl hydroxylase, 460
nickel, 323, 326, 341 Propionibacterium shermanii, 305
Pollution of nickel, 322 Propionyl
Polycistin-2, 94 -carnithine in blood, 311
Poly-glutamine neurological diseases, 117 -CoA carboxylase, 306
Polymerases -CoA metabolism, 306
DNA, 151, 486 Prostaglandin, 217
Polyphenols, 252 Prostate cancer, 511
Polyuria, 41, 45, 67 Prostatitis, 15
Porcine liver, 314 Proteases, 102, 117, 119, 121, 126, 420
Porphyria, 265 Protein(s) (see also individual names), 205
Porphyrins, 6, 142, 160, 235, 297, 315 CblC, 299–302
Positron emission tomography (PET), 200 EF-hand, 84, 90, 100, 105
Potassium, K+, 29–45, 50, 60, 63, G, 96, 118, 303
88, 210, 215 Hfe, 257, 265
absorption, 34 natural resistance-associated macrophage
arsenite, 492 (NRAMP), 5, 206, 208
blood levels, 41 prion (PrPc), 372, 379, 407
channels, 54, 111 -RNA interactions, 508
deficiency, 31, 35 S100, 52
-deficient diet, 42 seleno-. See Selenoproteins
excretion, 34, 35, 44 SIBLING family, 87
homeostasis, 32–37, 41 Protein data bank, 91
intake, 34, 35, 61 PDB 2BB5, 299
membrane action, 32, 33 PDB 1BMT, 304
permanganate, 200 PDB 1K7Y, 304
secretion, 34 PDB 2PMV, 299
supplementation, 60 PDB 1Q8J, 304
Potentials PDB 3SC0, 299
electrical, 31, 32, 42, 51 PDB 2XIQ, 304
membrane, 31, 94, 96, 107, 110, 206, 215, Protein dephosphorylation,
243, 488 103–106, 399
redox, 148, 233–235, 267, 271, 274, 361, Protein kinase(s), 92, 158, 324
366, 479, 502 A (PKA), 104, 105
Poultry industry, 491 B (PKB), 154–156, 159
PPi. See Pyrophosphate (PPi) C (PKC), 52, 65, 66, 92, 104
P450 reductase, 301, 307, 308 C signaling, 55, 65
Pre-eclampsia, 56 Protein phosphatase-1, 205
Index 565

Protein phosphorylation, 103–106, 405 R


Protein tyrosine phosphatase (PTPase), Rabbits, 21, 326, 459
154, 158, 159, 393, 406 Radicals
Proteinuria, 45 5′-deoxyadenosyl, 427
Proteobacteria, 349 free, 4, 14, 201, 214, 217, 244, 270, 271,
Proteolysis, 101 279, 374, 395, 465
Proton hydroxyl, 146, 214, 233, 234, 271, 329,
gradient, 99, 480, 486 373, 374, 465
pump inhibitors (PPI), 58, 71–73 superoxide, 20, 214
Protonation constant (see also Acid tyrosyl, 237
dissociation constants), 267 Ramalina celastri, 164
Protoporphyrin IX, 252 Rapamycin, 116
Protozoa(n), 7, 346 Rat(s), 21, 42, 161, 173–178, 185, 192, 204,
parasites, 141, 163, 336, 346 208, 217, 218, 273, 326, 348, 399, 436,
Provisional Tolerable Daily Intake for 460, 467, 484–487, 519, 520, 522
inorganic arsenic (PTDI), 481, 484 adipocytes, 155, 188
PrPc. See Prion protein (PrPc) BB, 462
Psammomys obesus, 519 B12-deficient rats, 305, 309, 313
Pseudomonas, 282 Belgrade, 264, 265
aeruginosa, 161, 337 brain, 423
fluorescens, 488, 489 diabetic, 153, 177
PTDI. See Provisional Tolerable Daily Intake genome, 422
for inorganic arsenic (PTDI) GR, 483
Pterin synthesis, 427–428 JCR:LA-cp, 180, 185
Pteris liver, 309, 311, 503
ensiformis, 485 models, 146, 180, 181, 313
vittata, 485 vitamin E-deficient, 501
PTH. See Parathyroid hormone (PTH) Wistar, 175
PTPase. See Protein tyrosine phosphatase RDA. See Recommended daily
(PTPase) allowance (RDA)
P-type ATPase, 367–369 Reactive
transmembrane, 209 nitrogen species (RNS), 3, 4, 465
Pulmonary oxygen species (ROS), 3, 61, 66, 119,
carcinogen, 453 126, 140, 146–148, 157, 158, 166,
diseases, 39 190, 214–216, 323, 325, 327, 329,
edema, 465 334, 362, 366, 377, 402, 435, 442,
infections, 13 460, 465–497, 511, 513
malfunctions, 144 Realgar, 479, 490
Purine-related biomarkers, 422 Receptor-operated Ca2+ channels (ROCCs), 96
Purkinje cells, 117 Recommended daily or dietary allowance
Pyridoxal isonicotinoyl hydrazine (RDA) for, 455, 501, 502, 524
(PIH), 282 cobalamin, 296
Pyrophosphate (PPi), 87, 88, 427 magnesium, 51
Pyruvate selenium, 501, 502
carboxylase, 20, 204, 205 Red arsenic, 479
decarboxylase, 205 Red blood cells, 9, 21, 181, 244, 261, 263, 348
dehydrogenase, 107, 481 transfusions, 9
dehydrogenase kinase, 328 Redox
Pythium, 9 agents, 148
cycle, 235, 271, 274, 394
potentials, 148, 233–235, 267, 271, 274,
Q 361, 366, 479, 502
Quartz, 452, 456, 463 Reference daily intake for Mn, 202
Quinines, 214 Refinery workers, 325
566 Index

Regeneration of tissues, 462 Ryanodine receptor (RyR), 94, 109, 120, 121,
Regulation of 124, 516
iron metabolism, 244–246 channels, 95, 98, 99, 109, 110
magnesium transport, 55
the calcium signal, 97–99
Renal S
cancer, 331 Saccharomyces cerevisiae, 364
cells, 52 Safe Drinking Water Act, 521
disease, 44, 45, 57, 67, 255, 442 Salivary glands, 192, 399
dysfunction, 20 Salmonella, 39, 282, 373
failure, 7, 18, 38–41, 44, 45, 57, 421 enterica serovar Typhimurium, 339
Renin, 37, 42–44, 67 typhii, 8
-angiotensin-aldosterone system, 32, 44 Salt marsh grass, 485
Reperfusion injury, 161 Salvarsan®, 478, 491
Reproductive SAM. See S-Adenosyl-methionine
function, 324 Sandflies, 164
system, 348, 402–403 Sarcoplasmic reticulum, 93, 95
Respiratory Sardinia, 264
alkalosis, 43 SARS. See Severe acute respiratory syndrome
chain, 94, 97, 107, 117, 119, 235, 362, (SARS)
396, 402 SCD. See Systemic contact dermatitis (SCD)
tract, 144, 325, 401 Schizophrenia, 311, 404, 441
tract infections, 11, 12, 407 Sea
Retina(l), 210, 403 squirts, 141, 143
degeneration, 280, 363 urchins, 112
Retinopathy, 64, 153, 185 Seafood, 252, 481
Reverse phase chromatography, 434 Seawater, 142, 417, 457
Rheumatism, 165 Seaweed, 296, 299, 480
Rheumatoid arthritis, 254, 255 Sec. See Selenocysteine (Sec)
Rhinitis, 144 Second messengers, 93, 95, 99, 112, 324
Rhizopus, 8, 9 Seizures, 31, 205, 402, 426, 435, 436, 440, 441
oryzae, 8, 9 SELECT. See The Selenium and Vitamin E
Rhodospirillum rubrum, 343 Cancer Prevention Trial (SELECT)
Ribonucleotide reductase, 237, 238, 270, Selenide, 505, 514
282, 283 dimethyl-, 523
RNA, 3, 51, 102, 149, 245, 246, 308, 314, Seleninic acid, 510
315, 392 Selenite, 505, 512, 513, 517, 520, 521, 524
-dependent ATPases, 508 Selenium, Se, 4, 13–19, 22, 58, 174, 340, 476,
m-, 66, 263, 264, 344, 506–508 484, 499–526
75
onco-micro, 406 Se labeling, 506
protein interactions, 507 administration, 17
ribosomal (rRNA), 508 absorption, 521
small interfering (siRNA), 114, 508 bioavailability, 517
tRNA[Ser]Sec, 503–505, 509, 514, 519 biomarker, 524
ROCCs. See Receptor-operated Ca2+ channels blood, 15, 517
(ROCCs) deficiency, 15–17, 501, 513, 517–520,
Rodents, 95, 174, 185, 217, 264, 273, 400, 522–524
459, 463, 464, 467 -deficient diet, 518, 520
models, 193, 203, 250 -deficient sheep, 514
ROS. See Reactive oxygen species (ROS) -depleted soil, 501
Rotenone, 119 excretion, 523
Roxarsone, 478, 491 exhalation, 523
Ruminants, 296, 418, 440 in infectious diseases, 15–18
Russia, 521 lowest observed adverse effect level, 518
Index 567

-related diseases, 516–518 Serine, 104, 154, 308, 311, 503, 508
supplementation, 15–17, 501, 511, 516, hydroxymethyltransferase, 309
517, 521, 524 metabolism, 308
therapy, 17 Serine/threonine phosphatase, 205
toxicity, 501, 517, 518, 524 Serotonin, 251, 422
transporter, 523 Serpentine, 323, 452
transport in mammals, 521–523 SerRS. See Seryl-tRNA synthetase (SerRS)
Selenocysteine (Sec), 502–503, 506, 510, Sertoli cells, 363, 522
512–514, 517, 521 Serum
biosynthesis, 503–505, 509 albumin, 15, 145
insertion sequence (SECIS), 500, 502, B12, 313
506–510 calcium, 201
methyl-, 521, 523 cholesterol, 201
synthetase (SecS), 505, 509, 518 chromium, 20, 179
tRNA, 503–505 cobalamin, 312
Selenocysteyl copper, 4, 18
-tRNA, 507–509 creatinine, 44
-tRNA[Ser]Sec, 505 magnesium, 53, 56–60, 62, 64, 68, 71–73
Selenomethionine, 16, 502, 503, 517, 521, 524 phosphorus, 201
Selenophosphate synthetase (SPS), 505 selenium, 14–16
Selenoprotein(s), 15, 16, 502–516, 518, sodium, 31, 38, 40, 45
519–525 trace elements, 4
15kDa (Sep15), 513, 515 transferrin, 238, 239, 283
biosynthesis, 508, 513 zinc, 4, 18
H (SelH), 506, 515 Seryl-tRNA, 503, 507, 508
I (SelI), 506 synthetase (SerRS), 503
K (SelK), 506, 516 Severe acute respiratory syndrome (SARS),
M (SelM), 515 159, 161
mRNA, 506–508 Sheep, 296, 305, 313, 372, 374,
N (SelN), 506, 516 379, 514
O, 515 Shigella, 39, 40, 282, 340
P (Sepp1), 15, 16, 506, 514, 518, 519, Shock, 44
522–524 SHR rats, 62
S (SelS), 506, 515, 516, 519 SH-SY5Y cells, 216, 218
T (SelT), 506, 515 Siberia, 517
V (SelV), 506, 514 SIBLING family of proteins, 87
W (SelW), 514 Sickle cell disease, 14, 22, 258, 262, 408
Selenosis, 516–518 Sideroblastic anemia, 263–265
Seminal fluid, 402 Siderocalin, 6, 283
Senile plaques, 213, 376, 377 Siderochelin, 283
Sensors Siderophores, 3, 6, 9, 261, 270, 271, 273,
Ni2+/Co2+, 348 282, 283
Sensory function, 324 Signaling, 55, 89, 91, 92, 98, 112, 152, 189,
Sep15 knockout mice, 513 334, 361, 366, 398, 518
Sepp1. See Selenoprotein P (Sepp1) agents, 88–93
Sepp1-knockout mice, 518, 522 calcium(II), 395
Sepsis, 6, 11, 13, 15, 401 pathways, 54, 66, 72, 115, 329–331, 393,
Septic 396, 409
mice, 6 Signal transduction, 154, 324, 396
patients, 14 Silica
shock, 16, 22, 66 bioavailability, 455
Sequence alignment of MOCS1A, 433 content of water, 454
SERCA pump, 93, 98, 109, 113, 114, crystalline, 453, 463, 464, 468
124, 125 -deficient diet, 463
568 Index

Silica (cont.) intake, 30, 34, 36, 37, 40, 61


from drinking water, 461 membrane action, 32, 33
gel, 452 metabolism, 348
-induced carcinogenesis, 465 Na+/Ca2+ exchangers, 86, 93, 94, 100, 109,
nanoparticles, 465–467 115, 119
Silicates, 322, 452, 453, 455, 456, 461, 463, Na+/Mg2+ exchanger, 53–55, 62,
467, 468 63, 65
alumino-, 452, 456, 461, 468 physiology, 32–37
Silicic acid, 453, 455–457, 459, 461, 463, -potassium ATPase, 37, 43
468, 469 -potassium ATPase (Na+,K+-ATPase),
Silicon(es), 451–469 36, 37, 43, 216
absorption, 464 -potassium pump, 31, 32, 118, 165
biomarker, 455 secretion, 35–37
carbide, 452 selenite, 17, 517, 521
dioxide (SiO2), 453–456, 463 sulfide, 514
distribution, 453–456 wasting, 67
implants, 453 Soft tissues, 50, 51
in urine, 455 Soil selenium, 521
Silicosis, 453, 463–467 Solar cells, 452
Silymarin, 218 Solid tumors, 70
Single-photon emission computed tomography Somatostatin, 403
(SPECT), 200 South Africa, 253
Singlet oxygen, 161 South America, 521
SiO2. See Silicon dioxide (SiO2) South East Asia, 261
SIRS. See Systemic inflammatory response Spartina alterniflora, 485
syndrome (SIRS) Spasticity, 518
Site-directed mutagenesis, 425 Sperm, 112, 313, 402, 511, 520
Skeletal maturation, 313, 512, 522
development, 396, 458–459 Spermidine catecholamides, 282
muscle diseases, 124–126 Spinal cord, 116, 118, 121, 212, 251,
muscles, 42, 50, 51, 63, 65, 95, 104, 108, 378, 432
109, 124, 179, 186, 187, 331, 370, 512, Spironolactone, 59, 68
519, 522 Spleen, 15, 146, 164, 516
Skin, 13, 31, 41, 85, 86, 144, 163, 282, 332, S100 protein, 52
334, 335, 337, 373, 374, 396, 403, 422, SPS. See Selenophosphate synthetase (SPS)
457, 463, 464, 468, 477, 482, 493, 523 Squamous cell carcinoma, 511
eczematous, 332 Stability constants (see also Affinity constants,
Small interfering RNA (siRNA), 114, 508 Binding constants, and Formation
Small intestine, 10, 18, 19, 21, 51, 86, 176, constants), 234, 238, 267, 269, 482
203, 250, 369, 370, 399, 417 conditional or apparent, 177, 238, 405
Smelters, 201 π-Stacking, 159
Smokers, 511 Staphylococcus, 282
Smooth muscles, 30, 62, 65, 66, 104, 108–110, aureus, 5, 6, 8, 13, 161, 342
399, 460 Starfish, 112
SO. See Sulfite oxidase (SO) Starvation, 4, 6, 42, 345, 488
SOD1. See Superoxide dismutase 1 (SOD1) Steel, 174, 452
SOD2. See Superoxide dismutase 2 (SOD2) production, 200
SOD1-deficient mice, 373 Stem cell
Sodium, Na+, 30–45, 53, 59–63, 67, 88, 95, disorder, 262
99, 119, 142 transplantation, 8
absorption, 37 Sterility, 191, 418
channel, 32, 118, 119 Steroid(s), 218, 235
excretion, 30, 35–37, 39, 44 hormone, 482, 483, 486
homeostasis, 34, 36, 37 STIM protein, 97, 98, 120
Index 569

Stomach, 18, 42, 101, 140, 144, 148, with silicon, 458
174, 192, 250, 296, 299, 338, 345, Swayback, 372, 374
346, 399 Sweat, 35, 283, 334
Store-operated channels (SOCCs), Sweden, 164
97, 98, 209 α-Syn, 119
calcium, 98, 120, 206 Synaptic vesicles, 105, 401, 402
plasma membrane, 93 Synaptotagmins, 91, 110, 111
Streptococcus, 282 α-Synuclein, 119, 212, 216, 284, 377, 378
pneumoniae, 8 Syphilis, 477, 491
Streptomyces, 150 Systemic
antibioticus, 273 contact dermatitis (SCD), 335
coelicolor, 343 inflammatory response syndrome (SIRS),
Streptozotocin (STZ), 155, 181 3, 4, 18
rats, 155 iron overload, 255–266, 284
Stroke, 65, 262, 276, 402
Strontium, Sr2+, 208
STZ. See Streptozotocin (STZ) T
Substitution therapy, 438, 439 T4. See Thyroxine (T4)
Succinate, 435 Tachycardia, 43, 58
dehydrogenase, 246 Taiwan, 479, 482, 483
Succinyl-CoA, 306 Talc, 452, 456
Sucrose, 6 Tamoxifen, 218
Sulfate, 45, 83, 146, 325, 326, 417, 425, 429, Tanning of leather, 200
435, 440 cTannins, 252
transporters, 417 Tanzania, 7
Sulfhydryl groups, 233, 406, 423 Tau protein, 120, 406, 407
Sulfide, 141, 329, 340, 423–425, 429, 441, Taurine, 61, 423, 436, 439, 440, 485
479, 490, 505, 514, 520 TB. See Tuberculosis (TB)
di-, 350, 512, 513 TC. See Transcobalamin (TC)
hydrogen, 441 TCA cycle, 205, 246, 306
nickel, 322, 325, 326, 329 T-cells, 17, 101, 105, 106, 160, 163,
Sulfite 323, 325, 332–335, 347,
accumulation, 425, 435, 436 400, 401, 467
toxicity, 423–426, 435 T1DM. See Type 1 diabetes (T1DM)
Sulfite oxidase (SO), 418, 419, 423–426 T2DM. See Type 2 diabetes (T2DM)
deficiency, 424–426, 439–440 Tea, 153, 155, 252
S-Sulfocysteine (SSC), 436–440 Teeth, 42, 83, 85–87, 421
Sulfuric acid manufacturing, 501 Tellurium, 501
Supercoiled plasmid DNA, 163 TEs. See Trace elements (TEs)
Superoxide, 4, 146, 147, 158, 163, 205, 214, Testicular
215, 233, 361, 362, 420, 422 cancer, 157, 158
detoxification, 361 damage, 310, 313
metabolism, 336 Testis, 105, 514–516, 519, 521, 522
radicals, 20, 214 Tetany, 41, 58, 68
Superoxide dismutases (SOD), 15, 205, 215, Tetrahydrobiopterin, 439
329, 342, 361, 362, 367, 370 Tetrahydrofolate
1 (SOD1), 121, 122, 212, 361, 362, 365, methyl-, 306–311
367, 377, 379 Tetrapyrrole ring, 297
2 (SOD2), 3, 4, 20, 22, 205, 212, 213, Tetrathiomolybdate, 378, 418, 440
362, 367 Tf. See Transferrin (Tf)
3 (SOD3), 362, 373 TfR. See Transferrin receptor (TfR)
Supplementation TGR. See Thioredoxin/glutathione
of vitamin B, 458 reductase (TGR)
of vitamin K, 458 Thailand, 253
570 Index

Thalassemia, 8, 258, 260–262, 264–266, 272, excito-, 102, 117, 118, 122, 205, 212,
274, 276, 277 218, 375
α-, 259, 261 nanocompounds containing cobalt, 315
β-, 259–262, 266 Toxicology of
major, 260, 261, 264, 272, 274 silica, 463–467
Thapsigargin, 114 silicon, 463–467
Therapeutic agents, 33, 172, 264, 477, 491 Toxins, 6, 12, 33, 208, 345
Thermotoga martima, 304, 308 TPC. See Two-pore channel (TPC)
The Selenium and Vitamin E Cancer TPN. See Total parenteral nutrition (TPN)
Prevention Trial (SELECT), 518 TR. See Thioredoxin reductase (TR)
Thiamine deficiency, 42, 58 Trace elements (TEs), 3, 4, 15, 17, 18, 22,
Thiazide diuretics, 38, 39, 43, 58, 60, 68 172–174, 176, 380, 457, 459, 476
Thiocyanate, 517 Traditional Chinese medicine, 477, 492
Thiol(s), 3, 214, 216, 306, 366, 395, 408, 436, Transcobalamin (TC), 299, 300
481–483, 502, 511, 512, 520 receptor, 300, 313, 332, 400
-disulfide oxidoreductase, 368 Transcription factor(s), 4, 100, 101, 105, 324,
Thioredoxin, 510, 512, 515 327, 329–331, 347, 391, 392, 396, 406,
glutathione reductase (TGR), 512 462, 466, 509
reductase (TR), 512 NFAT, 106
Thiosemicarbazones, 162, 282 SMAD4, 248
Thiosulfate, 424, 425, 438, 440, 441 Transferases
Threonine, 51, 104, 114, 205, 206 adenosyl-, 302, 309
Thrombosis, 399 adenylyl-, 429
Thymidine 5′-monophosphate (dTMP), arsenic methyl-(As3MT), 481, 490
308, 312 -cob(I)alamin adenosyl-, 302
Thymidylate synthase, 309 glutathione S-, 69
Thymulin, 12 glycosyl, 205
Thymus, 400, 515 3-mercaptopyruvate sulfur-(MSPT),
Thyroid 423, 424
atrophy, 517 Transferrin (Tf), 6, 9, 19, 21, 144–146,
Thyroid hormone(s), 511, 512 176–179, 186, 188, 202, 203, 208, 238,
deiodinases, 511–512 241–242, 244, 247, 248, 250, 255, 256,
receptor, 511, 512 265, 272, 277, 283, 284, 372, 407
Thyroxine (T4), 254, 512 Transferrin receptor (TfR), 20, 206, 208, 209,
Tissue 238, 241, 242, 244, 246
adipose, 145, 146, 405, 512, 519 knockout mice, 265
distribution of vanadium, 141 Transfusion therapy, 262
magnesium, 52, 59, 63 Transgenic mice, 217
TNF. See Tumor necrosis factor (TNF) Transglutaminase, 21, 203
Tobramycin, 58 Transient receptor potential channels
Tocolytic, 66, 70, 71 (TRP), 51
Tolerable intake for molybdenum, 418 Trans-membrane proton gradient, 480, 486
Tolerable upper intake level (TUL), 455 Transmissible spongiform
of selenium, 518 encephalopathies, 379
TonB-dependent transport of nickel, 342 Transplants, 59, 106, 254
Torula yeast, 174, 501 Transport of
Total parenteral nutrition (TPN), 13, 18, calcium, 93, 94, 106, 208
179–180, 371, 408, 484, 485 chromium, 176, 179
Toxemia, 38 iron, 176, 241, 284
Toxicity of iron-loaded transferrin, 241–242
arsenate, 481, 486 manganese, 20, 98, 202, 206–210
arsenite, 481 nickel, 323, 341, 342
chelators, 270–272 oxygen, 250, 362
drugs, 421 zinc(II), 401
Index 571

Trauma, 3, 4, 16, 18, 20, 22, 43, 250, 254 Type 2 diabetes (T2DM), 43, 63, 64, 102,
Traumatic brain injury, 402 152–154, 180–182, 184, 185, 404, 405,
Treatment of 462, 467, 516, 518, 519
cancer, 156–159, 231, 492 Tyrosinase, 361, 363–364, 368, 375
childhood diarrhea, 12 Tyrosine kinase, 153, 186, 189,
malaria, 13 400, 406, 493
manganism, 217 Tyrosine phosphatases, 158, 159, 161
MoCD type A, 438 phosphatase-1B (PTP-1B), 150, 154, 156,
MoCD type B, 439 186, 399, 405
molybdenum cofactor deficiency, 437–440 Tyrosyl radical, 237
sulfite oxidase deficiency, 439–440
Tremolite, 463
Triatominae, 163 U
Triazoles, 273 Ubiquitin, 492
Tricarboxylic acid cycle (TCA), 205, 246 UNICEF. See United Nations Children’s
Trichosporon, 9 Fund (UNICEF)
Trichostatin A, 328 United Kingdom, 17, 264, 454
Triglycerides, 180–182, 348, 459 United Nations Children’s Fund
Trimethylselenonium, 523 (UNICEF), 482
TR3-knockout mice, 512 United States, 34, 173, 184, 455, 459,
tRNA[Ser]Sec, 503–505, 509, 514, 519 501, 521
Tropomyosin, 108, 109, 123 Urea
Troponin, 52, 108, 109 cycle, 205
TRP. See Transient receptor potential channels hydrolysis, 336, 338
(TRP) Urease, 323, 336, 338–339, 342, 343,
TRPM6, 51, 52, 54, 55, 62, 63, 68, 69, 72 345–347, 349
TRPM7, 51, 52, 55, 62, 63, 72, 206 chaperones, 345
Trypanasoma cruzi, 163, 164, 346 Uric acid, 3, 419–422, 436, 438, 442
Trypanosome infections, 491 accumulation, 421
Trypanosomiasis, 15, 162 Urinary
American, 162 chromium loss, 20, 175, 177, 178
Tuberculosis (TB), 4, 7, 16, 19, 162, 165, 254, excretion of zinc, 10
283, 336, 346, 407 potassium, 42
Tubular necrosis, 59 selenium, 523, 524
Tubulins, 149 sulfite level, 436, 438
TUL. See Tolerable upper intake level (TUL) xanthine level, 436
Tumor(s), 165, 325–327, 330, 331, 408, Urinary tract
468, 511 calculi, 421
breast, 156 infections, 19, 336
cells, 69, 70, 72, 141, 147, 156, 157, Urogenital system, 348
164, 281, 282 Urolithiasis, 421, 464
necrosis factor (TNF), 4, 64, 190, Urothione, 442
396, 466 UV/Vis, 361
solid, 70
Tumor suppressor, 102, 327, 330, 511, 513
genes, 158, 327, 328, 331 V
Tungstate(VI), 160 Vaccine, 347, 468
Turkey, 40, 252 Valine metabolism, 311
Two-pore channel (TPC), 94, 95, 99, 118 Vanadate(V), 141–144, 146–161, 164, 165,
Type 1 copper enzymes, 361 187, 191
Type 2 copper enzymes, 361 inorganic, 158
Type 3 copper enzymes, 361 -phosphate antagonism,
Type 1 deiodinase (DI1), 506, 511–512 141, 147–152
Type 1 diabetes (T1DM), 63, 152, 181, 405 Vanadinite, 143, 164
572 Index

Vanadium (in), V, 140–166 E, 4, 17, 518


antidiabetic compounds, 154 E-deficient rats, 501
antiparasitic properties, 162 Vitamin B12, 297–310, 484
blood, 146 bioavailability, 296
cardio-protective effects, 160 deficiency, 296, 297, 300, 310–314,
cycling, 142–147 371, 372
food, 143 deficient rats, 305, 309, 313
freshwater, 142 VOCCs. See Voltage-operated Ca2+ channels
intake, 140, 143, 144, 146 (VOCCs)
minerals, 143 Volcanic emissions, 143, 322
nitrogenase, 140, 141 Voltage-dependent anion channels (VDAC),
overload, 143, 144 97, 520
oxides, 143, 144, 146, 164 Voltage-gated K+ channels, 111
oxides in breathing air, 146 Voltage-operated Ca2+ channels (VOCCs),
poisoning, 146 96, 111
rocks, 142, 143 Voltage-regulated
seawater, 142 Ca2+ channels, 206
sediments, 142, 143 channels, 208, 209
VOx, 143, 144, 164 Vomiting, 31, 38, 39, 41, 45, 56, 118, 375, 481
Vanadium(II), 143
Vanadium(III), 141, 143, 147
Vanadium(IV), 141–148, 151–154, 157, 158, W
160, 162, 165, 166 Watson-Crick base pairs, 506
Vanadium(V), 141–154, 156, 157, 161, 162 Welders, 201, 211, 212
Vanadocene, 156, 158, 159, 165 Welding fumes, 216, 217
Vanadyl sulfate, 153 Western blotting, 520
Vascular Wheat, 454, 485, 521
disease, 459, 460, 482 Whipple’s disease, 59
dysfunction, 62 White muscle disease, 501
endothelial growth factor (VEGF), 330 WHO. See World Health Organization (WHO)
smooth muscle cells, 62, 65, 66 Whole blood, 15, 21, 217
Vasopressin, 32, 53, 55, 59 Wistar rats, 175
VDAC. See Voltage-dependent anion channel Wood preservative, 491
(VDAC) World Health Organization (WHO), 253, 260,
VEGF. See Vascular endothelial growth factor 345, 346, 391, 502, 524
(VEGF) Wound healing, 13, 462–463
Verapamil, 125 Wriggle Sagami mouse model, 117
Vertebrates, 5, 6, 8, 85, 141, 206, 323, 336,
338, 425, 514
Vibrio X
cholerae, 39 Xanthine
parahemolyticus, 342 accumulation, 420, 421
vulnificus, 9 dehydrogenase (XDH), 419–422
Vietnam, 253 oxidase (XO), 191, 417–421, 430,
Viomycin, 58 435, 442
Viral Xanthine oxidoreductase (XOR), 419–422,
infections, 15, 517 431
liver disease, 399 deficiency, 420
Viruses, 17, 141, 159–161, 165, 517 Xanthinuria
Vitamin(s), 3, 310, 390, 422, 453, 458, 484 type 1, 420, 421
B6, 58 type 2, 421
C, 4, 17, 253, 422 XDH. See Xanthine dehydrogenase (XDH)
D, 52, 58, 84–86, 459 Xenobiotics, 33, 36, 235, 422, 423
dietary supplements, 53 Xeroderma pigmentosum group A, 483
Index 573

XO. See Xanthine oxidase (XO) deficiency, 391, 395, 396, 398–408
XOR. See Xanthine oxidoreductase (XOR) diet, 404
X-ray excretion, 10
absorbance, 188 finger proteins, 10, 392
X-ray crystal structure of finger transcription factors, 396
arsenate, 489 free, 394, 395, 408
cobalamin, 297 growth, 403
phosphate, 489 homeostasis, 10, 366, 391, 394, 395, 398,
401–404, 406–408
infectious diseases, 11–14
Y metabolism, 11, 391, 395, 399, 405, 408, 409
YAC128Q mouse model, 213 -metallo β-lactamases, 11
Yeast, 182, 218, 367, 368, 504, 521, 524 metalloproteins, 392, 398
Brewer’s, 174, 183, 186, 501 -metallothionein (Zn-MT), 11
torula, 174, 501 overload, 372, 395
Yellow arsenic, 479 proteome, 392
Yersinia, 9, 282, 340, 342 regulatory, 393
enterocolitica, 8 release, 393
signaling, 393, 394, 396, 400, 401
sulfate, 12
Z supplementation, 11–14, 396, 399, 401,
Zebra fish, 264, 265 402, 408
Zeolites, 458, 468, 469 therapy, 408
Zinc(II) (in), Zn2+, 4, 5, 10–15, 17–19, 21, 22, transporters, 11, 208, 399–401, 404–406
88, 149, 153, 208, 213, 231, 250, 266, vesicles, 393, 394
267, 271, 323, 326, 336, 337, 342, 346, Zincuria, 399
348, 361, 362, 366, 367, 371, 376, 380, ZIP. See Zrt-,Irt-like proteins (ZIP)
390–409, 429, 482–484, 492 Zip family, 394
acetate, 14 Zn-MT. See Zn-metallothionein (Zn-MT)
binding constant, 405 ZnT family, 394
biomarker, 396, 399, 407 Zrt-,Irt-like proteins (ZIP), 407
blood, 395, 401, 404 Zucker diabetic fatty rats, 175, 177, 180, 185

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