Professional Documents
Culture Documents
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
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
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
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
October 2005,
October 2008,
and August 2011
Preface to Volume 13
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
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
Titles of Volumes 1–44 in the Metal Ions in Biological Systems Series ........... xxi
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
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
xxi
xxii Titles of Volumes 1–44 in the Metal Ions in Biological Systems Series
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>
xxiii
xxiv Contents of Volumes in the Metal Ions in Life Sciences Series
Subject Index
Subject Index
xxvi Contents of Volumes in the Metal Ions in Life Sciences Series
Subject Index
Subject Index
Subject Index
Subject Index
Subject Index
Subject Index
Subject Index
Subject Index
Subject Index
Subject Index
Subject Index
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
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
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.
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.
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].
1.6 Calprotectin
2 Iron
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.
2.3.2 Malaria
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].
2.3.4 Diabetes
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].
3 Zinc
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
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.
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.
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].
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].
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,
14 Carver
4 Selenium
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].
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.
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
18 Carver
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
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.
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].
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
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
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].
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
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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1 Metal Ions and Infectious Diseases. An Overview from the Clinic 25
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
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.
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.
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
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.
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.
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+.
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.
2.3.1 Potassium
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
3.1 Hyponatremia
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
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
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.
(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
4.2 Hyperkalemia
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
References
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
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.
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.
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].
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.
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].
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]).
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
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
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.
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
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
mEq/L: altered atrioventricular conduction and heart block at 15.0 and 25.0 mEq/L,
and cardiac arrest at >25mEq/L.
3.2 Hypomagnesemia
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.
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.
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
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
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
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].
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
3.2.6 Inflammation
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.
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].
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.
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
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
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78 Romani
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
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.
1 Introduction
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
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.
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
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).
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
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.
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
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.
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.
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
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
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
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.
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
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.
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
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
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].
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
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.
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.
5.1.1 Ataxia
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
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
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].
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].
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].
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.
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.
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.
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
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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136 Brini, Ottolini, Calì, and Carafoli
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.
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
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
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
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.
(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
( )
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
OH2
O O
H2O OH2
V H2O V OH2 H2O V O
H2O OH2
H2O OH2 H2O
OH2 OH2
OH2 OH2
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.
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.
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)
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
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.
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
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.
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.
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).
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.
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.
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
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.
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
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
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.
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?
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
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
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
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.
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.
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
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
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?
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
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.
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.
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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196 Vincent
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
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
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
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].
2 Manganese Transport
?
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.
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].
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
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.
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
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).
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.
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].
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
7.3 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
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
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
References
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
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
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.
1 Introduction
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
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
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
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
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
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].
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.
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
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
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].
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 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
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
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).
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
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.
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
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
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].
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].
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
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.
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).
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
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
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.
ferroportin gene. Affected individuals express high hepcidin levels and the disease
displays phenotypic heterogeneity [68].
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.
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].
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).
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].
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].
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.
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].
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
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).
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
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
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
O O O
O O O N O O
Fe Fe Fe
O O N O O O
O O O
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.
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
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].
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].
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
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].
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.
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].
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).
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
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
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
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
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
7.1 Tuberculosis
7.2 Malaria
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
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F. M. Torti, Cancer Res. 2011, 71, 6728–6737.
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Anderson, G. D. McLaren, Humana Press, New York, 2012, 477–495.
247. D. B. Lovejoy, D. R. Richardson, Curr. Med. Chem. 2003, 10, 1035–1049.
248. H.M. Hanauske-Abel, M.-H. Park, A.-R. Hanauske, A.M. Popowicz, M. Lalande, J.E. Folk,
Biochim. Biophys. Acta, 1994, 1221, 115–124.
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251. J. Gao, D. R. Richardson, Blood 2001, 98, 842–850.
252. J. Yuan, D. B. Lovejoy, D. R. Richardson, Blood 2004, 104, 1450–1458.
253. D. Fu, D. R. Richardson, Blood 2007, 110, 752–761.
254. E. D. Weinberg, Biochim. Biophys. Acta 2009, 1790, 600–605.
255. N. H. Carbonetti, S. Boonchai, S. H. Parry, V. Vaisanenrhen, T. K. Korhonen, P. H. William,
Infection and Immunity 1986, 51, 966–968.
256. S. Ford, R. A. Cooper, R. W. Evans, R. C. Hider, P. H. Williams, Eur. J. Biochem. 1988, 178,
477–481.
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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.
1 Introduction
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.
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
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.
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.
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.
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
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
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.
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
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
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.
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
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.
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
3.3 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.
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.
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
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].
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
Abbreviations
Acknowledgment The author acknowledges Dr. C. L. Elmore (US Food and Drug Administration)
for reading and editing the English.
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320 Yamada
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
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
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.
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
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.
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
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.
(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
(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
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
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].
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
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.
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].
Figure 3 Reaction catalyzed by acireductone dioxygenase, and schematic structure of the nickel-
containing active site.
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
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
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].
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
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].
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].
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.
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.
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].
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
Figure 7 Reaction catalysed by methyl coenzyme-M reductase, and schematic structure of the
nickel-containing active site.
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
References
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
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.
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.
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].
2.1.3 Ceruloplasmin
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].
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
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].
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).
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.
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
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
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].
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
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].
3.1.1 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
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].
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].
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].
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].
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].
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].
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].
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
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].
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].
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
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
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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11 Copper: Effects of Deficiency and Overload 387
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
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.
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.
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].
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.
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
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].
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
TNFα TNFα
TNFα
TNF Receptor
K63
Tab2 or 3 TAK1
Targets mRNA for degradation K63
Zn
TNFα Zn
Nup475/TTP/Tis11
o
em
o
IKKα
γ/N
IKKα P
/N
26S Proteasome
K
Kγ
IK
IKKβ
K63
IK
IKKβ
K63 Activated IKK Complex
IKK Complex
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.
398 Maret
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.
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.
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
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].
400 Maret
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].
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.
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
402 Maret
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].
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.
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].
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].
404 Maret
4 Zinc in 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,
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].
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
4.2.2 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
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
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
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Chapter 13
Molybdenum in Human Health and Disease
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
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
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.
1 Introduction
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].
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.
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].
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).
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].
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.
2.1.4 Hyperuricemia
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.
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].
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.
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).
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.
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].
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
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].
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.
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.
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].
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].
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].
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].
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
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
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
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.
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
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
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.
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
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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13 Molybdenum in Human Health and Disease 447
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
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.
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
2 Silicon Biochemistry
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.
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
456 Martin
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].
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.
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
458 Martin
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].
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
460 Martin
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].
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].
462 Martin
3.4 Diabetes
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.
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].
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.
(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].
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
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
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.
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
Abbreviations
References
1. L. Jurkic, I. Cepanec, S. Pavelic, K. Pavelic, Nutr. Metab. 2013, 10, 2, DOI: 10.1186/1743-7075-10-2.
2. C. Exley, J. Inorganic. Biochem. 1998, 69, 123–139.
3. S. Sjoberg, J. Non-Crystalline Solids 1996, 196, 51–57.
4. C. Perry, Prog. Mol. Subcell. Biol. 2009, 47, 295–313.
5. K. Martin, J. Nutr. Health Aging 2007, 11, 94–97.
6. R. Jugdaohsingh, J. Nutr. Health Aging 2007, 11, 99–110.
7. A. Elmore, Cosmetic Ingredient Review Expert Panel, Int. J. Toxicol. 2003, 22, 37–102.
470 Martin
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
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
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.
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.
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
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
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].
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
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].
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
3.3.1 Microorganisms
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
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
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
P
O1
O3
O4
b Oδ1
D62
Cγ
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
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
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
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.
References
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
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
1 Introduction
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
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
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]:
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:
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).
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.
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
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
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.
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].
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
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.
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.
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].
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].
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].
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.
The functions of SelO and SelI are unknown. SelI localizes in the ER.
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].
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.
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.
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].
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 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].
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.
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
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.
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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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
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
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
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
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
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
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
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
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