Reactive Species Detection in Biology: From Fluorescence to Electron Paramagnetic Resonance Spectroscopy

Reactive Species Detection in Biology

From Fluorescence to Electron Paramagnetic Resonance Spectroscopy

Frederick A. Villamena

Department of Biological Chemistry and Pharmacology, The Ohio State University, OH, United States

Table of Contents

Cover image

Title page

Copyright

Dedication

Preface

Chapter 1. Introduction

References

Chapter 2. Chemistry of Reactive Species

Abstract

2.1 Introduction

2.2 Redox Chemistry

2.3 Properties of Reactive Species

References

Chapter 3. Reactive Species in Biological Systems

Abstract

3.1 Introduction

3.2 Extracellular Milieu

3.3 Membrane-Bound Enzymes

3.4 Cytosolic Enzymes

3.5 Organelle Enzymes

References

Chapter 4. Fluorescence Technique

Abstract

4.1 Introduction

4.2 Fluorescence Spectroscopy and Microscopy

4.3 Chemistry of Redox Detection by Fluorescence

4.4 Classification of Fluorescent RS Probes by Specificity

4.5 Considerations in Fluorescence Probe Application

References

Chapter 5. EPR Spin Trapping

Abstract

5.1 Introduction

5.2 Electron Paramagnetic Resonance Spectroscopy

5.3 Chemistry of Spin Trapping

5.4 Classification of Spin Traps

5.5 Kinetics and Thermodynamics of Spin Trapping and ph effect

5.6 Kinetics and Thermodynamics of Adduct Decay

5.7 Biostability and Cytotoxicity of Spin Traps

5.8 Synthesis of Spin Traps

5.9 Interpretation of EPR Spectra

5.10 Applications of Spin Trapping

References

Chapter 6. UV–Vis Absorption and Chemiluminescence Techniques

Abstract

6.1 Introduction

6.2 Superoxide Radical Probes

6.3 Hydroxyl Radical Probes

6.4 Antioxidant Capacity Assays

6.5 Nitric Oxide and Metabolites Probes

6.6 Thiols Probes

6.7 Peroxides Probes

6.8 Chemiluminescence

References

Chapter 7. Electrochemical, Mass Spectroscopic, Immunochemical, and Nuclear Magnetic Resonance Techniques

Abstract

7.1 Introduction

7.2 Electrochemical Techniques

7.3 Mass Spectroscopy

7.4 Immunochemical Technique

7.5 Nuclear Magnetic Resonance Spectroscopy and Imaging

References

Index

Copyright

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ISBN: 978-0-12-420017-3

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Dedication

To Prof. DeLanson R. Crist, my mentor, father, and friend

And to my parents, Prisco and Tess, for all your love and sacrifices.

Preface

Frederick A. Villamena, Columbus, OH, United States

Life is a sea of electrons that when in the state of equilibrium thrives but causes havoc when perturbed. But this oversimplistic analogy leaves a multitude of unanswered questions and is ridden with complexities. It is encouraging that current advancements in biochemistry and biological chemistry have allowed modern-day biomedical investigators to understand some of these complexities and get ever closer to unraveling some of the most fundamental questions in disease development. Most often overlooked are the analytical tools employed to probe these mysteries. A research area by itself, it owes its modern progress to chemists, biologists, engineers, and biomedical researchers for the development of innovative molecular probes and imaging agents, efficient methodologies, and state-of-the-art instrumentation. This book is therefore a tribute to them: without their tireless dedication, our understanding of disease pathogenesis would not be in the state it is today. At the heart of this perturbation of a sea of electrons is the reduction and oxidation chemistry that involves a variety of players, the most important of which is molecular oxygen; when not properly metabolized, it results in the production of reactive species. These reactive species are like an army of destructive forces that destroy, inactivate, or cause only partial functioning of key biomolecular systems essential for normal functions of living systems. Knowledge of reactive species’ location, their origin, and their identity as well as their target molecules and molecular trails they leave as biomarkers are critical and can only be accomplished through the use of both earlier and modern sophisticated analytical tools. Reactive species detection is therefore important for identifying specific molecular and cellular pathways leading to oxidative stress that could lead the way to the development of genetic, molecular, and pharmacological approaches, as well as diagnostic tools to prevent or ameliorate free radical–mediated diseases.

This book is basically divided into four major parts: introduction, chemistry of reactive species, their biology, and their modes of detection. Chapter 1, Introduction, provides a historical account of how oxygen and its reactive metabolites have been implicated in the pathogenesis of diseases. It also describes how important discoveries of the last century were aided by the use of analytical tools to characterize reactive species as important biological mediators and how bioassays can complement such findings. Chapter 2, Chemistry of Reactive Species, will only present biologically relevant reactions of reactive species in an effort to give readers a sense of their relative reactivity, specificity, and selectivity to various biomolecules. In Chapter 3, Reactive Species in Biological Systems, shows various sources of reactive species in biological settings, either from exogenous sources or as generated from enzymes, and it will discuss various sources of reactive species production from cellular compartments. Lastly, Chapters 4, Fluorescence Technique; Chapter 5, EPR Spin Trapping; Chapter 6, UV–Vis Absorption and Chemiluminescence Techniques; Chapter 7, Electrochemical, Mass Spectroscopic, Immunochemical, and Nuclear Magnetic Resonance Techniques; showcase conventional and modern analytical techniques that are often if not widely employed in the detection of biological reactive species such as fluorescence, electron paramagnetic resonance (EPR) spin trapping, UV–Vis light and chemiluminescence, and electrochemical, mass spectrometric, immunochemical, and other magnetic resonance techniques with the exception of some techniques that are too specialized (e.g., EPR and positron emission tomography imaging) or redundant in their principles and chemistry to those already discussed here.

I hope readers will find this first edition a one-stop kind of reference material for fundamental principles, limitations, and applications of the various analytical technique used in reactive species detection. Since this book will only cover so much information, readers are encouraged to consult the references cited herein for more detailed discussions and descriptions of the topics discussed.

This book would not have been possible without the dedication and passion of investigators working in the field of free radical research, whether in the synthesis of new molecules or the development of methodologies and instrumentations, as well as biomedical researchers who continuously validate these tools for their effective application in biological systems.

October, 2016

Chapter 1

Introduction

Respiration has been commonly known for centuries as an essential process for survival because it provides the fuel for the normal functioning of animal organs. This fuel found in air was first known as dephlogisticated air on its discovery in the 1770s by scientists Joseph Priestly and Carl Wilhelm Scheele.¹,² Antoine Lavoisier later coined the term oxygen and extended the theory of combustion to introduce the idea of respiration as a biological process in which inhaled oxygen is used in the oxidation of carbon and hydrogen from food to give carbonic acid, thus relating respiration as a combustion process.³,⁴ Studies from 1940s and 1950s on the enzymatic metabolism of oxygen provided molecular bases for oxygen’s diverse biological functions through enzyme-catalyzed transfer of oxygen atom to a substrate, or electron-transfer reactions to oxygen leading to the formation of reactive oxygen species (ROS) or water.⁵ This enzyme-catalyzed reduction of oxygen was later found to have beneficial as well as detrimental effects on cellular function. Without a doubt, the progress made in understanding oxygen metabolism owes a debt to the development of electrochemical techniques for analyzing oxygen in biological fluids and tissues as well as whole animals. Oxygen sensor development was described⁶ dating as far back as 1938 when the first biological application of platinum electrodes was demonstrated for the purpose of monitoring oxygen to study photosynthesis, and this was followed by the use of a Clark-type electrode that allowed for the measurement of oxygen tension (pO2) in an in vivo system, air, blood, and cell cultures, which then led to further innovation that exhibited high accuracy.

The absorption of oxygen and its transformation to carbonic anhydride (carbon dioxide) during respiration has thus been the paradigm of oxygen metabolism.⁷ In the early 1900s, the relationship between oxygen and disease was suggested by Todd,⁸ whereby the human body is in a state of chemical equilibrium between the processes of oxidation and reduction; when this equilibrium shifts toward the formation of more reduced species than oxidized ones, the body could lose resistance to diseases, and hence resupplying the body with oxygen in the form of ozonized air or oxidized oils is used as a therapeutic means of counteracting diseases such as tuberculosis or Bright’s disease. Oxygen therapy was employed for a variety of diseases; e.g., patients with respiratory disease such as pneumonia exhibited excellent therapeutic effect when such therapy was introduced soon after diagnosis.⁹ It also became apparent that oxygen exhibits toxicity against bacterial pneumococcus type I¹⁰ as well as in protozoans,¹¹ brain respiration,¹²,¹³ or in whole animals causing pulmonary damage.¹⁴ In humans, oxygen results in the reduction of blood-flow rate to the brain when inhaled at high atmospheric pressure,¹⁵ causing cerebral complications as well as diminished overall cardiac output and changes in alveoli that result in edema, transudation, and fibrinous deposits.¹⁶

The formation of hydroxyl radicals from water under ionizing radiation had long been implicated for the radicals’ biological actions and toxicity.¹⁷ Soon thereafter, chemical agents that have radiation-like properties were implicated in the initiation¹⁸ of cancer or tumor inhibition via chromosome alteration through formation of free radicals.¹⁹ Evidence supported the idea that free radicals formed radiolytically were toxic because they were found to diffuse inside the cells when generated extracellularly.²⁰ Moreover, it was also demonstrated that x-ray radiation inhibited glutathione metabolism inside the cells, and this inhibition was decreased at low oxygen concentration and on addition of catalase, which suggested the involvement of oxygen-derived reactive species such as H2O2.²¹ The link between radiation and oxygen levels on their cellular toxicity had become more apparent by their inactivation of T2 bacteriophage and by the observation that thiol compounds such as thiourea could compete with oxygen-derived radicals, thereby protecting the phages from radiation.²² Using electron paramagnetic resonance (EPR), radiation damage to DNA or RNA was reported to produce paramagnetic nucleic acids at 77K.²³ This finding was consistent with the increased effect of radiation on DNA inactivation in the presence of oxygen and protection in the presence of the thiol cysteamine²⁴,²⁵ with mutations successfully induced in the cell nucleus of onion rootlets, e.g., by hydroxyl radical and x-irradiation.²⁶

While the effect of irradiation-mediated free radical formation on nucleotides was not as pronounced as in proteins and peptides, it became clear that free radicals formed enzymatically could have profound consequences on protein function. Metabolic hydroxylation of aromatic amino acids has long been suspected as a biosynthetic process for the conversation of phenylalanine to tyrosine, tyrosine to 3,4-dihydroxyphenylalanine, kynurenine to 3-hydroxykynurenine, and tryptophan to 5-hydroxytryptophan.²⁷ In the 1950s, metabolic hydroxylation of aromatic compounds such as N-2-fluorenylacetamide in guinea pigs and rats was demonstrated and believed to be a detoxification mechanism.²⁸ This conversion was duplicated in cell-free in vitro studies involving ferrous ion, ascorbic acid, oxygen, and a chelating agent (ethylenediaminetetraacetic acid) under physiological conditions, showing that hydroxylation of aromatic compounds could indeed be mediated by free radial reaction, specifically that of hydroxyl radicals.²⁹ Not long after, it was proposed that biological hydroxylation occurs via activation of oxygen by peroxidase³⁰,³¹ and by other hydroxylating systems found in liver microsomes that require triphosphopyridine nucleotides and oxygen for their activity.³² Altogether, Harman³³ proposed the role of oxygen, metals ions, oxidative enzymes, and radiation on the development of age-related and degenerative diseases through generation of reactive oxygen species, and these propositions became the foundation of today’s widely accepted free radical theory of aging. Subsequently, the link between free radicals and the development of atherosclerosis,³⁴,³⁵ cancer,³⁶ and neurodegeneration³⁷ was proposed, and the role of proper nutrition and lower metabolic demand were seen as essential for the slower progression of free radical–mediated reactions in the body.³⁸,³⁹ While free radicals such as semiquinones were identified using EPR as integral components of the mitochondrial respiratory chain,⁴⁰,⁴¹ evidence for the production of ROS such as superoxide and hydrogen peroxide by mitochondria through electron transfer had become more compelling.⁴²–⁴⁴

Although the existence of superoxide as an inorganic species is known dating as far back as the late 1890s, its paramagnetic character was not established until the 1930s.⁴⁵ For the next four decades, studies on superoxide were mostly focused on their chemistry with metals and nonmetals and, for the first time, its characterization through EPR spectroscopy.⁴⁶ Only in the late 1960s did the idea become acceptable that superoxide could also be generated in biological system, an idea helped by the discovery of superoxide generation from enzymatic systems. The idea was first introduced by McCord and Fridovich,⁴⁷ whose seminal study demonstrated the production of superoxide from xanthine oxidase and xanthine; they found this formed species capable of reducing cytochrome c and initiating the sulfite oxidation reaction. In further support of this evidence, superoxide formation from xanthine oxidase was confirmed using EPR spectroscopy at pH 10; signal intensity was shown to be dependent on oxygen concentration and not the enzyme itself.⁴⁸ The oxygen origin of superoxide from xanthine oxidase was unequivocally confirmed using ¹⁷O-labeled O2 and EPR spectroscopy giving the 11-line or 6-line EPR spectra for the formed ¹⁷O2•– or ¹⁷O¹⁶O•–, respectively.⁴⁹ Generation of superoxide from oxygen was achieved through electrolytic reduction of oxygen in aqueous solution.⁵⁰ In addition, electrochemically generated chemiluminescence of lucigenin showed evidence of superoxide-mediated light emission, paving the way for the development of chemiluminescence probes for superoxide.⁵¹ Excited-state oxygen resulting from the oxidation of xanthine by xanthine oxidase can also induce chemiluminescence via recombination of ROS, probably that of superoxide,⁵² and this finding was further supported by evidence showing that singlet oxygen–sensitized fluorescence from organic compounds is mediated by superoxide, which suggests the possible enzyme-mediated formation of singlet oxygen.⁵³

The chemistry of superoxide enzymatic formation and decomposition then became of interest to investigators who wanted to know whether the mechanism is ligand mediated or metal mediated. It was demonstrated that superoxide is decomposed by erythrocuprein and ferricytochrome c and is formed during the oxidation of reduced flavin⁵⁴ rather the iron heme of flavoproteins.⁵⁵ This was later supported by studies showing that the one-electron reduction of oxygen by reduced flavins and quinones results in the formation superoxide.⁵⁶ However, the formation of superoxide from the reaction of oxygen with reduced iron-sulfur proteins from plant ferredoxins that are flavin free was also reported,⁵⁷ indicating that oxygen reduction can occur via electron transfer not only from organic radicals but also from low-valent metal ions.⁵⁶ The enzyme superoxide dismutase (SOD) was then proposed to catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and had been a gold standard as a competitive inhibitor for the investigation of superoxide-mediated reactions such as the oxidation of epinephrine to adrenochrome by xanthine oxidase and the reduction of ferricytochrome c or tetranitromethane.⁵⁸ The reduction of ferricytochrome c was found to be augmented by electron carriers such as flavin adenine dinucleotide, menadione, or flavin mononucleotide.⁵⁹ The characterization of SOD in bovine heart and its ubiquity in mammalian tissues suggest the important role SOD plays in the regulation of ROS in biological systems.⁶⁰ It became clearer that the cause of oxygen toxicity is not oxygen itself but the oxygen-derived reactive species such as superoxide, hydrogen peroxide, and hydroxyl radicals.

Detection of reactive species in biological systems could date as far back as the late 1800s and early 1900s through visual observations of color changes in test tubes or spot plates. For example, the detection of hydrogen peroxide in plants and milk employed the use of various reagents that impart color changes with oxidation.⁶¹–⁶³ The introduction of commercial ultraviolet visible spectrum (UV vis) spectrophotometers in the early 1940s by Arnold O. Beckman⁶⁴ allowed for the detection of oxygen-derived radicals (e.g., superoxide and hydroxyl) in biological systems. Since then, ROS detection focused on the use of spectrophotometric techniques that allow detection at the UV region and providing a more accurate, reproducible, time-saving, and reduced sample size for ROS analysis. Detection of superoxide became possible through superoxide dismutation by superoxide dismutase or reduction by ferricytochrome c.⁵⁶,⁵⁹,⁶⁵ Several other techniques has been employed for the analysis of H2O2 and superoxide radical such as electrochemistry,⁶⁶ fluorescence,⁶⁷ chemiluminescence,⁶⁸ and EPR spin trapping.⁶⁹ Hydroxyl radical detection involved the analysis of gaseous substances such as methane on hydroxyl radical reaction with dimethyl sulfoxide from human phagocytes as analyzed by mass spectroscopy⁷⁰ or ¹⁴C-carbon dioxide from the decarboxylation of [¹⁴C]benzoic acid on oxidation by hydroxyl radicals, also from human ganulocytes, using an ionization chamber-electrometer.⁷¹ Concurrently, the hydroxyl radical was identified through hydroxylation of 5,5-dimethyl-pyrroline N-oxide (DMPO) by EPR spin trapping in respiring rat heat mitochondria⁴⁴ or through hydroxylation of salicylate as measured by colorimetric or gas chromatographic (GC) assays.⁷²

Analytical techniques have a profound utility in resolving some of the most confounding questions in science but sometimes do not provide unequivocal evidence of their formation and therefore need to be complemented by biological assays. For example, the question whether the endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). Their pharmacological properties were found to be similar⁷³,⁷⁴ as well as their in vivo half-life of 3–5 s⁷³ or 41 s in Krebs solution.⁷⁵ However, subsequent findings show that the amount of nitric oxide detected by chemiluminescence was significantly less than that required to account for the detector vessel relaxation,⁷⁶ suggesting that NO is not the sole component of EDRF. Through the use of the same chemiluminescence technique, it was proposed that EDRF resembles that of S-nitrosocysteine (or S-nitrosothiols in general) in terms of its vasodilator potencies,⁷⁷ which is further supported by previous findings that show that exogenously introduced nitrosocysteine appears to be more similar to EDRF than free NO in terms of vasodilation per amount of contained NO.⁷⁸ Other perplexing data revealed through continuous-flow spectrophotometric detection using diazotization reaction and did not detect nitrogen oxides and lacked the specificity to distinguish NO from other nitrogen oxides released from cultured endothelial cells by bradykinin, adenosine triphosphate, or calcium ionophore, whereas the bioassay readily detected EDRF release.⁷⁹ Using EPR or spin trapping, the adduct formed from activation of muscarinic receptors by carbamylcholine did not resemble the free radicals originating from NO or hydroxylamine but is similar to that of L-arginine-NADPH-Ca²+–derived NO using 3,5-dibromo-4-nitrosobenzene sulfonate as a spin trap,⁸⁰ which further supports that L-arginine is the precursor of EDRF.⁸¹ However, the triplet signal observed for hemoglobin-NO (Hb-NO) complex cannot be duplicated from effluents collected from acetylcholine-stimulated release of EDRF from intact femoral arteries.⁸² EPR spin-trapping studies of NO using Hb corroborated the previous findings that EDRF resembles S-nitrosothiols and not NO at concentrations required to cause vasorelaxation similar to EDRF.⁸³ In spite of these equivocal data, bioassay methods provided evidence that supports initial findings that NO is EDRF such that it causes increased intracellular cGMP response.⁸⁴ For example, evidences derived from chemical and biological assays showed that EDRF inhibition by L-NMMA and L-NAME⁸⁵; potentiation of vasorelaxation by PDE isoenzymes⁸⁶; augmentation of EDRF activity through increased O2⁸⁷; inactivation of EDRF by pyrogallol or superoxide, stabilization by SOD and inhibition by oxyHb or K+⁷⁴; dependence of EDRF production from Ca²+ concentration and CaM⁸⁸; EDRF inhibition of platelet activity⁸⁹; and dependence of nitrite levels on EDRF production in biological fluids,⁹⁰,⁹¹ therefore, further supporting the fact that EDRF components is NO-derived. Other evidence also links EDRF to other substances such as the nonprostanoid endothelin-1⁹² and most recently H2S.⁹³

The interplay between superoxide and nitric oxide in biological system has a profound implication on the normal function of the cell. Superoxide radical dismutation to hydrogen peroxide ultimately leads to the formation of hydroxyl radicals via Fe-catalyzed or uncatalyzed Haber-Weiss processes.⁹⁴ The formation of these reactive intermediates, although critical in the modulation of cellular function, signaling, and immune response, in unregulated levels are detrimental to the integrity of key biomolecular systems and lead to cellular injury. Similarly, normal levels of nitric oxide are important mediators of physiological functions but when below or above normal levels could compromise cellular functions due to low NO bioavailabity or could cause toxicity, respectively. The paradoxical effect of NO at high levels confounds investigators since one would expect a more beneficial effect, but evidence showed that mice with long-term exposure to nitric oxide suffered degenerative and necrotic changes followed by bronchiolar epitheliums of the alveolar,⁹⁵ lysis of tumor cells on activation of endothelial cells by cytokines,⁹⁶ or chemically generated NO from NO-donating drugs causes pancreatic islet cells lysis in a time- and concentration-dependent manner.⁹⁷ The link between oxygen, nitric oxide, and superoxide toxicity in vivo was hypothesized in the seminal work by Oury et al. through the use of transgenic mice models of overexpressing human extracellular superoxide dismutase, endogenous nitric oxide synthase (NOS) inhibition and hyperbaric oxygen conditions, and showed that nitric oxide plays an important role in decreasing oxygen toxicity in the central nervous system by oxygen-mediated NO inactivation perhaps via superoxide formation.⁹⁸ Although the reaction of superoxide and NO could be considered protective due to NO’s sequestration of superoxide, the product formed from the reaction of these two paramagnetic species (i.e., peroxynitrite)⁹⁹ was proposed to be oxidative in nature by decomposing two potent oxidants (i.e., hydroxyl and nitric oxide), and this could have significant implications in the initiation of oxidative damage as mediated by activated leukocytes and myocardial or neural ischemia or reperfusion injury,¹⁰⁰–¹⁰⁵ as well as in the formation of atherosclerotic lesions.¹⁰⁶–¹⁰⁸ Although it was proposed a decade earlier that peroxynitrite is produced from direct oxidation of NO by oxymyoglobin,¹⁰⁹ this opened up the possibility that peroxynitrite may be produced in biological systems. Beckman et al.¹¹⁰ proposed the implication of peroxynitrite formation for endothelial injury through production of hydroxyl radical. Following their discoveries, mechanistic insights into the peroxynitrite cytotoxicity had become clearer by demonstrating its reactivity with protein thiols,¹¹¹ its formation of protein nitration,¹¹² its role in the initiation of lipid peroxidation¹¹³ and DNA oxidation.¹¹⁴ As with other reactive species, increasing evidence of peroxynitrite importance in the initiation of oxidative damage has become increasingly apparent and necessitating the development of analytical tools for its detection in biological systems. Early evidence of peroxynitrite formation in chemical systems mainly involved high-performance liquid chromatography (HPLC) analysis of hydroxylation of phenylalanine to yield isomeric mixtures of tyrosine products along with products of the nitration of phenylalanine and tyrosine, suggesting the formation of hydroxyl and nitrogen dioxide radical intermediates.¹¹⁵,¹¹⁶ EPR spin-trapping studies also confirmed the formation of these decomposition species using DMPO as a spin trap ¹¹⁷ or using oxyhemoglobin photometrically.¹¹⁸ However, in vivo detection of peroxynitrite proved to be a challenge due to its high reactivity, hence short in vivo half-life,¹¹⁹ but with the development of antinitrotyrosine antibodies, the in vivo evidence of peroxynitrite formation can be correlated with levels of nitrotyrosine in tissues as analyzed by immunohistochemical techniques.¹²⁰,¹²¹ Other techniques had since been applied for the analysis of peroxynitrite in vivo using fluorescent probes¹²² or HPLC,¹²³,¹²⁴ GC, or mass spectroscopic (MS) assay.¹²⁵

This introduction gives us just a short glimpse of the history of oxygen, reactive species derived from it, and the importance of detection techniques, whether crude or state of the art, in the understanding of their role in biology. Through the design of innovative analytical tools such as instrumentation and probes for reactive species detection, we are getting closer to unraveling the mysteries underlying the role of oxygen and its reactive derivatives in the pathogenesis of diseases that remain to be fully understood and in developing innovative therapeutic strategies to minimize if not prevent oxygen- or reactive species–mediated cellular injuries.

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