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THE HANDBOOK OF REDOX BIOCHEMISTRY Ian N. Acworth, D.Phil., Oxon.

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  • THE HANDBOOK OF REDOX BIOCHEMISTRY

    Ian N. Acworth, D.Phil., Oxon.

  • This book is dedicated to Emma Louise and Kimberly Ann. I would like to thank the following people. For their help with editing Debbie Aldrich, Kim Acworth, John Waraska, Bruce Kristal and Paul Gamache. For their support Scott Freeto, Bruce Bailey, Wayne Matson, Walter DiGiusto, M. Bogdanov, and many other members of ESA, Inc. For his advice, willingness to help, and for the Sunrise Free Radical Schools Prof. Garry Buettner. For continued help Dr. Ken Hensley, Dr. K. Williamson, and Prof. R. Floyd (Oklahoma Medical Research Foundation).

    I would like to acknowledge all researchers in this field without whose work this handbook could not have been completed.

    ESA, Inc. 22 Alpha Road

    Chelmsford, MA 01824-4171 USA (978) 250-7000

    Sales (800) 959-5095 Fax (978) 250-7090

    www.esainc.com An ISO 9001 Company

    ESA Analytical, Ltd. Brook Farm, Dorton

    Aylesbury, Buckinghamshire HP18 9NH

    England, UK 01844 239381

    Fax 01844 239382

    ii

  • Preface

    It has been several years since I co-wrote The Handbook of Oxidative Metabolism with my colleague Dr. Bruce Bailey, showing the use of electrochemical approaches in the study of free radical production, macromolecular damage and antioxidant protection. Since 1995, thousands of copies of the Handbook have been requested. It has been translated into other languages. It has even been used as a basic course work at university. But the field has moved rapidly and the original Handbook is now dated. I have now updated the old Handbook and renamed it The Handbook of Redox Biochemistry for reasons explained in the text. Although now greatly expanded it is, by necessity, selective in content. For readers wanting a more in depth view of the whole field I refer them to the excellent books by Halliwell and Gutteridge, Gilbert and Colton and several others mentioned in the reference section accompanying each chapter. I would very much appreciate having any errors, omissions or new findings brought to my attention. I can be contacted on: [email protected] Ian Acworth August 2003

    iii

  • CONTENTS

    Frontis i-ix Chapter 1. Introduction. Oxygen Toxicity From Microbes To Man. 2 Why Is Oxygen Toxic? 8 Free Radical Pro-Oxidants. 9 Reactive Oxygen Species, Reactive Nitrogen Species And Other Pro-Oxidants 17 How Do Aerobic Organisms Survive Even When Pro-Oxidants Are Being Continuously Produced? 18 Why Use Electrochemical Detection? 23 Conclusions. 24 References. 25 Chapter 2. The Chemistry Of Reactive Species. Oxygen And The Reactive Oxygen Species (ROS). 37 1. Oxygen. 37 Properties. 37 Formation. 37 Chemical Reactions And Biological Significance 38 2. Ozone. 42 Properties. 42 Formation. 42 Chemical Reactions And Biological Significance. 42 Measurement. 45 3. Singlet Oxygen. 46 Properties. 46 Formation. 46 Chemical Reactions And Biological Significance 48 Measurement. 49 4. Superoxide (Radical Anion). 49 Properties. 49 Formation. 50 Electron Transport Chains. 50 Immune Defense. 54

    Enzymes Reactions. 54 Oxygen-Heme Interaction 54 Metal-Catalyzed Auto-Oxidation 54 Chemical Reactions 55

    Biological Significance. 56 The Pro. 56 The Con. 58 Control. 59

    Measurement. 59 Auto-Oxidation And Redox Cycling 59 5. Hydrogen Peroxide. 63 Properties. 63 Formation. 63 Chemical Reactions And Biological Significance. 64 Measurement. 66 6. The Hydroxyl Free Radical. 67

    iv

  • Properties. 67 Formation. 67 Chemical Reactions And Biological Significance. 67 Measurement. 70 EPR. 70 HPLC. 72 Nitrogen And The Reactive Nitrogen Species (RNS). 82

    1. Nitrogen. 82 Properties. 82 Formation. 82 Chemical Reactions. 82

    2. The Oxides Of Nitrogen. 84 2.1 Nitric Oxide. 85 Physical Properties. 85 Formation. 86 Chemical Reactions And Biological Significance. 89 Measurement. 94 2.2 The Nitroxyl Anion And Nitrosonium Ion. 97 2.3 Peroxynitrite. 98 Properties. 98 Formation. 98 Chemical Reactions And Biological Significance. 100 Measurement. 106 2.4 Nitrosoperoxycarbonate And Nitrocarbonate. 106 2.5 Nitrogen Dioxide, The Nitronium Cation, And Nitrite. 107 Properties. 107 Formation. 107 Chemical Reactions And Biological Significance. 107 Measurement. 109

    2.6 The Higher Oxides Of Nitrogen Dinitrogen Trioxide, Dinitrogen Tetroxide And Dinitrogen Pentoxide. 110 Properties. 110

    Formation. 110 Chemical Reactions And Biological Significance. 110 Measurement. 111

    2.7 S-Nitrosothiols. 112 Properties. 112 Formation. 112 Chemical Reactions And Biological Significance. 113 Measurement. 114 Halogenated Reactive Species (RHS). 114 1. Chlorine And Hypochlorous Acid. 114 Properties. 114

    Formation. 114 Chemical Reactions And Biological Significance. 116 Measurement. 118

    2. Nitrosyl Chloride, Nitryl Chloride And Related Compounds. 118 Properties. 118 Formation. 118 Chemical Reactions And Biological Significance. 119 Measurement. 120 Sulfur, Thiols And Thiyl Radicals (Some Reactive Sulfur Species [RSS]). 121 Properties. 121

    Chemical Reactions And Biological Significance. 121 Measurement. 125 Carbonyl Compounds. 126

    v

  • Properties. 126 Formation. 126 Chemical Reactions And Biological Significance. 128 Measurement. 129 The Pro-Oxidant Activity Of Low Molecular Weight Compounds And Other Xenobiotics 131 References. 134 Appendix 2.1 Background To Electrode Potentials. 150 Thermodynamics Of Reversible Cells. 150 Standard Electrode Potentials. 153 Some Comments On SEPs. 155 Coupled Redox Reactions. 158 References. 158 Appendix 2.2 Background To Kinetics. 159 First-Order Processes. 160 Second-Order and Pseudo-First-Order Processes. 160 Some Published Second-Order Rate Constants. 161 Measurement Of Reaction Order And Reaction Rates. 165 References. 165 Appendix 2.3 Background To The White Blood Cell. 167 Granulocytes. 168 Lymphocytes. 168 Monocytes. 169 Chapter 3. Damage And Repair. DNA 171 Introduction. 171 The DNA Molecule. 171 DNA Damage. 175 The Consequences Of Oxidative DNA Damage. 185 Repair Of ROS/RNS-Induced Damage. 187 Base Excision Repair. 188 Nucleotide Excision Repair. 190 Mitochondrial DNA Repair. 190 Single Strand DNA Damage And PARP Activation. 190

    What Do The Levels Of DNA Adducts Mean? 191 Steady State Levels. 191 Total Adduct Levels. 198 Measurement Of DNA Damage. 203 Gas- And Liquid-Chromatography-Mass Spectrometry. 205 HPLC. 206

    Postlabeling assays. 208 Immunochemical detection. 209

    The Measurement Of 8-Hydroxy-2deoxyguanosine In Urine. 210 DNA Damage In Health And Disease. 213

    Amino Acids And Proteins. 214 Introduction 214

    Protein Molecular Structure 215 Pro-oxidants And Protein Damage. 218

    The Indirect Pathway. 218 The Direct Pathway. 222

    Oxidative Damage To Tyrosine. 226 Protein Repair And Degradation. 230 Amino Acid And Protein Damage In Aging And Disease. 234

    vi

  • Measurement Of Amino Acid And Protein Damage. 237 Whole Protein. 237 Protein Hydrolysates. 240

    Measurement Of Free Modified Amino Acids And Modified Residues In Whole Proteins And Protein Hydrolysates 241 1. Protein Carbonyls. 241

    2. Methionine Sulfoxide. 241 3. 2-Oxohistidine. 241 4. Tyrosine Markers. 242 3-Nitrotyrosine. 242 3-Chlorotyrosine. 246 Dityrosine. 248 Other Tyrosine Oxidation Products. 248 Lipids. 248

    Introduction. 248 Structure Of Biological Membranes. 249 Lipid Damage. 251 The Role Of Metals In Lipid Peroxidation. 256 Lipid Oxidation Products. 257 Malondialdehyde. 260 4-Hydroxyalkenals. 262 Other Reactive Carbonyls. 264 Cholesterol Oxidation. 264

    The Isoprostanes. 266 Lipid Repair. 268

    Lipid Damage And Disease. 269 Measurement Of Lipid Damage. 270 Diene Conjugates. 272 TBAR. 272 Carbohydrates. 276 Introduction. 276

    Ribose And Deoxyribose Damage. 277 Glycation, Glyoxidation, Advanced Glycation End Products (AGES) And Age-Related Pigments. 278

    References. 280 Appendix 3.1 Typical DNA Extraction And Hydrolysis. 305 DNA Extraction Procedure. 305 DNA Hydrolysis Procedure. 307 Chapter 4. Protection. Introduction. 309 Enzymes. 310 Catalases. 315 Peroxidases. 317

    The Biological Significance Of Catalase And Glutathione Peroxidase. 319 Glutathione-S-Transferase. 320

    Heme Oxygenases. 320 Superoxide Dismutases. 321

    The Catabolism Of Nitric Oxide. 323 Sequestration Of Metal Ions. 324 The Metabolism Of Iron And Copper. 326

    Iron And Copper Species As Pro-Oxidants. 329 Measurement Of Iron And Copper. 330 Low Molecular Weight Molecules. 330

    vii

  • Water-soluble antioxidants. 330 Albumin. 330 Ascorbic Acid. 340 Antioxidant Properties. 340 Pro-oxidant Properties. 344 Measurement. 345 Thiols. 346 1. Glutathione. 346 Biological Roles Of Glutathione. 347 Protection. 347 Detoxification And Bioactivation. 349 Cofactor. 350

    Storage Of Cysteine In A Non-Toxic Form. 351 Amino Acid Transport. 351

    Regulation. 351 Compartmentalization. 352

    Conditions And Diseases Affecting Glutathione. 352 Measurement Of Glutathione And Its Disulfide. 352.

    2. Homocysteine. 355 3. Miscellaneous Endogenous Sulfur-Containing

    Compounds. 358 Uric Acid. 359

    Formation. 359 Xanthine Oxidase And Tissue Injury. 360 Antioxidant And Pro-Oxidant Activities. 361 Measurement. 363 Fat-Soluble Antioxidants. 363 Carotenoids 363 Carotenoids And Disease. 364

    Antioxidant And Pro-Oxidant Activities Of Carotenoids. 365

    Retinoids. 367 The Biological Activity Of The Retinoids. 368 Antioxidant And Pro-Oxidant Activities Of The Retinoids. 369

    Measurement Of Carotenoids And Retinoids. 369 Quinones And Hydroquinones. 372

    Coenzyme Q (Ubiquinone, Ubiquinol). 372 Biology Of Coenzyme Q. 373

    Antioxidant And Pro-Oxidant Activities Of Coenzyme Q 375

    Measurement Of Coenzyme Q. 377 Plastoquinone. 378 Vitamin K. 378 Pyrroloquinoline Quinone. 380

    Tocopherols 380 Biology Of Tocopherols 382

    Antioxidant, Pro-Oxidant And Other Reactions Of The Tocopherols 383 Tocopherol And Disease. 387

    Measurement Of Tocopherols And Their Metabolites. 387 Other Endogenous And Exogenous Metabolites Proposed As Antioxidants. 391

    Bile Pigments. 391 Biogenic Amines. 391 Estrogen. 395

    viii

  • Histidine Derivatives. 396 Indoles And Related Compounds. 397 -Ketoacids. 398 -Lipoic, Dihydrolipoic Acids And Analogs. 399 Melanins 401 Melatonin. 402 Phytochemicals. 406

    Simple Phenolic Acids. 406 Flavonoids. 410

    Phytoestrogens. 415 Resveratrol. 418 Phytic Acid. 419 Sulfur-Containing Compounds. 419 Pteridines. 420

    Antioxidant Therapy. 421 Enzymes. 421 Chelators. 422 Low Molecular Weight Molecules. 424 Estimating The Total Antioxidant Capacity. 432 Antioxidants As Food Preservatives. 438 References. 441 Index. 480

    ix

  • Chapter 1 Introduction

    Mention a pure science such as chemistry or biology and most people will have a fair idea about the subject matter. Unfortunately, for those interested in studying the effects of reactive species on living organisms, no succinct and accurate descriptor of this field exists. Several general titles have been used over the years including free radical biology, redox chemistry and redox biology, yet none of them do justice to this complex, multi-disciplined field. While free radical biology ignores the fact that many chemical species being studied are not free radicals, redox chemistry implies a disregard for any biological aspects. Oxidative metabolism has been used but this is usually associated with energy metabolism. Although still not perfect I prefer the term Redox Biochemistry. I will discuss free radicals and redox reactions in greater detail below.

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  • OXYGEN TOXICITY FROM MICROBES TO MAN

    Oxygen is toxic to aerobic (and anaerobic) organisms, yet paradoxically oxygen is essential for their survival. Today terrestrial aerobes (both animals and plants) have successfully adapted to live in an atmosphere composed of approximately 21% oxygen and can survive minor fluctuations in the level of respired oxygen without disastrous consequences. True anaerobes, on the other hand, tolerate oxygen poorly, and some cannot survive even a brief exposure to atmospheric oxygen (Table 1.1). Anaerobes were the first living organisms on the planet. These evolutionary simple organisms show a wide range of oxygen tolerance. Strict or obligate anaerobes will only grow if oxygen is absent. While some obligate anaerobes are killed almost immediately following exposure to oxygen (aerophobic) (e.g., Clostridia species) others can survive for many days but cannot reproduce (e.g., Bacteroides fragilis). Another group of organisms, microaerophiles actually require some oxygen for growth but cannot survive when exposed to atmospheric oxygen concentrations. Most bacteria that reduce nitrate (producing nitrite, nitrous oxide or nitrogen) are called facultative anaerobes as they are not affected by exposure to oxygen and in fact will preferentially use oxygen, rather than nitrate, during respiration. Anaerobes can be found in any environment where oxygen levels are decreased to less toxic levels including muds and other sediments; bogs and marshes; polluted waters; certain sewage-treatment systems; rotting material; deep underground areas such as oil pockets; the sources of springs; decaying teeth and gangrenous wounds; the colon; and inappropriately canned foods. Rather than using oxygen during respiration (they usually lack terminal cytochromes that transfer electrons to oxygen) they use other electron acceptors such as ferric ions, sulfate or carbon dioxide which become reduced to ferrous ions, hydrogen sulfide and methane, respectively, during the oxidation of NADH (reduced nicotinamide adenine dinucleotide is a major electron carrier in the oxidation of fuel molecules) (Figure 1.1). Oxygen is toxic to anaerobes as it can affect the organisms internal homeostasis by altering its reductive capacity, consuming compounds such as NAD(P)H, thiols and other chemicals essential for biosynthetic reactions and inactivating key enzymes.

    Although anaerobes had free range during the early stages of the evolution of living organisms, this was eventually curtailed by the success of oxygen-producing photosynthetic plants. With the levels of oxygen rising in the atmosphere, anaerobes had three choices, adapt, find niches where oxygen would not penetrate, or die. Organisms eventually evolved that not only survived in an oxygen-enriched atmosphere but prospered. Evidence suggests that the atmospheric oxygen levels have fluctuated markedly over time, increasing from 15-18% in the late Devonian to as high as 35% in the late Carboniferous and early Permian periods. This hyperoxia has been suggested to be one of the

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  • possible causes of the mass extinction of terrestrial vertebrates (Graham et al. 1995). Atmospheric oxygen finally stabilized at todays level (at least to date).

    1) Sulfate Reduction (e.g., Desulfovibrio (water- logged soils), Desulfomaculum (spoilage of canned foods),

    ATP + SO42- Adenosine phosphosulfate (APS) + PPi

    Step 1: Sulfate is activated.

    Step 2: A hydrogenase splits molecular hydrogen. Reduction of APS produces sulfite.

    APS + H2 SO32- + AMP + H2OCyt c3

    Step 3: Electrons derived from hydrogen reduce sulfite to hydrogen sulfide

    SO32- + 6H+ + 6e- H2S + H2O + 2OH-

    Desulfomonas (intestines), Archaeglobus (a thermophile)):

    2) Methanogenesis pathway (e.g., Methanebacterium thermoautotrophicum )

    Carbon Dioxide Formylmethanofuran N5-Formyl-5,6,7,8-tetrahydromethanopterin

    N5, N10-Methenyltetrahydromethanopterin

    N5, N10-Methylenetetrahydromethanopterin

    5-Methyl-5,6,7,8-tetrahydromethanopterin

    Methane

    Methanofuran+ 2H+ + 2e-

    H2O H4MPT Methano-furan H2O

    F420H2F420

    F420H2

    F420

    CoM H4MPT

    Methyl-Coenzyme M

    HTP

    CoM-S-S-HTP

    CO2 + 8H+ + 8e- = CH4 + 2H2O Figure 1.1 Anaerobic Metabolism.

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  • Glucose

    Glucose 6-phosphate

    Fructose 6-phosphate

    Fructose 1,6-bisphosphate

    Dihydroxyacetone phosphate 2 x Glyceraldehyde 3-phosphate

    2 x 3-Phosphoglyceroyl phosphate

    2 x 3-Phosphoglycerate

    2 x 2-Phosphoglycerate

    2 x Phosphoenolpyruvate

    2 x Pyruvate2 xLactate

    Glycogen

    ATP

    ADP

    ATP

    ADP

    2ATP

    2ADP

    2ATP

    2ADP

    2NAD+ + 2Pi

    2NADH

    -ATP

    -ATP

    +2ATP

    +2ATP

    +2ATP

    2NAD+ 2NADH2CO2

    2 x Ethanal

    2 x Ethanol

    2NAD+

    2NADH

    Substrate-levelphosphorylation

    =

    Triglycerides

    Glycerol

    Fatty Acids

    ATP

    ADP

    NAD+NADH + H+

    Someaminoacids

    To Tricarboxylic Acid Cycle

    AnaerobicGlycolysis

    Anaerobic Fementation

    Figure 1.2 The Glycolytic Pathway And The Production Of ATP.

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  • Group Oxidizing Conditions

    Reducing Conditions

    Effect of Oxygen Example

    Aerobe- Obligate

    Growth No growth Essential Many bacteria, most fungi, algae, protozoa, all higher plants and animals

    Aerobe-Facultative

    Growth Growth Not required but better if oxygen is present

    Bacteria such as enteric and pathogenic species; some protozoa, yeasts (e.g., Saccharomyces) and fungi

    Anaerobe-aerophobic (obligate; strict)

    Death Growth Harmful Many bacteria some protozoa. Bacteroides, Clostridia, Fusobacterium, Methanobacterium, and Ruminococcus

    Anaerobe-aerotolerant (moderate)

    Growth Growth Not required but better if oxygen is present

    Bacteroides fragilis, Treponema pallidum

    Microaerophile Growth if oxygen level is not too high

    Growth if oxygen level not too low

    Required but at only low levels

    Campylobacter jejuni

    Table 1.1 The Effects Of Environment And Oxygen On Growth Of Aerobes And Anaerobes Facultative aerobes (Table 1.1) can survive in the presence or absence of oxygen. They obtain their energy either by oxidative phosphorylation or fermentation and do not require oxygen for synthesis. When oxygen is lacking this group of organisms can oxidize some organic compounds (which act as both electron donors and acceptors) with a small release of energy, in a process called fermentation. A variety of compounds can be fermented including most sugars, many amino acids, some organic acids, purines, pyrimidines and a variety of miscellaneous products. The energy is captured as two molecules of adenosine triphosphate (ATP) in a process termed substrate level phosphorylation. ATP is the cells immediate energy providing molecule and is used for growth, movement, and in biochemical processes e.g., biosynthesis and maintenance of ionic gradients. The stepwise breakdown of glucose into pyruvate is called glycolysis and occurs in both facultative and obligate aerobes (Figure 1.2). In fermentation, pyruvate produced by glycolysis is converted to ethanol or lactate (Figure 1.3). In the presence of oxygen however, glycolysis is

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  • followed by aerobic respiration and pyruvate is completely oxidized to carbon dioxide and water (Figure 1.4). The oxidation of pyruvate takes place in a series of steps called the tricarboxylic acid cycle (TCA) (also called the citric acid or Krebs cycle) that occurs in the mitochondrion (Figure 1.4). During aerobic respiration the oxidation of glucose generates 36 molecules of ATP. Two ATP molecules are generated by substrate level phosphorylation (part of the cytosolic glycolytic pathway) and two are produced by substrate level phosphorylation occurring in the mitochondrion. However, the vast majority, thirty-two ATP molecules, are produced by mitochondrial oxidative phosphorylation when electrons are transferred from NADH or flavin adenine dinucleotide (reduced) (FADH2) to oxygen by a series of electron carriers. Thus it can be seen that aerobic respiration generates much more energy than anaerobic processes. For example, if pyruvate is completely oxidized by the TCA cycle then yeast will be able to form 19 times more energy from a given amount of glucose when growing aerobically than when growing anaerobically.

    GLUCOSE Glycolysis

    PYRUVATE

    CO2 + H2O

    AEROBICOxidativePhosphorylation(TCA/electron

    transport)

    LACTATE

    ACETALDEHYDE ETHANOL

    ANAEROBIC

    Figure 1.3 The Metabolic Fate Of Pyruvate.

    Obligate aerobes (e.g., higher plants and animals) use oxygen in respiration and for the biosynthesis of a variety of biomolecules. All higher organisms are obligate aerobes but they can make use of both anaerobic and aerobic processes. For example, many tissues such as the red blood cell, the cornea of the eye, the skin, the kidney medulla and type IIb (fast twitch-glycolytic) skeletal muscle fibers make use of anaerobic glycolysis. Here the two molecules of ATP produced by the anaerobic conversion of glucose to lactate is sufficient to supply most of these tissues normal energy needs. However, as the average human requires more than 40kg/day of ATP, and as much as 0.5kg/minute when undergoing strenuous exercise, anaerobic respiration simply cannot keep pace with this demand. Rather, higher organisms must obtain the vast majority of their energy from aerobic respiration, and that is why oxygen is essential for their survival.

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  • Pyruvate

    Acetyl CoA

    Citrate

    Isocitrate

    a-ketoglutarate(2-oxoglutarate)

    SuccinylCoA

    Succinate

    Fumarate

    Malate

    Oxaloacetate

    NAD+

    NADH + CO2

    O2

    H2O

    3ATPNAD+

    NADH + CO2

    O2

    H2O

    3ATP

    GDP + PiGTP + CoASH

    Substrate-levelPhosphorylation

    GDP

    ADP

    ATP

    FADFADH2

    O2

    H2O

    2ATP

    H2O

    NAD+

    NADH

    O2

    H2O

    3ATP

    Electron transport chain

    CoA + NAD+

    NADH + CO2

    Fatty Acids

    xNADH, xFADH2

    Acetoacetyl CoA

    Amino Acids

    AminoAcids

    Glycolysis

    Figure 1.4 The Tricarboxylic Acid Cycle. Obligate aerobes are very oxygen sensitive. A total lack of oxygen is referred to as anoxia and rapidly results in cell death. For example, brain damage can result from perhaps as little as three minutes of anoxia. An acute decrease in respired oxygen leads to hypoxia, a situation where oxygen is still delivered to the tissue, but at a rate insufficient to maintain normal cellular processes. The effects of hypoxia depend upon the tissue and the degree and duration of the hypoxic event. For example, the brain is a very aerobic tissue and is exquisitely sensitive to oxygen tension. In higher animals an acute reduction in arterial oxygen tension

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  • leads to altered mental function, analgesia and loss of muscle coordination (Blass and Gibson (1979); Gibson and Blass (1976); Gibson et al. (1978; 1981)). A more marked drop can result in unconsciousness, progressive depression of the central nervous system, circulatory failure and death.

    Ischemia is a consequence of mechanical disruption of blood flow to a tissue resulting in decreased oxygen, glucose and ATP levels. For example, the occlusion of essential blood vessels to the heart (a consequence of atherosclerosis and/or blood clots) results in ischemia. This leads to myocardial damage and heart attack. It has been estimated that irreversible myocardial damage can occur after about 20 minutes of ischemia (Sobel (1974)). The affected tissue eventually dies.

    Exposure to elevated levels of oxygen results in hyperoxia and is deleterious to aerobic microorganisms, plants and animals. The growth of aerobic bacteria is inhibited following exposure to pure oxygen. Plants show decreased chloroplast development and leaf damage when exposed to oxygen levels above normal. Animals exposed to 100% oxygen show a variety of symptoms depending upon the duration of exposure (Crapo et al. (1980); Francica et al. (1991)). Humans suffer chest soreness, coughing and sore throats following several hours of exposure to pure oxygen. Longer periods cause alveolar damage, edema and permanent irreversible lung damage. Hyperoxia also leads to damage to most of the major organs. Unfortunately, earlier this century unintentional retinal damage and blindness (retrolental fibroplasia) was caused to premature babies when they were maintained on high oxygen levels in their incubators. Fortunately, the level of oxygen to which premature babies are exposed is now more carefully monitored. It should be noted, however, that hyperoxia can also be beneficial. For example, hyperbaric oxygen is used to treat gangrene because of its toxicity to the obligate anaerobes that cause it. Correct oxygen tension is important to deep sea divers, astronauts, mountain climbers, athletes going from low to high elevations and those undergoing general anesthesia. Oxygen tension is also important in preventing the growth of harmful anaerobic pathogens in canned and bottled foods and beverages.

    WHY IS OXYGEN TOXIC?

    Over the years, several theories have been put forward to explain oxygens toxicity. This subject was reviewed recently by Gilbert (1999) so only an overview will be presented here.

    One early hypothesis as to oxygens toxicity was that oxygen exerted its action through enzyme inhibition. For example, oxygen can inhibit nitrogenase and the first enzyme in the dark reactions of photosynthesis,

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  • ribulose 1,5-bisphosphate decarboxylase, and at high concentrations some thiol-containing enzymes (Haugaard (1946); Stadie et al. (1944)). However, enzyme inhibition is far too slow and limited to explain oxygens toxic effect, and not all enzymes are affected by oxygen.

    Abundant evidence showed that irradiation caused DNA damage and cancer through a free radical mechanism and that oxygen had a sensitizing effect (von Sonntag (1991) and references therein).

    In the mid 1950s Gerschman and Gilbert proposed that oxygen, itself a diradical, may exert its toxic action through the formation of free oxygen radicals. These could then damage biologically important macromolecules such as DNA, proteins and lipids (see Gerschman (1981); Gerschman et al. (1954); and reviews by Gilbert (1999); Halliwell and Gutteridge (1993)). This breakthrough proposal, however, was initially strongly criticized by researchers who proposed that free radicals were far too reactive to exist in any great quantity in biological materials. These objections were finally laid to rest by the detection of free radicals both in dry biological tissues and in living organisms by electron spin resonance (Commoner et al. (1954, 1957)).

    In 1954 Harman developed his free radical theory of aging that postulated that a single common process, modifiable by genetic and environmental factors, was responsible for the aging and death of all living things (Harman (1956; 1992a,b)). His theory proposed that the accumulating irreversible damage to biologically important macromolecules over time led to disease and aging.

    Free radicals were further implicated by the discovery of the enzyme superoxide dismutase (SOD). Fridovich theorized that the superoxide radical anion was the major toxic form of oxygen and that SOD protected against it (Fridovich (1983, 1986a,b); McCord and Fridovich (1969)). The superoxide theory of oxygen toxicity, though not completely correct, was responsible for a great deal of experimental work and a better understanding of the field as a whole (reviewed in Halliwell and Gutteridge (1993)).

    We now know that oxygen mediates its toxic effects through a variety of compounds, not just free radicals, many of which contain other atoms in addition to oxygen. The properties of these species will be dealt with in Chapter 2.

    FREE RADICAL PRO-OXIDANTS. The term radical originally used by chemists referred to an ionic group that had either positive or negative charges associated with it (e.g., carbonate, sulfate etc.). A free radical is now defined as an atom or molecule that has one or more unpaired electrons (i.e., electrons that occupy atomic or molecular orbitals by themselves) and is capable of independent existence. In the strictest sense the free of free radical, is redundant. It may come as some surprise that oxygen is a

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  • free radical (in fact a diradical) as are metals that have incomplete 3d shells (e.g., transition metals and their various oxidation (valency) states) (Table 1.2). Scandium 1s22s22p63s2 3p63d14s2 Sc3+

    (NT) 3p6

    Titanium 1s22s22p63s23p63d24s2 Ti2+ 3p6d2 Ti3+ 3p6d1

    Vanadium 1s22s22p63s23p63d34s2 V2+ 3p6d3 V3+ 3p6d2

    Chromium 1s22s22p63s23p63d44s2 Cr2+ 3p6d4 Cr3+ 3p6d3 x 1s22s22p63s23p63d54s1 Mn2+ 3p6d5 Mn3+ 3p6d4

    Iron 1s22s22p63s23p63d64s2 Fe2+ 3p6d6 Fe3+ 3p6d5

    Cobalt 1s22s22p63s23p63d74s2 Co2+ 3p6d7 Co3+ 3p6d6

    Nickel 1s22s22p63s23p63d84s2 Ni2+ 3p6d8

    Copper 1s22s22p63s23p63d104s1 Cu2+ 3p6d9 Cu+ (NT)

    3p6d10

    Zinc (NT) 1s22s22p63s23p63d104s2 Zn2+ (NT)

    3p6d10

    Table 1.2 The Electronic Configuration Of The Atoms Of First Transition Series And Some Of Their Ions. (NT non-transition. Note that NT compounds are also non-radicals.) Free radicals can be formed when a non-radical either gains or loses a single electron (Table 1.3). Free radicals can be formed during homolytic fission of covalent bonds. The energy required to cause bond dissociation can be brought about by several different processes, including exposure to heat or electromagnetic radiation, or by chemical reaction. Remember that covalent bonds are formed when two atoms share electrons (usually one from each atom). During homolytic fission one electron of the bonding pair is retained by atom A, while the other is retained by atom B forming the free radicals A and B, respectively. During homolysis of water, for example, the hydroxyl free radical (HO) and the hydrogen atom (H) are produced. Radical reactions are much more common in the gas phase and at high temperatures, e.g., combustion. Readers should be aware that many radical reactions found in the literature (especially chemistry texts) may be for gas phase reactions and are not always applicable to biological systems. Having said this, gas phase free radical chemistry is extremely important to those investigating the effects of atmospheric pollution and cigarette smoke on biological systems.

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  • 1. Heat. Radicals produced during combustion or by heating in absence of oxygen, e.g., CC, CH bonds typically require 450-600oC 2. Electromagnetic radiation. Including ionizing irradiation (e.g., x-rays, -rays) and photolysis (e.g., UV absorption)

    3. Redox reactions. Radicals are produced in reactions involving one-electron transfer: inorganic ions (e.g., ArN2+ + Cu+ Ar + N2 + Cu2+; Sandmeyer reaction) metals (e.g., H2O2 + Fe2+ Fe3+ + HO + OH-, Fenton reaction) electrolysis (e.g., 2RCO2- - e- 2RCO2 RR; Kolbe synthesis) hydroquinone-semiquinone-quinone systems (e.g., production of superoxide

    from oxygen by ubiquinol/ubiquinone redox couple)

    4. Enzymatic. Radicals are produced by the action of peroxidases (e.g., horseradish peroxidase) or oxidases (e.g., xanthine oxidase)

    5. Chemical. By the reaction of hydroxyl free radical with a variety of substrates By the reaction of peroxynitrite with a variety of substrates As part of enzyme catalyzed reactions By reactions involved in the generation of O2- during mitochondrial respiration By the reaction of oxygen with other radicals: Production of lipid peroxyl radical when oxygen reacts with an alkyl radical Production of peroxynitrite radical when oxygen reacts with nitric oxide By thermal decomposition of azo initiators (R-N=N-R): 2,2-azo-bis(2-amidinopropane) dihydrochloride [AAPH] for aqueous systems 2,2-azo-bis(2,4-dimethylvaleronitrile) [AMVN] for lipophilic systems By thermal decomposition of organic peroxides: Di-tert-butyl peroxide Dibenzoyl peroxide

    6. Ultrasound. Also called sonochemical production. Primary radicals (e.g., H and HO) are produced due to pyrolysis of molecules located within collapsing cavitation microbubbles, while secondary radicals are formed by hydrogen abstraction or addition of primary radicals to other molecular species

    7. Lithotripsy. Radicals are produced when high-energy shock waves are used to destroy solid objects, e.g., kidney stones

    8. Lyophilization. Radicals can be produced by freeze-drying/thawing processes

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  • Table 1.3 Free Radicals Can Be Produced In A Variety Of Ways. This table summarizes both in vitro and in vivo approaches for free radical production. (Crum et al. (1987); Doss and Swartz (1984); Fuciarelli et al. (1995); Halliwell and Gutteridge (1999); Heckly and Dimmick (1967); Hendrickson et al. (1970); Kondo et al. (1993); Misik and Riesz (1999); Misik et al. (1996, 1999); Morgan et al. (1988); Ostrowski (1969); Seel et al. (1991); Suhr et al. (1994); Vreugdenhil et al. (1991); Worthington et al. (1997)).

    A wide variety of radicals can exist (Table 1.4). Like any other chemical, radicals show a broad spectrum of physical and chemical properties. Some are stable and unreactive, whereas others react extremely rapidly. Some are hydrophobic while others hydrophilic. Radicals may share certain common characteristics and can be grouped together as presented in the following table. Unfortunately, as will be readily apparent such classification is not perfect as some radicals can belong to more than one category. For example, some sigma radicals are also carbon-centered monoradicals.

    Radical Examples (sigma) H (hydrogen atom), R (carbon-centered radical), R3C (pi) Ascorbyl, Tocopheryl, NAD

    Monoradicals R, R3C, NO Polyradicals O2 (a diradical) Carbon centered R, R3C Oxygen centered LO2

    Sulfur centered RS, RSO2

    Nitrogen centered NO, R2NO, NO2 Reducing CO2-, PQ-

    Oxidizing HO, LO2

    Metal Cu2+, Fe2+, Fe3+

    Table 1.4 Different Types Of Radicals. R is used as an abbreviation for an alkyl group, L represents a lipid (e.g., fatty acid). Based on an original by G.R. Buettner.

    Of all the radicals that can be formed sigma () radicals (e.g., the methyl radical, CH3) are generally much more reactive than pi () (e.g., the tocopherol-derived radical, tocopheryl) as their lone electron cannot be spread throughout the molecule (delocalized). -Radicals are generally less reactive than ones because the lone electron is not confined to just one atom, but is delocalized through the conjugated -bond system (Sykes (1975)). A physiological consequence is that -radicals play an important role in initiating lipid peroxidation while chain-breaking antioxidants prevent lipid peroxidation by reacting with the -radicals forming a much less energetic and less dangerous -radical species.

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  • Most free radicals have very short lifetimes. Without stabilizing features (e.g., delocalization or steric hindrance) they decompose rapidly, often in the absence of external agents. Decomposition is usually through:

    1. Unimolecular reactions (e.g., fragmentation or rearrangement), 2. Bimolecular reactions between radicals including dimerization (e.g., the

    formation of peroxynitrite from nitric oxide and the superoxide radical anion or the formation of hydrogen peroxide from two hydroxyl free radicals) or disproportionation (e.g., the formation of hydrogen peroxide and oxygen from two hydroperoxyl radicals1) which can involve electron or hydrogen atom transfer or

    3. Bimolecular reactions between radicals and other molecules (e.g., addition, displacement, or atom [often H] abstraction). Further information can be found in good chemistry texts.

    Phase Example Initiation

    Fe2+ + H2O2 Fe3+ + HO + OH-

    LH + HO L + H2O

    Propagation

    L + O2 LO2 LO2 + LH LO2H + L

    Termination

    L + L LL (dimerization) LO2 + L LL + O2 2LO2 non-radical products 2C2H5 C2H6 + C2H4 (disproportionation)

    reduced oxidized

    Table 1.5 The Three Phases Of Chain Reactions. (L represents a lipid undergoing peroxidation.)

    In biological systems the most infamous free radical cascade is the lipid peroxidation chain reaction (Table 1.5). Here a single initiation process can lead to the destruction of many poly-unsaturated fatty acid molecules. Unfortunately, not only does this affect membrane fluidity and thus many biochemical processes, but it can also lead to the production of cytotoxic carbonyl breakdown products (Chapter 3). Lipid peroxidation is also the major process responsible for food spoilage. Like any other chain reaction, lipid peroxidation consists of three phases termed a) initiation, b) propagation and c) termination. Biological systems are equipped with several mechanisms designed to prevent lipid peroxidation. Such processes include prevention of radical formation (inhibiting initiation) or 1 Note during disproportionation one species is reduced while the other is oxidized. involving electron or hydrogen atom transfer),

    WWW.ESAINC.COM 13

  • interception of fatty acid radicals once formed (inhibiting propagation). Biological systems are also capable of repairing damage that occurs. Several techniques can be used to measure free radicals. Electron paramagnetic resonance [EPR] (also called electron spin resonance, or ESR) is a very useful technique and is the only way to directly measure radicals. EPR makes use of the fact that the unpaired electron in a free radical has spin (either +1/2 or 1/2) and thus behaves as a small magnet (i.e., is paramagnetic). When placed in an external magnetic field the unpaired electron can align itself, either parallel or antiparallel, to that field (i.e., the free electron only has two possible energy levels). Exposure to electromagnetic radiation of the correct energy will move the electron from the lower energy level to a higher excited one. Thus an absorption spectrum is obtained which can be used for quantitation as well as gaining information about the environment surrounding the free radical (see Halliwell and Gutteridge (1993)). Direct EPR methods have a sensitivity limit of 0.1nmol/L and have been used extensively for in vitro work (e.g., to study the mechanism of enzyme action) but are often not selective enough for most in vivo work. Some researchers are, however, developing these techniques. Many free radicals are too reactive (e.g., HO) and have too short a half-life for direct EPR methods. This can be overcome by using spin-trap agents that react with the free radical to produce a longer-lived species that is still paramagnetic (Figure 1.5). Interestingly, spin traps are also proving to be beneficial in the treatment of diseases thought to involve oxidative stress where they probably act to scavenge damaging free radicals. For example, -phenyl-tert-butylnitrone (PBN) is being used at pharmacological levels to decrease ischemia-reperfusion injury in brain (Floyd (1990), Folbegrova et al. (1995)) and dog heart (Bolli et al. (1988)); reduce the size of liver edema in carbon tetrachloride intoxicated rats (Towner et al. (1993)); reduce the mortality associated with endotoxic shock in rodents (Miyajima and Kotake (1997) and references therein) and prolong the life span of the senescence-accelerated mouse model (Edamatsu et al. (1995)). The correct choice of a spin-trap agent is important. The ideal spin-trap should readily and specifically react with the radical of interest. It must also produce an adduct of sufficient longevity which possesses a characteristic EPR spectrum. It should never decompose during experimentation producing free radicals (see Halliwell and Gutteridge (1993)). Further limitations are placed upon a spin-trap by biological systems. The ideal reagent must not be toxic and should readily pass though any biological barrier (e.g., the blood-brain barrier) to reach the site of free radical production. A major problem with some spin-trap adducts is that they can be reduced in vivo by cellular reducing agents such as ascorbic acid and thiols, resulting in the production of diamagnetic (non EPR active) species.

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  • CH3

    CH3

    CH3

    N=O + R CH3

    CH3

    CH3

    NR

    O

    tert -Nitrosobutane Radical(less stable)

    Spin-trap adduct(more stable)

    CH=NO

    C(CH3)3

    +

    Phenyl- tert- butylnitronePBN

    -

    + HO CH-N

    OH

    O

    C(CH3)3

    -

    Spin-trap adduct

    PBN-OH Figure 1.5 Spin Traps React with Free Radicals to Produce Paramagnetic Products that can be Measured using EPR. A different approach to spin trapping is radical scavenging. Here the free radical reacts with an aromatic scavenging agent (e.g., salicylic acid). The aromatic-radical adduct can then be quantified using HPLC-based techniques. This approach is much more versatile than spin trapping as neither the scavenging agent nor the product needs to be a radical. Scavengers are usually less toxic than spin traps. Furthermore, as scavenging agents and products are electrochemically active they can be measured at biologically relevant levels using HPLC with electrochemical detection (see ESA Application Notes: 70-1749 Hydroxyl Free Radical Measurement; 70-4820 Alternative Method for Hydroxyl Free Radical Measurement). The use of aromatic scavenging agents will be revisited in Chapter 2.

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  • Reactive Oxygen Species (ROS)

    Reactive Nitrogen Species (RNS)

    Other

    A) Free Radicals Alkoxyl

    LO

    Nitric Oxide (monoxide)

    NO

    Carbon-centered Radicals

    e.g., CCl3

    Hydroperoxyl HO2 Nitrogen Dioxide NO2 Disulfide Radical

    RSSR-

    Hydroxyl HO Peroxynitrite radical ONO2 Hydrogen Atom H Peroxyl LO2 Thiyl Radical RS Superoxide O2- B) Non Radicals Hydrogen Peroxide

    H2O2

    Alkyl Peroxynitrite

    LO2NO-

    Aldehydes (e.g., 4-hydroxy-nonenal)

    RCHO

    Lipid Peroxides LO2H Chloramine NH2Cl Disulfide RSSR Oxygen O2 Dinitrogen Pentoxide N2O5 Hypohalous

    Acid e.g., HOCl and HOBr

    Ozone O3 Dinitrogen Tetroxide N2O4 Hypothio-cyanic Acid

    HOSCN

    Singlet Oxygen 1g O2

    Dinitrogen Trioxide N2O3 Malon-dialdehyde

    CHO- CH2CHO

    Singlet Oxygen 1g+ O2

    Nitrate NO3- Transition metal ions

    e.g., Fe2+, Fe3+

    Nitrite NO2- Nitrocarbonate O2NOCO2- Nitronium (Nitryl) NO2+ Nitrosonium (Nitrosyl) NO+ Nitrosoperoxycarbonate ONO2CO2- Nitrosonium Chloride NOCl Nitroxyl NO- Nitronium Chloride NO2Cl Peroxynitrite ONO2- Taurine

    monochloramine -SO3(CH3)2-NHCl

    Thionitrites (S-nitrosothiols)

    RSNO

    Table 1.6 The Different Pro-Oxidants And Other Species Of Importance To Biological Systems. (L alkyl; 1g and 1g+ represent the two forms of singlet oxygen; X a radical species).

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  • REACTIVE OXYGEN SPECIES, REACTIVE NITROGEN SPECIES AND OTHER PRO-OXIDANTS.

    Although often referred to as free radicals, many of the compounds of interest to the field of redox biochemistry are not free radicals and include many non-radical species (Table 1.6). The term reactive (or reduced) oxygen species (ROS) is also commonly used despite the fact that not all of the oxidizing species are reactive (e.g., the hydroxyl free radical is typically ten million times more reactive and much less selective than hydrogen peroxide), or are produced by the reduction of oxygen (e.g., ozone and singlet oxygen are not reduced forms of oxygen). Furthermore, the use of the term ROS does not take into account that many species contain nitrogen, chlorine or sulfur. Reactive nitrogen species (RNS) is commonly used to distinguish those compounds that contain nitrogen in addition to oxygen, again with disregard for the variation in reactivity between members of the group. As no suitable descriptors can be found, I will use the word pro-oxidant.

    Pro-oxidant Species Comments Ferryl species Essential to catalytic activity of cytochrome P450

    and peroxidases. Hydrogen peroxide The explosive oxidation of hydroquinone by

    hydrogen peroxide in the presence of catalase and peroxidase is used to generate a hot defensive spray by the bombardier beetle.

    Hydrogen peroxide and tyrosine radicals

    Required for the production of thyroxine by the thyroid peroxidase enzyme.

    Hydrogen peroxide Estrogen-induced uterine peroxidase activity plays a role in estrogen catabolism and may confer bactericidal activity too.

    Hydrogen peroxide Involved in the bioluminescence of several animal species.

    Hydrogen peroxide, phenoxyl radicals Involved in the formation of lignin. Oxidation and polymerization of tyrosine and phenylalanine residues catalyzed by peroxidases bound to the plant cell wall.

    Hydrogen peroxide With peroxidases are used by fungi to degrade lignin.

    Hydrogen peroxide Involved in fruit ripening. Hydrogen peroxide Fertilization of sea urchin eggs causes the rapid

    uptake of oxygen and production of hydrogen peroxide that is used by a peroxidase to produce tyrosyl radicals from tyrosine residues. These radicals readily dimerize to dityrosine cross-linking a fertilization membrane that prevents further spermatozoa from entering the egg.

    Hydrogen peroxide, superoxide and nitric oxide

    Redox regulation of gene expression, signal transduction and intracellular redox signaling. Activation of a transcription factor such as SoxS

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  • leads to the stimulation of transcription thereby permitting bacteria to gain resistance to oxidants, antibiotics and immune cells that generate nitric oxide. Nitric oxide can activate the Ras oncoprotein by S-nitrosylation of essential cysteine residue, stimulating GTPase activity and downstream signaling through activation of extracellular signal regulated kinase (ERK kinase). Hydrogen peroxide produced by a plasma membrane-bound NAD(P)H oxidase is activated by insulin and may act as an intracellular signal for this hormone promoting uptake of glucose and preventing triglyceride hydrolysis in adipocytes. Platelet derived growth factor uses hydrogen peroxide as intracellular messenger.

    Lipid peroxides and carbonyl metabolites

    Possibly act as antifungal and antibacterial agents protecting damaged plants from infection.

    Lipid centered radicals Prostaglandin and leukotriene metabolism. Nitric oxide Retrograde neurotransmitter. Nitric oxide Bone synthesis, degradation and remodeling. Nitric oxide, (nitrosothiols) endothelial-derived relaxing factor

    Blood pressure regulation.

    ROS, RNS, HOBr, HOCl, Cl2 Immune system defense. Tyrosine, tryptophan, glycine and thiyl radicals

    Essential to catalytic activity of several enzymes such as ribonucleoside diphosphate reductase and pyruvate dehydrogenase.

    Vitamin K hydroquinone and semiquinone

    Required for carboxylation of glutamate to -carboxylglutamic acid by microsomal glutamic acid carboxylase. Important in blood clotting.

    Table 1.7 Pro-oxidants Are Beneficial Too. (Halliwell and Gutteridge (1999) and references therein; and other references at the end of this chapter).

    HOW DO AEROBIC ORGANISMS SURVIVE EVEN WHEN PRO-OXIDANTS ARE BEING CONTINUOUSLY PRODUCED?

    The cells of aerobes are constantly being exposed to pro-oxidants. Consequently, their DNA, proteins, and lipids are continuously being damaged. During evolution one option would have been to prevent the formation of pro-oxidant species. This, however, would be virtually impossible to achieve in an oxygen-enriched environment as pro-oxidants are unavoidable side reactions of other important biochemical processes. Instead nature accepted that pro-oxidants would be produced so protective mechanisms evolved to repair and replace damaged molecules. In addition we are equipped with a suite of antioxidant defenses designed to prevent the formation of pro-oxidants, or to

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  • intercept and destroy them if formed. Interestingly, aerobes also make good use of pro-oxidants as messengers, signals and defense molecules (Table 1.7).

    Under normal conditions the production of pro-oxidants is presumed to be in balance with antioxidant defenses. However, the overproduction of pro-oxidants and/or decreased antioxidant protection can lead to tissue damage and disease. Thus, in individuals with a genetic predisposition or for those exposed to environmental stressors such as cigarette smoke, sunlight and pollution, the pro-oxidant/antioxidant balance can be upset (Figure 1.6). The overproduction of pro-oxidant species or the failure of antioxidant defenses results in a condition called oxidative stress, a causal, or at least ancillary, factor in the pathology of many diseases (Sies (1985, 1997)).

    AntioxidantsFoodsVitamins-H2O Sol.Fat Soluble Vit.Dietary Sup.Small MoleculesEnzymes

    AntioxidantsFoodsVitamins-H2O Sol.Fat Soluble Vit.Dietary Sup.Small MoleculesEnzymes

    Cell DamageActivation

    CellRepair

    Deactivation

    Stateof

    Oxidative Stress

    OxidantsSmoking

    Cell ActivityPollutantsRadiationUV Light

    Cellular Injury

    Oxidative BalanceOxidative Balance

    Figure 1.6. Oxidative Balance Between Pro-Oxidant And Antioxidant Species. Normally The Production Of Oxidants Is Matched By Antioxidant Defenses. Under Some Circumstances Oxidant Production Can Overwhelm These Defenses Resulting In Oxidative Stress, Cellular Damage And Disease.

    A continuously growing list of diseases and conditions, especially those involving inflammation, are reported to be associated with oxidative stress (Table 1.8). It is interesting to note that a number of these diseases are being treated by manipulation of antioxidant levels or by the use of drugs with antioxidant activity (Sies (1991)).

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  • Disease/condition Reference Abetalipoproteinaemia Mimo (1992) Active pulmonary sarcoidosis Calhoun et al. (1988) Adult respiratory distress syndrome Ballmer et al. (1994); Choi and Alam (1996) AIDS/HIV Baruchel and Wainberg (1992); Dobmeyer et al.

    (1997); McLemore et al. (1998); Revillard (1991) Aging Balin and Allen (1986); Beckman and Ames (1998);

    Benzi and Moretti (1995); Bohr and Anson (1995); Cutler (1991); Harman (1988, 1992a,b); Hocman (1981); Knight (1995); Lebel and Bondy (1992); Leibovitz and Siegel (1980); Nohl (1993); Papa and Skulachev (1997); Scarfiotti et al. (1997); Simic (1992); Sohal (1993); Suzuki (1993)

    Alcohol related diseases Goebel and Schneider (1981); Guemouri et al. (1993); Lieber (1997); Thome et al. (1997); Zhao et al. (1996)

    Alzheimers disease Beal (1997); Behl et al. (1994); Choi (1995); Markesbery (1997); McIntosh et al. (1997); Swerdlow et al. (1997); Volicer and Crino (1990)

    Amyotrophic lateral sclerosis Beal (1997); Chou (1997); Migheli et al. (1994) Apoptosis Monti et al. (1992); Samali et al. (1996); Slater et al.

    (1995); Stoian et al. (1996) Arthritis Biemond et al. (1988); Greenwald (1991); Kaur et al.

    (1996); Moulton (1996); Schiller et al. (1996); Stichtenoth and Frolich (1998)

    Asbestosis Kamp and Weitzman (1997); Kamp et al. (1992); Lenz et al. (1996); Rom et al. (1987)

    Asthma Hilterman et al. (1997); Smith et al. (1997) Atherosclerosis Bankson et al. (1993); Devaraj and Jialal (1996);

    Gambhir and Gambhir (1997); Napoli (1997) Autoimmune diseases (general) Bashir et al. (1993); Yoshida and Gershwin (1993) Autoimmune vasculitis Bashir et al. (1993); Belch et al. (1989); Bruce et al.

    (1997) Battens disease Clausen et al. (1988); Garg et al. (1982) Behcet's disease Ohno et al. (1997); Pronai et al. (1990) Blooms syndrome Emerit and Cerutti (1981) Bone disease (general) Ralston (1997) Bronchopulmonary dysplasia Banks et al. (1998) Cancer Bankson et al. (1993); Borek (1993); De Flora et al.

    (1991); Emerit (1994); Hochstein and Atallah Klotz (1998); Hocman (1981); Oberley and Buettner (1979); Oberley and Oberley (1997); Ockner et al. (1993); Palmer and Paulson (1997); Pryor (1997); Slaga (1995); Troll (1991); Trush and Kensler (1991); Weinberg (1996)

    Cardiovascular disease De Meyer and Herman (1997); Marin and Rodriguez-Martinez (1997); Welch and Loscalzo (1994)

    Cataracts Bhuyan et al. (1986); Niwa and Iizawa (1994); Varma et al. (1984, 1995); Walsh and Patterson (1991); Zigler and Hess (1985)

    Chediak-Higashi syndrome Falloon and Gallin (1986); Quie (1997); Volkman et al. (1984)

    Chronic granulomatous disease Umeki (1994); Volkman et al. (1984) Crohns disease Allgayer (1991); Baldassano et al. (1993); Curran et al.

    (1991); Kimura et al. (1997); McKenzie et al. (1996); Rachmilewitz et al. (1997); Solis-Herruzo et al. (1993)

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  • Cystic fibrosis Brown et al. (1994, 1995, 1996); Graseman et al. (1998); Percival et al. (1995); Portal et al. (1995); Winklhofer-Roob (1994); Worlitzsch et al. (1998)

    Diabetes Dandona et al. (1997); Giugliano et al. (1995); Semenkovich and Heinecke (1997); Wolff et al. (1991)

    Downs syndrome Brugge et al. (1992); Kedziora and Bartosz (1988); Lott (1982); Reiter et al. (1996)

    Duchennes muscular dystrophy Burr et al. (1987); Dioszeghy et al. (1989); Haycock et al. (1996); Ragusa et al. (1997)

    Exercise Fielding and Meydani (1997); Higuchi et al. (1985); Ji (1996); Lawson et al. (1997); Leeuwenburgh et al. (1994); Ortenblad et al. (1997); Packer (1997)

    Favism Gaetani et al. (1996); Mavelli et al. (1984); Musci et al. (1987); Winterbourn et al. (1986)

    Friedreichs ataxia Rotig et al. (1997) Gastritis Beno et al. (1993, 1994); Durak et al. (1994); Mannick

    et al. (1996) Gerstmann-Straussler Syndrome Migheli et al. (1994) Glomerular injury Rohrmoser and Mayer (1996) Gout Marcolongo et al. (1988); Rosen et al. (1986) Guillain Barre syndrome Gutowski et al. (1998) Hashimotos thyroiditis Bagchi et al. (1990); Sugawara et al. (1988); Szabo et

    al. (1996) Hemolytic diseases Fritsma (1983); Lachant and Tanaka (1986); Stack et

    al. (1989); Stocks et al. (1971); Vertongen et al. (1981); Winterbourn (1990); Yenchitsomanus and Wasi (1983)

    Hepatitis Arthur et al. (1985); Biasi et al. (1994); Biemond et al. (1988); Bonkovsky et al. (1997); De Maria et al. (1996); Yu et al. (1997)

    Huntingtons disease Beal (1995, 1996, 1997); Bondy (1995); Borlongan et al. (1996); Browne et al. (1997); Shapira (1996)

    Hutchinson-Gilford syndrome Goldstein (1971) Hypercholesterolaemia Cohen (1995); Devaraj and Jialal (1994); Harrison and

    Ohara (1995); Verhaar et al. (1998); Wennmalm (1994) Hypersensitivity pneumonitis Calhoun (1991) Idiopathic hemochromatosis Britton and Brown (1985); Gutteridge et al. (1985);

    Houglum et al. (1997); Selden et al. (1980); Young et al. (1994)

    Inborn errors of metabolism Bird et al. (1995); Blau et al. (1996); Brown and Squier (1996); Delgado and Calderon (1979); Jansen and Wanders (1997); Kavanagh et al. (1994); Loscalzo (1996); Moyano et al. (1997); Patel and Leonard (1995); Pitkanen and Robinson (1996); Prohaska (1986); Quie (1977); Welch et al. (1997); Whitin and Cohen (1988); Yoshida et al. (1995)

    Infectious mononucleosis Hokama et al. (1986); Niwa et al. (1984); Ritter et al. (1994)

    Inflammation (general) Billiar (1995); Chapple (1997); Cirino (1998); Connor and Grisham (1996); Dallegri and Ottonello (1997); Halliwell et al. (1988); Morris et al. (1995); Parke and Parke (1996); Pyne (1994); Southorn and Powis (1988); Stichtenoth and Frolich (1998); Trenam et al. (1992); Weitzman and Gordon (1990); Winrow et al. (1993); Winyard and Blake (1997)

    Inflammatory bowel disease Buffinton and Doe (1995); Macdonald (1998)

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  • Ischemia/reoxygenation injury; reperfusion injury

    Ar' Rajab et al. (1996); Bulkley (1994); Flaherty and Weisfeldt (1988); Gutteridge and Halliwell (1990); Hudson (1994); Johnson and Weinberg (1993); Maxwell (1997); McCord (1987); Szabo (1996); Waxman (1996); Weight et al. (1996)

    Kashin-Beck disease Peng et al. (1992); Wu and Xu (1987) Keshan disease Hensrud et al. (1994); Levander et al. (1997) Leprosy Agnihotri et al. (1996); Sethi et al. (1996); Sharp and

    Banerjee (1985) Liver disease (general) Abrams et al. (1995) Lupus Belmont et al. (1997); Benke et al. (1990); Cooke et al.

    (1997); Mohan and Das (1997); Suryaprabha et al. (1991)

    Macular degeneration Anderson et al. (1994); Nicolas et al. (1996); Van der Hagen et al. (1996)

    Malaria Delmas-Beauvieux et al. (1995); Ginsburg and Atamna (1994); Mishra et al. (1994); Postma et al. (1996); Vennerstrom and Eaton (1988)

    Motor neuron disease Anderson et al. (1997); Donohoe and Brady (1996); Lyras et al. (1996); Morrison (1995); Sendtner and Thoenen (1994); Shaw et al. (1995); Wong and Borchelt (1995); Zeman et al. (1994)

    Multiple sclerosis Calabrese et al. (1994); Clausen et al. (1997); Cooper et al. (1997); Hooper et al. (1998); Langemann et al. (1992); Nagra et al. (1997); Parkinson et al. (1997)

    Neuronal ceroid lipofuscinosis Garg et al. (1982); Gutteridge et al. (1983); Marklund et al. (1981); Santavuori et al. (1989)

    Pancreatitis Sanfey (1986) Parkinsons disease Beal (1997); Cadet and Brannock (1998); Ciccone

    (1998); Di Momte et al. (1992); Fahn and Cohen (1992); Gerlach et al. (1994); Hirsch et al. (1997); Jenner (1996); Jenner and Olanow (1996); Koller (1997); Owen et al. (1997); Simonian and Coyle (1996); Youdin et al. (1988, 1990)

    Periodontal disease Ellis et al. (1998); Kimura et al. (1993); Moore et al. (1994); Scmidt et al. (1996)

    Porphyria Monteiro et al. (1986, 1989); Thunell et al. (1997) Prion Diseases Brown et al. (1997); Wiseman and Goldfarb (1996) Renal dialysis Biasioli et al. (1997); Cristol et al. (1994);

    Westhuyzen et al. (1995) Retrolental fibroplasia Anderson et al. (1994); Cunningham (1987); Johnson

    et al. (1974); Southorn and Powis (1988) Rheumatic diseases Miesel et al. (1996) Salmonella typhimurium infection Mehta et al. (1998) Septic shock Brigham (1991); Goode and Webster (1993); Keusch

    (1993); Kilbourn et al. (1997); Kuhl and Rosen (1998); Novelli (1997); Taylor and Piantadosi (1995)

    Skin inflammation Trenam et al. (1992) Smoking Cantin and Crystal (1985); Chow (1993); Crystal

    (1991); Kohlmeier and Hastings (1995); McCusker (1992); Pryor, W.A. (1997); Rahman and MacNee (1996)

    Stroke Chang et al. (1998); Fisher and Bogousslavsky (1998); Keli et al. (1996); Mattson (1997); Meldrum (1995)

    Transplantation Hernandez and Granger (1988); Keith (1993); Lehr and

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  • Messmer (1996); McCord (1985); Meyer et al. (1998); Paller (1992); Toledo-Pereyra (1991)

    Ulcerative colitis Keshavarzian et al. (1997); Holmes et al. (1998); Lundberg et al. (1994); McKenzie et al. (1996); Ramakrishna et al. (1997); Reimund et al. (1998); Sedghi et al. (1994)

    Viral infection Peterhans (1997) Werners syndrome Marklund et al. (1981) Wilsons disease Britton and Brown (1995); Carmichael et al. (1995);

    Ogihara et al. (1995); Sokol et al. (1994) Xeroderma pigmentosum Crawford et al. (1988); Runger et al. (1995);

    Schallreuter et al. (1991) Table 1.8 Diseases And Conditions Associated With Oxidative Stress.

    WHY USE ELECTROCHEMICAL DETECTION? Oxidation can be defined as a gain in oxygen, a loss of hydrogen, a loss of protons or the loss of electrons. Conversely, reduction is the loss of oxygen, a gain of hydrogen, or the gain of electrons. The two processes are complementary and no oxidation process can take place without a corresponding reduction; these complimentary reactions are typically referred to as REDuction-OXidation or REDOX reactions. Of all the different detectors that are used in the study of redox biochemistry, perhaps the most useful is the electrochemical detector (ECD). This detector actually measures the flow of electrons (current) when an electron-rich compound loses electrons to the working electrodes surface while this compound undergoes oxidation (conversely, electron-poor compounds can also be measured as they accept electrons from the working electrodes surface while undergoing reduction). When coupled to the high resolution achievable with high-performance liquid chromatography (HPLC) an analytical instrument is produced that can be used to measure many different pro-oxidant, antioxidant, and damaged species (Chapter 2 and 3). Electrochemical detection is one of the most sensitive and selective detection techniques available for use with HPLC. The theory behind it has been extensively reviewed elsewhere (Acworth and Bowers (1997) and references therein; Acworth et al. (1997a,b,c; 1998)). Of all the ECDs on the market place, ESAs coulometric detectors are the most sensitive and selective, and are virtually maintenance free. ESA, Inc., offers two electrochemical detectors (Figure 1.7). The Coulochem detector offers a high-sensitivity DC mode along with pulsed and cyclic capabilities. The CoulArray is the only electrochemical detector that can work with even the most aggressive gradients. Practical examples using these detectors will be presented throughout this handbook.

    WWW.ESAINC.COM 23

  • Figure 1.7 The Coulochem III (Upper Figure) And CoulArray Detectors.

    WWW.ESAINC.COM 24

    http://www.esainc.com/products/HPLC/EC_Detectors/esa_coulochemIII.htmlhttp://www.esainc.com/products/HPLC/EC_Detectors/esa_coularray.htmhttp://www.esainc.com/products/HPLC/EC_Detectors/esa_coulochemIII.htmlhttp://www.esainc.com/products/HPLC/EC_Detectors/esa_coularray.htm
  • CONCLUSIONS.

    Oxygen is toxic and exerts its toxicity through the production of a variety of pro-oxidant species. During evolution living organisms either remained anaerobic surviving in oxygen poor conditions or became aerobic, adapting to the increased atmospheric levels of oxygen. Aerobic organisms tolerate continued production of pro-oxidants and have evolved mechanisms to repair or remove damaged molecules or to prevent the formation and to intercept and deactivate the pro-oxidant species. Normally there is a balance between production of pro-oxidant species and destruction by the antioxidant defenses. However, under certain conditions this balance is upset in favor of overproduction of the pro-oxidants leading to oxidative stress and disease. HPLC-ECD is one of the most sensitive analytical techniques for the measurement of pro-oxidants, antioxidants, and damage markers.

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