Handbook
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Series Introduction
Oxygen is a dangerous friend. Overwhelming evidence indicates that oxidative stress can lead to cell and
tissue injury. However, the same free radicals that are generated during oxidative stress are produced
during normal metabolism and thus are involved in both human health and disease.
Free radicals are molecules with an odd number of electrons. The odd, or unpaired, electron is highly
reactive as it seeks to pair with another free electron.
Free radicals are generated during oxidative metabolism and energy production in the body.
Free radicals are involved in:
Enzyme-catalyzed reactions Electron transport in
mitochondria Signal transduction and gene expression
Activation of nuclear transcription factors Oxidative
damage to molecules, cells, and tissues Antimicrobial
action of neutrophils and macrophages Aging and disease
Normal metabolism is dependent on oxygen, a free radical. Through evolution, oxygen was chosen
as the terminal electron acceptor for respiration. The two unpaired electrons of oxygen spin in the same
direction; thus, oxygen is a biradical, but not a very dangerous free radical. Other oxygen-derived free
radical species, such as superoxide or hydroxyl radicals, formed during metabolism or by ionizing
radiation are stronger oxidants and are therefore more dangerous.
In addition to research on the biological effects of these reactive oxygen species, research on reactive
nitrogen species has been gathering momentum. NO, or nitrogen monoxide (nitric oxide), is a free radical
generated by NO synthase (NOS). This enzyme modulates physiological responses such as vasodilation
or signaling in the brain. However, during inflammation, synthesis of NOS (iNOS) is induced. This iNOS
can result in the overproduction of NO, causing damage. More worrisome, however, is the fact that
excess NO can react with superoxide to produce the very toxic product peroxynitrite. Oxidation of lipids,
proteins, and DNA can result, thereby increasing the likelihood of tissue injury.
Both reactive oxygen and nitrogen species are involved in normal cell regulation, in which oxidants
and redox status are important in signal transduction. Oxidative stress is increasingly seen as a major
upstream component in the signaling cascade involved in inflammatory responses, stimulating adhesion
molecule and chemoattractant production. Hydrogen peroxide, which breaks down to produce hydroxyl
radicals, can also activate NF-KB, a transcription factor involved in stimulating inflammatory responses.
Excess production of these reactive species is toxic, exerting cytostatic effects, causing membrane
damage, and activating pathways of cell death (apoptosis and/or necrosis).
Virtually all diseases thus far examined involve free radicals. In most cases, free radicals are
secondary to the disease process, but in some instances free radicals are causal. Thus, there is a delicate
balance between oxidants and antioxidants in health and disease. Their proper balance is essential for
ensuring healthy aging.
The term oxidative stress indicates that the antioxidant status of cells and tissues is altered by
exposure to oxidants. The redox status is thus dependent on the degree to which a cell’s components are
in the oxidized state. In general, the reducing environment inside cells helps to prevent oxidative damage.
In this reducing environment, disulfide bonds (S—S) do not spontaneously form because sulfhydryl
groups kept in the reduced state (SH) prevent protein misfold- ing or aggregation. This reducing
environment is maintained by oxidative metabolism and by the action of antioxidant enzymes and
substances, such as glutathione, thioredoxin, vitamins E and C, and enzymes such as superoxide
dismutase (SOD), catalase, and the selenium-dependent glutathione and thioredoxin hydroperoxidases,
which serve to remove reactive oxygen species.
Changes in the redox status and depletion of antioxidants occur during oxidative stress. The thiol
redox status is a useful index of oxidative stress mainly because metabolism and NADPH- dependent
enzymes maintain cell glutathione (GSH) almost completely in its reduced state. Oxidized glutathione
(glutathione disulfide, GSSG) accumulates under conditions of oxidant exposure, and this changes the
ratio of oxidized to reduced glutathione; an increased ratio indicates oxidative stress. Many tissues contain
large amounts of glutathione, 2-4 mM in erythrocytes or neural tissues and up to 8 mM in hepatic tissues.
Reactive oxygen and nitrogen species can directly react with glutathione to lower the levels of this
substance, the cell’s primary preventative antioxidant.
Current hypotheses favor the idea that lowering oxidative stress can have a clinical benefit. Free
radicals can be overproduced or the natural antioxidant system defenses weakened, first resulting in
oxidative stress, and then leading to oxidative injury and disease. Examples of this process include heart
disease and cancer. Oxidation of human low-density lipoproteins is considered the first step in the
progression and eventual development of atherosclerosis, leading to cardiovascular disease. Oxidative
DNA damage initiates carcinogenesis.
Compelling support for the involvement of free radicals in disease development comes from
epidemiological studies showing that an enhanced antioxidant status is associated with reduced risk of
several diseases. Vitamin E and prevention of cardiovascular disease is a notable example. Elevated
antioxidant status is also associated with decreased incidence of cataracts and cancer, and some recent
reports have suggested an inverse correlation between antioxidant status and occurrence of rheumatoid
arthritis and diabetes mellitus. Indeed, the number of indications in which antioxidants may be useful in
the prevention and/or the treatment of disease is increasing.
Oxidative stress, rather than being the primary cause of disease, is more often a secondary
complication in many disorders. Oxidative stress diseases include inflammatory bowel diseases, retinal
ischemia, cardiovascular disease and restenosis, AIDS, ARDS, and neurodegenerative diseases such as
stroke, Parkinson’s disease, and Alzheimer’s disease. Such indications may prove amenable to antioxidant
treatment because there is a clear involvement of oxidative injury in these disorders.
In this new series of books, the importance of oxidative stress in diseases associated with organ
systems of the body will be highlighted by exploring the scientific evidence and the medical applications
of this knowledge. The series will also highlight the major natural antioxidant enzymes and antioxidant
substances such as vitamins E, A, and C, flavonoids, polyphenols, carotenoids, lipoic acid, and other
nutrients present in food and beverages.
Oxidative stress is an underlying factor in health and disease. More and more evidence indicates that
a proper balance between oxidants and antioxidants is involved in maintaining health and longevity and
that altering this balance in favor of oxidants may result in pathological responses causing functional
disorders and disease. This series is intended for researchers in the basic biomedical sciences and
clinicians. The potential for healthy aging and disease prevention necessitates gaining further knowledge
about how oxidants and antioxidants affect biological systems.
Rapid progress in the application of antioxidant substances and other micronutrients warranted a
revision and update of Handbook of Antioxidants, highlighting new fundamental studies on food-derived
antioxidants and biomarkers, vitamins E and C, coenzyme Q, carotenoids, flavonoids and other
polyphenols, antioxidants in beverages and herbal products, the thiol antioxidants glutathione and lipoic
acid, melatonin, selenium, and nitric oxide. Handbook of Antioxidants: Second Edition, Revised and
Expanded, is an authoritative volume regarding the chemical, biological, and clinical aspects of
antioxidant molecules. The individual chapters provide an in-depth account of the current knowledge of
vitamins or other naturally occurring antioxidant compounds and discuss critically the new aspects of
antioxidant therapy. We are delighted to have been involved with this project and are grateful to the
authors in this volume for their outstanding contributions.
Lester Packer
Enrique Cadenas
Preface
The Handbook of Antioxidants: Second Edition, Revised and Expanded, is an authoritative treatise on the
chemical, biological, and clinical aspects of antioxidant molecules. Each chapter provides an in-depth
account of the current knowledge of vitamins or other naturally occurring antioxidant compounds and
discusses critically the new aspects of antioxidant therapy.
About 100 million Americans are now using food supplements that have antioxidant activ ity, and
there is an urgent need for providing the scientific community and the general public with the most
current information available.
The biochemistry of reactive oxygen species is an important field with vast implications. Whereas
oxygen is an essential component for living organisms, the generation of reactive oxygen species seems
to be commonplace in aerobically metabolizing cells. Cells convene substantial resources to protect
themselves against the potentially damaging effects of reactive species. The first line of defense against
these free radicals is composed by enzymes, such as superoxide dismutase, glutathione peroxidase, and
catalase, and several vitamins and micronutrients, which actively quench these free radical species or are
required as cofactors for antioxidant enzymes. The cellular antioxidant status and its role in fighting
progression of certain disease processes associated with oxidative stress have gained potential therapeutic
significance in view of the beneficial effects of free-radical-scavenging drugs or antioxidants. Likewise,
epidemiological studies emphasize the relevance of antioxidant vitamins and nutrients in health issues
and/or prevention of chronic and degenerative diseases of aging.
Rapid progress in the application of antioxidant substances and other micronutrients warranted a
revision of Handbook of Antioxidants. This updated edition highlights new fundamental studies on foodderived antioxidants and biomarkers, vitamins E and C, coenzyme Q, carotenoids, flavonoids and other
polyphenols, antioxidants in beverages and herbal products, the thiol antioxidants glutathione and lipoic
acid, and melatonin, selenium, and nitric oxide.
We are delighted to have been involved with this project and thank the contributors to this volume
for their outstanding efforts.
Enrique Cadenas
Lester Packer
ix
Contents
Series Introduction......................................................................................................................................3
Preface....................................................................................................................................................... ix
Contents...................................................................................................................................................... x
Contributors............................................................................................................................................lxix
Food-Derived Antioxidants: How to Evaluate Their Importance in Food and In Vivo.........................lxxiii
Barry Halliwell...........................................................................................................................................lxxiii
I.INTRODUCTION....................................................................................................................................lxxiii
II.HOW TO DEFINE AN ANTIOXIDANT.............................................................................................lxxvi
x
III.ANTIOXIDANT CHARACTERIZATION IN VITRO.....................................................................lxxvii
A.Assays of “Total Antioxidant Activity”................................................................................................lxxvii
B.Scavenging of Reactive Nitrogen Species..............................................................................................lxxix
C.Scavenging of Reactive Oxygen Species................................................................................................lxxx
D.Effects of Antioxidants on Phagocytes.......................................................................................................26
xi
E.Heme Proteins as Pro-Oxidants...................................................................................................................27
F.Singlet Oxygen.............................................................................................................................................28
IV.WHAT DO WE LEARN FROM IN VITRO ANTIOXIDANT CHARACTERIZATION?..................28
A.DNA.............................................................................................................................................................29
B.Lipids: Lipid Peroxidation..........................................................................................................................29
xii
C.Proteins: Damage by Reactive Species.......................................................................................................31
V.CONCLUSION...........................................................................................................................................32
REFERENCES................................................................................................................................................32
2................................................................................................................................................................ 46
Measurement of Total Antioxidant Capacity in Nutritional and Clinical Studies........................................46
Guohua Cao.....................................46
Ronald L. Prior................................46
I.INTRODUCTION.................................46
II.OVERVIEW OF TOTAL ANTIOXIDANT CAPACITY ASSAY
METHODOLOGY....................................47
A.Methods Using Oxidants That Are Not Necessarily
Pro-Oxidants...................................47
B.Methods Using Oxidants That Are Pro-Oxidants. 48
III.APPLICATION OF TOTAL ANTIOXIDANT CAPACITY
ASSAYS IN NUTRITIONAL AND CLINICAL STUDIES.....49
A.Sample Preparation...........................49
xiii
C.Manual Procedure.............................50
D.Using H2O2-Cu2+ as Pro-oxidants in the ORAC
Assay..........................................51
E.ORAC Measured in Plasma or Serum: Effects of PCA
or Acetone Treatment...........................51
IV.FACTORS TO BE CONSIDERED IN TOTAL ANTIOXIDANT
CAPACITY ASSESSMENT............................51
A.Diets........................................51
B.Physical Activity............................52
C.Diseases and Genetics........................52
V.INTERPRETATION OF PLASMA OR SERUM TOTAL
ANTIOXIDANT CAPACITY...........................52
REFERENCES.....................................52
3................................................................................................................................................................ 54
Quantification of Isoprostanes as Indicators of Oxidant Stress In Vivo.......................................................54
Jason D. Morrow, William E. Zackert, Daniel S. Van
der Ende, Erin E. Reich, Erin S. Terry, Brian Cox,
Stephanie C. Sanchez, Thomas J. Montine, and L.
Jackson Roberts................................54
I.INTRODUCTION.................................54
II.HISTORICAL PERSPECTIVES.....................55
III.MECHANISM OF FORMATION OF THE ISOPROSTANES. 56
IV.FORMATION OF ISOPROSTANES IN VIVO...........56
V.RELEVANCE OF THE DISCOVERY OF ISOPROSTANES...62
A.Analytical Ramifications.....................62
VI.METHOD OF ANALYSIS OF THE ISOPROSTANES......64
VII.QUANTIFICATION OF ISOPROSTANES AS AN INDEX OF
OXIDANT STRESS.................................64
xiv
A.In Vitro Studies.............................64
B.F2-Isoprostane Quantification in Animal Models
of Oxidant Stress..............................65
C.Quantification of F2-Isoprostanes to Assess the
Role of Oxidant Injury in Human Diseases.......67
VIII.SUMMARY...................................69
ACKNOWLEDGMENTS................................70
REFERENCES.....................................70
4................................................................................................................................................................ 73
Efficacy of Vitamin E in Human Health and Disease...................................................................................73
Sharon V. Landvik..............................73
Lester Packer..................................73
I.INTRODUCTION.................................73
II.FUNCTIONS...................................74
III.CLINICAL DEFICIENCY STATES.................74
IV.REQUIREMENTS................................74
V.CANCER.......................................75
VI.CARDIOVASCULAR DISEASE......................85
VII.AGING......................................89
VIII.CATARACTS.................................90
IX.AIR POLLUTION...............................93
X.STRENUOUS EXERCISE...........................94
XI.SUMMARY.....................................96
REFERENCES.....................................96
5.............................................................................................................................................................. 106
xv
Vitamin E Bioavailability, Biokinetics, and Metabolism............................................................................106
Maret G. Traber...............................106
I.INTRODUCTION................................106
II.FACTORS THAT INFLUENCE BIOAVAILABILITY.....107
A.Intestinal Absorption.......................107
III.VITAMIN E BIOKINETICS.....................109
IV.DISTRIBUTION TO TISSUES....................109
A.Vitamin E Delivery to Tissues...............109
B.Tissue Vitamin E............................109
V.METABOLISM..................................110
A.Chroman Ring Oxidation......................110
B.Unoxidized Metabolites......................110
VI.CONCLUSIONS................................111
REFERENCES..............................................................................................................................................112
Biological Activity of Tocotrienols..............................................................................................................108
Stefan U. Weber and Gerald Rimbach.........................................................................................................108
xvi
I.INTRODUCTION......................................................................................................................................108
II.VITAMIN E ISOFORMS AND THEIR NATURAL SOURCES..........................................................108
III.ANTIOXIDANT ACTIVITY OF TOCOTRIENOLS............................................................................110
IV.ABSORPTION AND DISTRIBUTION IN TISSUES...........................................................................112
V.INHIBITION OF CHOLESTEROL SYNTHESIS..................................................................................112
xvii
VI.INHIBITION OF LDL OXIDATION.....................................................................................................113
VII.ANTICARCINOGENIC PROPERTIES...............................................................................................113
VIII.NEUROPROTECTION........................................................................................................................113
IX.SKIN PROTECTION AGAINST OXIDANTS.....................................................................................114
X.CONCLUSIONS AND FUTURE DIRECTIONS...................................................................................114
xviii
ACKNOWLEDGMENT...............................................................................................................................114
REFERENCES..............................................................................................................................................114
7.............................................................................................................................................................. 116
Vitamin C: From Molecular Actions to Optimum Intake............................................................................116
Sebastian J. Padayatty, Rushad Daruwala, Yaohui Wang, Peter K. Eck, Jian Song, Woo S. Koh, and Mark
Levine............................................................................................................................................................116
I.DEFICIENCY OF VITAMIN C................................................................................................................117
xix
A.Introduction...............................................................................................................................................117
B.Scurvy and the Discovery of Ascorbic Acid............................................................................................117
C.Symptoms and Signs of Scurvy................................................................................................................117
II.PROPERTIES AND FUNCTIONS OF VITAMIN C.............................................................................118
A.Ascorbic Acid as a Vitamin......................................................................................................................118
xx
C.Ascorbate Is a Cofactor for Enzymes.......................................................................................................120
D.Nonenzymatic Functions of Vitamin C....................................................................................................120
III.PHYSIOLOGY OF VITAMIN C............................................................................................................122
A.Tissue Distribution of Vitamin C.............................................................................................................122
B.Transport and Accumulation of Vitamin C..............................................................................................122
xxi
C.Biochemical Function in Relation to Concentration................................................................................128
D.Steady-State Plasma Concentration in Relation to Dose.........................................................................128
E.Vitamin C Intake Versus Plasma Concentration:.....................................................................................129
Results of Dietary Surveys............................................................................................................................129
F.Depletion-Repletion Studies......................................................................................................................129
Dose, mg/day..................................133
Dose (mg/day).................................134
xxii
G.Bioavailability of Oral Vitamin C............................................................................................................134
I. Food Sources of Vitamin C.......................................................................................................................139
J. Vitamin C Intake in the United States......................................................................................................139
K. Recommended Dietary Allowance for Vitamin C..................................................................................139
REFERENCES..............................................................................................................................................140
8.............................................................................................................................................................. 145
xxiii
Vitamin C and Cardiovascular Diseases......................................................................................................145
Anitra C. Carr and Balz Frei.........................................................................................................................145
I.INTRODUCTION......................................................................................................................................145
A.Vitamin C Is a Potent Water-Soluble Antioxidant...................................................................................145
Dehydroascorbic Acid (DHA)....................147
II.LIPID PEROXIDATION..........................................................................................................................150
xxiv
A.Vitamin C Inhibits In Vitro Lipid Peroxidation.......................................................................................150
B.Vitamin C Inhibits In Vivo Lipid Peroxidation........................................................................................151
III.CELLULAR MECHANISMS.................................................................................................................152
A.Vitamin C Attenuates Cellular Oxidant Production................................................................................152
B.Vitamin C Reduces Cellular Adhesion.....................................................................................................153
xxv