Naturally Occurring Antioxidants

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Chapter 108 Naturally Occurring Antioxidants

A growing body of research implicates excessive oxidative damage in multiple disease processes as well as aging. This chapter examines the impact of free radicals and pro-oxidants, and the role of antioxidants in countering oxidative stress and imbalanced redox regulation.

image Free Radicals and Nonradical Oxidants

Normal Metabolism Generates Reactive Oxygen Species

Reactive oxygen species (ROS) represent more or less reactive oxidizing agents, whether or not they are free radicals. They include NO, O2, and H2O2. Reactive nitrogen species (RNS) include peroxynitrite and its reaction products, such as NO2. Multiple cellular processes yield ROS.

The Free Radical Theory of Aging

According to this seminal proposal, free radicals from normal metabolism or external sources gradually overcome antioxidant mechanisms, leading to cumulative oxidative damage to essential cellular elements: proteins, DNA, and lipids.3 Accordingly, increased prevalence of chronic degenerative diseases can be viewed in terms of irreversible cellular deficits. The consequences of oxidative stress are often subtle: increased membrane fluidity and damage to membrane receptor proteins may alter cellular regulatory mechanisms such as signal transduction, inactivation of proteins required for ATP production, or calcium homeostasis. The more or less continuous production of ROS by activated phagocytes during chronic, possibly low-level, inflammation may eventually deplete antioxidant defenses, allowing ROS to attack cells with ensuing tissue injury.

More than 100 conditions provide circumstantial evidence linking oxidative damage to disease processes, although correlation cannot distinguish cause or consequence. This list includes several forms of cancer,4,5 atherosclerosis,6,7 hypertension, diabetes mellitus,8 cataracts,9 inflammation and autoimmune disease,10,11 lung disease,12 neurologic disorders including Alzheimer’s disease and Parkinson’s diease,13,14 hepatitis,15 obesity,16 fibromyalgia and chronic fatigue syndrome,17 in addition to cell death18 and attributes of aging.19,20 Psychological stress and responses to social stressors can affect antioxidant enzymes.21

Mitochondrial Deterioration

Mitochondrial deterioration poses a test of the free radical theory. An estimated 3% of oxygen molecules passing through mitochondria is converted superoxide, due to leakage of the electron transport chain.22 Mitochondria also produce H2O2 and lipid peroxides, which can damage highly susceptible mitochondrial DNA and alter mitochondrial membrane permeability to release apoptogenic substances such as cytochrome c. Paradoxically, mice lacking the mitochondrial defensive enzyme, manganese superoxide dismutase (MnSOD), have increased pathology and increased DNA damage, yet life span is unchanged.23

Hydroxyl Radicals and Peroxynitrite

Peroxynitrite from Superoxide and Nitric Oxide

High, sustained levels of NO and superoxide are associated with tissue toxicity, cancer, and inflammatory conditions, such as arthritis, juvenile diabetes, and ulcerative colitis.24 Oxidative stress can result from increased production of superoxide, which reacts spontaneously with NO to produce peroxynitrite (ONOO). Excessive superoxide can come from upregulated xanthine oxidase or cytochrome P450. ROS uncouple nitric oxide synthase (NOS) to produce superoxide, not NO. Thus, exposure of human endothelial cells to lysophosphatidylcholine leads to downregulation of endothelial NOS and SOD, resulting in a superoxide overload.25,26 Under inflammatory conditions, simultaneous production of superoxide and NO can increase 1000-fold, and production of peroxynitrite can increase by a million-fold.27

Reactive Oxygen Species: Function as Broad-Spectrum Antibiotics

Infection, toxic exposure, ischemia, and trauma activate phagocytic cells—macrophages, monocytes, neutrophils, and eosinophils to create ROS.28 The binding of immune complexes, bacterial endotoxins, or other inflammatory agents to cell surface receptors triggers a respiratory burst, a localized production of ROS able to oxidize viruses and bacteria. Excessive superoxide from NADPH oxidase undergoes dismutation to H2O2 via SOD. Myeloperoxidase in lysosomes then converts H2O2 and the chloride ion to hypochlorite, which spontaneously produces highly reactive chloramines from amines.

Oxidative Stress and Cell Signaling Cascades

Physiologic levels of ROS influence cell regulatory processes as diverse as apoptosis, cell growth, and chemotaxis.29,30 Many signal cascades are sensitive to redox balance and can be modulated by ROS and antioxidants. Essential to these pathways are multiple protein kinases, whose phosphorylation products are often other kinases or kinase inhibitors. Related protein phosphatases reverse those effects.

Transcriptions Factors

Nuclear factor-κB (NF-κB) and activating protein-1 (AP-1) help regulate inflammation and the response to oxidative stress. The factors bind to antioxidant response elements (AREs) in the promoter regions to induce transcription of inflammatory cytokines and adhesion molecules.

Nuclear Factor-κB

Proinflammatory cytokines and lipopolysaccharide (LPS) promote NF-κB activation. NF-κB upregulation may contribute to chronic oxidative stress and diseases, such as rheumatoid arthritis, hypertension, and even overweightness and obesity.31 In contrast, transcription factors can boost defenses against ROS, depending upon which signaling cascade is activated, to increase transcription of glutathione-S transferase (GSH S-transferase), metallothionein-1, and manganese-dependent SOD.

Protein Kinases and Protein Phosphatases

The kinase family includes the protein kinase C (PKC) group, which includes serine/threonine kinases that are dependent on calcium and phospholipids; the mitogen-activated protein kinase (MAPK) family (extracellular signal-regulated kinases, Jun N-terminal kinase [JNK], and p38 kinases); and tyrosine protein kinases. Related phosphatases reverse their actions. Members of these broad families can participate in post-transcriptional control due to their redox-sensitive cysteinyl domains.34 As an example, vasocontraction occurs when angiotensin II binds to its receptor and activates PKC, in turn activating NADPH oxidase to produce superoxide, which destroys NO. As another example, H2O2 from SOD can oxidize catalytic cysteinyl moieties of tyrosine phosphatases, thereby preventing inactivation of tyrosine kinases, including Src, to stimulate AP-1.33

Calcium Homeostasis

Transcription factors may be regulated through calcium signaling mechanisms, and H2O2 can stimulate calcium release from mitochondria.33 H2O2 can increase L-type calcium channels and calcium influx by vascular smooth muscle cells linked to hypertension.36

Specific Reactive Oxygen Species as Secondary Messengers for Signaling Cascades

Nitric Oxide

NO is synthesized by NOS, and physiologic NO levels regulate multiple processes; thus, NO produced by the endothelial isoform (eNOS) regulates vasodilation. In contrast, large amounts of NO are produced during inflammation by phagocytic cells by an inducible isoform (iNOS) in response to proinflammatory cytokines.41 NO is involved in S-nitrosylation of proteins by reacting with cysteinyl residues to form S-nitrothiols, which possess vasodilation activity and resist superoxide in plasma. As an example, ROS such as H2O2 can inhibit protein tyrosine phosphatase and its downstream actions via activation of NOS, with increased NO production and reversible active site cysteinyl S-nitrosylation.42 High levels of NO induce apoptosis by stimulating release of cytochrome c from mitochondria, activating the AKDK1, cyclic dependent kinase 1 (CDK1) and JNK pathway, leading to active caspases that trigger cell death.43

Hydrogen Peroxide

The primary source of H2O2 is superoxide, constitutively produced by mitochondria and NADPH oxidases. To control H2O2, catalase and peroxidases specifically degrade H2O2, whereas SOD limits superoxide to curtail H2O2 accumulation. A high steady-state level of H2O2, associated with inflammation, increases production of NF-κB and AP-1. In post-transcriptional mechanisms, H2O2 oxidizes cysteinyl moieties in redox switching enzymes. This process activates tyrosine kinases and inhibits related phosphatases,44 leading to nuclear factor translocation and increased expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), to promote neutrophil binding. ROS, like H2O2, can oxidize GSH peroxidase, peroxiredoxins (Prx), and inactive transcription factors directly by the formation of protein disulfides or sulfenic acid derivatives.45

Regulatory Roles for Superoxide and Peroxynitrite

The importance of superoxide in regulatory cascade mechanisms is restricted by its short half life and limited diffusion. Studies with SOD inhibitors suggest that superoxide may mediate sympathetic excitation in rabbit brain and regulate cardiovascular responses to stress.46 Some authors propose that peroxynitrite participates in control mechanisms.47 At low concentration in vitro, peroxynitrite may modulate signaling pathways by nitrating tyrosine residues in target enzymes, such as tyrosine kinases. The inhibition of phosphotyrosine-dependent signaling to block cell adhesion molecule production suggests a role in modulating signal transduction. Furthermore, peroxynitrite can promote tyrosine phosphorylation (activation) in various cell types, for example, by activating MAPKs or by inhibiting phosphotyrosine phosphatases. This upregulation can be transient and reversible, and achievable at relatively low peroxynitrite concentrations in vitro.48

Laboratory Assessment of Oxidative Stress

Currently used methods measure a small fraction of potential oxidation products, given the wide range of ROS and possible cell targets. These markers include urinary and/or plasma products of lipid peroxidation, such as F2 isoprostanes,49 and aldehydes, such as malondialdehyde from lipid fragmentation; products of protein degradation as protein carbonyls and nitrosylated adducts; and products of purine oxidation, such as 8-oxo-2-deoxyguanosine.19,50,51 Inflammation and oxidative stress are related mechanistically. The C-reactive protein serves as a marker for systemic inflammation, as well as liver dysfunction,52 and can be reduced by antioxidant supplementation.53 Gamma-glutamyltransferase (γ-glutamyltranspeptidase [GTT]) is linked to the redox state and to GSH/ oxidized GSH (GSSH) balance, therefore it promotes GSH production. GTT may be useful in assessing oxidative stress associated with type 2 diabetes, the metabolic syndrome,54 and Alzheimer’s disease,55 in addition to coronary heart disease and stroke.56 The ferric ion reducing ability (FRAP) assay has been used to correlate serum total antioxidant capacity (TAC) with metabolic risk factors. Ingestion of foods with high TAC values reduced markers of systemic inflammation and liver dysfunction.52,57

image Antioxidants

Antioxidants inhibit the oxidation of target molecules by radicals and ROS.58 There is an apparent “pecking order” among antioxidants; some are more readily oxidized than others and will be consumed rapidly unless replenished or recycled.59 Certain antioxidants are preventive inhibitors that block the initiation of free radical attack. Preventive inhibitors include defensive enzymes such as catalase, SOD, and peroxidases (GPx), as well low-molecular-weight compounds, including reduced GSH. Beta-carotene, chelating agents such as organic acids, and plant polyphenols represent preventive antioxidants when they quench singlet oxygen or sequester metal ion catalysts. Antioxidants can function as chain breakers, which convert free radicals to stable products and thus block free radical chain reactions. Vitamin E and ascorbic acid are chain-breaking antioxidants.

Laboratory Assessment of Antioxidant Activity

Studies of ROS and radical quenching by antioxidants frequently employ single time points (end-point assays). A more reliable approach evaluates the IC50, the concentration of antioxidant yielding 50% inhibition of a given oxidant or radical. It also is important to compare antioxidant activities in several assay systems. Studies showed that procyanidolic oligomers (PCOs) found in numerous foods and used in supplements (e.g., pine bark and grape seed extract) effectively quench diphenyl picrylhydrazyl radicals.60 Galloyl catechins were shown to be more effective than simple catechins in inhibition of NADPH-dependent lipid peroxidation of rat liver microsomes.61

The oxygen radical absorbance capacity (ORAC) is often used to compare antioxidant activity of foods and complex mixtures. This assay measures the decay in fluorescence (possibly from peroxyl radicals), and results are expressed as “trolox equivalents.”62 ORAC values for foods have been published by the U.S. Food and Drug Administration (FDA).63 In addition the FRAP assay is sometimes used. This method assesses ferric to ferrous reduction to evaluate TAC. The TAC content of more than 3000 foods and supplements has been published.64 To establish cell functionality, preliminary studies explored the feasibility of measuring antioxidant protection of erythrocytes and chemoattractant activity of polymorphs after oral supplementation.65 Cellular antioxidant activity (CAA), as measured by the ability to quench peroxyl radical-induced fluorescence of a fluorescein probe in cultured hepatoma cells, was developed to account for update and metabolism of antioxidants.66 It is worth noting that CAA values of flavonoids do not correlate with ORAC values.

Enzymes as Antioxidants

Superoxide Dismutase: Antioxidant Role for Manganese, Copper, and Zinc

SODs rapidly convert superoxide to H2O2. The mitochondrial enzyme (SOD2) requires manganese, whereas the cytoplasmic (SOD1) and extracellular (SOD3) enzymes require both copper and zinc. SOD2 is induced during acute inflammation,67 whereas SOD3 in vessel walls plays a major role in regulating vascular ROS.68 Knockout mice lacking SOD2 die several days after birth due to massive oxidative damage. Mice lacking SOD1 develop ROS-related pathologies with reduced lifespan. SOD may be effective in treating experimental ulcerative colitis.69 High risk, premature infants treated with prophylactic recombinant human SOD1 had reduced early pulmonary injury at 1 year.70 Preliminary evidence suggests that SOD can reduce free radical damage to skin after radiation treatment for breast cancer.71

Several lines of evidence suggest an involvement of SOD in neurologic abnormalities. Mutations in the SOD1 gene account for 20% of patients with a familial dominant form of amyotrophic lateral sclerosis, although the mechanism is unknown.72 Over-expression of SOD2 in a mouse model of Alzheimer’s disease was shown to reduce brain superoxide and prevent memory losses.73

Catalase

This iron-dependent enzyme converts H2O2 to diatomic oxygen extremely efficiently. It occurs widely in cells and is a component of peroxisomes. Animal studies with catalase, usually in conjunction with SOD, suggest protection against ischemic injury to the lung,74 intestinal ROS damage,75 and radiation.76 Examination of human atherosclerotic coronary arteries revealed that vascular antioxidant enzymes, including catalase, were selectively elevated in smooth muscle cells and macrophages in atherosclerotic lesions.77 In contrast, knockout mice lacking catalase were phenotypically normal, possibly due to effective scavenging by peroxidases.78

Redox Regulation: The Glutathione System

Glutathione

This sulfhydryl-reducing agent is a tripeptide containing cysteine that occurs in millimolar concentrations in most cells, where it acts as a detoxifying agent, assists amino acid transport, and quenches free radicals, in addition to regulating the internal redox environment of cells. Together with ascorbate, GSH participates in the regeneration of vitamin E, which emphasizes the cooperation of antioxidants. Reduced GSH reacts directly with singlet oxygen, hydroxyl radicals, and superoxide radicals to form oxidized glutathione (GSSG). With aging, there is a general decline in GSH levels, and low GSH levels are associated with a variety of chronic conditions, such as diabetes, age-related macular degeneration, gastrointestinal disorders, and neurodegenerative diseases.89

GSH reductase reduces GSSG with NADPH, linking thiol regulation to a robust glucose metabolism. GSH reductase helps maintain a high GSH/GSSG ratio. Oxidative stress reduces this ratio, activates transcription factors, and increases production of IL-1 and TNF.90 NADPH and GSH are cofactors for other reductases that help regenerate tocopherol and ascorbate, demonstrating the principle that overall metabolic balance is a prerequisite for adequate antioxidant defense.

GSH S-transferase adds GSH to potentially toxic substances and is considered a component of phase II detoxification enzymes, although it also functions in the synthesis of prostaglandins and leukotrienes. Like GSH peroxidases and Prxs, GSH S-transferase requires an ample supply of reduced GSH.

Redox Regulation: The Thioredoxin System: Antioxidant Role for Selenium

Thioredoxin (TRX) is a small sulfhydryl protein that acts as a reducing agent for Prxs and ribonucleotide reductase, and TRX also promotes the activation of several transcription factors.91 TRX reductase is a selenocysteine-containing enzyme that specifically catalyzes the reduction of TRX. Together, TRX and TRX reductase represent a widespread endogenous redox-regulating system with multiple roles in intracellular signaling and resistance to oxidative stress. Thus, thioredoxin activates apoptosis signal-regulating kinase-1. It reduces redox factor-1 (REF-1), which then reduces oxidized (inactive) transcription factors such as NF-κB and Nref-2. Apurinic/apyrimidinic endonuclease 1 (APE-1), a dual functional DNA repair enzyme that can also reduce transcription factors (such as AP-1 and p53) is regenerated by thioredoxin, linking DNA repair to cellular redox balance.92

Antioxidant Enzymes in Response to Oxidative Stress

Antioxidant enzyme levels tend to decrease with age-related conditions.93,94 In contrast, several antioxidant enzymes are often induced during oxidative stress. In human atherosclerotic coronary arteries, catalase, GSH peroxidase, and SOD were upregulated in atherosclerotic lesions, possibly as the result of oxidative stress.77 These enzymes are part of a battery of proteins regulated by defensive genes. Nuclear transcription factors such as Nfr2 can be regarded as a cell-sensing system for oxidative and electrophilic stress, which is responsive within 15 minutes after exposure to a stressor.95 Transactivation of AREs is regulated by redox balance, in which nuclear factor Nrf2 migrates to the nucleus in response to ROS, where it activates genes responsible for a battery of antioxidant enzymes, including Prx-6, GPx, GSH transferase, TRX, as well as phase II detoxifying enzymes.32

Micronutrients as Antioxidants

Vitamin C

Ascorbic acid can react with a wide range of ROS, including superoxide, singlet oxygen, hypochlorite, and sulfur radicals.96 Ascorbic acid is an efficient chain-breaking antioxidant in human plasma,97 where it protects lipids and membranes by scavenging peroxyl and hydroxyl radicals.98 The recommended dietary allowance (90 mg for men >19 years old; 75 mg for women >19 years old) is probably an underestimate. Healthy young women may require at least 400 mg/day of ascorbic acid according to tissue saturation studies.99 Vitamin C can reduce heavy metal toxicity.100 Dehydroascorbate is reduced back to ascorbate via GSH and NADPH. In animal models, high levels of ascorbate compensated for low GSH production and vice versa. A combination of ascorbate with vitamin E is possibly more effective than ascorbate alone for older adults.101 Ascorbic acid functions with GSH and lipoic acid to regenerate α-tocopherol.

Cardiovascular Disease

Observational studies suggest that ascorbic acid has, at best, modest effects on the risk of coronary heart disease. Although a meta-analysis found no benefit with vitamin C supplementation on survival and cardiovascular disease (CVD) risk,102 others reported benefits with high supplemental vitamin C.103 The Nurses’ Health Study suggested that vitamin C intake of more than 359 mg/day (diet plus supplements) reduced the risk of CVD.104 For diabetic postmenopausal women, high vitamin C supplementation correlated with an increased risk of CVD mortality.105 The Physicians’ II study found that vitamin C supplementation at 500 mg/day was not cardioprotective for middle-aged men.106

Hypertension

In young healthy women, a higher level of plasma vitamin C was associated with decreased blood pressure.107 A randomized controlled study of hypertensives supplemented with ascorbic acid 500 mg/day found a significant reduction of blood pressure only during the first month of treatment, suggesting a short-term benefit.108

Cancer

High ascorbic acid intake has been linked to a reduced risk of cancers of the oral cavity and esophagus,109 ovaries,110 stomach,111 and colon. However, pooled analysis of eight prospective studies found vitamin C intake unrelated to lung cancer.112 Treatment of male physicians aged equal to or more than 50 years with 500 mg ascorbic acid daily for 8 years found no reduced risk of prostate cancer or total cancer.113 Several studies suggested that extracellular ascorbate at millimolar concentrations (achieved via intravenous or intraperitoneal administration) acted as an antitumor agent in animals and humans via a pro-oxidant mechanism. A plausible mechanism involves ascorbate reduction of transition metals (cupric to cuprous, ferric to ferrous), which react with oxygen, yielding superoxide and H2O2, to which tumor cells are susceptible.114

Neurodegeneration

The Rotterdam Study suggested a high intake of ascorbic acid and vitamin E was associated with a reduced risk of Alzheimer’s disease, especially among cigarette smokers.115 Although ascorbate in plasma or cerebrospinal fluid of 32 patients with Alzheimer’s disease did not correlate with cognitive decline over 1 year, the ratio of cerebrospinal fluid ascorbate/plasma ascorbate increased, which was possibly related to an impaired ability of the brain to concentrate neuroprotective nutrients.116

Vitamin C: A Cell Response Modifier?

Vitamin C was able to increase the efficiency of generating mouse and human pluripotent stem cells from somatic cells, in part by attenuating cell senescence.119 In other research, expression of five functional protein groups related to signaling, apoptosis, and activated transcription, among others, was detected in vitamin C–treated T cells.120 Generally, vitamin C appears to function primarily as an antioxidant in modulating redox balance in a variety of situations, such as attenuating cytochrome P450 detoxification or plasma C-reactive protein levels.

Vitamin E (Tocopherols)

Vitamin E refers to four tocopherols (α, β, γ, δ) and tocotrienols (α, β, γ, δ) that possess an unsaturated isoprenoid side chain. Vitamin E represents a primary chain-breaking antioxidant of lipids, lipoproteins, and membranes, where it acts as a peroxyl radical scavenger, creating a tocopheryl radical. This radical will decompose unless converted back to tocopherol by ascorbic acid, GSH, and coenzyme Q10 (CoQ10), illustrating that antioxidant defenses complement one another.97,121 Vitamin E can exacerbate hypertension in susceptible people. High levels may antagonize other fat-soluble vitamins, thus decreasing bone mineralization. This vitamin may be contraindicated in patients receiving anticoagulants or in those with a vitamin K deficiency.

Vitamin E supplements often incorporate synthetic α-tocopherol, a mixture of D and L isomers, but only the D form (RRR-α-tocopherol) is active in the body. Esterified α-tocopherol is considerably more stable than unesterified tocopherol, and supplements often use esters of vitamin E, which are readily hydrolyzed and absorbed. As with other fat-soluble vitamins, supplementation is prudent in patients with malabsorption syndromes.

Immunity

Supplementing healthy elderly people (consuming an otherwise typical diet) with 60 to 800 mg vitamin E improved several aspects of cell-mediated immunity within 6 to 12 months.122 In other research, ingestion of 268 mg/day of natural vitamin E for 8 months significantly reduced serum immunoglobulin-E, with apparent normalization of most lesions in patients with atopic dermatitis.123

Cardiovascular Disease

Hypothetically, the oxidation of low-density lipoprotein (LDL) and other lipoproteins can initiate atherosclerosis. Increasing dietary vitamin E intake to more than 20 times the usual intake in a Western-type diet was suggested to reduce the risk of heart disease and its manifestations, such as myocardial infarction, among low-risk populations.124 However, randomized, placebo-controlled clinical trials with α-tocopherol (200 to 800 IU/day) in Western populations have been mainly negative.125

Only 5 of 12 clinical studies on vitamin antioxidants and CVD reported reduced cardiovascular death and nonfatal myocardial infarction. These include the Cambridge Heart and Antioxidant Study (CHAOS), the Womens’ Health Study (reduced risk of sudden death from CVD), and the Secondary Prevention with Antioxidants of CVD in End Stage Renal Disease (SPACE) study of hemodialysis patients, who were at high risk of oxidative stress. Inconsistent results with vitamin E may reflect compounding variables including the heterogeneity of risk factors for coronary heart disease, treatment for late course of disease versus treatment for early-onset oxidative stress,126 and genotypic differences among subgroups, for example, the haptoglobin 2-2 genotype in type 2 diabetic patients.127 More generally, it is unrealistic to expect that dosing with one or two antioxidants can substitute for multiple synergistic factors that support interlocking regulatory pathways in reducing risks of chronic degenerative disease.

Neurodegenerative Disease

A high intake of vitamin E and vitamin C was found to be associated with a reduced risk of Alzheimer’s disease according to the Rotterdam study.115 In a controlled trial of patients with moderately severe Alzheimer’s disease, α-tocopherol administered at a daily level of 2000 IU delayed the onset of severe dementia or death in comparison with controls.128 However, a controlled clinical study of patients with mild cognitive impairment found that treatment with 2000 IU vitamin E failed to slow mental decline.129

Cancer

Effects of antioxidant supplementation often depend on the subject’s nutritional status. Clinical studies with vitamin E illustrate this principle. The Shanghai Breast Cancer Study observed a 20% reduction in breast cancer risk with vitamin E supplementation among women with low dietary intake.130 The Linxian General Population Nutrition Intervention Trial, which included adults deficient in several micronutrients, examined the effects of 50 mcg/day of selenium, 30 mg/day of vitamin E, and 15 mg/day of β-carotene. Supplementation led to decreased all-cause mortality, including overall cancer risk.131 In contrast, U.S. studies of vitamin E on cancer rates reported negative results for breast cancer,132 endometrial cancer,133 lung cancer (pooled analysis of eight studies),134 or cancer incidence and cancer mortality.135 Although earlier studies suggested an inverse association between vitamin E intake and risk of prostate cancer, the Physician’s Health Study II of men aged fifty years or older found no reduction in risk with vitamin E supplementation at 400 IU after eight years.113 Supplementation of men equal to or greater than fifty-five years with vitamin E 400 IU per day for up to twelve years increased the risk of prostate cancer by 17%. Men who took vitamin E together with selenomethionine (200 mcg per day) had no higher risk than those taking a placebo.136 Compounding variables in such studies included status of vitamin D, ω-6 fatty acids, and γ-tocopherol, testosterone status in addition to polymorphisms for α-tocopherol–associated protein.137

Gamma-Tocopherol

Although the typical U.S. diet supplies twice as much γ-tocopherol as the α form or tocotrienols, α-tocopherol predominates in the body, due to selection by liver tocopherol-transfer protein. Gamma-tocopherol is also rapidly degraded. It selectively blocks reactive nitrogen species and complements antioxidant actions of α-tocopherol.139 Gamma-tocopherol can protect pancreatic β-cells against NO inhibition in vitro.140 Mice supplementation with a mixture of α- and γ-tocopherol (not α-tocopherol alone) reduced age-related transcriptional changes in the brain.141 Clinical findings using γ-tocopherol are limited. Patients with metabolic syndrome treated with γ-tocopherol 800 mg and α-tocopherol 800 mg/day for 6 weeks had significantly decreased markers of inflammation and oxidative stress (high-sensitivity C-reactive protein and nitrotyrosine) compared with placebo.142 The EPIC Study found no correlation between plasma levels of either α- or γ-tocopherol and risk of prostate cancer.143

Tocotrienols

Tissue distributions of tocotrienols and tocopherols differ, which is perhaps indicative of selective mechanisms in various tissues.144 Tocotrienols can induce apopotosis in prostate cancer and breast cancer cells.145 Tocotrienols can suppress inflammatory markers, such as IL-6, TNF-α, and NO, by stimulated macrophages.146 Clinical studies are in their infancy. Hypercholesterolemic subjects treated with mixtures of tocotrienols daily for 28 days did not have improved serum lipid or glucose concentrations or lipid peroxidation (urinary 8-iso-prostaglandin F2a).147 In contrast, treatment of healthy adults with tocotrienols 160 mg/day for 6 months according to a randomized, double-blind protocol resulted in reduced DNA damage.148

Vitamin E: A Specific Cellular Response Modifier

Several studies suggest that vitamin E administration can increase expression of genes related to Th1/Th2 ratios in older mice.149 Alpha-tocopherol can block upregulation of NF-κB, PKC, and p38 MAPK pathways to prevent inflammatory cytokine production in stimulated human monocytes and in human dendritic cells.150 Variation in cytokine responses to vitamin E in elderly populations seems to reflect polymorphisms in cytokine genes.151 Nonetheless, such observations reflect the altered redox state of cells and tissues. Because other antioxidants yield similar effects as α-tocopherol, some authors contend that vitamin E protects membrane domains and polyunsaturated fatty acid pathways from ROS as its underlying mechanism of action, rather than involvement in specific signaling actions.152

Carotenoids

These plant pigments are conveniently divided into carotenes and xanthophylls (oxygenated carotenes). The best-known carotenoid, β-carotene, represents only 25% to 33% of plasma carotenoids. In general, carotenoids are versatile antioxidants. Beta-carotene is especially effective at low oxygen tension, as found in tissues.153 Increased carotenoid levels have been associated with decreased LDL oxidation.154 They can quench ROS produced during inflammation and modulate levels of proinflammatory prostaglandins and leukotrienes.155,156

Lung Cancer

Although retrospective studies suggested an inverse correlation, analysis of pooled results from six prospective cohort studies found no relationship between dietary β-carotene and lung cancer risk reduction.157 A recent systematic review of prospective studies concluded that dietary intake of total carotenoids did not significantly reduce the risk of lung cancer.158 Supplementation was studied in three large randomized controlled trials (RCTs). The two studies (Alpha-Tocopherol, beta Carotene Cancer Prevention Trial and the beta Carotene and Retinol Efficacy Trial) reported an increased risk of lung cancer in high-risk groups supplemented with β-carotene. In contrast, the Physicians’ Health Study (only 11% smokers) found no effect with β-carotene supplements.

Breast Cancer

The inverse relationship between consumption of carotenoid-rich fruits and vegetables for the risk of breast cancer was strongest among premenopausal women.160 For women who were smokers and did not use supplements, dietary α-carotene and β-carotene intake was inversely associated with the risk of breast cancer.161 In contrast, the Women’s Health Study found no link between lycopene intake and reduced risk of breast cancer.

Adult Macular Degeneration

The xanthophylls lutein and zeaxanthin are the only carotenoids in the retina and macula, where they filter blue light. Cross-section and retrospective case–control studies suggested that increased consumption of carotenoids, especially lutein and zeaxanthin, correlated with a lower risk of advanced, age-related macular degeneration.162 Individuals with intermediate risk of adult macular degeneration (AMD) or with advanced AMD in one eye should consider using a combination of vitamin C, vitamin E, β-carotene, and zinc, as recommended by the Age-Related Eye Disease Study (AREDS). A large scale clinical trial of lutein, zeaxanthin, and ω-3 fatty acids is in progress (AREDS-2).163

Coenzyme Q10 (Ubiquinone)

Ubiquinones contain side chains with isoprene units, and the predominant form in humans is ubiquinone 10 (CoQ10). It functions as an essential electron carrier in mitochondrial ATP production. As the only lipophilic antioxidant synthesized in the body, CoQ10 also stabilizes membranes and functions as an important antioxidant that can recycle α-tocopherol.166 Ubiquinol, the reduced form of CoQ10, protects LDL against lipid peroxidation.167 CoQ10 synthesis requires vitamins B2, B6, B12, and folate, and synthesis may not be optimal in people with a low intake of these important vitamins.

Cardiovascular Disease

In a mouse model for atherosclerosis, large doses of CoQ10 reduced plaque formation.169 In a RCT of patients at risk for CVD, 6 month treatment with a combination of vitamins C and E, CoQ10, and selenium increased arterial elasticity, lowered glycosylated hemoglobin, and increased high-density lipoprotein cholesterol compared with controls.170 CoQ10 has been used with conventional therapy in treatment of congestive heart failure. Studies employing doses ranging 100 to 200 mg/day for up to 3 months reported minor improvements.171 Drugs such as β-blockers and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors lower serum levels of CoQ10.172 However, the necessity of CoQ10 supplementation in these situations remains uncertain. A meta-analysis of 12 clinical trials of various protocols suggested a reduction in elevated systolic and diastolic blood pressure among hypertensive patients supplemented with CoQ10.173 Large, well-controlled trials of CoQ10 are needed. Results of a multinational RCT of patients with cardiac failure (New York Heart Association Classes III & IV) receiving standard therapy and 300 mg of CoQ10 daily can be expected in the near future.174

Thiols as Antioxidants

Alpha-Lipoic Acid

As a coenzyme of pyruvate dehydrogenase and α-ketoglurate dehydrogenase, α-lipoic acid (ALA) is required in mitochondrial catabolism of carbohydrates and fatty acids. ALA also scavenges hydroxyl radicals, peroxynitrite, superoxide, and peroxyl radicals, and it can regenerate endogenous antioxidants, including ascorbic acid and GSH.

Diabetes

Meta-analysis of four RCTs with intravenous ALA indicated significant improvement in diabetic neuropathy.175 Whether oral ALA supplementation offers benefits is less clear. The highest tissue level likely attainable from oral doses is less than 10% of other intracellular antioxidants such as GSH.176 Using a rat model of diabetes, investigators showed that month long ALA intraperitoneal treatment worsened energy balance and increased fat accumulation.177

Aging

Animal studies suggested that high-dose ALA and/or acetyl-L-carnitine can improve brain function,178 reduce brain lipid peroxidation, and increase brain antioxidant enzyme activities in the brains of aged rats,179 especially in the hippocampus.180 Aged mice supplemented orally with ALA had increased glucose tolerance, energy expenditure, and skeletal muscle mitochondria biogenesis, but also had increased loss of lean body mass.181 It is not clear that these results can be extrapolated clinically.

Cardiovascular Disease

Feeding high-dose ALA also reduced myocardial oxidant production to levels observed in healthy young rats.182 ALA supplementation was used in mouse models of ischemia reperfusion and atherosclerosis, where it slowed the formation of atherosclerotic lesions.183 In a rat model of diabetes, intraperitoneal ALA reduced cardiac apoptosis, caspase expression, and enhanced MnSOD activity and GSH levels.184

N-Acetylcysteine and Glutathione

GSH synthesis is regulated in part by cysteine availability. As a supplement, N-acetylcysteine is an effective precursor for GSH. It is deacetylated in the gut to free cysteine, and oral supplementation can efficiently raise intracellular GSH189 in model systems and in healthy individuals.190 N-acetylcysteine would be expected to play a supportive role to improve GSH status and maintain cell redox balance, counter ROS, and attenuate drug toxicity as a conjugating agent in drug metabolism.191 Orally administered GSH may increase intestinal GSH levels via a GSH transport system.192

Oxidative Stress

Supplementation of healthy, trained men with N-acetylcysteine 1800 mg/day for 3 days before exhaustive resistance exercise significantly increased plasma thiols and TAC and reduced lipid peroxidation and protein oxidation.193 A RCT of hypertensive patients with type 2 diabetes reported that 6 month treatment with N-acetylcysteine and arginine significantly reduced systolic blood pressure and lowered levels of oxidized LDL, C-reactive protein, VCAM, and protein oxidation (plasma nitrotyrosine), suggesting improved endothelial function along with reduced oxidative stress.194

Minerals and Oxidative Stress

Magnesium

As a cofactor for nearly all ATP-requiring reactions, magnesium is integral to intermediary metabolism. Low magnesium status is linked to inflammation and activation of the systemic stress response. Subclinical magnesium deficiency is common in the United States, and magnesium deficiency is correlated with chronic diseases associated with aging.195

In rodents, low magnesium status caused release of substance P, leading to increased circulating inflammatory cells, cytokines, and ROS, with depletion of antioxidants associated with cardiomyopathy.196 Furthermore, magnesium deficiency promoted senescence in primary human fibroblasts.197 Experimental magnesium deficiency created systemic inflammation with leukocyte and macrophage activation, inflammatory cytokine release, formation of acute phase proteins, and oxidative stress. Possible mechanisms included opening calcium channels and activation of NF-κB.198 High fructose intake, for example, from excessive high fructose corn syrup intake, coupled with low magnesium status, could set the stage for oxidative stress, insulin resistance, and metabolic syndrome in susceptible individuals.199

Zinc

Beyond its role in SOD, zinc is required by many transcription factors, including tumor suppression protein p53. “Zinc fingers”—domains that bind multiple zinc atoms—stabilize these proteins. In addition to stabilizing membranes, zinc is involved in apoptosis, hormone release, and nerve transmission.200 Subclinical zinc deficiency manifests as inflammation and oxidative stress, and it often accompanies aging.201 That zinc deficiency diminishes immune function, especially Th1 activity, is well established. Zinc is essential for IL-2 mediated T-cell activation. The mechanism seems to rely on activation of NF-κB, resulting in increased expression of TNF-α, IL-1B, and IL-8 cytokines.202 Although a RCT of subjects over 65 years old found that supplementation with zinc 25 mg/day for 3 months increased levels of cytotoxic T cells and CD4 T cells, other clinical studies reported negative results.203,204

Metabolites as Secondary Antioxidants

L-Carnitine

This metabolite is synthesized mainly by the liver and kidney, then transported to tissues using fatty acids and carbohydrate as fuel. Fatty acid transport into mitochondria relies on a carnitine shuttle for β-oxidation. A decline in carnitine correlates with the age-related decline in mitochondrial energy production and increased ROS.205 After demonstrations that supplementation with acetyl-L-carnitine reversed age-related decline in tissue carnitine level in rats, later experiments showed that animals supplemented with high doses of ALA and acetyl-L-carnitine for 1 month exhibited improved substrate binding of brain carnitine acetyltransferase,178,206 improved memory,207 and decreased DNA damage.208 Whether such improvements with high doses of acetyl-L-carnitine translate into long-term benefits for patients remains to be investigated.

Melatonin: A Hormone with Antioxidant Potential

In addition to helping set the circadian rhythm and sleep patterns, this hormone also acts as an antioxidant in vitro and in vivo.213 In senescence-accelerated mice, long-term administration of physiologic levels of melatonin corrected hepatic mitochondrial dysfunction, suggesting that melatonin may reduce oxidative damage associated with aging.214 Melatonin can act by inhibiting the expression of eNOS in laboratory animals.215

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