Hydroxyl Radicals from H₂O₂ and Superoxide

H₂O₂ is not very reactive; however, it spontaneously forms hydroxyl radicals in the presence of iron and copper ions. Superoxide is not highly reactive, either, and usually superoxide concentrations in the cytoplasm and mitochondria are held in check by superoxide dismutases (SODs), which yield H₂O₂. Superoxide also rapidly forms hydroxyl radicals in the presence of transition metal ions. Because hydroxyl radicals are such powerful oxidants, they probably diffuse only several angstroms before attacking cell constituents and forming characteristic decomposition products of lipids, proteins, and DNA.

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

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.

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

Proinflammatory Cytokines

Transcription factors can be activated by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α). Inhibition of NF-κB and AP-1 activation block transcription of proinflammatory cytokines, including interleukin (IL)-1β, IL-2, IL-4, IL-6, and TNF-α.

Inflammatory Eicosanoids

The arachidonate cascade features proinflammatory hydroperoxides and endoperoxide eicosanoids: prostaglandins such as PGG₂, PGH₂, and PGE₂ (via COX); leukotrienes; and hydroxyeicosatetraenoic acid (via lipoxygenase). COX-2 can be induced by superoxide, H₂O₂, and inflammatory cytokines such as IL-1 and TNF-α. Stimulated lipoxygenase can trigger ROS production through sequential activation of GTPase (Rac), PKC, phosphorylation of the NOx subunit of NADPH oxidase, leading to activation of plasma membrane NADPH oxidase and ultimately superoxide synthesis.³⁵

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

Apoptosis and Cell Cycle Regulation

Programmed cell death is regulated by complex pathways involving transcription factors; therefore, it too relies on the cellular redox balance. Oxidative stress triggers apoptosis in several model systems, and apoptosis may be regulated by antioxidants.^37,38 Conditions ranging from diabetes mellitus and heart failure to HIV infection may entail altered apoptosis.³⁹

Regulation of Xenobiotic, Phytochemical, and Carcinogen Metabolism

ROS modulate components of the cytochrome P450 system to activate potential carcinogens. Phase II detoxification enzymes (sulfotransferases, quinone reductase, GSH S-transferase, uridine diphosphate glucuronyl transferase) can block carcinogenic effects and modulate detoxification.⁴⁰

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

Glutathione Peroxidases: Antioxidant Role for Selenium

GPxs reduce H₂O₂ as well as lipid peroxides in the presence of reduced GSH. Unlike catalase and SOD, GPxs require selenocysteine. GPxs exist as several isoforms. GPx1 is a cytoplasmic isoform that prefers H₂O₂ as a substrate. GPx4 has a high affinity for lipid hydroperoxides, reducing membrane lipid peroxides to nontoxic fatty acid alcohols.⁷⁹ Antioxidant enzymes, including extracellular GSH peroxidase, are induced by oxidative stress associated with lung diseases.⁸⁰ Mice lacking GPx1 have a normal life span; however, they develop early cataracts. In contrast, GPx4 knockout mice do not survive early embryonic development.⁸¹ Curiously, reduced levels of GPx4 may increase the mouse life span.⁸²

Peroxiredoxin

This group of six thiol peroxidases reduces H₂O₂, lipid peroxide, and peroxynitrite via an active site cysteinyl, –SH. The PrxII isoform is one of the most abundant proteins in erythrocytes. Prxs control intracellular levels of H₂O₂ to regulate cell signaling in various cell types. Mice lacking PrxI or PrxII develop severe hemolytic anemia and are prone to cancer due to elevated H₂O₂. Prx1 knockout mice exhibit atherosclerotic lesions due to endothelial cell dysfunction.⁸³ NO may protect macrophages against oxidative and nitrosative stress by inducing Prx.⁸⁴

Ferritin, Transferrin, and Ceruloplasmin

Free iron and copper ions catalyze the conversion of H₂O₂ to hydroxyl radicals; therefore, proteins that bind these ions help protect tissues against ROS. Iron stored in ferritin does not participate in generation of free radicals. Under normal circumstances, little unbound iron is present in cells. However, with chronic inflammation, unbound iron may be released from ferritin, posing a potential hazard. Iron storage disease is linked to oxidative damage. Transferrin (which has a high affinity for iron) and ceruloplasmin (which binds copper) can be considered part of the antioxidant defenses.⁸⁵

Metallothionein

This cysteine-rich protein binds many metals. It is induced by cytokines, toxic metals, and oxidative stress. Mitochondrial-specific ROS generators can increase the production of metallothionein in the liver by 3.7- to 11.8-fold in mice, far more than SOD or GSH peroxidase.⁸⁶ Similarly acute ethanol-induced hepatotoxicity and associated oxidative stress were curtailed in mice that had been genetically manipulated to overexpress metallothionein, compared with wild-type mice.⁸⁷

Selenoprotein P

Nearly 60% of plasma selenium is represented by selenoprotein P, which transports selenium to tissues. In vitro, selenoprotein P protects against peroxynitrite-induced damage and reduces phospholipid hydroperoxides.⁸⁸

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.

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

Cataract

Although some investigations reported the risk of cataracts and retinal damage with lowered vitamin C status, a prospective study employing 500 mg of vitamin C, 400 IU of vitamin E, and 15 mg of β-carotene found no effect on the development or progression of cataracts.¹¹⁷

Diabetes

A 12-year prospective study of nondiabetic participants in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk Study noted those with the highest 20% of plasma vitamin C had a 62% lower risk of developing type 2 diabetes compared with those in the lowest 20%.¹¹⁸

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.

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

Diabetes

Whether α-tocopherol can benefit diabetes has not been established through well-controlled clinical studies. Treatment of elderly patients with impaired glucose regulation with α-tocopherol 1000 IU and vitamin C 1000 mg/day reduced markers of oxidative stress and inflammation while improving insulin sensitivity.¹³⁸

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

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.

Prostate Cancer

A meta-analysis of 11 case–control studies and 10 prospective studies found that the highest intake of lycopene or tomatoes accounted for a 11% to 19% reduction in prostate cancer risk.¹⁵⁹ Dietary lycopene intake was not related to prostate cancer risk in a more recent large prospective study.¹⁴³

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.

Cardiovascular Disease

Although several prospective studies found that higher carotenoid-rich foods correlated with reduced risk of CVD, others did not.¹⁰³ Four RCTs found no effect of oral β-carotene, ranging from 20 to 50 mg/day, in preventing CVD.

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

Immunity

CoQ₁₀ may enhance the immune system. Supplementation of healthy volunteers with CoQ₁₀ 3 mg/kg daily for 12 weeks was found to increase white cell levels and decrease lymphocyte DNA damage.¹⁶⁸

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

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

Obesity

Preliminary data are suggestive. A study of 1127 of obese and pre-obese subjects supplemented for 4 months with ALA 800 mg/day reported significant reduction in weight, blood pressure, body mass index, and abdominal circumference.¹⁸⁵

Neurologic Disease

Clinical results are preliminary. Patients with varying stages of Alzheimer’s disease administered 600 mg/day of ALA for 48 months showed slowed disease progression in patients with mild, not moderate, dementia.¹⁸⁶

Alpha-Lipoic Acid: A Cell Response Modifier?

ALA can induce several phase 2 antioxidant and thiol-protective enzymes, including enzymes needed for GSH synthesis. Old rats treated with ALA intraperitoneally were found to have increased γ-glutamylcysteine ligase (GGL) activity, reflecting induced Nrf2 binding to ARE and increased transcriptional levels of GGL subunits, thus reversing the age-related losses.¹⁸⁷ ALA activated protein kinase B (Akt) to increase survival of cultured neuronal cells exposed to oxidative stress.¹⁸⁸

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

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

Does a Bilirubin–Biliverdin Cycle Protect Against Reactive Oxygen Species?

Bilirubin bound to albumin can act as an antioxidant in vitro, although the in vivo mechanism is unclear. Earlier cell culture studies suggested a protective role of bilirubin–biliverdin cycling via heme oxygenase and biliverdin reductase. However, more recent data indicated that bilirubin is degraded by ROS, rather than being converted to biliverdin and recycled.²¹²

Dietary Patterns

For typical Western diets, the estimated daily consumption of total flavonoids is in the range of 100 to 200 mg,²¹⁹ although intake reflects cultural and even regional preferences. As examples, simple phenolic acids (caffeic acid, ferulic acid, gallic acid, and coumaric acids) accounted for 75% of total phenolic intake among Finnish adults, whereas flavonoids accounted for 24% of the total.²²⁰ In an adult Spanish population, the mean intake of total flavonoids was shown to be 313 mg/day, with proanthocyanidins accounting for 60% of the total.²²¹

Flavonoid Bioavailability/Biotransformation

The uptake of individual flavonoids is generally quite limited. Most flavonoids have very low water solubility. Phosphatidylcholine complexes of poorly soluble flavonoids may improve uptake.²²² Absorbed aglycone flavonoids are rapidly conjugated (sulfates, glucuronides) or methyated by intestinal and liver systems, then excreted. Such flavonoid derivatives may have different biological effects than parent compounds. Additionally, gut bacteria can degrade polyphenols by ring session, and absorbed monophenols may act differently than parent compounds. Enterohepatic and enteric recycling of flavonoid metabolites is another complication.²²³ Analysis of plasma and urine of healthy volunteers 2 hours after ingesting 200 mg quercetin or green flavonoids failed to detect increases in any of the 71 aromatic metabolites observable at 5 hours after ingestion, suggesting that flavonoid metabolites are not responsible for the rapid responses to ingested flavonoid-rich foods or beverages.²²⁴

Are Flavonoids Important Antioxidants In Vivo?

In vitro, various flavonoids scavenge a variety of radicals^225,226 and block LDL oxidation.²²⁷ As chelators, flavonoids can inhibit spontaneous generation of hydroxyl radicals²²⁸ catalyzed by copper and iron ions. Although data are conflicting, it appears that flavonoids and their derivatives probably make small contributions as direct antioxidants in vivo.²²⁹ The observed increased antioxidant capacity in plasma after ingestion of fruits, fruit juice, and flavonoid-rich foods may be due to increased production of the antioxidant uric acid, rather than due to flavonoids.^230,231 Peak plasma concentrations of flavonoids and their metabolites after acute ingestion are often less than 1 µmol/L, several orders of magnitude lower than plasma or intracellular concentrations of ascorbic acid, uric acid, and GSH.

Soy

Soy isoflavones can suppress tumors in animal models. Genistein can induce apoptosis via downregulation of Bcl-2 and Bcl-XL, antiapoptotic proteins in human hepatoma cell lines,²³⁶ as well as in breast, prostate, pancreatic, and non-small lung cancer cells. Genistein also inhibits activation of NF-κB and Akt signaling pathways that normally balance cell survival and apoptosis.²³⁷ A recent meta-analysis suggested a significant association between consumption of soy-based foods and reduction of the risk of prostate cancer.²³⁸ Epidemiologic studies on the relationship between a reduced risk of breast cancer and soy intake have yet to provide definitive evidence for taking soy supplements. RCTs are in progress.

Tea

Green tea contains epigallocatechin, epigallocatechin-3 gallate (EGCG), and epicatechin-3 gallate. These flavonoids may block signal transduction pathways and induce apoptosis in various tumor cells, including skin, prostate, colon, breast, and lung.²³⁹ EGCG blocked the growth of human cervical cancer cells via cell cycle arrest and induced apoptosis.²⁴⁰ It did so by inhibiting tyrosine kinases, thus blocking MAPK signaling pathways and NF-κB and AP-1.²⁴¹ Green tea catechins can limit DNA oxidation, lipid peroxidation, and free radical production in smokers.²⁴²

Analysis of nearly 200 epidemiologic studies examined associations between green tea consumption and risks of cancer at different sites and found that the majority of reports did not show a preventive effect. Inhibition with green tea consumption was more frequent among Asian populations according to case–control studies.^239,243 Tea consumption may help specific subgroups, rather than a general population, such as lowering the risk of breast cancer only among women with low catechol-O-methyl transferase.²⁴⁴ Intervention studies are a more effective way to establish protective effects of tea on cancer risk. In a double-blind study, patients at risk for prostate cancer treated with green tea catechins 600 mg/day for 1 year had a cancer rate of 3% compared with a 30% rate for the placebo group.²⁴⁵ A prospective study of patients after resection for colon cancer or polypectomy employed a daily dose of EGCG 20 mg and apigenin 20 mg for up to 4 years. The recurrence rate of neoplasia was 7% for treated patients versus 47% for untreated patients.²⁴⁶

Antioxidant-Rich Beverages

Short-term investigations suggest that regular consumption of fairly large amounts of cocoa, green and black tea, wine, grape juice, and soy can improve vascular and endothelial cell functions. Whether these results translate into long-term reductions in risk of CVD is not known.

Inflammation

Experiments with guinea pig epidermal cells suggest that flavonoids, such as quercetin, can inhibit COX-2 and 5-lipoxygenase pathways to curtail production of inflammatory eicosanoids.²⁶⁶ Flavonoids may reduce expression of adhesion molecules needed to recruit white cells to arterial walls, a property unrelated to their antioxidant potential. Quercetin and kaempferol inhibited adhesion molecule expression induced by TNF-α in human aortic endothelial cells. Pre-exposure of hepatocytes to flavonoids blocked this inhibitory effect, suggesting that liver biotransformation reduces the ability of flavonoids to influence endothelial cells.²⁶⁷ Quercetin supplementation of healthy volunteers with quercetin (500 or 1000 mg) together with vitamin C (125 mg or 250 mg) daily over 12 weeks significantly increased plasma quercetin without changing plasma oxidant capacity (FRAP and ORAC).²⁶⁸

Oxidative Stress

Stress induced by strenuous exercise increased plasma cortisone and lipid peroxides and decreased SOD, GPx, and catalase in rat brains. Treatment with quercetin significantly increased these activities, while lowering plasma peroxides.²⁶⁹ In contrast, long-term quercetin supplementation (1000 mg/day) in athletes did not protect against exercise-induced oxidative stress, although supplementation might lead to a modest improvement in maximal oxygen uptake in untrained individuals.^270,271

Cardiovascular Disease

Short-term consumption of flavonoid-rich foods and beverages can improve CVD risk factors, including endothelial function in healthy volunteers and patients with coronary artery disease. Interest was stimulated by the Zutphen study, which reported an inverse relationship between the incidence of stroke and coronary artery disease and consumption of flavonoids, particularly quercetin.²³⁵

Using a RCT with crossover design, healthy men who ingested 200 mg of quercetin experienced lowered plasma endothelin-1 and increased plasma nitrite and S-nitrothiols, which are biomarkers of NO production.²⁷²

Chemoprevention

Quercetin can induce apoptosis and stimulate production of antioxidant enzymes such as NADPH quinone oxidoreductase activity in human breast cancer cells.^273,274 With induced lung carcinogenesis in mice, quercetin pretreatment effectively prevented losses of SOD, catalase, GPx, GSH S-transferase, GSH reductase, while limiting tumor formation.²⁷⁵ A case–control study found an inverse relationship between the intake of quercetin-rich foods and lung cancer risk, especially for heavy smokers. This effect corresponded to upregulation of GSH transferase isozymes and downregulation of cytochrome P450 isoforms.²⁷⁶

Pine Bark Extract

Pine bark extract (PBE) represents a complex mixture of polyphenols enriched in procyanidolic oligomers (PCOs), containing two to seven monomers of catechin and epicatechin. PCOs are effective antioxidants in vitro and can protect and/or regenerate α-tocopherol and vitamin C. PBE can also upregulate antioxidative enzymes in cultured cells.²⁷⁷

Oxidative Stress

Wine flavonoids including PCOs are effective antioxidants in vitro. They can reduce lipid peroxides and decrease susceptibility of isolated LDL to oxidation. Grape seed extract (GSE) treatment led to significant postprandial reduction in plasma lipid hydroperoxides and increased antioxidant capacity in healthy volunteers.²⁸³ Male smokers administered GSE had significantly decreased platelet reactivity compared with control subjects given a placebo.²⁸⁴ Type 2 diabetic patients received GSE 600 mg/day according to a controlled double-blind protocol. After 4 weeks, significant decreases were observed for plasma fructosamine and high-sensitivity C-reactive protein, and significant increases were seen for whole blood GSH compared with placebo.²⁸⁵

Neurologic Disease

After animal experiments indicating that GSEs can limit lipid peroxidation in the brain,²⁸⁶ GSE polyphenols were found to inhibit protofibril formation and protofiber oligomerization in a mouse model of Alzheimer’s disease.²⁸⁷

Chemoprevention

GSE is able to inhibit proliferation of a variety of cancer cell lines involving regulatory targets such as MAPKs, NF-κB, phosphoinositide 3-kinases (PI 3-kinases), and angiogenesis.²⁸⁸ Furthermore, pretreatment with GSE protected rodents against experimentally induced hepatotoxicity, pulmonary toxicity, and doxorubicin-induced cardiotoxicity by suppressing oxidative stress.^286,289

Silymarin.

Silymarin, a flavonoid concentrate from milk thistle, and silibinin, a principal constituent of silymarin, can scavenge ROS and decrease inflammation.

Resveratrol

Grapes, blueberries, and peanuts contain resveratrol and methylated resveratrol belonging to the stilbene family of polyphenols. Resveratrol can act as an antioxidant to protect human erythrocytes against H₂O₂–induced lipid peroxidation in vitro.²⁹⁶ Stilbenes exhibit low solubility and rapid clearance, and ingestion of acute doses of resveratrol by healthy subjects achieved only nanomolar plasma concentrations, concentrations several orders of magnitude less than those used for in vitro or animal studies.

Ginkgo biloba

A variety of evidence suggests benefits of Ginkgo biloba extract for vascular insufficiency, cerebral insufficiency, and improved cognition. Most studies utilized a standardized extract, EGb 761.

Curcumin

Curcuminoids are bright yellow pigments found in turmeric, a family of lipophilic diketones. Curcumin, the principal curcuminoid, is poorly absorbed with serum concentrations in the nanomolar to micromolar range after oral ingestion of up to 8 g.³¹⁵ Extending the observation that curcumin scavenges ROS and RNS in vitro, consumption of 200 mg/day of curcuminoids decreased serum lipid peroxide levels and LDL lipid peroxidation in healthy subjects.³¹⁶

Garlic

Many health attributes of garlic are derived from multiple organosulfur compounds, including allicin and its derivatives in garlic. Some may influence antioxidant status.

Mechanism

Depending upon the experimental design, polyphenols can inhibit COX, lipooxygenase, telomerase, xanthine oxidase, cytochrome P450, and protein glycation, as well as modulating drug transport systems and platelet function. Flavonoids may influence multiple signaling pathways, making it difficult to assign specific actions. As an example, red wine polyphenols administered to rats for 16 weeks inhibited chemically induced colon cancer.³³¹ In the process, responses to oxidative stress were downregulated. Microarray analysis of cellular changes after polyphenol treatment for 14 days revealed the following frequencies of downregulated genes: receptors and signal transduction, 22%; transport and binding proteins, 14%; inflammatory and immune response, 11%; metabolic enzymes, 4%; gene expression/control, 4%; xenobiotic metabolism, 3%; and cell cycle regulation, 3%. In the final analysis, observed protective effects of flavonoid-rich foods in humans may be independent of flavonoids.

Does Vitamin E Increase Mortality Risk

Meta-analysis of 68 randomized trials found that supplementation up to 800 IU/day did not change the risk of all-cause mortality.¹⁰² A more recent meta-regression analysis of 29 clinical trials representing a total of 138,462 participants and vitamin E dosages ranging from 16.5 to 5000 IU/day found no causal relationship between high-dose vitamin E and mortality.³⁶⁹ Indiscriminant use of high doses of vitamin E is not recommended.^136,370 Vitamin E can interfere with vitamin K-dependent clotting factors, and high doses increase the risk of bleeding in at-risk individuals.

Does Excessive Beta-Carotene Contribute to Disease?

Beta-carotene may act as a pro-oxidant for those at risk for oxidative stress (smoking, alcohol consumption); β-carotene may also raise the risk of lung cancer in high-risk populations unless protected by antioxidants like vitamin E.³⁷¹ When β-carotene is supplemented during oxidative stress, cleavage products are produced, which are genotoxic, and these may account for the carcinogenic effects observed in the Alpha Tocopherol, Beta Carotene Cancer Prevention Study (ATBC) and Beta Carotene and Retinol Efficacy Trial (CARET).³⁷² High levels of β-carotene may be contraindicated in smokers with myocardial infarctions. In one study, the rate of fatal coronary heart disease rose in patients receiving 20 mg/day β-carotene (with or without α-tocopherol).³⁷³ Carotenoids generally lose their effectiveness at high concentrations and at high partial pressures of oxygen. This feature may account for the possible benefits of consumption of carotenoid-rich foods, although therapeutic doses of isolated carotenoids may be deleterious.³⁷⁴

Can Antioxidants Contribute to Oxidative Stress?

The potential pro-oxidant activity of polyphenolic radicals is well documented.³⁷⁵ Flavonoids are metabolized and detoxified by liver enzymes, and pharmacologic doses can induce phase I detoxification enzymes, increasing the ability to transform toxins. The tradeoff lies in the possibly greater sensitivity to mutagens. Vitamins C and E exhibit pro-oxidant activity in vitro under certain conditions. When exposed to catalytic levels of iron or copper ions, ascorbate promotes the formation of H₂O₂ and hydroxyl radicals.^376,377 In vitro studies suggest that ascorbic acid can decompose lipid peroxides to genotoxins.³⁷⁸ In vivo, these effects would likely require high ascorbate concentrations coupled with the depletion of tocopherol.³⁷⁹ Excessive dietary iron or iron accumulation could promote ascorbate-induced oxidation, and ascorbate could act as an oxidant in the presence of iron or copper released during inflammation or injury. In a RCT, hypertensive patients were treated with 500 mg ascorbic acid and/or 1000 mg grape seed polyphenols daily for 6 weeks. Although ascorbic acid alone reduced elevated systolic pressure, the combination significantly increased both systolic and diastolic blood pressure.³⁸⁰

Antioxidants as Adjuvant Cancer Therapy

A systematic review found that 24 of 33 randomized, controlled studies reported decreased toxicity of chemotherapy with concomitant use of antioxidants, including vitamin E and vitamin C, with one study of vitamin A reporting increased toxicity.³⁸¹ As a recent example, a study of pediatric patients with acute lymphoblastoma being treated with chemotherapy and concurrent supplementation with 400 IU vitamin E and 600 mg N-acetylcysteine daily for 28 days found that children taking the supplements experienced less liver toxicity, fewer blood abnormalities, and fewer blood transfusions.³⁸² The use of α-tocopherol succinate to inhibit tumor growth in vivo remains unproven. Because this derivative possesses no antioxidant activity, presumably its inhibition of cancer cell proliferation and induction of apoptosis in tumor cells relies on other mechanisms.