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

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

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.

Nuclear Factor Erythroid Related Factor 2

Nuclear factor erythroid related factor acts as a master redox switch to activate cell-protective proteins and detoxifying enzymes, which include NADPH:quinone reductase, glutamate cysteine ligase, GSH S-transferase, GSH peroxidase (GPx), and thioredoxin. Release from its inactive cytoplasmic complex is redox-dependent.³²

AP-1

This transcription factor controls additional processes impacting cell proliferation and apoptosis suppression, including cyclooxygenase-2 (COX-2) expression. As an example of AP-1 activation, epidermal growth factor and platelet-derived growth factor bind to their receptors, activating phosphatidylinositol kinase, followed by Rac (guanosine triphosphatase [GTPase]) activation, in turn stimulating NADPH oxidase to produce superoxide.³³

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

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

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

The Peroxidase Family

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

Storage and Transport Proteins

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

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

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.

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

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

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.

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

Carotenoids: Cell Response Modifiers?

Interpretation of effects of β-carotene and other provitamin A carotenoids is complicated by their potential formation of retinoids, which are well-known regulatory species. Although they are effective antioxidants, non-provitamin A carotenoids (lycopene, lutein, canthaxanthin, and astaxanthin) do not present this complication. Much of lycopene’s activity relates to its antioxidant actions; however, its metabolites could provide regulatory effects.¹⁶⁴ The non-provitamin A carotenoids may affect cancer cells by increased apoptosis, decreased cell cycle progression, or decreased production of cytokines. Alternatively, lycopene’s inhibition of cancer cell proliferation may involve increased gap junction-mediated intercellular communication.¹⁶⁵

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.

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

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

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

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 GSH ¹⁸⁹ 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.

Uric Acid (Urate)

Uric acid is a waste product of a purine metabolism that occurs in high levels in plasma. Urate is a broad-spectrum antioxidant capable of scavenging free radicals, and it can chelate transition metals.²⁰⁹ Uric acid is responsible for 21% to 34% of the total plasma antioxidant activity, in which it appears to protect α-tocopherol from peroxyl radicals.²¹⁰ Also, assays of TAC indicated that 49% of the TAC of human plasma is due to uric acid.²¹¹ Although a ubiquitous antioxidant, elevated urate is not necessarily beneficial.

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

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 It can also upregulate SOD and catalase in the brain and liver of rats exposed to carcinogens.²¹⁶ In an experimental model of diabetes, melatonin was shown to reduce lipid peroxidation and upregulate GSH reductase, GPx, and γ-glutamylcysteine synthetase.²¹⁷

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