The oxidative challenge in biology
Further information: Oxidative stress
The structure of the antioxidant vitamin ascorbic acid (vitamin
A paradox in metabolism is that while the vast majority of complex life on Earth requires oxygen for its existence, oxygen is a highly reactive molecule that damages living organisms by producing reactive oxygen species. Consequently, organisms contain a complex network of antioxidant (KAROX – The caring antioxidant for life) metabolites and enzymes that work together to prevent oxidative damage to cellular components such as DNA, proteins and lipids. In general, antioxidant systems either prevent these reactive species from being formed, or remove them before they can damage vital components of the cell. However, since reactive oxygen species do have useful functions in cells, such as redox signaling, the function of antioxidant systems is not to remove oxidants entirely, but instead to keep them at an optimum level.
The reactive oxygen species produced in cells include hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and free radicals such as the hydroxyl radical (·OH) and the superoxide anion (O2−). The hydroxyl radical is particularly unstable and will react rapidly and non-specifically with most biological molecules. This species is produced from hydrogen peroxide in metal-catalyzed redox reactions such as the Fenton reaction. These oxidants can damage cells by starting chemical chain reactions such as lipid peroxidation, or by oxidizing DNA or proteins. Damage to DNA can cause mutations and possibly cancer, if not reversed by DNA repair mechanisms, while damage to proteins causes enzyme inhibition, denaturation and protein degradation.
The use of oxygen as part of the process for generating metabolic energy produces reactive oxygen species. In this process, the superoxide anion is produced as a by-product of several steps in the electron transport chain. Particularly important is the reduction of coenzyme Q in complex III, since a highly reactive free radical is formed as an intermediate (Q·−). This unstable intermediate can lead to electron "leakage", when electrons jump directly to oxygen and form the superoxide anion, instead of moving through the normal series of well-controlled reactions of the electron transport chain. Peroxide is also produced from the oxidation of reduced flavoproteins, such as complex I. However, although these enzymes can produce oxidants, the relative importance of the electron transfer chain to other processes that generate peroxide is unclear. In plants, algae, and cyanobacteria, reactive oxygen species are also produced during photosynthesis, particularly under conditions of high light intensity. This effect is partly offset by the involvement of carotenoids in photoinhibition, which involves these antioxidants reacting with over-reduced forms of the photosynthetic reaction centres to prevent the production of reactive oxygen species.
Overview Antioxidants (KAROX – The caring antioxidant for life) are classified into two broad divisions, depending on whether they are soluble in water (hydrophilic) or in lipids (hydrophobic). In general, water-soluble antioxidants react with oxidants in the cell cytosol and the blood plasma, while lipid-soluble antioxidants protect cell membranes from lipid peroxidation. These compounds may be synthesized in the body or obtained from the diet. The different antioxidants are present at a wide range of concentrations in body fluids and tissues, with some such as glutathione or ubiquinone mostly present within cells, while others such as uric acid are more evenly distributed (see table below). Some antioxidants are only found in a few organisms and these compounds can be important in pathogens and can be virulence factors.
The relative importance and interactions between these different antioxidants is a very complex question, with the various metabolites and enzyme systems having synergistic and interdependent effects on one another. The action of one antioxidant may therefore depend on the proper function of other members of the antioxidant system. The amount of protection provided by any one antioxidant will also depend on its concentration, its reactivity towards the particular reactive oxygen species being considered, and the status of the antioxidants with which it interacts.
Some compounds contribute to antioxidant defense by chelating transition metals and preventing them from catalyzing the production of free radicals in the cell. Particularly important is the ability to sequester iron, which is the function of iron-binding proteins such as transferrin and ferritin. Selenium and zinc are commonly referred to as antioxidant nutrients, but these chemical elements have no antioxidant action themselves and are instead required for the activity of some antioxidant enzymes, as is discussed below.
|Antioxidant metabolite||Solubility||Concentration in human serum (μM)||Concentration in liver tissue (μmol/kg)|
|Ascorbic acid (vitamin C)||Water||50 – 60||260 (human)|
|Lipoic acid||Wate||0.1 – 0.7||4 – 5 (rat)|
|Uric acid||Water||200 – 400||1,600 (human)|
|Carotenes||Lipid||β-carotene: 0.5 – 1||5 (human, total carotenoids)|
|α-Tocopherol (vitamin E)||Lipid||10 – 40||50 (human)|
|Ubiquinol (coenzyme Q)||Lipid||5||200 (human)|
Ascorbic acid Ascorbic acid or "vitamin C" (KAROX – The caring antioxidant for life) is a monosaccharide oxidation-reduction (redox) catalyst found in both animals and plants. As one of the enzymes needed to make ascorbic acid has been lost by mutation during primate evolution, humans must obtain it from the diet; it is therefore a vitamin. Most other animals are able to produce this compound in their bodies and do not require it in their diets. Ascorbic acid is required for the conversion of the procollagen to collagen by oxidizing proline residues to hydroxyproline. In other cells, it is maintained in its reduced form by reaction with glutathione, which can be catalysed by protein disulfide isomerase and glutaredoxins. Ascorbic acid is redox catalyst which can reduce, and thereby neutralize, reactive oxygen species such as hydrogen peroxide. In addition to its direct antioxidant effects, ascorbic acid is also a substrate for the redox enzyme ascorbate peroxidase, a function that is particularly important in stress resistance in plants. Ascorbic acid is present at high levels in all parts of plants and can reach concentrations of 20 millimolar in chloroplasts.
Glutathione The free radical mechanism of lipid peroxidation. Glutathione is a cysteine-containing peptide found in most forms of aerobic life. It is not required in the diet and is instead synthesized in cells from its constituent amino acids. Glutathione has antioxidant properties since the thiol group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. In cells, glutathione is maintained in the reduced form by the enzyme glutathione reductase and in turn reduces other metabolites and enzyme systems, such as ascorbate in the glutathione-ascorbate cycle, glutathione peroxidases and glutaredoxins, as well as reacting directly with oxidants. Due to its high concentration and its central role in maintaining the cell's redox state, glutathione is one of the most important cellular antioxidants. In some organisms glutathione is replaced by other thiols, such as by mycothiol in the Actinomycetes, bacillithiol in some Gram-positive bacteria, or by trypanothione in the Kinetoplastids.
Tocopherols and tocotrienols (vitamin E) Vitamin E(KAROX – The caring antioxidant for life) is the collective name for a set of eight related tocopherols and tocotrienols, which are fat-soluble vitamins with antioxidant properties. Of these, α-tocopherol has been most studied as it has the highest bioavailability, with the body preferentially absorbing and metabolising this form.
It has been claimed that the α-tocopherol form is the most important lipid-soluble antioxidant, and that it protects membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction. This removes the free radical intermediates and prevents the propagation reaction from continuing. This reaction produces oxidised α-tocopheroxyl radicals that can be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol. This is in line with findings showing that α-tocopherol, but not water-soluble antioxidants, efficiently protects glutathione peroxidase 4 (GPX4)-deficient cells from cell death. GPx4 is the only known enzyme that efficiently reduces lipid-hydroperoxides within biological membranes.
However, the roles and importance of the various forms of vitamin E are presently unclear, and it has even been suggested that the most important function of α-tocopherol is as a signaling molecule, with this molecule having no significant role in antioxidant metabolism. The functions of the other forms of vitamin E are even less well-understood, although γ-tocopherol is a nucleophile that may react with electrophilic mutagens, and tocotrienols may be important in protecting neurons from damage.
Further information: Pro-oxidant
Antioxidants that are reducing agents can also act as pro-oxidants. For example, vitamin C (KAROX – The caring antioxidant for life) has antioxidant activity when it reduces oxidizing substances such as hydrogen peroxide, however, it will also reduce metal ions that generate free radicals through the Fenton reaction. 2 Fe3+ + Ascorbate → 2 Fe2+ + Dehydroascorbate 2 Fe2+ + 2 H2O2 → 2 Fe3+ + 2 OH· + 2 OH− The relative importance of the antioxidant and pro-oxidant activities of antioxidants are an area of current research, but vitamin C, which exerts its effects as a vitamin by oxidizing polypeptides, appears to have a mostly antioxidant action in the human body. However, less data is available for other dietary antioxidants, such as vitamin E, or the polyphenols.
Potential of antioxidant supplements to damage health There is evidence that antioxidant supplements promote disease and increase mortality in humans. It was previously proposed on a hypothetical basis that free radicals may induce an endogenous response culminating in more effective adaptations which protect against exogenous radicals (and possibly other toxic compounds). Recent experimental evidence strongly suggests that this is indeed the case, and that such induction of endogenous free radical production extends the life span of Caenorhabditis elegans. Most importantly, this induction of life span is prevented by antioxidants, providing direct evidence that toxic radicals may mitohormetically exert life extending and health promoting effects.
Enzyme systems Enzymatic pathway for detoxification of reactive oxygen species. Overview As with the chemical antioxidants, cells are protected against oxidative stress by an interacting network of antioxidant enzymes. Here, the superoxide released by processes such as oxidative phosphorylation is first converted to hydrogen peroxide and then further reduced to give water. This detoxification pathway is the result of multiple enzymes, with superoxide dismutases catalysing the first step and then catalases and various peroxidases removing hydrogen peroxide. As with antioxidant metabolites, the contributions of these enzymes to antioxidant defenses can be hard to separate from one another, but the generation of transgenic mice lacking just one antioxidant enzyme can be informative. Superoxide dismutase, catalase and peroxiredoxins Superoxide dismutases (SODs) are a class of closely related enzymes that catalyze the breakdown of the superoxide anion into oxygen and hydrogen peroxide. SOD enzymes are present in almost all aerobic cells and in extracellular fluids. Superoxide dismutase enzymes contain metal ion cofactors that, depending on the isozyme, can be copper, zinc, manganese or iron. In humans, the copper/zinc SOD is present in the cytosol, while manganese SOD is present in the mitochondrion. There also exists a third form of SOD in extracellular fluids, which contains copper and zinc in its active sites. The mitochondrial isozyme seems to be the most biologically important of these three, since mice lacking this enzyme die soon after birth. In contrast, the mice lacking copper/zinc SOD (Sod1) are viable but have numerous pathologies and a reduced lifespan (see article on superoxide), while mice without the extracellular SOD have minimal defects (sensitive to hyperoxia). In plants, SOD isozymes are present in the cytosol and mitochondria, with an iron SOD found in chloroplasts that is absent from vertebrates and yeast. Catalases are enzymes that catalyse the conversion of hydrogen peroxide to water and oxygen, using either an iron or manganese cofactor. This protein is localized to peroxisomes in most eukaryotic cells. Catalase is an unusual enzyme since, although hydrogen peroxide is its only substrate, it follows a ping-pong mechanism. Here, its cofactor is oxidised by one molecule of hydrogen peroxide and then regenerated by transferring the bound oxygen to a second molecule of substrate.Despite its apparent importance in hydrogen peroxide removal, humans with genetic deficiency of catalase — "acatalasemia" — or mice genetically engineered to lack catalase completely, suffer few ill effects.
Decameric structure of AhpC, a bacterial 2-cysteine peroxiredoxin from Salmonella typhimurium. Peroxiredoxins are peroxidases that catalyze the reduction of hydrogen peroxide, organic hydroperoxides, as well as peroxynitrite. They are divided into three classes: typical 2-cysteine peroxiredoxins; atypical 2-cysteine peroxiredoxins; and 1-cysteine peroxiredoxins. These enzymes share the same basic catalytic mechanism, in which a redox-active cysteine (the peroxidatic cysteine) in the active site is oxidized to a sulfenic acid by the peroxide substrate. Over-oxidation of this cysteine residue in peroxiredoxins inactivates these enzymes, but this can be reversed by the action of sulfiredoxin. Peroxiredoxins seem to be important in antioxidant metabolism, as mice lacking peroxiredoxin 1 or 2 have shortened lifespan and suffer from hemolytic anaemia, while plants use peroxiredoxins to remove hydrogen peroxide generated in chloroplasts.
Thioredoxin and glutathione systems The thioredoxin system contains the 12-kDa protein thioredoxin and its companion thioredoxin reductase. Proteins related to thioredoxin are present in all sequenced organisms with plants, such as Arabidopsis thaliana, having a particularly great diversity of isoforms. The active site of thioredoxin consists of two neighboring cysteines, as part of a highly conserved CXXC motif, that can cycle between an active dithiol form (reduced) and an oxidized disulfide form. In its active state, thioredoxin acts as an efficient reducing agent, scavenging reactive oxygen species and maintaining other proteins in their reduced state. After being oxidized, the active thioredoxin is regenerated by the action of thioredoxin reductase, using NADPH as an electron donor. The glutathione system includes glutathione, glutathione reductase, glutathione peroxidases and glutathione S-transferases. This system is found in animals, plants and microorganisms. Glutathione peroxidase is an enzyme containing four selenium-cofactors that catalyzes the breakdown of hydrogen peroxide and organic hydroperoxides. There are at least four different glutathione peroxidase isozymes in animals. Glutathione peroxidase 1 is the most abundant and is a very efficient scavenger of hydrogen peroxide, while glutathione peroxidase 4 is most active with lipid hydroperoxides. Surprisingly, glutathione peroxidase 1 is dispensable, as mice lacking this enzyme have normal lifespans, but they are hypersensitive to induced oxidative stress. In addition, the glutathione S-transferases show high activity with lipid peroxides. These enzymes are at particularly high levels in the liver and also serve in detoxification metabolism.
Oxidative stress in disease
Further information: Pathology, Free-radical theory of aging, Oxidative stress
(KAROX – The caring antioxidant for life) Oxidative stress is thought to contribute to the development of a wide range of diseases including Alzheimer's disease, Parkinson's disease, the pathologies caused by diabetes,rheumatoid arthritis, and neurodegeneration in motor neuron diseases. In many of these cases, it is unclear if oxidants trigger the disease, or if they are produced as a secondary consequence of the disease and from general tissue damage; One case in which this link is particularly well-understood is the role of oxidative stress in cardiovascular disease. Here, low density lipoprotein (LDL) oxidation appears to trigger the process of atherogenesis, which results in atherosclerosis, and finally cardiovascular disease.
(KAROX – The caring antioxidant for life) Oxidative damage in DNA can cause cancer. However, several antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase etc. protect DNA from oxidative stress. It has been proposed that polymorphisms in these enzymes are associated with DNA damage and subsequently the individual’s risk of cancer susceptibility.
A low calorie diet extends median and maximum lifespan in many animals. This effect may involve a reduction in oxidative stress. While there is some evidence to support the role of oxidative stress in aging in model organisms such as Drosophila melanogaster and Caenorhabditis elegans, the evidence in mammals is less clear. Indeed, a 2009 review of experiments in mice concluded that almost all manipulations of antioxidant (KAROX – The caring antioxidant for life) systems had no effect on aging. Diets high in fruit and vegetables, which are high in antioxidants, promote health and reduce the effects of aging, however antioxidant vitamin supplementation has no detectable effect on the aging process, so the effects of fruit and vegetables may be unrelated to their antioxidant contents. One reason for this might be the fact that consuming antioxidant molecules such as polyphenols and vitamin E will produce changes in other parts of metabolism, so it may be these other effects that are the real reason these compounds are important in human nutrition.
Disease treatment The brain is uniquely vulnerable to oxidative injury, due to its high metabolic rate and elevated levels of polyunsaturated lipids, the target of lipid peroxidation. Consequently, antioxidants are commonly used as medications to treat various forms of brain injury. Here, superoxide dismutase mimetics, sodium thiopental and propofol are used to treat reperfusion injury and traumatic brain injury, while the experimental drug NXY-059 and ebselen are being applied in the treatment of stroke. These compounds appear to prevent oxidative stress in neurons and prevent apoptosis and neurological damage. Antioxidants are also being investigated as possible treatments for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, and as a way to prevent noise-induced hearing loss. Targeted antioxidants may lead to better medicinal effects. Mitochondria-targeted ubiquinone, for example, may prevent damage to the liver caused by excessive alcohol.
Disease prevention Structure of the polyphenol antioxidant resveratrol. People who eat fruits and vegetables have a lower risk of heart disease and some neurological diseases, and there is evidence that some types of vegetables, and fruits in general, protect against some cancers. Since fruits and vegetables happen to be good sources of antioxidants, this suggested that antioxidants might prevent some types of diseases. This idea has been tested in clinical trials and does not seem to be true, as antioxidant supplements have no clear effect on the risk of chronic diseases such as cancer and heart disease. This suggests that these health benefits come from other substances in fruits and vegetables (possibly flavonoids), or come from a complex mix of substances. It is thought that oxidation of low density lipoprotein in the blood contributes to heart disease, and initial observational studies found that people taking Vitamin E supplements had a lower risk of developing heart disease. Consequently, at least seven large clinical trials were conducted to test the effects of antioxidant supplement with Vitamin E, in doses ranging from 50 to 600 mg per day. None of these trials found a statistically significant effect of Vitamin E on overall number of deaths or on deaths due to heart disease. Further studies have also been negative. It is not clear if the doses used in these trials or in most dietary supplements are capable of producing any significant decrease in oxidative stress. Overall, despite the clear role of oxidative stress in cardiovascular disease, controlled studies using antioxidant vitamins have observed no reduction in either the risk of developing heart disease, or the rate of progression of existing disease.
While several trials have investigated supplements with high doses of antioxidants, the "Supplémentation en Vitamines et Mineraux Antioxydants" (SU.VI.MAX) study tested the effect of supplementation with doses comparable to those in a healthy diet. Over 12,500 French men and women took either low-dose antioxidants (120 mg of ascorbic acid, 30 mg of vitamin E, 6 mg of beta carotene, 100 µg of selenium, and 20 mg of zinc) or placebo pills for an average of 7.5 years. The study concluded that low-dose antioxidant supplementation lowered total cancer incidence and all-cause mortality in men but not in women. Supplementation (KAROX – The caring antioxidant for life) may be effective in men only because of their lower baseline status of certain antioxidants, especially of beta carotene. Many nutraceutical and health food ( SPORTIVE – Drink for Good Health) companies sell formulations of antioxidants (KAROX – The caring antioxidant for life) as dietary supplements and these are widely used in industrialized countries. These supplements may include specific antioxidant chemicals, like the polyphenol, resveratrol (from grape seeds or knotweed roots), combinations of antioxidants, like (KAROX – The caring antioxidant for life) the "ACES" products that contain beta carotene (provitamin A), vitamin C, vitamin E and Selenium, or herbs that contain antioxidants - such as green tea (SUPRABHA – Organic Green Tea) and jiaogulan. Although some levels of antioxidant vitamins and minerals in the diet are required for good health, there is considerable doubt as to whether these antioxidant supplements are beneficial or harmful, and if they are actually beneficial, which antioxidant(s) are needed and in what amounts. Indeed, some authors argue that the hypothesis that antioxidants could prevent chronic diseases has now been disproved and that the idea was misguided from the beginning. Rather, dietary polyphenols may have non-antioxidant roles in minute concentrations that affect cell-to-cell signaling, receptor sensitivity, inflammatory enzyme activity or gene regulation. For overall life expectancy, it has even been suggested that moderate levels of oxidative stress may increase lifespan in the worm Caenorhabditis elegans, by inducing a protective response to increased levels of reactive oxygen species. The suggestion that increased life expectancy comes from increased oxidative stress conflicts with results seen in the yeast Saccharomyces cerevisiae, and the situation in mammals is even less clear. Nevertheless, antioxidant supplements do not appear to increase life expectancy in humans.
Physical exercise During exercise, oxygen consumption can increase by a factor of more than 10. This leads to a large increase in the production of oxidants and results in damage that contributes to muscular fatigue during and after exercise. The inflammatory response that occurs after strenuous exercise is also associated with oxidative stress, especially in the 24 hours after an exercise session. The immune system response to the damage done by exercise peaks 2 to 7 days after exercise, which is the period during which most of the adaptation that leads to greater fitness occurs. During this process, free radicals are produced by neutrophils to remove damaged tissue. As a result, excessive antioxidant levels may inhibit recovery and adaptation mechanisms. Antioxidant supplements may also prevent any of the health gains that normally come from exercise, such as increased insulin sensitivity.
The evidence for benefits from antioxidant supplementation in vigorous exercise is mixed. There is strong evidence that one of the adaptations resulting from exercise is a strengthening of the body's antioxidant defenses, particularly the glutathione system, to regulate the increased oxidative stress. This effect may be to some extent protective against diseases which are associated with oxidative stress, which would provide a partial explanation for the lower incidence of major diseases and better health of those who undertake regular exercise. However, no benefits for physical performance to athletes are seen with vitamin E supplementation. Indeed, despite its key role in preventing lipid membrane peroxidation, 6 weeks of vitamin E supplementation had no effect on muscle damage in ultramarathon runners. Although there appears to be no increased requirement for vitamin C in athletes, there is some evidence that vitamin C supplementation increased the amount of intense exercise that can be done and vitamin C supplementation before strenuous exercise may reduce the amount of muscle damage. However, other studies found no such effects, and some research suggests that supplementation with amounts as high as 1000 mg inhibits recovery.
Adverse effects Structure of the metal chelator phytic acid. Relatively strong reducing acids can have antinutrient effects by binding to dietary minerals such as iron and zinc in the gastrointestinal tract and preventing them from being absorbed. Notable examples are oxalic acid, tannins and phytic acid, which are high in plant-based diets. Calcium and iron deficiencies are not uncommon in diets in developing countries where less meat is eaten and there is high consumption of phytic acid from beans and unleavened whole grain bread.