Space-filling model of the antioxidant metabolite glutathione. The yellow sphere is the redox-active sulfur atom that provides antioxidant activity, while the red, blue, white, and dark grey spheres represent oxygen, nitrogen,
hydrogen, and carbon atoms, respectively.
An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions
by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols.
Although oxidation reactions are crucial for life, they can also be damaging;
hence, plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases. Low levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells.
As oxidative stress might be an important part of many human diseases, the use of antioxidants in pharmacology is intensively studied, particularly as treatments for stroke and neurodegenerative diseases. However, it is unknown whether oxidative stress is the cause or the consequence of disease.
As part of adaptation for life plants began
producing antioxidants such as ascorbic acid ( Vitamin C), polyphenols, flavonoids and tocopherols. The term antioxidant originally was used to refer specifically to a chemical that prevented the consumption of oxygen.
Early research on the role of antioxidants in biology focused on their use in preventing the oxidation
of unsaturated fats, which is the cause of rancidity.
The possible mechanisms of action of antioxidants were first explored when it was recognized that a substance with anti-oxidative activity is likely to be
one that is itself readily oxidized. Research into how vitamin E prevents the process of lipid peroxidation led to the identification of antioxidants as reducing agents that prevent oxidative reactions, often by scavenging reactive oxygen species before they can damage cells.
The oxidative challenge in biology
The structure of the antioxidant vitamin ascorbic acid (vitamin C).
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 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.
Antioxidants 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.
Ascorbic acid or "vitamin C" 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. 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.
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, or by trypanothione in the Kinetoplastids.
Melatonin is a powerful antioxidant that can easily cross cell membranes and the blood-brain barrier. Unlike other antioxidants, melatonin does not undergo redox cycling, which is the ability of a molecule to undergo repeated reduction and oxidation. Redox cycling may allow other antioxidants (such as vitamin C) to act as pro-oxidants and promote free radical formation. Melatonin, once oxidized, cannot be reduced to its former state because it forms several
stable end-products upon reacting with free radicals. Therefore, it has been referred to as a terminal (or suicidal) antioxidant.
Tocopherols and tocotrienols (vitamin E)
Vitamin E 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.
Antioxidants that are reducing agents can also act as pro-oxidants. For example, vitamin C 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 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, for example, appears to have a mostly antioxidant action in the body. However, less data is available for other dietary antioxidants, such as vitamin E, or the polyphenols.
Enzymatic pathway for detoxification of reactive oxygen species.
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
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.
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 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.
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.
It is thought that oxidation of low density lipoprotein in the blood contributes to heart disease.
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.
nutraceutical and health food companies sell formulations of antioxidants 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 the "ACES" products that contain beta carotene (provitamin A),
vitamin C, vitamin E and Selenium, or herbs that contain antioxidants -
such as green tea and jiaogulan. Dietary polyphenols may have non-antioxidant roles in minute concentrations that affect cell-to-cell
signaling, receptor sensitivity, inflammatory enzyme activity or gene regulation.
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
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.
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.
Nonpolar antioxidants such as eugenol—a major component of oil of cloves—have toxicity limits that can be exceeded with the misuse of undiluted essential oils. Toxicity associated with high doses of water-soluble antioxidants such as ascorbic acid are less of a concern, as these
compounds can be excreted rapidly in urine.
Measurement and levels in food
Fruits and vegetables are good sources of antioxidants.
Measurement of antioxidants is not a straightforward
process, as this is a diverse group of compounds with different reactivities to different reactive oxygen species. In food science, the oxygen radical absorbance capacity (ORAC) has become the current industry standard for assessing antioxidant strength of whole foods, juices
and food additives. Other measurement tests include the Folin-Ciocalteu reagent, and the Trolox equivalent antioxidant capacity assay.
Antioxidants are found in varying amounts in foods such as vegetables, fruits,
grain cereals, eggs, meat, legumes and nuts. Some antioxidants such as lycopene and ascorbic acid can be destroyed by long-term storage or prolonged cooking. Other antioxidant compounds are more stable, such as the polyphenolic antioxidants in foods such as whole-wheat cereals
and tea. The effects of cooking and food processing are complex, as these processes can also increase the bioavailability of antioxidants, such as some carotenoids in vegetables. In general, processed foods contain fewer antioxidants than fresh and uncooked foods, since the preparation processes
may expose the food to oxygen.
containing high levels of these antioxidants
C (ascorbic acid)
Fruits and vegetables
antioxidants (resveratrol, flavonoids)
Tea, coffee, soy, fruit, olive oil, chocolate, cinnamon, oregano and red wine
Carotenoids (lycopene, carotenes, lutein)
Fruit, vegetables and
Other antioxidants are not vitamins and are instead
made in the body. For example, ubiquinol (coenzyme Q) is poorly absorbed from the gut and is made in humans through the mevalonate pathway. Another example is glutathione, which is made from amino acids. As any glutathione in the gut is broken down to free cysteine, glycine and glutamic acid before being absorbed, even large oral doses have little effect on the concentration of glutathione in the body. Although large amounts of sulfur-containing amino acids such as acetylcysteine can increase glutathione, no evidence exists that eating high levels of these glutathione precursors is beneficial for healthy adults. Supplying more of these precursors may be useful as part of the treatment of some diseases, such as acute respiratory distress syndrome, protein-energy malnutrition, or preventing the liver damage produced by paracetamol overdose.
Other compounds in the diet can alter the levels of antioxidants by acting as
pro-oxidants. Here, consuming the compound causes oxidative stress, which the body responds to by inducing higher levels of antioxidant
defenses such as antioxidant enzymes. Some of these compounds, such as isothiocyanates and curcumin, may be chemopreventive agents that either block the transformation of abnormal cells into cancerous cells, or even kill existing cancer cells.