Ten misconceptions about antioxidants
2013; Elsevier BV; Volume: 34; Issue: 8 Linguagem: Inglês
10.1016/j.tips.2013.05.010
ISSN1873-3735
AutoresAalt Bast, Guido R.M.M. Haenen,
Tópico(s)Free Radicals and Antioxidants
Resumo•Opinions on antioxidants are highly polarized, leading to misunderstanding in the field.•Specific antioxidants may have specific effects and side effects.•With their pleiotropic modes of action, antioxidants should be considered bioactives. Oxidative damage is a common cellular event involved in numerous diseases and drug toxicities. Antioxidants prevent or delay oxidative damage, and therefore there has been extensive research into the discovery of natural and newly designed antioxidants. Initial excitement regarding the potential health benefits of antioxidants has diminished. Currently, it is even claimed that antioxidants increase mortality. The antioxidant pendulum appears to swing from healthy to toxic and from general panacea to insignificant ingredient. Owing to the polarity of views towards antioxidants, nutritional recommendation ranges from advice to increase antioxidant status in plasma to the notion that it is a useless measurement. Such views, lacking sufficient scientific support, lead to misconceptions, which in our opinion hinder the rational use of food supplements and impedes the design and development of new antioxidant drugs. As a result, good opportunities might easily be missed. Oxidative damage is a common cellular event involved in numerous diseases and drug toxicities. Antioxidants prevent or delay oxidative damage, and therefore there has been extensive research into the discovery of natural and newly designed antioxidants. Initial excitement regarding the potential health benefits of antioxidants has diminished. Currently, it is even claimed that antioxidants increase mortality. The antioxidant pendulum appears to swing from healthy to toxic and from general panacea to insignificant ingredient. Owing to the polarity of views towards antioxidants, nutritional recommendation ranges from advice to increase antioxidant status in plasma to the notion that it is a useless measurement. Such views, lacking sufficient scientific support, lead to misconceptions, which in our opinion hinder the rational use of food supplements and impedes the design and development of new antioxidant drugs. As a result, good opportunities might easily be missed. Few scientific subjects have generated as many controversial opinions as antioxidants have. The topic is discussed not only in the scientific literature but also in the lay press. A Google search combining the words ‘antioxidant’ and ‘health’ gives over 82 million hits, exemplifying the popularity of the subject. Amid the debate, claims about antioxidants can be strongly exaggerated and irrelevant and flawed arguments are even advanced to substantiate such claims. For example, a mixture of compounds is marketed as a ‘life extension formula’ because the compounds are antioxidants and it is claimed that long-term use of a soap that contains the antioxidant vitamin E is ‘excellent for stretch marks, wrinkles, and blemishes. The soap is also suitable for people with a sensitive skin, and beneficial for eczema or psoriasis’. An accurate risk–benefit analysis of antioxidants cannot be achieved in this way. A basic fact is that antioxidants are part of our daily diet in fruits, vegetables, beverages, spices, and herbs (Box 1), and antioxidant intake is the focus of increasing attention. More recently, designer foods have been enriched with antioxidants. Antioxidants are commonly taken as supplements, and there are also drugs with a clear antioxidant profile on the market [1Bast A. Antioxidant pharmacotherapy.Drug News Perspect. 1994; 7: 465-472Google Scholar]. For several decades, we have noticed that the antioxidant pendulum appears to swing vigorously from ‘only healthy’ to ‘extremely toxic’, and from ‘natural antioxidants are best’ to ‘antioxidants cannot act’. The squabbling parties do not seem to listen to counter-arguments. Erroneous statements are not corrected, and thus the pendulum oscillates to the extremes. This inevitably hampers research in the field and confuses both scientists and consumers. As a consequence, we might fail to spot opportunities for which antioxidants may aid in optimizing health.Box 1Redox balance between oxidants and antioxidantsWhat is an oxidant?From a chemical point of view, an oxidant takes up electrons. Biologically, oxidants damage biomolecules. They are reactive species that originate from various biological processes. Examples of oxidizing reactive species are the superoxide anion radical (O2•–), the hydroxyl radical (•OH), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl). Reactive oxygen species are formed as by-products of mitochondrial respiration and are also generated during inflammation from NADPH oxidase on phagocytes. Xanthine oxidase, the metabolism of arachidonic acid, cytochrome P450, nitric oxide synthase, and myeloperoxidase are also possible sources of reactive species. The biotransformation of drugs, such as the redox cycling drugs doxorubicin and nitrofurantoin, leads to the formation of reactive species.What is an antioxidant?From a chemical point of view, an antioxidant is a compound that prevents or delays the oxidation of another compound.Of two compounds, the one that becomes oxidized functions as an antioxidant for the other. In this sense, antioxidants are not very special. Biologically, however, the definition of an antioxidant requires that it should be active in protecting physiological targets (e.g., fatty acids, proteins, and DNA) at relatively low concentrations. Dietary antioxidants include vitamins E and C. Drugs such as the anesthetic propofol and the recently developed antifibrotic agent pirfenidone act as antioxidants. The mucolytic agent N-acetylcysteine acts as a precursor for the endogenous antioxidant glutathione. From a chemical point of view, an oxidant takes up electrons. Biologically, oxidants damage biomolecules. They are reactive species that originate from various biological processes. Examples of oxidizing reactive species are the superoxide anion radical (O2•–), the hydroxyl radical (•OH), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl). Reactive oxygen species are formed as by-products of mitochondrial respiration and are also generated during inflammation from NADPH oxidase on phagocytes. Xanthine oxidase, the metabolism of arachidonic acid, cytochrome P450, nitric oxide synthase, and myeloperoxidase are also possible sources of reactive species. The biotransformation of drugs, such as the redox cycling drugs doxorubicin and nitrofurantoin, leads to the formation of reactive species. From a chemical point of view, an antioxidant is a compound that prevents or delays the oxidation of another compound. Of two compounds, the one that becomes oxidized functions as an antioxidant for the other. In this sense, antioxidants are not very special. Biologically, however, the definition of an antioxidant requires that it should be active in protecting physiological targets (e.g., fatty acids, proteins, and DNA) at relatively low concentrations. Dietary antioxidants include vitamins E and C. Drugs such as the anesthetic propofol and the recently developed antifibrotic agent pirfenidone act as antioxidants. The mucolytic agent N-acetylcysteine acts as a precursor for the endogenous antioxidant glutathione. Here we discuss ten misconceptions and try to restore the balance. Antioxidants react with reactive oxygen species (ROS) and thus neutralize their chemical reactivity. It was suggested that this mechanism prevents the (cellular) damage induced by ROS. This led to the claim that ROS-mediated diseases could be treated. ROS are involved in numerous diseases [2Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine.4th edn. Oxford University Press, 2007Google Scholar]. Subsequently, it was suggested that antioxidants could prevent and treat many diseases [3Willcox J.K. et al.Antioxidants and prevention of chronic disease.Crit. Rev. Food Sci. Nutr. 2004; 44: 275-295Crossref PubMed Scopus (831) Google Scholar], which boosted antioxidant research. ROS play a role in chronic obstructive pulmonary disease (COPD), for example. Patients with COPD even exhale the oxidant (ROS) hydrogen peroxide. Another example is the suggested causative role of ROS in ischemia–reperfusion damage in brain, heart, and kidney. In atherosclerosis, low-density lipoprotein (LDL) is oxidized and is subsequently taken up by macrophages, leading to foam cell formation and eventually cardiovascular complications. Compounds that can give rise to oxygen radicals (nitrofurantoin, doxorubicin) can also induce tissue damage in lung and heart, respectively. Many studies on protection by antioxidants have been conducted. The expectations for antioxidants were set too high and it was apparent that these compounds cannot remedy everything. Moreover, unrealistic health claims disappointed consumers and scientists. Initial enthusiasm turned into disbelief, and some antioxidants, such as vitamin C, were even considered to be toxic [4Lee S.H. et al.Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins.Science. 2001; 292: 2083-2086Crossref PubMed Scopus (407) Google Scholar, 5Podmore I.D. et al.Vitamin C exhibits pro-oxidant properties.Nature. 1998; 392: 559Crossref PubMed Scopus (721) Google Scholar]. Regarding healthful effects, the positive attitude towards antioxidants was primarily based on in vitro experiments. Effective chemical scavenging activity of antioxidants in vitro led to extrapolation to a protective potential in vivo. Identifying the bioavailability of antioxidants has been largely neglected, although polyphenolic antioxidants appeared to have low bioavailability. In addition, bioaccessibility, corresponding to the fraction that becomes available for absorption, must be considered; in other words, the antioxidant has to be liberated from the food matrix [6Anson N.M. et al.Bioprocessing of wheat bran improves in vitro bioaccessibility and colonic metabolism of phenolic compounds.J. Agric. Food Chem. 2009; 57: 6148-6155Crossref PubMed Scopus (204) Google Scholar]. After absorption, first-pass metabolism can be very extensive, and currently there is more emphasis on the biological relevance of antioxidant metabolites [7Hollman P.C. et al.The biological relevance of direct antioxidant effects of polyphenols for cardiovascular health in humans is not established.J. Nutr. 2011; 141: 989S-1009SCrossref PubMed Scopus (346) Google Scholar]. We now realize that high chemical reactivity of the parent compound in vitro is not conclusive evidence that the compound can cure any disease associated with ROS. We are again observing overenthusiasm towards antioxidants with the discovery of their effects on gene expression by mechanisms other via an influence on the DNA sequence (i.e., epigenetics). Presented as a new unifying mechanism to putatively explain all the activities of antioxidants [8Malireddy S. et al.Phytochemical antioxidants modulate mammalian cellular epigenome: implications in health and disease.Antioxid. Redox Signal. 2012; 17: 327-339Crossref PubMed Scopus (97) Google Scholar], it seems that history repeats itself. A recent meta-analysis of selected randomized clinical trials concluded that antioxidant supplementation increased all-cause mortality [9Bjelakovic G. et al.Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis.J. Am. Med. Assoc. 2007; 297: 842-857Crossref PubMed Scopus (1872) Google Scholar]. However, this conclusion was refuted after re-examination showed that none of the studies had mortality as a primary outcome [10Biesalski H.K. et al.Reexamination of a meta-analysis of the effect of antioxidant supplementation on mortality and health in randomized trials.Nutrients. 2010; 2: 929-949Crossref PubMed Scopus (49) Google Scholar]. Despite the obvious criticism, the general unjustified and unbalanced notion that antioxidants could be highly unsafe remains. Instead of the polarized view whereby antioxidants are either good or bad, a high benefit–risk ratio would be a more appropriate approach to evaluate antioxidants. Such a ratio weighs both the pros and cons, and thus provides a more realistic and balanced view. Observational studies have suggested a protective effect of vitamin E intake on coronary heart disease [11Stampfer M.J. et al.Vitamin-E consumption and the risk of coronary-disease in women.N. Engl. J. Med. 1993; 328: 1444-1449Crossref PubMed Scopus (1924) Google Scholar]. However, many large, randomized, placebo-controlled trials reported disappointing results for the effect of vitamin E on risk of cardiovascular disease [12Vivekananthan D.P. et al.Use of antioxidant vitamins for the prevention of cardiovascular disease: meta-analysis of randomised trials.Lancet. 2003; 361: 2017-2023Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar, 13Eidelman R.S. et al.Randomized trials of vitamin E in the treatment and prevention of cardiovascular disease.Arch. Intern. Med. 2004; 164: 1552-1556Crossref PubMed Scopus (196) Google Scholar]. A critical evaluation of these studies suggested that a detailed analysis of the participants’ diets might lead to a different conclusion. It was suggested that some apparently healthy participants have higher rates of lipid peroxidation than others. These ‘rancids’ might show a higher risk of cardiovascular disease and would be the individuals who might benefit from additional antioxidants [14Halliwell B. The antioxidant paradox.Lancet. 2000; 355: 1179-1180Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar]. Similarly, the overall null effect of vitamin E on total stroke occurrence might simply be due to a broad definition of stroke and this it might not be possible to capture the differences in pathophysiology underlying various ischemic and hemorrhagic events. A meta-analysis of studies that investigated the effect of vitamin E on stroke indeed showed that it increased the risk of hemorrhagic stroke by 22% and reduced the risk of ischemic stroke by 10% [15Schürks M. et al.Effects of vitamin E on stroke subtypes: meta-analysis of randomised controlled trials.Br. Med. J. 2010; 314: c5702Crossref Scopus (267) Google Scholar]. Because of the unfavorable risk–benefit ratio, that is, a relatively small risk reduction for ischemic stroke and the generally more severe outcome for hemorrhagic stroke, the authors cautioned against indiscriminate widespread use of vitamin E [15Schürks M. et al.Effects of vitamin E on stroke subtypes: meta-analysis of randomised controlled trials.Br. Med. J. 2010; 314: c5702Crossref Scopus (267) Google Scholar]. Articles on antioxidants and mortality [9Bjelakovic G. et al.Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis.J. Am. Med. Assoc. 2007; 297: 842-857Crossref PubMed Scopus (1872) Google Scholar, 16Watson J. Oxidants, antioxidants and the current incurability of metastatic cancers.Open Biol. 2013; 3: 120144Crossref PubMed Scopus (283) Google Scholar] have received much attention. For example, it has recently been argued that antioxidant use is more likely to cause than to prevent cancer [16Watson J. Oxidants, antioxidants and the current incurability of metastatic cancers.Open Biol. 2013; 3: 120144Crossref PubMed Scopus (283) Google Scholar]. Blockage of oxidant-driven apoptosis in cancer cells by antioxidants was presented as a potentially hazardous phenomenon. Because antioxidants do not have to have a beneficial effect per se, it is important to identify groups such as ‘rancids’ who might benefit from antioxidants. We should not place too much credence in unbalanced alarming news. As the founding father of orthomolecular medicine, Linus Pauling advocated the use of the antioxidant vitamin C in doses of 1000 mg [17Pauling L. Vitamin C and the Common Cold. W.H. Freeman, 1970Google Scholar] to optimize health, which vastly exceeds the recommended dietary allowance of 75–90 mg/day [18Institute of Medicine Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. National Academy Press, 2000Google Scholar]. A solid scientific justification with clinical studies supporting the assumed health benefit of mega doses of vitamin C is, however, lacking. Moreover, the Renaissance physician Paracelsus noted more than 500 years ago that every compound has negative effects at a high dose. This also holds for antioxidants. Administration of high doses of antioxidants might explain the increased toxicity that is sometimes reported. For example, supplementation with 20 mg of β-carotene in male smokers increased the incidence of lung cancer by 18% [19The Alfa-Tocopherol, Beta Carotene Cancer Prevention Study GroupThe effect of vitamin E and beta carotene on the incidence of lung cancer and other cancer in male smokers.N. Engl. J. Med. 1994; 330: 1029-1035Crossref PubMed Scopus (4432) Google Scholar]. Note that the estimated average daily intake of β-carotene is only 2–7 mg [20Kousik A. et al.Intake of the major carotenoids and the risk of epithelial ovarian cancer in a pooled analysis of 10 cohort studies.Int. J. Cancer. 2006; 119: 2148-2154Crossref PubMed Scopus (42) Google Scholar, 21EFSA Panel on Food Additives and Nutritional Sources Added to Food Scientific opinion on the re-evaluation of mixed carotenes and beta-carotene as food additive.EFSA J. 2012; 10: 2593Google Scholar]. In hindsight it is astonishing that at the time of supplementation studies, essential information on the biotransformation of β-carotene was lacking [22Wang X.D. Absorption and metabolism of beta-carotene.J. Am. Coll. Nutr. 1994; 13: 314-325Crossref PubMed Scopus (84) Google Scholar]. Clearly, ‘the more the better’ is not the case. Identification of an optimal dose with a high benefit–risk ratio is required, along with adequate knowledge of the biotransformation of antioxidants. Antioxidants have the ability to donate electrons. This reducing power is essential in neutralizing radicals and other reactive species. In the presence of transition metal ions, electron donation may lead to a pro-oxidant effect. The effect of ascorbic acid on iron-induced lipid peroxidation in vitro is a very illustrative example of this effect [23Bast A. et al.Oxidants and antioxidants: state of the art.Am. J. Med. 1991; 91: 2S-13SAbstract Full Text PDF PubMed Scopus (533) Google Scholar]. Iron itself induces mild lipid peroxidation and in combination, ascorbic acid promotes the lipid peroxidation process by reducing the transition metal ion. However, at a relatively high concentration, ascorbic acid inhibits lipid peroxidation. Therefore, at low concentrations ascorbic acid behaves as a pro-oxidant but at high concentrations it becomes an antioxidant [24Halliwell B. Vitamin C: antioxidant or pro-oxidant in vivo.Free Radic. Res. 1996; 25: 439-454Crossref PubMed Scopus (472) Google Scholar]. This contradicts the idea that a high concentration of an antioxidant always has a pro-oxidant effect. The pro-oxidant activity of vitamin C in vivo has been the subject of well-publicized studies [4Lee S.H. et al.Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins.Science. 2001; 292: 2083-2086Crossref PubMed Scopus (407) Google Scholar, 5Podmore I.D. et al.Vitamin C exhibits pro-oxidant properties.Nature. 1998; 392: 559Crossref PubMed Scopus (721) Google Scholar]. Daily supplementation with 500 mg vitamin C for 6 weeks elevated plasma vitamin C levels by 60% [5Podmore I.D. et al.Vitamin C exhibits pro-oxidant properties.Nature. 1998; 392: 559Crossref PubMed Scopus (721) Google Scholar]. The authors suggested that vitamin C has pro-oxidant properties after observing levels of oxidized DNA bases measured in peripheral blood lymphocytes [5Podmore I.D. et al.Vitamin C exhibits pro-oxidant properties.Nature. 1998; 392: 559Crossref PubMed Scopus (721) Google Scholar]. Criticism included the notion that DNA oxidation did not occur in vivo but rather during DNA isolation. Moreover, the suggested increase in lymphocyte vitamin C levels was not checked [25Carr A. et al.Does vitamin C act as a pro-oxidant under physiological conditions?.FASEB J. 1999; 13: 1007-1024Crossref PubMed Scopus (735) Google Scholar]. A separate study demonstrated that vitamin C could react with lipid hydroperoxides, leading to DNA damaging agents [4Lee S.H. et al.Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins.Science. 2001; 292: 2083-2086Crossref PubMed Scopus (407) Google Scholar]. The in vitro study methodology did not take into account three important points: (i) the decomposition of lipid hydroperoxides by glutathione peroxidases in vivo, (ii) the excessively high concentration of lipid peroxides, and (iii) the fact that the formation of lipid hydroperoxides in vivo will occur only after vitamin C has been exhausted [25Carr A. et al.Does vitamin C act as a pro-oxidant under physiological conditions?.FASEB J. 1999; 13: 1007-1024Crossref PubMed Scopus (735) Google Scholar]. An authoritative review by Carr et al. concluded that the data on vitamin C and DNA oxidation in vivo are inconsistent and conflicting, but some of the discrepancies can be explained by flaws in experimental design and methodology [25Carr A. et al.Does vitamin C act as a pro-oxidant under physiological conditions?.FASEB J. 1999; 13: 1007-1024Crossref PubMed Scopus (735) Google Scholar]. There seems to be confusion between the high dose relationship to toxicity according to Paracelsus and the notion of pro-oxidant behavior. Nevertheless, this misconception persists. In general, antioxidants act by delaying or preventing the oxidation of other compounds. Therefore, the word antioxidant is used as a general descriptive term because antioxidants are in fact distinct chemical entities with different modes of action for their effects. Antioxidants can be thiols, phenols, and amines and may be either hydrophilic or lipophilic. In analogy to the term pharmacophore, we introduced the term nutricophore to indicate the functional moiety in a molecule and emphasize the diverse chemical properties of antioxidants [26Rezk B.M. et al.The antioxidant activity of ploretin: the disclosure of a new antioxidant pharmacophore in flavonoids.Biochem. Biophys. Res. Commun. 2002; 295: 9-13Crossref PubMed Scopus (240) Google Scholar, 27Heijnen C.G.M. et al.Protection of flavonoids against lipid peroxidation: the structure activity relationship revisited.Free Radic. Res. 2002; 36: 575-581Crossref PubMed Scopus (182) Google Scholar]. This diversity gives every antioxidant its unique (bio-)chemical profile, which is reflected in different sites of action and biological activities. Different antioxidants display different biological effects, and for specific pathological conditions the right antioxidant needs to be selected. In protecting LDL against oxidation, for example, the lipophilic and hydrophilic features of the antioxidant hydroxytyrosol are both involved in its activity, which is exhibited at the lipid–water interface. This misconception emanates from the fact that the reaction rate of radicals with endogenous (bio-)molecules is very high. This means that in order to protect, antioxidants have to react even faster with radicals, which, it is argued, is impossible. This is correct for extremely reactive hydroxyl radicals. However, lipid peroxyl radicals in membranes, which have a much longer half-life than hydroxyl radicals, can be neutralized by vitamin E [28van Acker S.A.B.E. et al.Molecular pharmacology of vitamin E: structural aspects of antioxidant activity.Free Radic. Biol. Med. 1993; 15: 311-328Crossref PubMed Scopus (281) Google Scholar]. Even for hydroxyl radicals, site-specific scavenging, for example by binding to iron and neutralizing the radical at the site of formation, may provide protection. In other words, primary protection, that is, preventing the formation of hydroxyl radicals, is possible. Antioxidants can also act indirectly. They can have very specific activities, such as inhibition of NADPH oxidase, regulation of redox-sensitive signal transduction pathways including transcription factors, and inhibition of poly(ADP-ribose)polymerase (PARP-1) [29Weseler A.R. et al.Oxidative stress and vascular function: implications for pharmacologic treatment.Curr. Hypertens. Rep. 2010; 12: 154-161Crossref PubMed Scopus (126) Google Scholar]. As well as direct scavenging, indirect antioxidant action through anti-inflammatory activity or induction of antioxidant protective factors can occur (Table 1). Without antioxidants, life in an aerobic environment would be impossible. Antioxidants do act. Their action has been established by determining their effect on biomarkers reflecting oxidative damage. Examples are given in Table 1.Table 1Balanced view of biomarkersaThe biological half-life of radicals and other reactive species is too short for direct detection. Therefore, evidence has to rely on indirect measurements. This is often based on the products generated when a biological target has been hit, that is, lipids, proteins, and DNA [69]. PUFA, polyunsaturated fatty acid. BiomarkerAdvantagesDisadvantagesLipidsF2-isoprostanes 60Morrow J.D. et al.Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers – smoking as a cause of oxidative damage.N. Engl. J. Med. 1995; 332: 1198-1203Crossref PubMed Scopus (1276) Google Scholar: prostaglandin-like products of primarily arachidonic acidHighly specificRequires specialized analytical skillsMalondialdehyde (MDA) 61Nielsen F. et al.Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors.Clin. Chem. 1997; 43: 1209-1214PubMed Google Scholar: reactive breakdown product of PUFAs formed during lipid peroxidationFrequently applied methodMDA is broken down by aldehyde dehydrogenasesLipid dienes 62Guichardant M. Lagarde M. Analysis of biomarkers from lipid peroxidation: a comparative study.Eur. J. Lipid Sci. Technol. 2009; 111: 75-82Crossref Scopus (27) Google Scholar: product of PUFAs formed during lipid peroxidationSimpleLow sensitivity and specificityEx vivo LDL oxidation 63Rietjens S.J. et al.New insights into controversies on the antioxidant potential of the olive oil antioxidant hydroxytyrosol.J. Agric. Food Chem. 2007; 55: 7609-7614Crossref PubMed Scopus (138) Google Scholar: antioxidants prevent oxidation of LDL isolated after antioxidant supplementationSemi-functional markerAmphiphilic antioxidants are lost during LDL isolationDNA8-Hydroxy-2′-deoxyguanosine 64Shigenaga M.K. Ames B.N. Assays for 8-hydroxy-2′-deoxyguanosine: a biomarker of in vivo oxidative DNA damage.Free Radic. Biol. Med. 1991; 10: 211-216Crossref PubMed Scopus (343) Google Scholar: damaged nucleotide formed by radical damage to DNASpecific and sensitiveProne to artifactsComet 65Collins A.R. The comet assay for DNA damage and repair.Mol. Biotechnol. 2004; 26: 249-261Crossref PubMed Scopus (2193) Google Scholar: DNA damage and strand breaksSemi-functional markerWell-standardized procedure requiredProteinsProtein carbonyls 66Dalle-Donne I. et al.Protein carbonyl groups as biomarkers of oxidative stress.Clin. Chim. Acta. 2003; 329: 23-38Crossref PubMed Scopus (1780) Google Scholar: protein oxidation productsRelatively stableHigh background and poor selectivityNitrotyrosine 67Van der Vliet A. et al.Nitrotyrosine as biomarker for reactive nitrogen species.Methods Enzymol. 1996; 269: 175-184Crossref PubMed Google Scholar: formed by reaction of reactive nitrogen species with tyrosineSpecificNitration of tyrosine during sample preparationProtein activity 68Aksenov M. et al.Oxidative modification of creatine kinase BB in Alzheimer's disease brain.J. Neurochem. 2000; 74: 2520-2527Crossref PubMed Scopus (236) Google Scholar: the activity of many proteins is reduced after reaction with reactive speciesSemi-functional markerLow sensitivitya The biological half-life of radicals and other reactive species is too short for direct detection. Therefore, evidence has to rely on indirect measurements. This is often based on the products generated when a biological target has been hit, that is, lipids, proteins, and DNA 69Halliwell B. Free radicals and antioxidants– quo vadis?.Trends Pharmacol. Sci. 2011; 32: 125-130Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar. PUFA, polyunsaturated fatty acid. Open table in a new tab Several methods [e.g., the Trolox equivalent antioxidant capacity (TEAC), oxygen radical absorbance capacity (ORAC) and ferric reducing ability of plasma (FRAP)] are used to determine overall antioxidant status in biological and food samples (Table 2). Antioxidant status in blood plasma was promoted as an ideal biomarker because it reflects the antioxidant capacity of all antioxidants present in a sample. The United States Department of Agriculture (USDA) even suggested a definite daily portion of ORAC units as dietary intake. This advice and the ORAC database for selected foods was withdrawn in 2010 because it was rightly regarded as biologically invalid [Oxygen Radical Absorbance Capacity (ORAC) of selected foods, Release 2, http://www.ars.usda.gov/services/docs.htm?docid=15866].Table 2Antioxidant activity: examples of enzymatic and non-enzymatic antioxidants, antioxidant capacity assays, and indirect antioxidant activity Enzymatic antioxidantsAntioxidant activityEssential groups in the active center (human origin)Superoxide dismutase (SOD) 70Fukai T. Ushio-Fukai M. Superoxide dismutases: role in redox signaling, vascular function, and diseases.Antioxid. Redox Signal. 2011; 15: 1583-1606Crossref PubMed Scopus (1260) Google Scholar2 O2–+2 H+→H2O2+O2Cu and Zn (SOD1, SOD3), Mn (SOD2)Glutathione perox
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