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Measuring reactive species and oxidative damage<i>in vivo</i>and in cell culture: how should you do it and what do the results mean?

Free radicals and other reactive species (RS) are thought to play an important role in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause. This article first reviews what is meant by the terms free radical, RS, antioxidant, oxidative damage and oxidative stress. It then critically examines methods used to trap RS, including spin trapping and aromatic hydroxylation, with a particular emphasis on those methods applicable to human studies. Methods used to measure oxidative damage to DNA, lipids and proteins and methods used to detect RS in cell culture, especially the various fluorescent "probes" of RS, are also critically reviewed. The emphasis throughout is on the caution that is needed in applying these methods in view of possible errors and artifacts in interpreting the results.

Measurement of reactive species

Abstract This chapter describes the fundamental principles behind the detection and measurement of reactive species (RS) in vivo, as well as the quantification of the damage that they can do to biomolecules (oxidative damage). The use of electron spin resonance (ESR) is described in detail, with a particular focus on spin traps such as PBN, DMPO and DEPMPO, TEMP, TEMPD, and POBN, and the technique of immuno-spin trapping. The detection of hydroxyl radicals by spin trapping, aromatic hydroxylation, deoxyribose degradation, and other techniques is presented, as is the detection of superoxide and nitric oxide by ESR and other techniques, both direct and indirect (e.g. Griess reaction). Peroxynitrite measurement is described, as is that of singlet O2 (using fluorescent probes, spin traps, scavengers and quenchers, monomol and dimol light emission, and deuterium oxide). The measurement of cellular redox state and peroxide levels is explained in detail, especially the use of roGFPs, cpYFP, Amplex red, and Hyper or boronate-based H2O2 detectors. Essential points to be considered in the use of DCFHDA and other ‘ROS probes’ (luminol, lucigenin, L-012, HPF, APF, dihydrorhodamine, dihydroethidium) are presented in detail. Biomarkers of oxidative damage to bilirubin (biopyrrins) and urate (allantoin) are presented. The techniques used to measure oxidative damage to DNA (strand breaks, 8-hydroxyguanine, formamidopyrimidines), lipids (peroxides, conjugated dienes, loss of PUFAs, TBA test, aldehydes, isofurans, isoprostanes), and proteins (carbonyls, oxidation products of specific amino acid residues) are discussed. Finally, the value and limitations of ‘total antioxidant capacity’ determinations in food and body fluids are presented.

Aromatic hydroxylation and nitration of phenylalanine and tyrosine by peroxynitrite

Peroxynitrite is a highly reactive species, generated from Superoxide and nitric oxide. Some effects of peroxynitrite are ascribed to the molecule itself, but decomposition products of the protonated form, peroxynitrous acid, may account for much of its reactivity in biological systems. Suggested products include highly‐reactive hydroxyl radicals, but thermodynamic calculations have been used to claim that free hydroxyl radicals cannot be formed from peroxynitrite. We utilized aromatic hydroxylation of phenylalanine as a specific detector of hydroxyl radicals, and found that incubation of phenylalanine with peroxynitrite leads to a small amount of p ‐, m ‐ and o ‐tyrosine, specific products of attack by this radical. Products of nitration of phenylalanine and tyrosine were also detected, as was dityrosine. Peroxynitrite decomposition generates several reactive species, including some that can nitrate aromatic rings. Formation of nitro‐aromatic compounds may be a useful marker of peroxynitrite generation in biological systems.

Oxygen radicals: a commonsense look at their nature and medical importance.

No abstract is provided for this article.

Polyphenols: antioxidant treats for healthy living or covert toxins?

Antioxidant defence mechanisms: From the beginning to the end (of the beginning)

When life first evolved on Earth, there was little oxygen in the atmosphere. Evolution of antioxidant defences must have been closely associated with the evolution of photosynthesis and of O2-dependent electron transport mechanisms. Studies with mice lacking antioxidant defences confirm the important roles of MnSOD and transferrin in maintaining health, but show that glutathione peroxidase (GPX) and CuZnSOD are not essential for everyday life (at least in mice). Superoxide can be cytotoxic by several mechanisms: one is the formation of hydroxyl radicals. There is good evidence that OH· formation occurs in vivo. Other important antioxidants may include thioredoxin, and selenoproteins other than GPX. Nitric oxide may be an important antioxidant in the vascular system. Diet-derived antioxidants are important in maintaining human health, but recent studies employing "biomarkers" of oxidative DNA damage are questioning the "antioxidant" roles of β-carotene and ascorbate. An important area of future research will be elucidation of the reasons why levels of steady-state oxidative damage to DNA and lipids vary so much between individuals, and their predictive value for the later development of human disease.

Reactive Oxygen Species, Antioxidants, and Acquired Immunodeficiency Syndrome

A cquired immunodeficiency syndrome (AIDS) results from A infection with a human immunodeficiency virus (HIV-1 or HIV-2) that eventually destroys a specific subset (CD4+) of helper T lymphocytes, so that the patient ultimately succumbs to opportunistic infections and/or certain neoplasms.¹A high proportion of, and perhaps all, HIV-seropositive patients will show disease progression. Thus, of a cohort of HIV-1—positive subjects who were followed up for 3 years, 19% developed AIDS-related complex (ARC) and 26% developed AIDS.²Also, 41% of those who remained asymptomatic showed laboratory evidence of decline of immunologic status.²The only drug currently approved for the treatment of AIDS is 3'-azido-3'-deoxythymidine (azidothymidine, or AZT, now called zidovudine), which is therapeutically effective but has significant time- and dose-related toxicity.^3,4 Recent reports have implicated<i>reactive oxygen species</i>both in the pathogenesis of HIV infection and in some of the side effects of drugs such as zidovudine.

Hydroxylation of aromatic compounds by reduced nicotinamide-adenine dinucleotide and phenazine methosulphate requires hydrogen peroxide and hydroxyl radicals, but not superoxide

1. A mixture of NADH and phenazine methosulphate hydroxylates aromatic compounds at acidic pH values. 2. Hydroxylation is inhibited by catalase and by scavengers of the hydroxyl radical (-OH) but not by superoxide dismutase. 3. It is concluded that neither O2 leads to nor HO2- is sufficiently reactive to hydroxylate aromatic rings.

Peroxisomes and Glyoxysomes

Elevated uric acid and impaired triglyceride metabolism predict mortality in women after ischemic stroke

Role of Free Radicals in the Neurodegenerative Diseases

No abstract is provided for this article.

A Metabolite Profiling Approach to Identify Biomarkers of Flavonoid Intake in Humans1–3

Flavonoids are phytochemicals that are widespread in the human diet. Despite limitations in their bioavailability, experimental and epidemiological data suggest health benefits of flavonoid consumption. Valid biomarkers of flavonoid intake may be useful for estimating exposure in a range of settings. However, to date, few useful flavonoid biomarkers have been identified. In this study, we used a metabolite profiling approach to examine the aromatic and phenolic profile of plasma and urine of healthy men after oral consumption of 200 mg of the pure flavonoids, quercetin, (-)-epicatechin, and epigallocatechin gallate, which represent major flavonoid constituents in the diet. Following enzymatic hydrolysis, 71 aromatic compounds were quantified in plasma and urine at 2 and 5 h, respectively, after flavonoid ingestion. Plasma concentrations of different aromatic compounds ranged widely, from 0.01 to 10 μmol/L, with variation among volunteers. None of the aromatic compounds was significantly elevated in plasma 2 h after consumption of either flavonoid compared with water placebo. This indicates that flavonoid-derived aromatic compounds are not responsible for the acute physiological effects reported within 2 h in previous human intervention studies involving flavonoids or flavonoid-rich food consumption. These effects are more likely due to absorption of the intact flavonoid. Our urine analysis suggested that urinary 4-ethylphenol, benzoic acid, and 4-ethylbenzoic acid may be potential biomarkers of quercetin intake and 1,3,5-trimethoxybenzene, 4-O-methylgallic acid, 3-O-methylgallic acid, and gallic acid may be potential markers of epigallocatechin gallate intake. Potential biomarkers of (-)-epicatechin were not identified. These urinary biomarkers may provide an accurate indication of flavonoid exposure.

Glutathione and ascorbic acid in spinach (<i>Spinacia oleracea</i>) chloroplasts. The effect of hydrogen peroxide and of Paraquat

The stroma of spinach chloroplasts contains ascorbic acid and glutathione at millimolar concentrations. [Reduced glutathione]/[oxidized glutathione] and [ascorbate]/[dehydroascorbate] ratios are high under both light and dark conditions and no evidence for a role of oxidized glutathione or dehydroascorbate in the dark-deactivation of fructose bisphosphatase could be obtained. Addition of H2O2 to chloroplasts in the dark decreases the above ratios, an effect that is reversed on illumination. Addition of Paraquat to illuminated chloroplasts caused a rapid oxidation of reduced glutathione and ascorbate, and apparent loss of dehydroascorbate. Paraquat rapidly inactivated fructose bisphosphatase activity, as assayed under physiological conditions.

Book Review

AbstractStress-Inducible Cellular Responses Eds. U. Feige, R. I. Morimoto, I. Yahara and 8. S. Polla Birkhauser Verlag, Basel, 1996

Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies?

Diets rich in polyphenols are epidemiologically associated with lower risk of developing some age-related diseases in humans. This apparent disease-protective effect of polyphenols is often attributed to their powerful antioxidant activities, as established in vitro. However, polyphenols can also exert pro-oxidant activities under certain experimental conditions. Neither pro-oxidant nor anti-oxidant activities have yet been clearly established to occur in vivo in humans, nor are they likely given the limited levels of polyphenols that are achievable in vivo after consumption of foods and beverages rich in them. Other actions of polyphenols may be more important in vivo. Many studies of the biological effects of polyphenols in cell culture have been affected by their ability to oxidise in culture media, and awareness of this problem can avoid erroneous claims.

Gender Differences in Steady-state Levels of Oxidative Damage to DNA in Healthy Individuals

Oxidative damage to DNA has often been used as a biomarker for oxidative stress and more specifically for cancer risk. Indeed, the measurement of oxidative damage to DNA, particularly of 8-hydroxyguanine (8OHG) and 8-hydroxy-2'-deoxyguanosine (8OHdG), has been adopted as a method for establishing the effects of antioxidant supplementation towards protection from certain cancers, cardiovascular and neuro-degenerative diseases, both in patients and healthy individuals. However, reported levels of 8OHdG or 8OHG vary considerably, possibly due to the different methodologies used, and only few data are available for the non-smoking and the female population. In this paper, steady-state levels of oxidative damage to DNA measured in a group of 20 males and 19 females are reported. Significant gender differences in levels of modified DNA bases such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine), 8-hydroxyadenine (8OHA) and 5-hydroxycytosine (5OHC), measured by gas chromatography-mass spectrometry (GC/MS), were observed. The results are discussed in relation to the Vitamin C and iron status of the subjects and to the existing, yet limited, literature data. The role of gender in predisposition to oxidative damage to DNA needs to be addressed in future studies.

Effect of oxidized glutathione on the inhibition of glucose-6-phosphate dehydrogenase by NADPH

Correspondence| March 15 1986 Effect of oxidized glutathione on the inhibition of glucose-6-phosphate dehydrogenase by NADPH Barry Halliwell Barry Halliwell 1Department of Biochemistry, King's College (KQC), Strand Campus, London WC2R 2LS, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1986) 234 (3): 741. https://doi.org/10.1042/bj2340741 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Barry Halliwell; Effect of oxidized glutathione on the inhibition of glucose-6-phosphate dehydrogenase by NADPH. Biochem J 15 March 1986; 234 (3): 741. doi: https://doi.org/10.1042/bj2340741 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1986 London: The Biochemical Society1986 Article PDF first page preview Close Modal You do not currently have access to this content.

The characterization of antioxidants

The role of antioxidants in nutrition is an area of increasing interest. Antioxidants are used (1) to prolong the shelf life and maintain the nutritional quality of lipid-containing foods, and (2) to modulate the consequences of oxidative damage in the human body. This review discusses what an antioxidant is and how the properties of antioxidants may be characterized.