F2-isoprostanes (F2-IPs) are formed by the free radicalcatalysed oxidation of arachidonic acid. The measurement of F2-IPs, especially 8-epi-PGF2α, is recognised as a reliable marker of lipid peroxidation and is currently used as a sensitive index of oxidative stress in vivo. The majority of 8-epi-PGF2α present in the circulation occurs in association with lipoproteins which are synthesised in the liver. Since lipoproteins are derived from dietary fatty acids and triglycerides, it is possible that 8-epi-PGF2α generated in polyunsaturated fatty acid-rich food (during initial processing/packaging or during meal preparation) may become incorporated within these lipoproteins during synthesis. In view of the growing use of 8-epi-PGF2α as a marker of lipid peroxidation in vivo in nutritional or clinical studies, it is therefore important to investigate the possibility that the circulating levels measured could be confounded by the presence of 8-epi-PGF2α in food. In this study we evaluated the levels of 8-epi-PGF2α present in several popular fastfoods, using a combination of solid phase extraction and gas chromatography-mass spectrometry. Fastfoods were selected to represent meals prepared from vegetable-, chicken-, fish- and meat-derived ingredients. Total (free + esterified) 8-epi-PGF2α levels ranged from 0.09 to 0.73 pmol/g (122–644 pmol/mmol arachidonic acid), with the highest levels present in beef-derived meals. Further investigation of hamburgers and cheeseburgers revealed 8-epi-PGF2α levels of 1.83 ± 0.24 and 0.84 ± 0.03 nmol/mmol arachidonic acid, respectively. Lower concentrations of vitamin E were found in the hamburgers. The postprandial contribution to plasma 8-epi-PGF2α levels following ingestion of 100 g portions of these fast-foods would therefore be expected to be no greater than the low picomole range, and would be unlikely to influence the normal endogenous levels of 8-epi-PGF2α, and those produced during oxidative stress.
The basic chemistry of the propagation of lipid peroxidation reactions has been known for years, but the mechanism of initiation of this process in biological membrane systems is still uncertain. Currently available assays for measuring peroxidation are reviewed - the more specific the assay used, the less peroxide is found in healthy human tissues and body fluids. Lipid peroxidation can arise as a consequence of tissue injury in many disease states and may sometimes contribute significantly to worsening the tissue injury.
A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O2 • , and H2O2. In the presence of iron salts, O2 • and H2O2 can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described.
The elastase-inhibitory activity of alpha 1-antiproteinase is inactivated by hydroxyl radicals (.OH) generated by pulse radiolysis or by reaction of iron ions with H2O2 in the presence of superoxide or ascorbate. Uric acid did not protect alpha 1-antiproteinase against inactivation by .OH in pulse radiolysis experiments or in the superoxide/iron/H2O2 system, whereas it did in systems containing ascorbic acid. We propose that radicals formed by attack of .OH on uric acid are themselves able to inactivate alpha 1-antiproteinase, but that these uric acid radicals can be 'repaired' by ascorbic acid.
Measurement of nitrotyrosine in biological fluids and tissues is increasingly being used to monitor the production of reactive nitrogen species in vivo. The detection of nitrotyrosine in vivo has been reported with the use of a variety of methods including immunoassay, HPLC and GLC/MS. The validity of HPLC and immunoassays have been questioned with regard to their selectivity and sensitivity limits. In principle, the measurement of nitrotyrosine by GLC/MS permits a highly specific, highly sensitive and fully quantitative assay. The nitration of tyrosine under acidic conditions in the presence of nitrite is well documented. Derivatization for the full quantification of nitrotyrosine by using GLC/MS can lead to the artifactual nitration of tyrosine if performed under acidic conditions in the presence of nitrite. We describe a novel alkaline method for the hydrolysis and derivatization of nitrotyrosine and tyrosine, and demonstrate its applicability to the measurement of plasma concentrations of both free and protein-bound nitrotyrosine and tyrosine. A detection limit of 1 pg for nitrotyrosine and 100 pg for tyrosine has been achieved. Our method allows, for the first time, the analysis of free and protein-bound nitrotyrosine and tyrosine in biological samples. The plasma concentrations (means±S.E.M.) of free tyrosine and nitrotyrosine in eight normal subjects were 12±0.6 μg/ml and 14±0.7 ng/ml respectively. Plasma proteins contained tyrosine and nitrotyrosine at 60.7±1.7 μg/mg and 2.7±0.4 ng/mg respectively.
The plant-derived phenolic compounds gossypol, quercetin and myricetin are powerful inhibitors of iron-induced lipid peroxidation in rat liver microsomes, under all five experimental conditions tested and at low micromolar concentrations (IC50 ⩽ 1.5 μM). However, they greatly accelerate the generation of hydroxyl radicals (.OH) from H2O2 in the presence of Fe3+-EDTA at pH 7.4, as measured by the deoxyribose assay. At 100 μm, the three phenolic compounds enhanced · OH formation up to eight-fold. The hydroxyl radical generation was inhibited by catalase and Superoxide dismutase, suggesting a mechanism in which the phenols oxidize to produce Superoxide radical, which then assists · OH generation from H2O2 in the presence of Fe3+-EDTA. At concentrations up to 75 μM, quercetin and myricetin also accelerate bleomycin-dependent DNA damage in the presence of Fe3+, possibly by reducing the Fe3+-bleomycin-DNA complex to the Fe2+ form. Hence these naturally-occurring substances can have pro-oxidant effects under some reaction conditions and cannot be classified simplistically as “antioxidants”.
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.<sup>1</sup>A 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.<sup>2</sup>Also, 41% of those who remained asymptomatic showed laboratory evidence of decline of immunologic status.<sup>2</sup>The 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.<sup>3,4</sup> 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.
Cell culture is widely used by biochemists and cell/molecular biologists, but the fluctuating (and often elevated) levels of O 2 to which cells in culture are exposed can affect many of their properties.So can the low level of antioxidants found in some cell culture media.Reagents, especially "antioxidants," added to cell culture media can react with the constituents of the media to produce H 2 O 2 and degradation products that can influence cell behavior.Several published papers describing the cellular effects of ascorbate, polyphenols, and carotenoids have, in fact, reported artifacts due to the actions of the degradation products of these "antioxidants."A greater awareness of the potential artifacts in cell culture studies is needed among the free radical/antioxidant community.
Photosynthesis and the biochemistry of the chloroplast are dealt with in this textbook, which is now available in an updated and corrected paperback edition. It places great emphasis on developments in our knowledge of the Calvin cycle and its regulation, C 4 and CAM plants, nitrogen and sulphur metabolism, phenolic compounds, photorespiration, and the toxic effects of oxygen on plant tissues.
