Measurement of oxysterols and alpha-tocopherol in plasma and tissue samples as indices of oxidant stress status.

Oxidant stress seems to play a role in several setting of human pathology, such as atherosclerosis, cancer, and aging. The study of oxidant stress in human disease should be based on the evaluation of either sensitive and specific markers of enhanced oxidant stress, such as oxysterols, or antioxidant defense, by measuring alpha-tocopherol. We have developed a rapid method to measure the oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol in plasma (50 healthy subjects) and tissue as an index of oxidant stress in vivo, and from the same sample alpha-tocopherol content. The mean plasma concentration of 7beta-hydroxycholesterol and 7-ketocholesterol was 4.6+/-1.1 and 13.4+/-7.6 ng/mL, respectively. Plasma alpha-tocopherol concentration was 5.8+/-1.0 micromol/mol cholesterol. Samples from atherosclerotic plaques contained 20 times more cholesterol, about 45 times higher oxysterols levels, and 600 times more alpha-tocopherol compared to normal arteries. No significant difference in cholesterol and oxysterol content was observed between cirrhotic and normal liver. However, cirrhotic liver contained significantly smaller concentration of alpha-tocopherol compared to normal liver. In conclusion, we have developed a rapid and reliable method for the assay of cholesterol oxidation products and alpha-tocopherol in plasma and tissue useful for estimation of oxidant stress/antioxidant balance.

Bioavailability of vitamin E as function of food intake in healthy subjects: effects on plasma peroxide-scavenging activity and cholesterol-oxidation products.

Clinical trials with vitamin E have yielded contrasting results. In these trials, the amount of vitamin E given was different, and the compliance was not assessed in all studies. In addition, the modality of intake, ie, in relation to food, was not specified in any trial. Vitamin E is lipophilic, and its absorption is expected to be increased by food. We studied the bioavailability of vitamin E in relation to food intake and the effect on the lipid peroxide-scavenging activity of plasma and on 7beta-hydroxycholesterol and 7-ketocholesterol (oxysterols) as markers of oxidant stress. Twenty healthy Italian subjects were randomly assigned to take vitamin E at 300 mg/d on an empty stomach (group A) or during dinner (group B) for 15 days. Plasma vitamin E markedly increased in group B (84%) compared with group A (29%). The lipid peroxide-scavenging activity of plasma increased significantly in group B (14%, P=0.005) but did not change in group A. All subjects showed very low levels of plasma oxysterols, which were not affected by vitamin E supplementation in either group. This study shows that plasma concentration of vitamin E and plasma antioxidant activity in response to oral supplementation are markedly affected by food intake. Healthy Italian subjects show very low levels of cholesterol oxidation products; these low levels are possibly related to the Mediterranean diet. To obtain maximal absorption, vitamin E must be given at meals. These data should be taken into account in clinical trials with vitamin E.

Low-density lipoprotein oxidation.

Free radical mediated oxidation of low-density lipoproteins (LDL), which has been extensively studied in the last two decades, plays a central role in the development of the atherosclerotic plaque. Oxidation involves the lipid moiety of LDL in a chain reaction mechanism. In the initial phase, free radicals preferentially attack highly oxidizable polyunsaturated fatty acids. Subsequent recruitment of other molecules includes cholesterol and phospholipids. The process of oxidation is counteracted by antioxidants present in LDL. By-products formed during oxidation of LDL lipids, which may have biological activity, react with amino acid residues of the LDL protein backbone with the consequent modification of chemical and immunological properties responsible for cellular receptor shift. Oxidation-altered apolipoprotein B of oxidized LDL is, in fact, recognized by the macrophage scavenger receptor responsible for foam cell formation. The mechanism of LDL oxidation and the impact on atherogenesis are discussed.

Fluorescence quenching of dipyridamole associated to peroxyl radical scavenging: a versatile probe to measure the chain breaking antioxidant activity of biomolecules.

Dypiridamole is a highly efficient chain breaking antioxidant (Iuliano et al., Free Radic. Biol. Med. 18 (1995) 239-247) with an aromatic ring system responsible for an intense absorption band in the 400-480-nm region and for an intense fluorescence. Dipyridamole fluorescence is quantitatively quenched upon reaction with peroxyl radicals. In the presence of a flux of peroxyl radicals generated by thermal dissociation of azo-initiators, dipyridamole fluorescence decays linearly, showing a first-order reaction with respect to peroxyl radicals, and zero-order with respect to dipyridamole. The pH optimum for the fluorescence quenching is in the 7-8 range, from pH 7 to 6, the decay of fluorescence rapidly decreases to became negligible below pH 5.5. Dipyridamole consumption is blocked in the presence of an added chain breaking antioxidant for a time that is proportional to the antioxidant concentration. This effect is shown for ascorbic acid, trolox, vitamin E, uric acid, and N, N'-diphenyl-p-phenylenediamine. The slope of the linear correlation relative to trolox allows calculation of the bimolecular rate constant for a given molecule and peroxyl radicals. Comparison of data obtained by the dipyridamole consumption are comparable to values obtained by the oxygen consumption method.