engleski

The optimization of the radical reaction assay in lipid biological samples: microprocedure for the lipid hydroperoxide assay

Radical-mediated oxidation products of lipids being as specific markers of oxidative damage in vivo have been studied for many years. One of these oxidative products are lipid hydroperoxides (LOOH) which are well known to initiate adverse free-radical chain reactions. The knowledge of LOOH reactions and their reaction mechanisms is necessary since lipid peroxidation process has a major impact on the pathology of many diseases with an oxidative aetiology, such as cancer, degenerative and inflammatory diseases.1 One of the pathways for excessive production of free radicals is decomposition of hydroperoxides of unsaturated fatty acids catalyzed by iron. Although both reactions of LOOH decomposition in the presence of iron realize in biological systems, reduction of LOOH with Fe2+ is more feasible than oxidation of LOOH with Fe3+: LOOH + Fe2+  LO• + Fe3+ + OH– The aim of this work was to reestimate the oxidation of Fe2+ by hydroperoxide of oleic and linoleic acid to Fe3+ and subsequent complexation of the latter by thiocyanate to develop a sensitive LOOH assay by optimizing the assay conditions. This spectrophotometric indirect method for quantitative determination of LOOH is used to follow accurately the initial or early stages of the lipid peroxidation processes. It is reliable assay and does not dependent on laborious and expensive equipment.2 Any sample containing LOOH is suitable for the ferric thiocyanate method. In the case of real biological samples as are tissues (e.g. brain, liver homogenate), cultured cells and fresh blood plasma) microprocedure was proposed. Using this method the estimated lowest detectable limit was about 170 pmol LOOH/ml of analyzed solution, which corresponded to about 50 pmol LOOH/mg lipid in complex natural mixtures.

lipid hydroperoxide ; ferrithiocyanate method ; free radical

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