THE MEASUREMENT OF LIPID PEROXIDATION: THE OPTIMIZATION OF THE LIPID HYDROPEROXIDE ASSAY

Physical and emotional stress, metabolic alterations, infections, carcinogenesis or inflammation are conditions that can trigger overproduction of reactive oxygen species. Increase of reactive oxygen species, free radicals and/or decreased antioxidant defense result in the misbalance in the cell redox reactions i.e. in the state termed the oxidative stress. The reactive oxygen species are continuously formed in small quantities during the normal metabolism of cell, however their overproduction is cytotoxic and damages macromolecules (DNA, proteins, sugars and lipids). Polyunsaturated fatty acids that are the main constituents of cell membranes are subject to free radicals induced-lipid peroxidation resulting in the destruction of biomembranes. Oxidative stress triggers a cellular stress response thereby activating a number of the redox-sensitive signaling cascades. A low levels oxidative stress induces protective effects but at high levels it may lead to more damaging effects. Quantification of lipid peroxidation or determination of the concentrations of lipid oxidation products is essential to assess the role of oxidative injury in pathophysiological disorders. One of initial products are lipid hydroperoxides (LOOH) which are well known to initiate further autoxidations during reactions of their production and decomposition. One of the pathways for excessive production of free radicals is decomposition of LOOH catalyzed by iron. The small pool of non-bound ferrous iron in cells is known to provide one of the active species involved in probably the most important route for free radical formation leading to increased levels of lipid peroxidation. 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+. Spectrophotometric quantitative determination of LOOH is used to follow accurately the initial or early stages of the lipid peroxidation process. The spectrophotometric method of analysis of LOOH is based on the oxidation of ferrous to ferric ion and subsequent complexation of ferric ion by thiocyanate.1 The aim of this work was a reestimation of the oxidation of ferrous ion by hydroperoxide of oleic and linoleic acid to optimize the assay conditions with special attention given to the rate of oxidation of ferrous ion with alkyl hydroperoxide affected by acid type and its concentration, thus improving acceleration of the oxidation and the sensitivity of the method. Special attention was given to the measurements of very low LOOH concentrations from biological materials.2 The lowest detectable limit was estimated to be about 170 pmol LOOH/ml of analyzed solution, which corresponded to about 50 pmol LOOH/mg lipid in complex natural mixtures.