Radiolabeled cholesterol as a reporter for assessing one-electron turnover of lipid hydroperoxides.

A novel approach for assessing the peroxidative chain initiation potency of lipid hydroperoxides has been developed, which involves use of 14C-labeled cholesterol (Ch) as a "reporter" lipid. Unilamellar liposomes containing 1-palmitoyl-2-oleoyl-phosphatidylcholine, [14C]Ch, and 3beta-hydroxy-5alpha-cholest-6-ene-5-hydroperoxide (5alpha-OOH) or 3beta-hydroxycholest-5-ene-7alpha-hydroperoxide (7alpha-OOH) [100:75:5, mol/mol] were used as a test system. Liposomes incubated in the presence of ascorbate and a lipophilic iron complex were analyzed for radiolabeled oxidation products/intermediates (ChOX) by means of silica gel high-performance thin layer chromatography with phosphorimaging detection. The following ChOX were detected and quantified: 7alpha-OOH, 7beta-OOH, 7alpha-OH, 7beta-OH, and 5, 6-epoxide. Total ChOX yield increased in essentially the same time- and [iron]-dependent fashion for initiating 5alpha-OOH and 7alpha-OOH. The initial rate of [14C]7alphabeta-OH formation was greatly diminished when GSH and ebselen (a selenoperoxidase mimetic) were present, consistent with the attenuation of one-electron peroxide turnover. [14C]Ch-labeled L1210 cells also accumulated ChOX when incubated with 5alpha-OOH-containing liposomes. The rate of accumulation was substantially greater for Se-deficient than Se-sufficient cells, indicating that peroxide-induced chain reactions were modulated by selenoperoxidase action. These results illustrate the advantages of the new approach for highly sensitive in situ monitoring of cellular peroxidative damage. Copyright 1999 Academic Press.