Ascorbate-enhanced lipid peroxidation in photooxidized cell membranes: cholesterol product analysis as a probe of reaction mechanism.

Cholesterol was used as an in situ probe for studying mechanisms of lipid peroxidation in isolated erythrocyte membranes subjected to different prooxidant conditions. The membranes were labeled with [14C]cholesterol by exchange with prelabeled unilamellar liposomes and photosensitized with hematoporphyrin derivative. Irradiation with a dose of blue light resulted in thiobarbituric acid-detectable lipid peroxidation that was increased markedly by subsequent dark incubation with 0.5-1.0 mM ascorbate (AH-). Ascorbate-stimulated lipid peroxidation was inhibited by EDTA, desferrioxamine (DOX) and butylated hydroxytoluene (BHT), suggesting that the process is free radical in nature and catalyzed by membrane-bound iron. Thin layer chromatography and radiometric scanning of extracted lipids from photooxidized membranes revealed that the major oxidation product of cholesterol was the 5 alpha-hydroperoxide (5 alpha-OOH), a singlet oxygen adduct. Post-irradiation treatment with AH-/Fe(III) resulted in an almost-total disappearance of 5 alpha-OOH and the preponderance of free radical oxidation products, e.g. 7-ketocholesterol, the epimeric 7 alpha-/7 beta-hydroperoxides (7 alpha-/7 beta-OOH) and their respective alcohols (7 alpha-/7 beta-OH). EDTA, DOX and BHT inhibited the formation of these products, while catalase and superoxide dismutase had no effect. These results are consistent with a mechanism involving 1-electron reduction of photogenerated hydroperoxides to oxyl radicals, which trigger bursts of free radical lipid peroxidation. Though generated in this system, partially reduced oxygen species, viz. superoxide, hydrogen peroxide and hydroxyl radical, appear to be relatively unimportant in the autoxidation process.