Role of Oxygen Radicals in Peroxidation of Docosahexaenoic Acid by Rat Brain Homogenate in vitro

Abstract

The effect of free radicals, enzyme inhibitors, and free radical scavengers on docosahexaenoic acid (22: 6w3) metabolism by rat brain homogenate was studied in vitro. Rat brain homogenate was incubated with [¹⁴C]-22: 6w3 at 37°C for 15 min, and hydroxylated (1–3) 22: 6w3 compounds were separated by reversed-phase high-performance liquid chromatography. Radioactivity of the fractions was measured by liquid scintillation counting. Ferrous iron, Fenton’s reagent, and hydrogen peroxide stimulated the formation of 22: 6w3 metabolites. When a boiled homogenate was incubated, the stimulatory effect of Fenton’s reagent on metabolite formation was decreased markedly and that of hydrogen peroxide disappeared completely. Both nordihydroguaiaretic acid (NDGA) and alpha-tocopherol inhibited the stimulatory effect of ferrous iron, Fenton’s reagent, and hydrogen peroxide. Superoxide dismutase (SOD) and indomethacin did not alter the hydrogen peroxide-induced stimulation of metabolite formation; however, mannitol potentiated this stimulation. These observations indicate that the mechanism of stimulation by ferrous iron and Fenton’s reagent is probably mediated by a lipid hydroperoxide. The data also suggest (a) the participation of lipoxygenase in the stimulation of polyunsaturated fatty acid peroxidation by ferrous iron and Fenton’s reagent, and (b) the importance of alpha-tocopherol as an effective antioxidant in the membrane lipid bilayer.