A possible mechanism for initiation of lipid peroxidation by ascorbate in rat liver microsomes

Abstract

The mechanism by which lipid peroxidation progresses has been known for years, but there is disagreement regarding the mode of its initiation. The aim of this study was to examine: (a) the role of endogenous iron in the initiation of ascorbate-induced lied peroxidation in microsomal and liposomal membranes; (b) the role of oxygen-free radicals in this process; and (c) the redox state of ascorbate during the course of lipid peroxidation. Asrorbate-induced lipid peroxidation was assessed by measuring hydroperoxide and thiobarbituric add reactive substances (MARS) formation in membranes after incubation in Tris-HO buffer (pH 7.4) for 15 min. To confirm the role of endogenous iron and oxygen-free radicals, the effect of iron chelating agents (EDTA and thiourea) and radical scavengers (benzoate, mannitol, catalase and SOD) on lipid peroxidation was examined. Spectrophotometric measurements and ESR spectra have made it possible to determine ascorbate concentration and its redox state. Ascorbate promoted lipid peroxidation in both rat liver micrsuomes and üposomes without addition of exogenous iron. Iron chelating agents such as EDTA and thiourea inhibited lipM peroxidation, while SOD, catalase, mannitol and benzoate had no effect. The addition of 5 μM Fe 2+ (or Fe 3+) to the incubation mixture did not significantly alter hydroperoxide production, but that of TBARS was increased. Lipid peroxidation significantly altered the fatty add profile in microsomes and liposomas, the. most affected being the C 20:4 and C 22:6 species. Ascorbate in Tris-HCI buffer (pH 7.4) autoxidized very slowly. Its oxidation was catalyzed by Fe 3+ ions at a rate determined by incubation time and iron concentration. In contrast, no ascorbate oxidation occurred in the presence of microsomes when lipid peroxidation was proceeding at a maximal rate. Under these conditions a typical ascorbyl radical ESR spectrum signal greater than that arising from ascorbate alone was obtained and the magnitude of this signal was unchanged by variations of microsome or ascorbate concentrations. A ferrous ion-ascorbyl radical complex was responsible for this signal. These results suggest that an asenrbate-microsomal iron complex is responsible for the initiation of lipid peroxidation, and that during this process ascorbate remains in its reduced form.