Microsomal lipid peroxidation. I. Characterization of the role of iron and NADPH.

The NADPH/iron-dependent peroxidation of lipids in rat liver microsomes was found to depend on the presence of free ferrous iron. High concentrations of Fe2+ initiated microsomal lipid peroxidation at an initial rate which was independent of NADPH. With Fe3+, lower Fe2+ concentrations, or longer incubation times, NADPH stimulated microsomal lipid peroxidation and this stimulation was blocked by cytochrome c, which is capable of accepting electrons from the microsomal NADPH—cytochrome P-450 reductase. NADPH did not induce peroxidation in the absence of iron, nor did ferric iron in the absence of NADPH. Thus the role of NADPH appears to be maintenance of iron in the reduced Fe2+ state. The presence of superoxide dismutase, catalase, or the hydroxyl radical scavengers ethanol or thiourea had little or no effect on the iron-initiated peroxidation, ruling out dependence on reduced states of oxygen. Complete chelation of iron by EDTA completely inhibited peroxidation and no stimulation by either EDTA or ADP was observed over the peroxidation produced by free iron alone. Preincubation of microsomes in the absence of iron did not enhance the rate of peroxidation upon subsequent addition of iron, suggesting that iron acts by initiating peroxidative decomposition of membrane lipids rather than by catalyzing the breakdown of preformed hydroperoxides. Liposomes of extracted microsomal lipid also underwent peroxidation in the presence of ferrous iron at a rate comparable to that of intact microsomes. Ascorbate stimulated iron-induced liposomal peroxidation but had no effect in the absence of iron. Hydrogen peroxide did not enhance iron-induced liposomal peroxidation and inhibited at high concentrations, ruling out a role for hydroxyl radicals produced by reduction of H2O2 by Fe2+.