Damaging effects of oxygen radicals on resealed erythrocyte ghosts

Resealed ghosts of human erythrocytes are sensitive to oxidative damage induced by xanthine oxidase acting on xanthine in the presence of iron. Damage was assessed in terms of lipid peroxidation and increased permeation of trapped markers, Na+ and glucose-6-P. Key findings are as follows. (a) Marker efflux from xanthine/xanthine oxidase/iron-treated ghosts accelerated after a lag, Na+ emerging far ahead of glucose-6-P. (b) Both effluxes and lipid peroxidation were stimulated by Fe(III) in a dose-dependent fashion and inhibited by chelating agents. (c) The antioxidant butylated hydroxytoluene effectively halted lipid peroxidation and net glucose-6-P efflux, but slowed Na+ efflux only partially. (d) Lipid peroxidation and marker release could be completely inhibited by superoxide dismutase or catalase, indicating that O2- and H2O2 are both required, possibly as precursors of OH. via the iron-catalyzed Haber-Weiss reaction (O2- + H2O2 leads to OH- + OH. + O2). (e) OH. scavengers, e.g. ethanol, mannitol, choline, had no protective effect against marker efflux and lipid peroxidation. Yet these agents did intercept OH. in the bulk medium, since they inhibited the degradation of 2-deoxyribose added as an extramembranous OH. probe. It is proposed that OH. produced on the membrane at iron binding sites reacts so rapidly with target molecules that scavengers cannot compete. (f) Desferrioxamine abolished all effects, including net egress of Na+. EDTA, while totally inhibitory toward lipid peroxidation and glucose-6-P release, diminished Na+ release partially, changing it to first order, approximately 3-fold faster than background. The latter response was totally inhibited by catalase, but only marginally by superoxide dismutase. This and other evidence suggests that different forms of membrane damage are responsible for enhanced permeation of the two markers; although glucose-6-P depends on lipid peroxidation, Na+ does not, certainly when EDTA is present.