Effects of ascorbate on membrane phospholipids and tocopherols of intact erythrocytes during peroxidation by t-butylhydroperoxide: comparison with effects of dithiothreitol.

Peroxidation of intact human erythrocytes by t-butylhydroperoxide (tBHP)) was studied. By incubation of the erythrocytes with 1 mM tBHP, reduced glutathione (GSH) was exhausted within 1 min, and tocopherols (Toc) and phospholipids (PL) decreased to nearly their lowest levels (in this study) within 5 min. The rate of decrease of alpha-Toc was faster than that of gamma-Toc, but alpha-Toc was never exhausted. The rates of decrease of Toc were faster than that of PL. Malondialdehyde increased slowly to reach a maximal value at 30 min. Methemoglobin (metHB) reached a maximum at 15 min. The maximal levels of these substances were maintained until 90 min incubation, which indicated that the peroxidation by tBHP had stopped spontaneously until at least 90 min. By the incubation with tBHP for 30 min, phosphatidylethanolamine (PE) and alpha-Toc decreased to about 70 and 30% of control levels, respectively, and gamma-Toc and GSH were almost exhausted. Ascorbate (0.1 mM) afforded protection of 92% to PE, 50% to alpha-Toc, and 65% to gamma-Toc against peroxidation, but ascorbate had no preventive effect at all on the formation of metHB and the decrease of GSH. These results may indicate that ascorbate-mediated protection of the membrane PL against the peroxidation depends primarily on Toc. On the other hand, dithiothreitol (DTT) (5 mM) almost completely prevented the formation of metHB, and DTT completely protected the PL and Toc against peroxidation, indicating the importance of sulfhydryl groups in erythrocytes.