Hemolysis and membrane lipid changes induced by xanthine oxidase in vitamin E deficient red cells

A procedure to induce hemolysis by the hypoxanthine-xanthine oxidase reaction was developed and applied to vitamin E deficient red blood cells (RBCs) in rats. The reaction system was as follows: 0.16 mM hypoxanthine, 0.05 U/ml xanthine oxidase in 2.5% RBC suspensions with an isotonic buffer (pH 7.4) containing 10 mM phosphate buffer and 125 mM saline (277 mOsm). Hemolysis was observed to depend on the vitamin E concentrations in the RBCs. Hemolysis was inhibited by catalase but not by SOD. After the reaction with vitamin E deficient RBCs, an increase in TBARS in the aqueous phase of the reaction mixture was observed. This accompanied the increase in fluorescent substances in the lipid extracts, in association with a significant decrease in the PE and PS of the RBCs, and a decrease in arachidonic acid in membrane lipids. The above changes were almost completely inhibited by tocopherol incorporated into vitamin E deficient RBCs.