Lipid peroxidation in hemoglobin-containing liposomes. Effects of membrane phospholipid composition and cholesterol content

Abstract

The effects of phospholipid-oxidation state and vesicle composition on lipid peroxidation in hemolysate-containing liposomes (hemosomes) were studied by the thiobarbituric acid assay. Liposomes (hemosomes) were prepared from egg phosphatidylcholine (PC) with either low (PC 0.08) or high (PC 0.66) oxidation indices reflecting low and high conjugated diene/lipid hydroperoxy contents. Thiobarbituric acid reactivity was negligible over 6 h at 38°C in buffer-containing (control) liposomes prepared from PC 0.08, whereas it was slightly increased in those prepared from PC 0.66. Encapsulated hemolysate had no effect in PC 0.08 liposomes, but significantly increased thiobarbituric acid reactivity in those prepared from PC 0.66. Inclusion of either phosphatidylethanolamine or phosphatidylinositol in the membrane further increased lipid peroxidation in hemosomes prepared from PC 0.66, whereas phosphatidic acid and phosphatidylserine were inhibitory. Inclusion of cholesterol in the membrane had no effect in PC 0.66 hemosomes, but significantly inhibited lipid peroxidation in the presence of phosphatidylethanolamine or phosphatidylinositol. The effects of phosphatidic acid and cholesterol were dose-dependent. Co-incorporation of cholesterol and phosphatidic acid or phosphatidylserine in the membrane resulted in almost complete elimination of hemoglobin (Hb)-induced lipid peroxidation. Lysophosphatidic acid had similar effect as phosphatidic acid, whereas lysophosphatidylserine exerted inhibition only in the presence of phosphatidylethanolamine. The rate of lipid peroxidation showed no correlation with the amount of encapsulated Hb, neither with the oxidation indices nor the polyunsaturated fatty acid contents of negatively charged phospholipids. The above findings suggest a possible role for the high cholesterol content and preferential localization of phosphatidylserine in the inner bilayer leaflet of erythrocyte membrane in protecting against Hb-induced lipid peroxidation in the membrane.