Photooxidation of cell membranes in the presence of hematoporphyrin derivative: reactivity of phospholipid and cholesterol hydroperoxides with glutathione peroxidase.
The susceptibility of photodynamically-generated lipid hydroperoxides to reductive inactivation by glutathione peroxidase (GPX) has been investigated, using hematoporphyrin derivative as a photosensitizing agent and the human erythrocyte ghost as a target membrane. Photoperoxidized ghosts were reactive in a glutathione peroxidase/reductase (GPX/GRD)-coupled assay only after phospholipid hydrolysis by phospholipase A2 (PLA2). However, enzymatically determined lipid hydroperoxide values were consistently approx. 40% lower than iodometrically determined values throughout the course of photooxidation. Moreover, when irradiated ghosts were analyzed iodometrically during PLA2/GSH/GPX treatment, a residual 30-40% of non-reactive lipid hydroperoxide was observed. The possibility that cholesterol product(s) account for the non-reactive lipid hydroperoxide was examined by tracking cholesterol hydroperoxides in [14C]cholesterol-labeled ghosts. The sum of cholesterol hydroperoxides and GPX/GRD-detectable lipid hydroperoxides was found to agree closely with iodometrically determined lipid hydroperoxide throughout the course of irradiation. Thin-layer chromatography of total lipid extracts indicated that cholesterol hydroperoxide was unaffected by PLA2/GSH/GPX treatment, whereas most of the phospholipid peroxides were completely hydrolyzed and the released fatty acid peroxides were reduced to alcohols. It appears, therefore, that the GPX-resistant lipid hydroperoxides in photooxidized ghosts were derived primarily from cholesterol. Ascorbate plus Fe3+ produced a burst of free-radical lipid peroxidation in photooxidized, PLA2-treated ghosts. As expected for fatty acid hydroperoxide inactivation, the lipid peroxidation was inhibited by GSH/GPX, but only partially so, suggesting that cholesterol hydroperoxide-derived radicals play a major role in the reaction.
Thomas
J P
JP
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Girotti
A W
AW
D001205:Ascorbic Acid / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002784:Cholesterol / Q000097:blood*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000378:metabolism*; D005980:Glutathione Reductase / Q000378:metabolism; D017324:Hematoporphyrin Derivative; D006415:Hematoporphyrins / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood*; D007501:Iron / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D009249:NADP / Q000097:blood; D010084:Oxidation-Reduction; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000097:blood*; D010777:Photochemistry; D011838:Radiation-Sensitizing Agents
D006415:Hematoporphyrins; D008081:Liposomes; D010743:Phospholipids; D011838:Radiation-Sensitizing Agents; D009249:NADP; D017324:Hematoporphyrin Derivative; D002784:Cholesterol; D006861:Hydrogen Peroxide; D007501:Iron; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; D010741:Phospholipases A; D054467:Phospholipases A2; D001205:Ascorbic Acid; D002118:Calcium