title: Lipid photooxidation in erythrocyte ghosts: sensitization of the membranes toward ascorbate- and superoxide-induced peroxidation and lysis.
abstract: The damaging effects of ascorbate (AH-) and superoxide (O-2) on resealed erythrocyte ghosts containing predetermined levels of lipid hydroperoxides (LOOHs) have been studied. Continuous blue light irradiation of membranes in the presence of protoporphyrin resulted in steadily increasing LOOH levels and enhanced release of a trapped marker, glucose 6-phosphate (G6P), after a 3- to 4-h lag. Neither superoxide dismutase (SOD) nor catalase inhibited these effects, ruling out O-2 and H2O2 as reactive intermediates. A 1-h light dose produced partially photoperoxidized ghosts, which, in the dark at 37 degrees C, released G6P no faster than unirradiated controls (approximately 7%/h). When xanthine oxidase plus xanthine (XO/X) was introduced as a source of O-2 and H2O2, the irradiated membranes lysed rapidly (t1/2 approximately 2 h). EDTA or SOD inhibited the reaction, whereas catalase had little or no effect. Unirradiated ghosts were not lysed by XO/X unless the system was supplemented with Fe(III), in which case total protection was afforded by SOD or catalase. In all experiments there was an excellent correlation between postirradiation lipid peroxidation (thiobarbituric acid reactivity) and G6P release. Similar observations were made with AH-. For example, dark incubation of photooxidized ghosts in the presence of 0.5 mM AH- resulted in rapid lysis (t1/2 approximately 1 h), which was stimulated approximately twofold by 50 microM Fe(III) and was inhibited by EDTA. By comparison, unirradiated ghosts showed no net peroxidation or lysis after 3 h exposure to Fe(III)/AH-. Neither SOD nor catalase protected against AH--stimulated damage. AH--promoted lipid peroxidation was inhibited by butylated hydroxytoluene, a lipophilic antioxidant, but was unaffected by 2,5-dimethylfuran or ethanol, singlet oxygen, and hydroxyl radical traps, respectively. These results suggest that a mechanism exists by which photogenerated LOOHs undergo redox metal-mediated reduction to alkoxy radicals (LO.), which trigger a burst of membrane-disrupting lipid peroxidation.
authors.forename: A W
authors.initials: AW
authors.lastname: Girotti
authors.forename: J P
authors.initials: JP
authors.lastname: Thomas
authors.forename: J E
authors.initials: JE
authors.lastname: Jordan
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology*; D002374:Catalase / Q000097:blood; D004492:Edetic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000097:blood; D006801:Humans; D066298:In Vitro Techniques; D008054:Lipid Peroxides / Q000097:blood*; D008066:Lipolysis / Q000187:drug effects; D010084:Oxidation-Reduction; D010777:Photochemistry; D011524:Protoporphyrins / Q000494:pharmacology; D012964:Sodium / Q000097:blood; D013482:Superoxide Dismutase / Q000097:blood; D013481:Superoxides / Q000494:pharmacology*; D019820:Xanthine; D014969:Xanthine Oxidase / Q000097:blood; D014970:Xanthines / Q000097:blood
chemical_list: D005958:Glucosephosphates; D008054:Lipid Peroxides; D011524:Protoporphyrins; D014970:Xanthines; D013481:Superoxides; D019820:Xanthine; D019298:Glucose-6-Phosphate; D004492:Edetic Acid; D012964:Sodium; C028025:protoporphyrin IX; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D001205:Ascorbic Acid