7-HYDROPEROXYCHOLESTEROL AND ITS PRODUCTS IN OXIDIZED LOW-DENSITY-LIPOPROTEIN AND HUMAN ATHEROSCLEROTIC PLAQUE

7-Hydroperoxycholesterols (7OOHs) are intermediates in cholesterol oxi dation and potential cytotoxins. A normal-phase HPLC method with UV (2 05 nm) detection was developed that could resolve 7 alpha OOH, 7 beta OOH, 7-ketocholesterol (7K), and the epimeric 7-hydroxycholesterols (7 OHs). 7OOH formation was investigated when LDL was exposed to four dif ferent oxidizing systems: Cu2+; Ham's F-10; mouse peritoneal macrophag es in Ham's F-10; and a metal-independent peroxyl-radical generating s ystem (AAPH). With all four oxidizing systems, 7OOH (both free and est erified, mostly as the beta-isomer) was the major oxysterol formed at early times, with 7K dominating at later stages (greater than or equal to 24 h) in Cu-oxLDL. When LDL was oxidized in the presence of cells there was transfer of free oxysterols from LDL to the cells. Negligibl e 7OOH, but significant amounts of 7OH, accumulated in the cells sugge sting efficient cellular reduction of 7OOH. Lipid extracts from eight plaque samples obtained from patients undergoing carotid endarterectom y were analyzed. Only trace amounts of 7OOH (<0.02% of total cholester ol) could be detected using this normal-phase HPLC method with UV dete ction or with a more sensitive reverse-phase method utilizing chemilum inescence detection. 7K was the major 7-oxygenated sterol detected, at least 20-fold in excess of that calculated for 7OOH, followed by 7 be ta OH and 7 alpha OH. The trace concentrations of 7OOH in plaque indic ate its lability in biological/cellular systems and may signify the ab ility of cells in the artery wall to metabolize it further.