Generation of 7-ketocholesterol by a Route Different from the Decomposition of Cholesterol 7-hydroperoxide

Abstract

7-Ketocholesterol (7C=O), one of free radical-mediated cholesterol oxidation products, has received much attention owing to its biologically active functions and potential contributions to various pathological situations. 7-Ketocholesterol (7C=O) is a one-electron reduced form of cholesterol 7-hydroperoxide (7-OOH) and possibly may be formed as a secondary product via 7-OOH, but the mechanism of 7C=O generation is not well understood. The large generation of 7C=O in cholesterol oxidation has been observed in the present study with azo radical initiator, AAPH and large unilamellar vesicles (LUVs) consisting of saturated or unsaturated phosphatidylcholine. The study has been thus made to determine the mechanism of formation by means of this oxidation system. Large formation of 7-oxygenated products, particularly 7C=O has been demonstrated with GC, GC/MS, TLC and HPLC. A time course study of these products has indicated the generation rates of 7C=O to be far higher than those of 7-OOH in saturated and unsaturated LUVs. Unsaturated phosphatidylcholine present in cholesterol oxidation has resulted in greater formation of 7-hydroxycholesterol (7-OH) and cholesterol 5,6-epoxide (EP) without affecting 7-OOH formation. The generation mechanism of 7C=O has been investigated with the antioxidants Ebselen, which reduces lipid hydroperoxide to the corresponding alcohol, and 2-keto-4-thiolmethyl butyric acid (KTBA), an alkoxyl radical scavenger. KTBA completely inhibited the formation of 7C=O, accompanied by the complete suppression of cholesterol oxidation in saturated LUV and over 95% suppression in unsaturated LUV. Ebselen had little effect on 7C=O formation in either LUV but had an effect on that of 7-OH. 7-Ketocholesterol (7C=O) formation would therefore appear to occur directly from cholesterol 7-alkoxyl radical by a route different from the decomposition of 7-OOH. The generation mechanism proposed for 7C=O is as follows; 1) cholesterol 7-alkoxyl radical formation by dyad termination reaction with the cholesterol 7-peroxyl radical, and 2) 7-ketocholesterol formation through β-scisson reaction of cholesterol 7-alkoxyl radical via an aldehyde intermediate.