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A second cholesterol-derived metabolite in addition to 7 alpha-hydroxycholesterol was observed to be produced from endogenous microsomal cholesterol in the presence of hamster liver microsomal fractions and NADPH, when analyzed by HPLC using the method of Ogishima and Okuda (Anal Biochem 158: 228-232, 1986). However, only 7 alpha-hydroxycholesterol was produced in the presence of rat hepatic microsomal protein fractions and NADPH. The second metabolite was facilely produced when endogenous 7 alpha-hydroxycholesterol was incubated with hamster liver microsomes and NADPH, but not with rat liver microsomes. The second metabolite derived from either endogenous cholesterol or exogenous 7 alpha-hydroxycholesterol contained three hydroxyl groups as shown by mass spectrometric analysis. After oxidation of the 3 beta-ol group by cholesterol oxidase, the metabolite comigrated with 7 beta-hydroxycholest-3-one on normal phase HPLC, but was resolved from both 7 alpha- and 7 beta-hydroxycholest-3-one on reverse phase HPLC. The data indicate that the second metabolite is a hydroxylated product of 7 alpha-hydroxycholesterol, possibly cholest-5-ene-3 beta,7 alpha, 12 alpha-triol. Cholestyramine feeding increased production of both 7 alpha-hydroxycholesterol and its metabolite from endogenous cholesterol by 3-fold in hamster liver microsomes in vitro. However, the direct conversion of 7 alpha-hydroxycholesterol to the metabolite by hamster liver microsomes was not increased appreciably after cholestyramine feeding (20-30%). The hydroxylation of 7 alpha-hydroxycholesterol was similar in characteristics to cholesterol 7 alpha-hydroxylase activity in that it was dependent on NADPH, was inhibited by several known P450 inhibitors, and was affected by an inhibitory autobody elicited against rat hepatic NADPH: cytochrome P450 oxidoreductase. 5,6- and 7,8-Benzoflavone were poor inhibitors (IC50 approximately 1 mM) of cholesterol 7 alpha-hydroxylase activity in liver microsomes from cholestyramine-fed rats, but caused a striking enhancement of the 7 alpha-hydroxylase activity of liver microsomes from untreated rats in vitro. In contrast, 7,8-benzoflavone inhibited cholesterol 7 alpha-hydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities of microsomes from normal and cholestyramine-fed hamsters. However, 5,6-benzoflavone stimulated cholesterol 7 alpha-hydroxylase activity in liver microsomes from normal and cholestyramine-fed hamsters, but inhibited 7 alpha-hydroxycholesterol hydroxylase activity by approximately 50%. These results suggest that hepatic cholesterol 7 alpha-hydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities apparently involve multiple forms of cytochrome P450 in untreated and cholestyramine-treated hamsters.
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