Characteristics of cholesterol 7α-hydroxylase and 7α-hydroxycholesterol hydroxylase activities of rodent liver

Abstract

A second cholesterol-derived metabolite in addition to 7α-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 7α-hydroxycholesterol was produced in the presence of rat hepatic microsomal protein fractions and NADPH. The second metabolite was facilely produced when endogenous 7α-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 7α-hydrocholesterol contained three hydroxyl groups as shown by mass spectrometric analysis. After oxidation of the 3β-ol group by cholesterol oxidase, the metabolite comigrated with 7β-hydroxycholest-3-one on normal phase HPLC, but was resolved from both 7α- and 7β-hydroxycholest-3-one on reverse phase HPLC. The data indicate that the second metabolite is a hydroxylated product of 7α-hydroxycholesterol, possibly cholest-5-ene-3β,7α,12α-triol. Cholestyramine feeding increased production of both 7α-hydroxycholesterol and its metabolite from endogenous cholesterol by 3-fold in hamster liver microsomes in vitro. However, the direct conversion of 7α-hydroxycholesterol to the metabolite by hamster liver microsomes was not increased appreciably after cholestyramine feeding (20–30%). The hydroxylation of 7α-hydroxycholesterol was similar in characteristics to cholesterol 7α-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 (IC 50 ≅ 1 mM) of cholesterol 7α-hydroxylase activity in liver microsomes from cholestyramine-fed rats, but caused a striking enhancement of the 7α-hydroxylase activity of liver microsomes from untreated rats in vitro. In contrast, 7,8-benzoflavone inhibited cholesterol 7α-hydroxylase and 7α-hydroxycholesterol hydroxylase activities of microsomes from normal and cholestyramine-fed hamsters. However, 5,6-benzoflavone stimulated cholesterol 7α-hydroxylase activity in liver microsomes from normal and cholestyramine-fed hamsters, but inhibited 7α-hydroxycholesterol hydroxylase activity by approximately 50%. These results suggest that hepatic cholesterol 7α-hydroxylase and 7α-hydroxycholesterol hydroxylase activities apparently involve multiple forms of cytochrome P450 in untreated and cholestyramine-treated hamsters.