Cholesterol and 27-hydroxycholesterol 7 alpha-hydroxylation: evidence for two different enzymes

The use of 2-hydroxypropyl-beta-cyclodextrin as a vehicle for solubilizing cholesterol and 27-hydroxycholesterol has led to a study of their rates of 7 alpha-hydroxylation in microsomal preparations from hamster liver and HepG2 cells. Addition of the vehicle alone to the cholesterol 7 alpha-hydroxylase assay always caused a several-fold increase in activity. Preloading the vehicle with cholesterol further augmented the rate of 7 alpha-hydroxycholesterol formation. Preloading the vehicle with 27-hydroxycholesterol or 27-hydroxycholestanol (molar ratio 1/1.2) minimally decreased cholesterol 7 alpha-hydroxylase activity (-12%), compared with preloading with cholestanol (-50%), a known competitive inhibitor of the enzyme. Microsomes from hamster liver yielded rates of 7 alpha,27-dihydroxcholesterol formation of 1.5 to 3.0 nmol/min per mg protein, compared with 0.3 nmol/min per mg protein for 7 alpha-hydroxycholesterol. Although cholesterol and cholestanol had minimal effects on the rate of 7 alpha-hydroxylation of 27-hydroxycholesterol, addition of an approximately equimolar amount of 27-hydroxycholestanol inhibited the rate of formation by 65%. Attempts to separate and identify the two C-27 sterol 7 alpha-hydroxylases chromatographically led to the finding that Emulgen 913 selectively inactivates 7 alpha-hydroxylation of 27-hydroxycholesterol. These results indicate that the metabolic pathway for bile acid synthesis from 27-hydroxycholesterol is not governed by cholesterol 7 alpha-hydroxylase.