Regulation of bile acid synthesis. Measurement of cholesterol 7 alpha-hydroxylase activity in rat liver microsomal preparations in the absence of endogenous cholesterol

Abstract

A rapid procedure was developed to measure hepatic cholesterol 7 alpha-hydroxylase activity in the absence of endogenous microsomal cholesterol. This method involves the preparation of an acetone powder from the microsomal fraction of rat liver that retains its cholesterol 7 alpha-hydroxylase activity and contains virtually no endogenous cholesterol. The enzyme activity is measured in the presence of labeled exogenous cholesterol as the only substrate source, and can be expressed in terms of picomoles of product formed when a simple isotope incorporation procedure is employed. Optimal assay conditions were determined and the reproducibility of the acetone powder cholesterol 7 alpha-hydroxylase assay was established. A comparison of the proposed method with the previously used double isotope derivative procedure showed comparable enzyme activities in control rats and both methods exhibited an increase in the rate of hydroxylation after cholestyramine treatment and a decrease following cholic acid treatment. In contrast, the acetone powder assay did not show any change in cholesterol 7 alpha-hydroxylase activity during cholesterol feeding. These findings suggest that bile acid feeding influences the amount of active cholesterol 7 alpha-hydroxylase present in the liver whereas cholesterol feeding does not.