Bile acid synthesis in man: assay of hepatic microsomal cholesterol 7~hydroxylase activity by isotope d ilution-mass spectrometry

The present work describes an accurate assay of the rate-limiting enzyme in bile acid synthesis, the cholesterol 7ahydroxylase, in human liver. The assay is based on isotope dilution-mass spectrometry, and endogenous microsomal cholesterol is used as the only substrate for the enzyme. Operative liver biopsies were obtained from patients undergoing elective cholecystectomy under highly standardized conditions. In ten gallstone patients, the enzyme activity of the microsomal fraction averaged 9.6 * 1.4 (mean * SEM) pmol. min-' . mg protein-' corresponding to a daily synthesis of about 0.5 mmol of bile acids. Three cholestyramine-treated patients displayed a fourfold higher enzyme activity. No evidence was obtained supporting the concept that the cholesterol 7a-hydroxylase is modulated by phosphorylation-dephosphorylation. Einarsson, K., B. Angelin, S. Ewerth, K. Nilsell, and I. Bjorkhem. Bile acid synthesis in man: assay of hepatic microsomal cholesterol 7ahydroxylase activity by isotope dilution-mass spectrometry, J. Lipid&, 1986. 27: 82-88. Supplementary key words phosphorylation cholestyramine phosphorylation deThe first step in the major pathway for conversion of cholesterol into bile acids is 7a-hydroxylation (1, 2). Early studies in the rat gave evidence that this is also the ratelimiting step in the biosynthesis of bile acids (3, 4). The enzyme has been found to be an NADPH-dependent mixed-function oxidase consisting of a specific species of cytochrome P-450 and NADPH-cytochrome P-450 reductase (5, 6). Since 7a-hydroxylation of cholesterol may also occur due to autooxidation or lipid peroxidation, it is difficult to assay the enzyme activity properly. Another complicating factor in the assay is that endogenous microsomal cholesterol serves as substrate and that it appears impossible to assay the activity under conditions of substrate saturation (1, 2). Several approaches have been tried, but the best assays are considered to be those based on measurement of the actual mass of 7a-hydroxycholesterol formed (1, 2), e.g., by isotope dilution-mass spectrometry as described by Bjorkhem and Danielsson (7). The occurrence of 7a-hydroxylase activity in human liver was first demonstrated by Bjorkhem et al. (8), and an enzyme assay based on conversion of labeled substrate was later described by Nicolau et al. (9). The activity in human liver was found to be about one magnitude lower than that in rat liver. This fact, in combination with the difficulty in obtaining sufficient amounts of human liver biopsies, has prevented a more thorough investigation of the properties and regulation of the human enzyme. In view of the great impact of abnormal cholesterol and bile acid metabolism in various human diseases, we considered it important to develop a more accurate method than those previously used for assay of the 7a-hydroxylase activity in human liver. In the present work, such an assay based on isotope dilution-mass spectrometry is described. With use of this assay we have studied the possibility that the cholesterol 7a-hydroxylase is modulated by phosphorylation and dephosphorylation as has been described for the HMG-CoA reductase in human liver (10). We have also determined the activity during treatment with cholestyramine, a drug known to stimulate bile acid biosynthesis in man (11, 12). MATERIALS AND METHODS