dihydroxycoprostane and 7-alpha-hydroxy-4-cholesten-3-one

Conversion of cholesterol into cholic acid in mammalian liver requires a 12alpha-hydroxylation step. Results have been presented suggesting that two different enzymes are involved in this hydroxylation with different activities towards the two steroids believed to be the physiological substrates for the enzyme, 7alpha-hydroxy-4-cholesten-3-one and 5beta-cholestane-3alpha,7alpha-diol. It is shown here that rabbit liver microsomes and partly purified sterol 12alpha-hydroxylase as well as COS cells transfected with a cDNA coding for this enzyme are able to catalyze 12alpha-hydroxylation of the two substrates at similar relative rates. Also 7alpha-hydroxycholesterol and 3alpha,7alpha-dihydroxy-5beta-cholestanoic acid are 12alpha-hydroxylated by the three systems. It is concluded that rabbit liver contains one major sterol 12alpha-hydroxylase with a broad substrate specificity.

Topics: Animals; Cholestanols; Cholestenones; Cholesterol; COS Cells; DNA, Complementary; Enzyme Inhibitors; Female; Gas Chromatography-Mass Spectrometry; Hydroxycholesterols; Hydroxylation; Microsomes, Liver; Rabbits; Steroid 12-alpha-Hydroxylase; Sterols; Substrate Specificity; Transfection

The synthesis of 2H4-labeled 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one is described. A mixture of these compounds, together with 2H3-labeled 5-cholestene-3 beta, 7 alpha-diol, was added to extracts of different subcellular fractions of liver. After purification by high performance liquid chromatography and conversion into trimethylsilyl ethers, the amounts of different endogenous unlabeled steroids were determined by selected ion monitoring. In normal liver, the concentration of 5-cholestene-3 beta, 7 alpha-diol (about 0.1-0.2 microgram/ml protein) was higher than the concentration of the other steroids (about 0.01-0.05 microgram/mg protein). The concentration of the different steroids was highest in the microsomal fraction of the liver homogenate. In a liver sample from a patient with cerebrotendinous xanthomatosis (CTX), the amounts of the 12 alpha-hydroxylated steroids were considerably higher than in the normal liver. The levels of 7 alpha-hydroxy-4-cholesten-3-one and 5 beta-cholestane-3 alpha, 7 alpha-diol were similar or only slightly higher than in the liver of the control patients. The concentration of 5-cholestene-3 beta, 7 alpha-diol was very high in the mitochondrial fraction of the CTX-liver. The findings are in accordance with the previous demonstration that the basic metabolic defect in CTX is a lack of the mitochondrial 26-hydroxylase. The results are further compatible with the contention that 7 alpha,26-dihydroxy-4-cholesten-3-one is an important intermediate in the normal bile acid biosynthesis.

Topics: Bile Acids and Salts; Brain Diseases, Metabolic; Cholestanols; Cholestenones; Female; Humans; Hydroxycholesterols; Lipid Metabolism, Inborn Errors; Liver; Mass Spectrometry; Middle Aged; Radioisotope Dilution Technique; Subcellular Fractions; Xanthomatosis