Faculty Bibliography

BACKGROUND & AIMS: Cyp 7-/- mice lack a functional cholesterol 7alpha-hydroxylase enzyme and develop cholestasis before up-regulation of 27-hydroxycholesterol 7alpha-hydroxylase activity. Because 7alpha-hydroxylation is not the initial step in this metabolic pathway, we tested the hypothesis that cholesterol 27-hydroxylase is expressed at an earlier step and leads to the production of monohydroxy bile acids. METHODS: Polymerase chain reaction with specific oligonucleotides was used to detect messenger RNA (mRNA) coding for cholesterol 27-hydroxylase in 5-day-old normal and Cyp 7-/- mice. Gas-liquid chromatography-mass spectrometry and reverse isotope dilution were used to identify intermediates in the cholesterol 27-hydroxylase metabolic pathway. Light and electron microscopy were used to evaluate the morphological appearance of the liver. RESULTS: mRNA for cholesterol 27-hydroxylase was identified in the liver and spleen. The monohydroxy bile acids 3beta-hydroxy-5-cholenoate and 3alpha-hydroxy-5beta-cholanoate together with their precursor, 27-hydroxycholesterol, were identified in liver homogenates. Cholestasis, present focally, was manifested as dilated bile canaliculi, partial loss of microvilli, and retention of electron-dense biliary material. CONCLUSIONS: The cholesterol 27-hydroxylase metabolic pathway of bile acid synthesis is expressed in neonatal life. The absence of 7alpha-hydroxylase activities unmasks the cholestatic potential of monohydroxy bile acids. The Cyp 7-/- knockout mouse mimics cholestatic events known to occur in humans and provides a unique opportunity for studying regulatory determinants

Human endothelium obtained from both the aorta and the pulmonary artery has been evaluated for the presence of the messenger RNA coding for the expression of sterol 27-hydroxylase. Unique oligomers were designed to detect the mRNA by reverse transcription followed by the polymerase chain reaction. The amplified product was sequenced and was found to be identical to the published sequence for nucleotides 491 to 802 of the human sterol 27-hydroxylase cDNA. Northern blot analysis confirmed the presence of 27-hydroxylase mRNA in pulmonary artery and aortic endothelium. As part of these studies, enzymatic activity was assayed in cultured arterial endothelium using cholesterol as a substrate and isotope ratio gas-liquid chromatography-mass spectrometry to identify the metabolites, 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, in the medium. Localization of sterol 27-hydroxylase to vascular endothelium indicates intracellular production of the biologically active metabolite 27-hydroxycholesterol

The report of a novel cytochrome P450 enzyme in mouse hippocampus (cyp7b) with close homology to cholesterol 7 alpha-hydroxylase led us to determine the substrate specificity with respect to 27-hydroxycholesterol, known to be a potent inhibitor of cholesterol synthesis. Transfection of 293/T-cells with PcDNA3.1(+)-mcyp7b was followed by metabolism of 2.5 microM 27-hydroxycholesterol to the 7 alpha-hydroxy intermediate, cholest-5-ene,3 beta,7 alpha,27-triol, with complete loss of down-regulation of cholesterol synthesis. Addition of 5 microM and 10 microM concentrations of the triol to HepG2 and CHO cells, respectively, also did not reduce cholesterol synthesis. The contrast between the biologic effect on cholesterol synthesis by these two C27 hydroxysterols and the wide tissue distribution of both cholesterol 27-hydroxylase and hydroxysterol 7 alpha-hydroxylase implies local regulatory effects prior to their further catabolism in the liver to chenodeoxycholic and cholic acids

Sterol 27-hydroxylase activity in bovine aortic endothelial (BAE) cells in culture has been compared with that in HepG2 cells and in Chinese hamster ovary (CHO) cells using identical culture conditions. The total enzyme activity of BAE cells (3.0 nmol/72 h per mg cell protein) was comparable with that of HepG2 cells (4.0 nmol/72 h per mg protein) and both values were significantly greater than that in CHO cells (0.002 nmol/72 h per mg protein). The enzyme was identified in the mitochondria extracted from BAE cells by Western blotting using an antibody of proven specificity, and its metabolites 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid were identified by mass spectrum analysis. The presence of the enzyme in endothelium provides a mechanism for preventing accumulation of intracellular cholesterol by initiating a pathway of bile acid synthesis different from that initiated by 7 alpha-hydroxylation of cholesterol in the liver