Journal of Lipid Research, Vol 34, 643-649, Copyright © 1993 by Lipid Research, Inc.

ARTICLES

Synthesis of 27-hydroxycholesterol in rat liver mitochondria: HPLC assay and marked activation by exogenous cholesterol

B Petrack and BJ Latario
Research Department, CIBA-GEIGY Corporation, Summit, NJ 07901.

Sterol 27-hydroxylase, the mitochondrial enzyme that catalyzes the first step in oxidation of the sterol side chain in hepatic bile acid synthesis, also catalyzes the synthesis of 27-hydroxycholesterol from cholesterol. We have developed a high performance liquid chromatography (HPLC) assay for this enzyme, using either endogenous or exogenous cholesterol as substrate and cholesterol oxidase to convert 27- hydroxycholesterol to 4-cholesten-27-hydroxy-3-one. The alpha,beta- unsaturated ketone product was separated by normal phase HPLC and quantitated via absorption at 240 nm. Addition of cholesterol dissolved in 2-hydroxypropyl-beta-cyclodextrin to the assay mixture raised the enzyme activity of rat liver mitochondria more than 10-fold. 2- Hydroxypropyl-beta-cyclodextrin itself was partially effective, apparently by making more endogenous cholesterol accessible to the enzyme. Availability of cholesterol to the enzyme limits synthesis of 27-hydroxycholesterol in rat liver. Using our assay to simultaneously determine the activities of cholesterol 7 alpha-hydroxylase and cholesterol 27-hydroxylase in rat liver homogenates, we demonstrated that the two enzymes are separately regulated.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C. R. Wooton-Kee, D. E. Cohen, and M. Vore Increased cholesterol 7{alpha}-hydroxylase expression and size of the bile acid pool in the lactating rat Am J Physiol Gastrointest Liver Physiol, April 1, 2008; 294(4): G1009 - G1016. [Abstract] [Full Text] [PDF]

				T. Li, W. Chen, and J. Y. L. Chiang PXR induces CYP27A1 and regulates cholesterol metabolism in the intestine J. Lipid Res., February 1, 2007; 48(2): 373 - 384. [Abstract] [Full Text] [PDF]

				X. Li, P. Hylemon, W. M. Pandak, and S. Ren Enzyme activity assay for cholesterol 27-hydroxylase in mitochondria J. Lipid Res., July 1, 2006; 47(7): 1507 - 1512. [Abstract] [Full Text] [PDF]

				D. Rodriguez-Agudo, S. Ren, P. B. Hylemon, K. Redford, R. Natarajan, A. Del Castillo, G. Gil, and W. M. Pandak Human StarD5, a cytosolic StAR-related lipid binding protein J. Lipid Res., August 1, 2005; 46(8): 1615 - 1623. [Abstract] [Full Text] [PDF]

				O. Guryev, R. A. Carvalho, S. Usanov, A. Gilep, and R. W. Estabrook A pathway for the metabolism of vitamin D3: Unique hydroxylated metabolites formed during catalysis with cytochrome P450scc (CYP11A1) PNAS, December 9, 2003; 100(25): 14754 - 14759. [Abstract] [Full Text] [PDF]

				Y. Ota, T.-A. Eto, S.-I. Tanaka, H. Sueta, H. Shiotsuki, Y. Maeda, M. Une, and K. Chijiiwa Assay method for mitochondrial sterol 27-hydroxylase with 7{alpha}-hydroxy-4-cholesten-3-one as a substrate in the rat liver J. Lipid Res., December 1, 2003; 44(12): 2400 - 2405. [Abstract] [Full Text] [PDF]

				N. B. Javitt 25R,26-Hydroxycholesterol revisited: synthesis, metabolism, and biologic roles J. Lipid Res., May 1, 2002; 43(5): 665 - 670. [Abstract] [Full Text] [PDF]

				M. Schwarz, D. W. Russell, J. M. Dietschy, and S. D. Turley Alternate pathways of bile acid synthesis in the cholesterol 7{alpha}-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding J. Lipid Res., October 1, 2001; 42(10): 1594 - 1603. [Abstract] [Full Text] [PDF]

				N. B. Javitt, Y. C. Lee, C. Shimizu, H. Fuda, and C. A. Strott Cholesterol and Hydroxycholesterol Sulfotransferases: Identification, Distinction from Dehydroepiandrosterone Sulfotransferase, and Differential Tissue Expression Endocrinology, July 1, 2001; 142(7): 2978 - 2984. [Abstract] [Full Text] [PDF]

				E. Hall, P. Hylemon, Z. Vlahcevic, D. Mallonee, K. Valerie, N. Avadhani, and W. Pandak Overexpression of CYP27 in hepatic and extrahepatic cells: role in the regulation of cholesterol homeostasis Am J Physiol Gastrointest Liver Physiol, July 1, 2001; 281(1): G293 - G301. [Abstract] [Full Text] [PDF]

				Z. Zhang, D. Li, D. E. Blanchard, S. R. Lear, S. K. Erickson, and T. A. Spencer Key regulatory oxysterols in liver: analysis as {{Delta}}4-3-ketone derivatives by HPLC and response to physiological perturbations J. Lipid Res., April 1, 2001; 42(4): 649 - 658. [Abstract] [Full Text]

				A. Honda, G. Salen, S. Shefer, Y. Matsuzaki, G. Xu, A. K. Batta, G. S. Tint, and N. Tanaka Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice: assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry J. Lipid Res., March 1, 2000; 41(3): 442 - 451. [Abstract] [Full Text]

				G. J. Schroepfer Jr. Oxysterols: Modulators of Cholesterol Metabolism and Other Processes Physiol Rev, January 1, 2000; 80(1): 361 - 554. [Abstract] [Full Text] [PDF]

				F. Lammert, D. Q-H. Wang, B. Paigen, and M. C. Carey Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: integrated activities of hepatic lipid regulatory enzymes J. Lipid Res., November 1, 1999; 40(11): 2080 - 2090. [Abstract] [Full Text]

				S. V. Bhagwat, G. Biswas, H. K. Anandatheerthavarada, S. Addya, W. Pandak, and N. G. Avadhani Dual Targeting Property of the N-terminal Signal Sequence of P4501A1. TARGETING OF HETEROLOGOUS PROTEINS TO ENDOPLASMIC RETICULUM AND MITOCHONDRIA J. Biol. Chem., August 20, 1999; 274(34): 24014 - 24022. [Abstract] [Full Text] [PDF]

				G. Xu, G. Salen, S. Shefer, G. S. Tint, L. B. Nguyen, T. T. Parker, T. S. Chen, J. Roberts, X. Kong, and D. Greenblatt Regulation of classic and alternative bile acid synthesis in hypercholesterolemic rabbits: effects of cholesterol feeding and bile acid depletion J. Lipid Res., August 1, 1998; 39(8): 1608 - 1615. [Abstract] [Full Text]

				I. A. Pikuleva, A. Babiker, M. R. Waterman, and I. Bjorkhem Activities of Recombinant Human Cytochrome P450c27 (CYP27) Which Produce Intermediates of Alternative Bile Acid Biosynthetic Pathways J. Biol. Chem., July 17, 1998; 273(29): 18153 - 18160. [Abstract] [Full Text] [PDF]

				L. Kritharides, M. Kus, A. J. Brown, W. Jessup, and R. T. Dean Hydroxypropyl-beta -cyclodextrin-mediated Efflux of 7-Ketocholesterol from Macrophage Foam Cells J. Biol. Chem., November 1, 1996; 271(44): 27450 - 27455. [Abstract] [Full Text] [PDF]

				R. S. Kushwaha, B. Guntupalli, E. M. Jackson, and H. C. McGill Jr Effect of Estrogen and Progesterone on the Expression of Hepatic and Extrahepatic Sterol 27-Hydroxylase in Baboons (Papio sp) Arterioscler. Thromb. Vasc. Biol., August 1, 1996; 16(8): 1088 - 1094. [Abstract] [Full Text]