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Title: Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous (4- sup 14 C)cholesterol as substrate

Abstract

The HPLC-spectrophotometric method for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous (4-{sup 14}C)cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous (4-{sup 14}C)-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase.

Authors:
; ; ; ; ;  [1]
  1. Medical College of Virginia-VCU, Richmond (USA)
Publication Date:
OSTI Identifier:
6983953
Resource Type:
Journal Article
Journal Name:
Analytical Biochemistry; (USA)
Additional Journal Information:
Journal Volume: 182:2; Journal ID: ISSN 0003-2697
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; HYDROXYLASES; LIQUID COLUMN CHROMATOGRAPHY; CARBON 14 COMPOUNDS; CHOLESTEROL; LIVER; MEASURING METHODS; RATS; SPECTROPHOTOMETRY; TRACER TECHNIQUES; ANIMALS; BODY; CHROMATOGRAPHY; DIGESTIVE SYSTEM; ENZYMES; GLANDS; HYDROXY COMPOUNDS; ISOTOPE APPLICATIONS; LABELLED COMPOUNDS; MAMMALS; ORGANIC COMPOUNDS; ORGANS; OXIDOREDUCTASES; RODENTS; SEPARATION PROCESSES; STEROIDS; STEROLS; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Hylemon, P B, Studer, E J, Pandak, W M, Heuman, D M, Vlahcevic, Z R, and Chiang, J Y. Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous (4- sup 14 C)cholesterol as substrate. United States: N. p., 1989. Web. doi:10.1016/0003-2697(89)90581-2.
Hylemon, P B, Studer, E J, Pandak, W M, Heuman, D M, Vlahcevic, Z R, & Chiang, J Y. Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous (4- sup 14 C)cholesterol as substrate. United States. https://doi.org/10.1016/0003-2697(89)90581-2
Hylemon, P B, Studer, E J, Pandak, W M, Heuman, D M, Vlahcevic, Z R, and Chiang, J Y. 1989. "Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous (4- sup 14 C)cholesterol as substrate". United States. https://doi.org/10.1016/0003-2697(89)90581-2.
@article{osti_6983953,
title = {Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous (4- sup 14 C)cholesterol as substrate},
author = {Hylemon, P B and Studer, E J and Pandak, W M and Heuman, D M and Vlahcevic, Z R and Chiang, J Y},
abstractNote = {The HPLC-spectrophotometric method for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous (4-{sup 14}C)cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous (4-{sup 14}C)-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase.},
doi = {10.1016/0003-2697(89)90581-2},
url = {https://www.osti.gov/biblio/6983953}, journal = {Analytical Biochemistry; (USA)},
issn = {0003-2697},
number = ,
volume = 182:2,
place = {United States},
year = {Wed Nov 01 00:00:00 EST 1989},
month = {Wed Nov 01 00:00:00 EST 1989}
}