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

Journal Article · Wed Nov 01 00:00:00 EST 1989 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(89)90581-2· OSTI ID:6983953

Hylemon, P B; Studer, E J; Pandak, W M; Heuman, D M; Vlahcevic, Z R; Chiang, J Y ^[1]

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1. Medical College of Virginia-VCU, Richmond (USA)

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.

• OSTI ID: 6983953
• Journal Information: Analytical Biochemistry; (USA), Vol. 182:2; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

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