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Determination of microsomal lauric acid hydroxylase activity by HPLC with flow-through radiochemical quantitation

Journal Article · · Anal. Biochem.; (United States)
; ; ; ;
An assay for the microsomal hydroxylation of lauric acid (LA), based on HPLC with flow-through radiochemical detection, has been developed. Conditions were optimized for resolution and quantitation of three microsomal metabolites of /sup 14/C-LA, one of which has not been reported previously as a metabolite of LA in mammalian microsomal incubations. These products, 12-(omega)-hydroxy-LA, 11-(omega-1)-hydroxy-LA, and a novel metabolite, 10-(omega-2)-hydroxy-LA, were isolated by HPLC and identified by gas chromatography/mass spectrometry. In the presence of NADPH, the formation of all three metabolites was linear with time and microsomal protein concentration. Hydrogen peroxide also supported the microsomal metabolism of LA, although the ratio of metabolites was substantially different than that produced by NADPH-supported microsomes. Several biochemical probes (metyrapone, ..cap alpha..-naphthoflavone, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, and 10-undecynoic acid) were used to dissociate the three LA hydroxylase activities. These experiments suggest that the site-specific hydroxylation (omega-, (omega-1), (omega-2)-) of LA may be catalyzed by different isozymes of cytochrome P-450.
Research Organization:
Smith Kline and French Labs., Swedeland, PA (USA)
OSTI ID:
7040028
Journal Information:
Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 170:1; ISSN ANBCA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHROMATOGRAPHY
DODECANOIC ACID
ENZYME ACTIVITY
ENZYMES
GAS CHROMATOGRAPHY
HYDROXYLASES
HYDROXYLATION
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
MASS SPECTROSCOPY
METABOLITES
MICROSOMES
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANOIDS
OXIDOREDUCTASES
SEPARATION PROCESSES
SPECTROSCOPY