Determination of microsomal lauric acid hydroxylase activity by HPLC with flow-through radiochemical quantitation
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), Vol. 170:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CARBON 14 COMPOUNDS
HYDROXYLATION
DODECANOIC ACID
MICROSOMES
ENZYME ACTIVITY
GAS CHROMATOGRAPHY
HYDROXYLASES
LIQUID COLUMN CHROMATOGRAPHY
MASS SPECTROSCOPY
METABOLITES
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHROMATOGRAPHY
ENZYMES
LABELLED COMPOUNDS
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANOIDS
OXIDOREDUCTASES
SEPARATION PROCESSES
SPECTROSCOPY
550201* - Biochemistry- Tracer Techniques