Determination of the cytochrome P-450 IV marker, omega-hydroxylauric acid, by high-performance liquid chromatography and fluorimetric detection.
The formation of omega-hydroxylauric acid from lauric acid is an indicator of the activity of cytochrome P-450 IV family proteins. The two main metabolites of lauric acid, (omega-1)-and omega-hydroxylauric acid, have been completely separated by reversed-phase high-performance liquid chromatography. Measurement of lauric acid hydroxylase activity in microsomal liver samples, based on derivatization of the substrate and metabolites with the fluorescent agent 4-bromomethyl-6,7-dimethoxycoumarin, is a precise method (coefficient of variation = 7.6 and 10% for omega and (omega-1) metabolites, respectively) with good sensitivity (signal-to-noise ratio in microsomal samples of untreated rats greater than 20). In microsomal fractions from livers of rats treated with di-(2-ethylhexyl)phthalate the extent of omega-hydroxylation of lauric acid increased dose-dependently (ca. ten-fold). The (omega-1)-hydroxylase activity was not altered. A strong correlation between immunochemically determined cytochrome P-450 IVA1 and lauric acid omega-hydroxylase activity was found (r = 0.94, n = 30).
Dirven
H A
HA
Department of Toxicology, University of Nijmegen, The Netherlands.
de Bruijn
A A
AA
Sessink
P J
PJ
Jongeneelen
F J
FJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007850:Lauric Acids / Q000032:analysis / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D007850:Lauric Acids; C030358:lauric acid; C031263:12-hydroxydodecanoic acid; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A