A gas chromatography-mass spectrometry method for the measurement of fatty acid omega and omega(-1) hydroxylation kinetics by CYP4A1 using an artificial membrane system.
A gas chromatography-mass spectrometry assay method for the analysis of lauric, myristic, and palmitic acids and their omega and omega(-1) hydroxylated metabolites from in vitro incubations of cytochrome P450 CYP4A1, involving solid-phase extraction and trimethysilyl derivatization, was developed. The assay was linear, precise, and accurate over the range 0.5 to 50microM for all the analytes. It has the advantages of a more rapid analysis time, an improved sensitivity, and a wider range of analytes compared with other methods. An artificial membrane system was optimized for application to purified CYP4A1 enzyme by investigating the molar ratios of cytochrome b(5) and cytochrome P450 reductase present in the incubation mixture. Using this method, the kinetics of omega and omega(-1) oxidation of lauric, myristic, and palmitic acids by CYP4A enzymes were measured and compared in rat liver microsomes and an artificial membrane system.
Holmes
Victoria E
VE
Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, NG7 2RD, Nottingham, UK.
Bruce
Mary
M
Shaw
P Nicholas
PN
Bell
David R
DR
Qi
Fan Ming
FM
Barrett
David A
DA
D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002849:Chromatography, Gas / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072470:Cytochrome P450 Family 4; D005227:Fatty Acids / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats
C465387:Cyp4a1protein, rat; D005227:Fatty Acids; D007850:Lauric Acids; C030358:lauric acid; D003577:Cytochrome P-450 Enzyme System; D000072470:Cytochrome P450 Family 4