A gas chromatography–mass spectrometry method for the measurement of fatty acid ω and ω−1 hydroxylation kinetics by CYP4A1 using an artificial membrane system

• Published in: Analytical biochemistry Vol. 325; no. 2; pp. 354 - 363
• Main Authors: Holmes, Victoria E, Bruce, Mary, Shaw, P.Nicholas, Bell, David R, Qi, Fan Ming, Barrett, David A
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 15-02-2004
• Subjects: Artificial membrane; CYP4A1; Fatty acids; GC–MS; Hydroxylation; Kinetics; GC–MS; CYP4A1; Artificial membrane; Kinetics; Hydroxylation; Fatty acids
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2003.10.046

A gas chromatography–mass spectrometry assay method for the analysis of lauric, myristic, and palmitic acids and their
ω and
ω
−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 50
μM 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
ω and
ω
−1 oxidation of lauric, myristic, and palmitic acids by CYP4A enzymes were measured and compared in rat liver microsomes and an artificial membrane system.