Abstract

We have reexamined the hydroxylation of [1-¹⁴C]-lauric acid by trout liver microsomes and reconstituted trout P450s using a new HPLC system that gave an improved separation of hydroxylauric acids. Under these conditions, hepatic microsomes from yearling juvenile trout were shown to form ω-, (ω-1)-, (ω-2)-, (ω-3)-, (ω-4)-, (ω-5)-, and (ω-6)-OH lauric acid oxidation products (12-OH, 11-OH, 10-OH, 9-OH, 8-OH, 7-OH, and 6-OH lauric acid, respectively) as verified by GC/MS analysis. Microsomes from male and female juvenile trout liver formed (ω-1)-OH lauric acid as the major metabolite (23–29% of total radioactivity) and no major differences were observed between males and females. By contrast, liver microsomes from 3-year-old sexually mature trout had substantially lower lauric acid hydroxylase activity than juvenile microsomes and produced small quantities of only the (ω-1)-, (ω-2)-, and (ω-6)-hydroxylation products. Moreover, microsomes from sexually mature female trout had markedly lower lauric acid hydroxylase activity than those from the sexually mature male trout. Rat liver microsomes were quite catalytically active but formed mostly the ω- and ω-1 lauric acid oxidation products. Lauric acid metabolism also was analyzed in reconstituted systems containing purified juvenile trout LMC1 (CYP2M1) and LMC2 (CYP2K1) and with hepatic microsomes from juvenile trout in the presence of rabbit polyclonal antibodies raised against the two purified trout P450s. CYP2M1 catalyzed the (ω-6)-hydroxylation of lauric acid while the trout CYP2K1 form produces mainly (ω-1)-OH lauric acid together with a smaller quantity of the (ω-2)-hydroxylation product. All of the trout and rat radiometric lauric acid metabolism results were confirmed by direct mass spectrometric analysis of derivatized lauric acid metabolism mixtures. Use of direct mass spectrometric analysis for the hydroxylated lauric acids offers considerable advantages since the method did not require use of a radioactive fatty acid, completely separated all of the lauric acid hydroxylation products, confirmed identification of each metabolite, and was more sensitive than the radiometric analysis method.