THE REGIOSPECIFIC HYDROXYLATION OF LAURIC ACID BY RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) CYTOCHROME P450S

Citation
Dr. Buhler et al., THE REGIOSPECIFIC HYDROXYLATION OF LAURIC ACID BY RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) CYTOCHROME P450S, Drug metabolism and disposition, 25(10), 1997, pp. 1176-1183
Citations number
34
Categorie Soggetti
Pharmacology & Pharmacy
ISSN journal
00909556
Volume
25
Issue
10
Year of publication
1997
Pages
1176 - 1183
Database
ISI
SICI code
0090-9556(1997)25:10<1176:TRHOLA>2.0.ZU;2-K
Abstract
We have reexamined the hydroxylation of [1-C-14]-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 omega-, (omega-1)-, (omega-2)-, (omega-3)-, (omega-4)-, (omeg a-5)-, and (omega-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 verifi ed by GC/MS analysis. Microsomes from male and female juvenile trout l iver formed (omega-1)-OH lauric acid as the major metabolite (23-29% o f total radioactivity) and no major differences were observed between males and females. By contrast, liver microsomes from 3-year-old sexua lly mature trout had substantially lower lauric acid hydroxylase activ ity than juvenile microsomes and produced small quantities of only the (omega-1)-, (omega-2)-, and (omega-6)-hydroxylation products. Moreove r, microsomes from sexually mature female trout had markedly lower lau ric acid hydroxylase activity than those from the sexually mature male trout. Rat liver microsomes were quite catalytically active but forme d mostly the omega- and omega-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 hepa tic microsomes from juvenile trout in the presence of rabbit polyclona l antibodies raised against the two purified trout P450s. CYP2M1 catal yzed the (omega-6)-hydroxylation of lauric acid while the trout CYP2K1 form produces mainly (omega-1)-OH lauric acid together with a smaller quantity of the (omega-2)-hydroxylation product. All of the trout and rat radiometric lauric acid metabolism results were confirmed by dire ct mass spectrometric analysis of derivatized lauric acid metabolism m ixtures. Use of direct mass spectrometric analysis for the hydroxylate d 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 ea ch metabolite, and was more sensitive than the radiometric analysis me thod.