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
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.