Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.
The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.
Fisher
Michael B
MB
Department of Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Jackson
David
D
Kaerner
Andreas
A
Wrighton
Steven A
SA
Borel
Anthony G
AG
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000408:Alamethicin / Q000494:pharmacology*; D001711:Biotransformation; D002853:Chromatography, Liquid; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism
D003374:Coumarins; D014535:Uridine Diphosphate Glucuronic Acid; D000408:Alamethicin; C017299:7-ethoxycoumarin; D009249:NADP; D015660:7-Alkoxycoumarin O-Dealkylase; D014453:Glucuronosyltransferase