PT  - JOURNAL ARTICLE
AU  - Michael B. Fisher
AU  - David Jackson
AU  - Andreas Kaerner
AU  - Steven A. Wrighton
AU  - Anthony G. Borel
TI  - 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
AID  - 10.1124/dmd.30.3.270
DP  - 2002 Mar 01
TA  - Drug Metabolism and Disposition
PG  - 270--275
VI  - 30
IP  - 3
4099  - http://dmd.aspetjournals.org/content/30/3/270.short
4100  - http://dmd.aspetjournals.org/content/30/3/270.full
SO  - Drug Metab Dispos2002 Mar 01; 30
AB  - 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. The American Society for Pharmacology and Experimental Therapeutics