Abstract
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.
Footnotes
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↵1 Current address: Discovery Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Pfizer, Inc., Groton, CT 06340.
- Abbreviations used are::
- P450
- cytochrome P450
- UGT
- UDP-glucuronosyltransferases
- 7-EC
- 7-ethoxycoumarin
- 7-HC
- 7-hydroxycoumarin
- LC
- liquid chromatography
- MS
- mass spectrometry
- UDPGA
- UDP-glucuronic acid
- HPLC
- high-performance liquid chromatography
- amu
- atomic mass unit
- Received September 14, 2001.
- Accepted December 6, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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