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Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Published on: March 18, 2012
Flavin-containing monooxygenase-mediated metabolism of N-deacetyl ketoconazole by rat hepatic microsomes
R J Rodriguez1, P J Proteau, B L Marquez
1College of Pharmacy, Oregon State University, Corvallis, Oregon 97331-3507, USA. Rosita.Rodriguez@orst.edu
Abstract:
Although ketoconazole is extensively metabolized by hepatic microsomal enzymes, the route of formation and toxicity of suspected metabolites are largely unknown. Reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice. DAK may be susceptible to successive oxidative attacks on the N-1 position by flavin-containing monooxygenases (FMO) producing potentially toxic metabolites. Previous laboratory findings have demonstrated that postnatal rat hepatic microsomes metabolize DAK by NADPH-dependent monooxygenases to two metabolites as determined by HPLC. Our current investigation evaluated DAK's metabolism in adult male and female rats and identified metabolites that may be responsible for ketoconazole's hepatotoxicity. DAK was extensively metabolized by rat liver microsomal monooxygenases at pH 8.8 in pyrophosphate buffer containing the glucose 6-phosphate NADPH-generating system to three metabolites as determined by HPLC. The initial metabolite of DAK was a secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole, which was confirmed by liquid chromatography/mass spectrometry and NMR spectroscopy. Extensive metabolism of DAK occurred at pH 8.8 in pyrophosphate buffer (female 29% and male 53% at 0.25 h; female 55% and male 57% at 0.5 h; and female 62% and male 66% at 1.0 h). Significantly less metabolism of DAK occurred at pH 7.4 in phosphate buffer (female 11%, male 17% at 0.25 h; female 20%, male 31% at 0.5 h; and female 27%, male 37% at 1 h). Heat inactivation of microsomal-FMO abolished the formation of these metabolites from DAK. SKF-525A did not inhibit this reaction. These results suggest that DAK appears to be extensively metabolized by adult FMO-mediated monooxygenation.
Insights
N-deacetyl ketoconazole (DAK) is extensively metabolized by adult rat flavin-containing monooxygenases (FMO). This metabolism produces potentially toxic metabolites, shedding light on ketoconazole
Area of Science:
- Pharmacology
- Biochemistry
- Toxicology
Background:
- Ketoconazole metabolism by hepatic enzymes is known, but metabolite formation and toxicity remain unclear.
- N-deacetyl ketoconazole (DAK) is a primary metabolite in mice, potentially undergoing further oxidation by flavin-containing monooxygenases (FMO) to toxic compounds.
- Previous studies showed postnatal rat hepatic microsomes metabolize DAK via NADPH-dependent monooxygenases.
Purpose of the Study:
- To investigate DAK metabolism in adult male and female rats.
- To identify metabolites potentially responsible for ketoconazole-induced hepatotoxicity.
Main Methods:
- Incubation of DAK with adult male and female rat liver microsomes at pH 8.8 (pyrophosphate buffer) and pH 7.4 (phosphate buffer) with a NADPH-generating system.
- Analysis of metabolites using High-Performance Liquid Chromatography (HPLC).
- Confirmation of metabolite structure using liquid chromatography/mass spectrometry and NMR spectroscopy.
- Assessment of flavin-containing monooxygenase (FMO) involvement via heat inactivation and SKF-525A inhibition studies.
Main Results:
- Rat liver microsomes produced three DAK metabolites at pH 8.8, with extensive metabolism observed (e.g., 66% in females, 66% in males at 1 hour).
- The primary metabolite identified was N-deacetyl-N-hydroxyketoconazole.
- Metabolism was significantly lower at pH 7.4 compared to pH 8.8.
- Metabolite formation was abolished by heat inactivation of microsomal FMO, and not inhibited by SKF-525A, indicating FMO-mediated metabolism.
Conclusions:
- Adult rat FMOs extensively metabolize DAK, primarily at alkaline pH.
- The identified N-deacetyl-N-hydroxyketoconazole is a key metabolite.
- These findings suggest FMO-mediated metabolism of DAK contributes to ketoconazole's hepatotoxicity.
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