Abstract
The volatile anesthetic halothane undergoes substantial biotransformation generating metabolites that mediate hepatotoxicity. Aerobically, halothane undergoes cytochrome P450-catalyzed oxidation to trifluoroacetic acid (TFA), bromide and a reactive intermediate that can acetylate liver proteins. These protein neo-antigens stimulate an immune reaction that mediates severe hepatic necrosis (“halothane hepatitis”). This investigation identified the human P450 isoform(s) that catalyze oxidative halothane metabolism. Halothane oxidation by human liver microsomes was assessed by TFA and bromide formation. Eadie-Hofstee plots of TFA and bromide formation were both nonlinear, suggesting the participation of multiple P450s. Microsomal TFA and bromide formation were inhibited 45 to 66% and 21 to 26%, respectively, by the P450 2A6 inhibitors 8-methoxypsoralen and coumarin, 84 to 90% by the P450 2E1 inhibitor 4-methylpyrazole and 55% by diethyldithiocarbamate, an inhibitor of both P450 2A6 and 2E1. Selective inhibitors of P450s 1A, 2B6, 2C9/10, 2D6 and 3A4 did not affect halothane oxidation. At saturating halothane concentrations (2.4 vol%) only cDNA-expressed P450 2A6 and 2B6 catalyzed significant rates of TFA and bromide formation, and P450 2E1 catalyzed comparatively minimal oxidation. Conversely, at subsaturating halothane concentrations (0.30 vol%), metabolism by P450 2E1 exceeded that by P450 2A6. Among a panel of human liver microsomes, there were significant linear correlations between halothane oxidation and P450 2A6 activity and protein content at saturating halothane concentrations (2.4 vol%), and a significant correlation between metabolite formation and P450 2E1 activity (but not P450 2A6 activity) at subsaturating concentrations (0.12 vol%). These experiments suggested P450 2A6 and 2E1 as the predominant catalysts at saturating and subsaturating halothane concentrations, respectively. Further kinetic analysis using cDNA-expressed P450 and liver microsomes clearly demonstrated that P450 2E1 is the high affinity/low capacity isoform (K m= 0.030-0.053 vol%) and P450 2A6 is the low affinity/high capacity isoform (K m = 0.77-1.2 vol%). Evidence was also obtained for substrate inhibition of P450 2E1. Thein vitro clearance estimates (Vmax/K m) for microsomal P450 2E1 (4.3-5.7 ml/min/g) were substantially greater than those for microsomal P450 2A6 (0.12-0.21). These clearances, as well as rates of apparent halothane oxidation predicted from kinetic parameters in conjunction with plasma halothane concentrations measured during clinical anesthesia in humans, demonstrated that both P450 2E1 and P450 2A6 participate in human halothane metabolism, and that P450 2E1 is the predominant catalytic isoform.
Footnotes
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Send reprint requests to: Dr. Evan Kharasch, University of Washington, Department of Anesthesiology, Box 356540, Seattle, WA 98195.
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↵1 This work was supported by grants from the National Institutes of Health (R01 GM48712, GM 48349) and by a Pharmaceutical Research and Manufacturers of America Foundation Faculty Development Award to E.D.K.
- Abbreviations:
- cDNA
- complementary deoxyribonucleic acid
- GC/MS
- gas chromatography-mass spectrometry
- HLM
- human liver microsomes
- HPLC
- high performance liquid chromatography
- IOD
- integrated optical density
- NADPH
- β-nicotinamide adenine dinucleotide, reduced form
- P450
- cytochrome P450
- TFA
- trifluoroacetic acid
- vol%
- headspace concentration (v/v)
- AST
- aspartate aminotransferase
- ALT
- alanine aminotransferase
- GST
- glutathioneS-transferase
- Received July 23, 1996.
- Accepted December 6, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
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