| Abstrakt: |
Studies of cytochrome P-4502E1 (CYP2E1)-mediated oxidation of ethanol have been hampered by the lack of a suitable probe for in vivo human studies. Chlorzoxazone, a prescribed skeletal muscle relaxant, is metabolized to 6-hydroxychlorzoxazone by CYP2E1 and has been advocated as a specific probe of this enzyme on the basis of microsomal studies. The applications of this probe may include delineating the contribution of CYP2E1 to in vivo human ethanol metabolism. However, the activity of nonmicrosomal enzymes in metabolizing chlorzoxazone is unknown. Alcohol dehydrogenase (ADH), predominantly a hepatic cytosolic enzyme, may be more important than CYP2E1 in the oxidation of ethanol to acetaldehyde. The contribution of catalase in the in vivo oxidation of ethanol to acetaldehyde is controversial. To determine if either of these enzymes metabolizes chlorzoxazone and whether ethanol oxidation by either enzyme is inhibited by chlorzoxazone or its metabolite, multiple in vitro studies were performed. ADH enzyme kinetics were performed with human recombinant β1β1 and β3β3 ADH with ethanol and chlorzoxazone (0.5 to 2.5 mM). Neither ADH isoenzyme exhibited NAD+-dependent oxidation of chlorzoxazone, but displayed Michaelis-Menten kinetics for ethanol with Km values of 89 μM and 34 mM, for β1β1, and β3β3, respectively. Typical in vivo concentrations of chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone, did not alter β1β1, or β3β3 ADH-mediated oxidation of ethanol to acetaldehyde. Studies of human hepatic nonmicrosomal enzyme activity were expanded to include all nonmicrosomal NAD+-dependent hepatic enzymes by starch gel electrophoresis assessment. Human hepatic enzymatic activity in the presence of chlorzoxazone was similar to that observed in the control sample (no added substrate), suggesting a lack of metabolism by NAD+-dependent enzymes. Similarly, human erythrocyte catalase, in the presence of a hydrogen peroxide generating system, did not metabolize chlorzoxazone. Furthermore, neither chlorzoxazone nor 6-hydroxychlorzoxazone altered the catalase-induced formation of acetaldehyde from ethanol. These data are consistent with chlorzoxazone as a specific probe of CYP2E1 that may be useful to alcohol researchers. [ABSTRACT FROM AUTHOR] |
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