IDENTIFICATION OF CYTOCHROME-P450 2E1 AS THE PREDOMINANT ENZYME CATALYZING HUMAN LIVER MICROSOMAL DEFLUORINATION OF SEVOFLURANE, ISOFLURANE, AND METHOXYFLURANE

Background. Renal and hepatic toxicity of the fluorinated ether volati le anesthetics is caused by biotransformation to toxic metabolites. Me tabolism also contributes significantly to the elimination pharmacokin etics of some volatile agents. Although innumerable studies have explo red anesthetic metabolism in animals, there is little information on h uman volatile anesthetic metabolism with respect to comparative rates or the identity of the enzymes responsible for defluorination. The fir st purpose of this investigation was to compare the metabolism of the fluorinated ether anesthetics by human liver microsomes. The second pu rpose was to test the hypothesis that cytochrome P450 2E1 is the speci fic P450 isoform responsible for volatile anesthetic defluorination in humans. Methods: Microsomes were prepared from human livers. Anesthet ic metabolism in microsomal incubations was measured by fluoride produ ction. The strategy for evaluating the role of P450 2E1 in anesthetic defluorination involved three approaches: for a series of 12 human liv ers, correlation of microsomal defluorination rate with microsomal P45 0 2E1 content (measured by Western blot analysis), correlation of defl uorination rate with microsomal P450 2E1 catalytic activity using mark er substrates (para-nitrophenol hydroxylation and chlorzoxazone 6-hydr oxylation), and chemical inhibition by P450 isoform-selective inhibito rs.Results. The rank order of anesthetic metabolism, assessed by fluor ide production at saturating substrate concentrations, was methoxyflur ane > sevoflurane > enflurane > isoflurane > desflurane > 0. There was a significant linear correlation of sevoflurane and methoxyflurane de fluorination with antigenic P450 2E1 content (r = 0.98 and r = 0.72, r espectively), but not with either P450 1A2 or P450 3A3/4. Comparison o f anesthetic defluorination with either para-nitrophenol or chlorzoxaz one hydroxylation showed a significant correlation for sevoflurane (r = 0.93, r = 0.95) and methoxyflurane (r = 0.78, r = 0.66). Sevoflurane defluorination was also highly correlated with that of enflurane (r = 0.93), which is known to be metabolized by human P450 2E1. Diethyldit hiocarbamate, a selective inhibitor of P450 2E1, produced a concentrat ion-dependent inhibition of sevoflurane, methoxyflurane, and isofluran e defluorination. No other isoform-selective inhibitor diminished the defluorination of sevoflurane, whereas methoxyflurane defluorination w as inhibited by the selective P450 inhibitors furafylline (P450 1A2), sulfaphenazole (P450 2C9/10), and quinidine (P450 2D6) but to a much l esser extent than by diethyldithiocarbamate. Conclusions. These result s demonstrate that cytochrome P450 2E1 is the principal, if not sole h uman liver microsomal enzyme catalyzing the defluorination of sevoflur ane. P450 2E1 is the principal, but not exclusive enzyme responsible f or the metabolism of methoxyflurane, which also appears to be catalyze d by P450s 1A2, 2C9/10, and 2D6. The data also suggest that P450 2E1 i s responsible for a significant fraction of isoflurane metabolism. Ide ntification of P450 2E1 as the major anesthetic metabolizing enzyme in humans provides a mechanistic understanding of clinical fluorinated e ther anesthetic metabolism and toxicity.