Cytochrome P450 and volatile anesthetic metabolism: From benchtop to bedside

The toxicity of volatile anesthetics is intimately related to their metabolism by cytochrome P450. The fluorinated ether anesthetics, such as methoxyflurane, undergo P450-catalyzed metabolism which liberates inorganic fluoride ion. Excessive fluoride production causes subclinical renal toxicity or overt renal failure. Halothane causes a rare but often fatal fulminant hepatic necrosis ('halothane hepatitis'). The initial event in halothane hepatitis is P450-catalyzed oxidative halothane metabolism to a reactive intermediate that trifluoroacetylates liver proteins. Milder, subclinical hepatic reactions also occur commonly after halothane. Such hepatotoxicity results from P450-mediated halothane activation to free radicals. Thus, the seminal event in volatile anesthetic toxification is P450-mediated metabolism. The main hypothesis we have tested is that specific isoforms of human P450 are responsible for anesthetic toxification, and that selective inactivation of these isoforms can be accomplished clinically to diminish anesthetic metabolism and toxicity. Human liver and kidney microsomes have been developed as in vitro models in our laboratory for human volatile anesthetic metabolism. Using human liver microsomes in vitro, we identified the principal human P450 isoforms catalyzing the oxidation of fluorinated ether anesthetics to inorganic fluoride ion in vitro. Enflurane, isoflurane, desflurane and sevoflurane are metabolized predominantly, in not exclusively, by P450 2E1. In contrast, methoxyflurane is metabolized by P450 2E1 as well as by several other P450 isoforms. We have also investigated volatile anesthetic metabolism by microsomes from human kidneys, the ultimate site of toxicity. Results showed that human kidney microsomes metabolize the nephrotoxin methoxyflurane to inorganic fluoride. Metabolism of sevoflurane, which is not nephrotoxic, was substantially less. Human kidneys contain little or no P450 2E1, but do contain P450 3A and other P450s. Since sevoflurane is metabolized predominantly by P450 2E1 but methoxyflurane is metabolized by several P450s, this may explain the comparatively minimal renal sevoflurane metabolism. These results suggest that if intrarenally generated fluoride or other metabolites are nephrotoxic, then renal metabolism may contribute to methoxyflurane nephrotoxicity. In contrast, the relative paucity of renal sevoflurane defluorination may explain the absence of sevoflurane nephrotoxicity, despite plasma fluoride concentrations which may exceed the nephrotoxic fluoride threshold of 50 μM seen with methoxyflurane. Using human liver microsomes in vitro, we also identified cytochrome P450 2E1 as the predominant P450 isoform catalyzing oxidative halothane activation to the reactive metabolite which forms trifluoroacetylated protein antigens. Conversely, P450s 3A4 and 2A6 have been identified as the major isoforms responsible for reductive halothane metabolism. This provided a definitive enzymatic basis for differentiating the two types of halothane hepatic toxicity. Having identified the function of various P450s in human anesthetic metabolism in vitro, the next objective was to verify their role in anesthetic metabolism in vivo. Disulfiram was developed as an effective clinical inhibitor of P450 2E1. Disulfiram pretreatment prior to surgery substantially inhibited the metabolism of enflurane, sevoflurane and isoflurane in surgical patients, confirming the role of P450 2E1 in anesthetic defluorination in vivo. For example, fluoride and hexafluoroisopropanol formation were inhibited 75% by disulfiram inhibition of P450 2E1. Disulfiram also inhibited trifluoroacetic acid production in patients anesthetized with halothane, demonstrating that P450 2E1 is the predominant enzyme responsible for human oxidative halothane metabolism in vivo. Inhibition of P450 2E1 by a single preoperative oral disulfiram dose dramatically diminished production of the halothane metabolite responsible for the neoantigen formation which initiates halothane hepatitis. Single-dose disulfiram may provide effective prophylaxis against halothane hepatitis. Thus, we can now identify patient populations and individuals potentially at risk for anesthetic toxicity. Clinical strategies may now be devised for avoiding specific anesthetic agents or administering protective adjuvants to diminish anesthetic toxicity.

Duke Scholars

Author Evan Kharasch Anesthesiology, General, Vascular, High Risk Transplant & Cr ...