The formation of cytochrome P-450 metabolic intermediate complexes in isolated hepatocytes.

This thesis examines the formation of cytochrome P-450 metabolic intermediate (MI) complexes in isolated hepatocytes. The validity of the model was established, and comparisons were made between MI complex formation in hepatocytes and microsomal suspension derived from untreated and Phenobarbital-pretreated rats. The rates of MI complex formation, the presence of the substrate optimum effect for certain substrates, and the stability of the MI complexes were compared. Information from these comparisons was used in the study of possible mechanisms for inconsistent observation on MI complex formation between microsomes and isolated perfused livers and whole animals. The relationship between substrate concentration and rate of reaction was established for N-hydroxyamphetamine, SKF 525-A and norbenzphetamine in hepatocytes from Phenobarbital-pretreated rats, as well as for N-hydroxyamphetamine and norbenzphetamine in hepatocytes from untreated rats. These relationships will provide a basis for comparison in future studies of MI complex formation in isolated perfused livers. The stability of MI complexes formed in vitro in microsomes was examined, and it was determined that the MI complexes are unstable under conditions. The rates of decay are influenced by temperature, and the degree of influence varies depending on the substrate from which the MI complex is formed. Hepatocytes isolated from drug-treated rats were found to contain similar amounts of MI complex as microsomes from identically treated animals. The implications of the results on possible mechanisms underlying the discrepant in vitro and in vivo observation are discussed.