PROPRANOLOL OXIDATION BY HUMAN LIVER-MICROSOMES - THE USE OF CUMENE HYDROPEROXIDE TO PROBE ISOENZYME SPECIFICITY AND REGIOSELECTIVITY AND STEREOSELECTIVITY

1. Three oxidations of the enantiomers of propranolol were studied in human liver microsomes under two reaction conditions. Previous in vitro studies had established that two of the livers were from poor metaboliser (PM) phenotypes for the debrisoquine 4-hydroxylase (cytochrome P-450IID6) and the remaining seven were from extensive metaboliser (EM) phenotypes. 2. In the presence of NADPH and oxygen 4- and 5-hydroxylation of propranolol occurred in microsomes from all nine livers, as did propranolol N-desisopropylation. R(+)-propranolol was oxidized preferentially along the three pathways, although enantio-selectivity observed for N-desisopropylation may have arisen not only from stereoselectivity in formation rates, but also from stereoselectivity in subsequent microsomal metabolism, possibly by monoamine oxidase. The involvement of monoamine oxidase in the further microsomal metabolism of N-desisopropylpropranolol was indicated by inhibition of the metabolism of this compound when incubated with phenelzine. 3. Cumene hydroperoxide has been proposed to support only the activity of cytochrome P450IID6. This is consistent with the observations that a) propranolol 4- and 5-hydroxylation occurred in microsomes from the EM livers only and b) side-chain oxidation was not observed under these conditions in either PM or EM livers. 4. Using cumene hydroperoxide to support the reactions, the 4-hydroxylation of propranolol showed little enantioselectivity, whereas S(-)-propranolol was 5-hydroxylated about twice as fast as the R(+)-enantiomer. There were highly significant correlations between the rates of 4- and 5-hydroxylation of R(+)-propranolol (r = 0.96, P < 0.00l, n = 7 livers) and of S(-)-propranolol (r = 0.98, P < 0.001). Both oxidations were described by single-site Michaelis-Menten kinetics. 5. The findings suggest that P450IID6 is involved in both the 4- and 5-hydroxylations of propranolol, but that these metabolites can also be formed by other P450 isoenzymes. It is confirmed that P450IID6 does not contribute to the N-desisopropylation of propranolol. Furthermore, the finding that mephenytoin did not inhibit the appearance of this metabolite is not consistent with the results of in vivo studies suggesting the involvement of the same enzyme in the side-chain oxidation of propranolol and the 4-hydroxylation of mephenytoin.