The kinetics of ethylmorphine N-demethylation in isolated hepatocytes: the effect of drug transport and oxygen concentration.

In previous studies we have observed that the Km for ethylmorphine N-demethylase was only 50 to 100 microM in the isolated hepatocyte as opposed to 250 to 340 microM in isolated microsomes. These data suggested that either the drug is actively transported into the cell or that the enzymes in the cell had a reduced turnover number. Our current study indicates that ethylmorphine is transported into the hepatocyte with a Km of 250 microM and Tm of 20 nmol/min 10(6) cells. On the basis of temperature and inhibitor studies, this would appear to be active transport. When the turnover number of the ethylmorphine N-demethylase was decreased by reducing the oxygen from 20% of the gas phase to 5%, the activity was partially uncompetitively inhibited with the Km decreasing from 182 to 105 microM. The Vm decreased from 1.85 nmol/min 10(6) to 0.71 nmol/min 10(6) cells. These data suggest that the low Km we initially observed was probably due to active transport of the substrate into the hepatocyte. Further, our observations indicate that the effects of oxygen pressure on the kinetic parameters should be considered in predicting in vivo kinetics from in vitro studies.