Drug Metabolism in Rat Hepatic Microsomal Preparations, Isolated Hepatocytes and Liver Slices.

1. Experiments were undertaken in order to assess the use of isolated rat hepatocytes as a tool in drug metabolism studies. To this end the rate of N-demethylation of ethylmorphine and hydroxylation of biphenyl, thiabendazol and benzo(a)pyrene were followed using isolated hepatocytes, liver slices and microsomal preparations. Also conjugation reactions were followed in the hepatocytes. 2. With ethyimorphine N-methylation and henzo(a)pyrene hydroxylation highest enzyme activities were observed in the microsomal preparations with slices showing more activity than did the hepatocytes. For biphenyl, however, the highest rates of hydroxylation were observed in hepatocytes. Isolated cells were shown to be capable of performing phase II reactions with the hydroxylated metabolites of biphenyl. This was characteristic of cells capable of excluding trypan blue (viable cells). Non-viable cells, produced by exposure to high biphenyl concentrations, although capable of phase I reactions were unable to conjugate the hydroxylated products. 3. As described by other workers, it was thought possible that the low enzyme activities observed in isolated hepatocytes could be due to leakage of one or more of the NADPH regenerating cofactors from the cells into the medium. In order to test this hypothesis a number of experiments were conducted in which cofactors were added to the hepatocytes incubation medium in an attempt to prevent such leakage. 4. It was shown that addition of NADPH and glucose 6-phosphate (independently and together) to the hepatocyte incubation medium greatly enhanced the hydroxylation of biphenyl hydroxylation and ethylmorphine N-demethylation, to levels in great excess of those found in microsomal preparations. Addition of glucose 6-phosphate dehydrogenase had no detectable effect. The results obtained from this study were very variable. It is suggested that this variation is the result of varying degrees of damage to the cell membrane. The concept of a cell being classed as being either non-viable or viable as a result of the dye exclusion test is too 'black and white'. The membrane probably undergoes a spectrum of changes, with the uptake of dye monitoring only a small change somewhere on this spectrum. 5. Experiments using radio-labelled substrates indicated that NADPH and glucose 6-phosphate were actively taken up from the medium by hepatocytes. It was also demonstrated that glucose 6-phosphate was taken up from a perfusion medium by an isolated perfused rat liver, although this was only very slight. It is possible that glucose 6-phosphate is only taken up by damaged cells, such as those produced during the isolation procedures. If this is the case, the controversy over whether or not benefit is gained by use of glucose 6-phosphate during post surgical shock to aid recovery may be resolved. 6. It is concluded that isolated rat hepatocytes used in the presence of glucose 6-phosphate is a useful tool in drug metabolism studies, for not only does it give higher rates of metabolism than microsomal preparations, but it is also capable of monitoring phase II reactions