Drug Oxidations Dependent on Cytochrome P‐450 in Isolated Hepatocytes

Isolated hepatocytes from male rats pretreated with phenobarbital were used to study metabolic effects associated with monooxygenase activity dependent on cytochrome P ‐450. The hepatocytes metabolized an added drug substrate, [ dimethylamino ‐ 14 C]aminopyrine (1 mM) at an N ‐demethylation rate of 1.31 ± 0.1 μmol × min −1 × g (dry weight of hepatocytes) −1 . The overall ATP/ADP ratios were 5.0 in the presence of aminopyrine, similar to the controls. 2. The levels of citrate and isocitrate decreased, and the citrate/2‐oxoglutarate ratio decreased to 55% of the control in the presence of drug substrates (aminopyrine, ethylmorphine, hexobarbital), correlating well with the decrease in the overall NADPH/NADP + ratio observed earlier with the perfused liver. It is concluded that supply of NADPH by extramitochondrial isocitrate dehydrogenase accompanies the NADPH supply by the pentosephosphate cycle, so that in spite of the larger capacity of the latter a portion of the reducing equivalents is derived from the mitochondria via the tricarboxylates even in the fed state. This results from the necessity of relieving the inhibition of glucose‐6‐phosphate dehydrogenase by NADPH for activation of the pentosephosphate cycle. 3. Subfractionation of hepatocytes from fed rats with the digitonin method revealed decreases of the citrate/2‐oxoglutarate ratios both in the particulate (mitochondrial) and the supernatant (cytosolic) fractions, corresponding to a positive shift in the NADPH redox potential of about 10 mV (or to a decrease of the free [NADPH]/[NADP + ] ratio to one‐half) in both compartments as calculated from the combined equilibria of NADP + ‐linked isocitrate dehydrogenase and aconitate hydratase. 4. Only in cells from fasted, not from fed rats, the rate of aminopyrine N ‐demethylation was diminished to 2/3 in the presence of aminotransferase inhibitors, suggesting a requirement for transamination steps in the transfer of NADPH‐reducing equivalents from mitochondria to cytosol under these conditions.