Effect of ethanol and metabolic substrates on the oxidation of aminopyrine, formaldehyde and formate by isolated hepatocytes

The production of [¹⁴C]O₂ from ¹⁴C-labeled aminopyrine involves the following sequence: Aminopyrine → formaldehyde → formate → CO₂. Ethanol has the potential to affect any of the above steps in this sequence. In isolated hepatocytes from fasted rats ethanol inhibited CO₂ production from aminopyrine, but not from formaldehyde or formate. Significant inhibition was found at low levels of ethanol that do not affect aminopyrine metabolism by isolated microsomes. There was only a slight accumulation of formaldehyde and formate during the oxidation of aminopyrine to CO₂, both in the absence or presence of ethanol. This suggests that step I is the rate-limiting step and the step most sensitive to ethanol. There was accumulation of formate during the oxidation of formaldehyde to CO₂. Ethanol decreased this formate accumulation suggesting some inhibition of the overall rate of formaldehyde oxidation. Addition of pyruvate, lactate, fructose, xylitol and sorbitol increased the rate of CO₂ production from aminopyrine, but not from formaldehyde or formate. This increase by the substrates is probably due to an increase in the availability of NADPH required for step I. There was a slight increase in the accumulation of formaldehyde or formate during the oxidation of aminopyrine to CO₂ in the presence of the substrates. Pyruvate and fructose nearly completely prevented the inhibition of CO₂ production from aminopyrine by ethanol. Partial prevention was noted with the other substrates. Reduction of the cellular redox state as a consequence of ethanol metabolism may interfere with the transport of NADPH out of the mitochondria and, consequently, decrease the availability of NADPH for step I. The metabolic substrates may provide NADPH via the pentose phosphate cycle and/or restore substrate shuttle intermediates depleted by ethanol metabolism. In addition, pyruvate and glyceraldehyde (from fructose metabolism) can directly reoxidize the ethanol-derived NADH. Taken as a whole, these results suggest that step I in the sequence aminopyrine to CO₂ appears to be the rate-limiting step in isolated hepatocytes from fasted rats and the step sensitive to inhibition by ethanol.