Establishment of steady-state metabolism of ethanol in perfused rat liver: the quantitative analysis using kinetic mechanism-based rate equations of alcohol dehydrogenase

Abstract

Alcohol dehydrogenase (ADH) catalyzes oxidation of ingested ethanol to acetaldehyde, the first step in hepatic metabolism. The purpose of this study was to establish an ex vivo rat liver perfusion system under defined and verified steady states with respect to the metabolites and the metabolic rates, and to quantitatively correlate the observed rates with simulations based on the kinetic mechanism-based rate equations of rat liver ADH. Class I ADH1 was isolated from male Sprague–Dawley rats and characterized by steady-state kinetics in the Krebs–Ringer perfusion buffer with supplements. Nonrecirculating liver perfusion with constant input of ethanol at near physiological hepatic blood flow rate was performed in situ. Ethanol and the related metabolites acetaldehyde, acetate, lactate, and pyruvate in perfusates were determined. Results of the initial velocity, product, and dead-end inhibition studies showed that rat ADH1 conformed to the Theorell–Chance Ordered Bi Bi mechanism. Steady-state metabolism of ethanol in the perfused liver maintained up to 3 h as evidenced by the steady-state levels of ethanol and metabolites in the effluent, and the steady-state ethanol disappearance rates and acetate production rates. The changes of the metabolic rates were qualitatively and in general quantitatively correlated to the results from simulations with the kinetic rate equations of ADH1 under a wide range of ethanol, in the presence of competitive inhibitor 4-methylpyrazole and of uncompetitive inhibitor isobutyramide. Preliminary flux control analysis estimated that apparent C ADH J in the perfused liver may approximate 0.7 at constant infusion with 1–2 mM ethanol, suggesting that ADH plays a major but not the exclusive role in governing hepatic ethanol metabolism. The reported steady-state rat liver perfusion system may potentially be applicable to other drug or drug–ethanol interaction studies.