Department of Surgery, Cathay General Hospital, Taipei, Taiwan.
Alcohol. 2010 Sep;44(6):541-51. doi: 10.1016/j.alcohol.2010.07.002. Epub 2010 Aug 19.
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 3h 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.
乙醇脱氢酶（ADH）催化摄入的乙醇氧化为乙醛，这是肝脏代谢的第一步。本研究的目的是建立一个在代谢物和代谢速率方面具有明确和验证的稳态的离体大鼠肝脏灌流系统，并根据大鼠肝脏 ADH 的基于动力学机制的速率方程的模拟来定量关联观察到的速率。从雄性 Sprague-Dawley 大鼠中分离出 I 类 ADH1，并在添加物的 Krebs-Ringer 灌注缓冲液中通过稳态动力学进行了表征。在近生理肝血流速率下进行非循环肝脏灌注，并持续输入乙醇。在灌流液中测定乙醇和相关代谢物乙醛、醋酸盐、乳酸盐和丙酮酸。初始速度、产物和末端抑制研究的结果表明，大鼠 ADH1 符合 Theorell-Chance 有序双-双机制。灌流肝脏中乙醇的稳态代谢可维持长达 3 小时，这可通过流出物中乙醇和代谢物的稳态水平以及稳态乙醇消失率和醋酸盐生成率来证明。在广泛的乙醇浓度下，与 ADH1 的动力学速率方程的模拟结果相比，代谢率的变化在定性和总体上具有定量相关性，存在竞争性抑制剂 4-甲基吡唑和非竞争性抑制剂异丁酰胺。初步通量控制分析估计，在以 1-2mM 乙醇恒速输注时，灌流肝脏中的表观 C（ADH）（J）可能接近 0.7，表明 ADH 在调节肝脏乙醇代谢中起主要作用，但不是唯一作用。报道的稳态大鼠肝脏灌流系统可能适用于其他药物或药物-乙醇相互作用研究。
