Tirona R G, Pang K S
Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
J Pharmacol Exp Ther. 1999 Sep;290(3):1230-41.
The conjugation kinetics of glutathione (GSH) and ethacrynic acid (EA) were studied in rat liver perfusion studies, where efficient removal occurred (steady-state extraction ratio E(ss), approximately 0.8-0.4 at concentrations ranging from 10-200 microM) despite the appreciable plasma protein binding. The declining E(ss) paralleled the saturation in GSH conjugate (EA-SG) formation; EA-SG primarily appeared in bile as the unchanged glutathionyl adduct (90%) and minimally as cleavage products. The GSH conjugation of EA in perfused liver was described by the constants K(m)(overall) of 67 microM and V(max)(overall) of 0.23 micromol/min/g liver. These differed from those observed for the bimolecular nonenzymatic (constant of 126 microM(-1) min(-1)) and enzymatic (K(m) for GSH and EA were 1.2 mM and 94 microM, respectively; V(max) of 533 nmol/min/mg liver cytosolic protein or 32 micromol/min/g liver) GSH conjugation of EA in vitro. But they were similar to those estimated for EA uptake in isolated rat hepatocytes by saturable (K(m)(uptake) = 57 microM, and V(max)(uptake) = 0.55 micromol/min/g liver) and nonsaturable (0.015 ml/min/mg) processes. At increasing EA concentrations (>25 microM), time-dependent changes were observed for E(ss) and EA-SG formation, which rapidly decreased with time after the attainment of steady state due to the rapid loss of cellular GSH. The composite data were described adequately by a physiological model that accounted for transport and the GSH-dependent conjugation of EA. The results suggest that the rate-limiting process for hepatic EA GSH conjugation is cellular uptake, but cosubstrate availability controls the rate of metabolism when GSH becomes depleted.
在大鼠肝脏灌注研究中，对谷胱甘肽（GSH）与依他尼酸（EA）的结合动力学进行了研究。尽管血浆蛋白结合显著，但仍发生了有效清除（稳态提取率E(ss)，在10 - 200 microM浓度范围内约为0.8 - 0.4）。E(ss)的下降与GSH共轭物（EA - SG）形成的饱和情况平行；EA - SG主要以未改变的谷胱甘肽加合物形式（90%）出现在胆汁中，以裂解产物形式出现的极少。灌注肝脏中EA的GSH结合情况由总体K(m)为67 microM和总体V(max)为0.23微摩尔/分钟/克肝脏来描述。这些与体外观察到的EA的双分子非酶促结合（常数为126 microM(-1)分钟(-1)）和酶促结合（GSH和EA的K(m)分别为1.2 mM和94 microM；V(max)为533纳摩尔/分钟/毫克肝脏胞质蛋白或32微摩尔/分钟/克肝脏）不同。但它们与通过可饱和（K(m)(摄取)=57 microM，V(max)(摄取)=0.55微摩尔/分钟/克肝脏）和非饱和（0.015毫升/分钟/毫克）过程估计的分离大鼠肝细胞中EA摄取情况相似。在EA浓度增加（>25 microM）时，观察到E(ss)和EA - SG形成随时间的变化，在达到稳态后，由于细胞内GSH的快速损失，它们随时间迅速下降。通过一个考虑了转运和EA的GSH依赖性结合的生理模型，能够充分描述这些综合数据。结果表明，肝脏中EA的GSH结合的限速过程是细胞摄取，但当GSH耗尽时，共底物的可用性控制着代谢速率。
