Distributed-in-space glutathione conjugation kinetics of ethacrynic acid in liver

grantor: University of TorontoThe factors affecting the hepatocellular glutathione (GSH) conjugation of ethacrynic acid (EA), a model substrate, in the formation of the glutathione adduct EA-SG were assessed for their influence on local GSH conjugation rates from the inlet to the outlet of the liver. These included plasma protein binding, sinusoidal uptake, spontaneous and enzyme-catalyzed conjugation, co-substrate (GSH) availability and EA-SG efflux at canalicular and sinusoidal membranes and their zonation within the acinus. The spontaneous coupling of EA with GSH was rapid, whereas the 'in vitro' conjugation catalyzed by the glutathione 'S'-transferases (GSTs) in rat liver was even faster. The conjugation rates greatly exceeded the EA uptake rate by saturable and non-saturable processes in isolated rat hepatocytes. When these ' in vitro' data were viewed with respect to events in whole liver (single-pass perfused rat liver), transport and not GSH conjugation was found to be rate-limiting in the rapid conversion of EA to EA-SG. In studies with isolated, enriched periportal and perivenous hepatocytes, EA uptake and EA-SG efflux were functionally similar among hepatocytes throughout the acinus. However, a shallow and increasing (portal to venous) GST activity gradient was observed in rat liver. When these zonal, uptake and metabolic 'in vitro' data were utilized to predict EA disposition in liver perfusions in the presence and absence of bovine serum albumin (BSA), a series compartment model was found to be most consistent with data obtained in the absence of albumin. The model predicted both a temporal and zonal depletion of GSH among hepatocytes and a change in the rate-limiting step with increasing perfusion time from uptake to co-substrate availability. The model was less capable of predicting the influence of high albumin protein binding on uptake and metabolism, unless a role of BSA in facilitating drug uptake was assumed. This investigation showed for the first time that, in addition to metabolic enzymes, flow and substrate binding, other important factors such as zonal transport, co-substrate, and GSH conjugate efflux further influence the distributed-in-space processing of acceptor substrates in GSH conjugation.Ph.D