title: Bimolecular glutathione conjugation kinetics of ethacrynic acid in rat liver: in vitro and perfusion studies.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
authors.forename: R G
authors.initials: RG
authors.lastname: Tirona
authors.forename: K S
authors.initials: KS
authors.lastname: Pang
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004232:Diuretics / Q000378:metabolism / Q000493:pharmacokinetics*; D004976:Ethacrynic Acid / Q000378:metabolism / Q000493:pharmacokinetics*; D005978:Glutathione / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008954:Models, Biological; D008956:Models, Chemical; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D004232:Diuretics; D005978:Glutathione; D004976:Ethacrynic Acid