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, ∼0.8–0.4 at concentrations ranging from 10–200 μM) 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 μM and V_max^overall of 0.23 μmol/min/g liver. These differed from those observed for the bimolecular nonenzymatic (constant of 126 μM⁻¹ min⁻¹) and enzymatic (K_m for GSH and EA were 1.2 mM and 94 μM, respectively; V_max of 533 nmol/min/mg liver cytosolic protein or 32 μmol/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 μM, and V_max^uptake = 0.55 μmol/min/g liver) and nonsaturable (0.015 ml/min/mg) processes. At increasing EA concentrations (>25 μM), 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.