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 Ess, ∼0.8–0.4 at concentrations ranging from 10–200 μM) despite the appreciable plasma protein binding. The declining Ess 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 Kmoverall of 67 μM and Vmaxoverall 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 (Km for GSH and EA were 1.2 mM and 94 μM, respectively; Vmax 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 (Kmuptake = 57 μM, and Vmaxuptake = 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 Ess 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.
Footnotes
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Send reprint requests to: Dr. K. Sandy Pang, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ontario, Canada M5S 2S2. E-mail: pang{at}phm.utoronto.ca.
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↵1 This work was supported by the Medical Research Council of Canada (MRC, MA9104). R.G.T. was supported by a fellowship from the Pharmaceutical Manufacturers Association of Canada—Health Research Foundation and MRC, and the University of Toronto Open Fellowship. This work was presented in part at the Annual Meeting of the American Society for Pharmacology and Experimental Therapeutics, 1997 (San Diego, CA).
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Received for publication December 18, 1998.
- Abbreviations:
- EA
- ethacrynic acid
- EA-CYS
- EA cysteine conjugate
- EA-CG
- EA cysteinylglycinyl conjugate
- EA-NAC
- EAN-acetylcysteine conjugate
- EA-SG
- ethacrynic acid GSH conjugate
- BSP
- bromosulfophthalein
- BUM
- bumetanide
- TCA
- taurocholic acid
- GSH
- reduced glutathione
- GSSG
- oxidized glutathione
- Accepted April 23, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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