Efficient hepatic uptake and concentrative biliary excretion of a mercapturic acid

The role of the liver in the disposition of circulating mercapturic acids was examined in anesthetized rats and in the isolated perfused rat liver using S-2,4-dinitrophenyl-N-acetylcysteine (DNP-NAC) as the model compound. When DNP-NAC was infused into the jugular vein (150 or 600 nmol over 60 min) it was rapidly and nearly quantitatively excreted as DNP-NAC into bile (42-36% of the dose) and urine (48-62% of dose). Some minor metabolites were detected in bile (<4%), with the major metabolite coeluting on HPLC with the DNP conjugate of glutathione (DNP-SG). Isolated rat livers perfused single pass with 3 microM DNP-NAC removed 72 +/- 9% of this mercapturic acid from perfusate. This rapid DNP-NAC uptake was unaffected by sodium omission, or by L-cysteine, L-glutamate, L-cystine, or N-acetylated amino acids, but was decreased by inhibitors of hepatic sinusoidal organic anion transporters (oatp), indicating that DNP-NAC is a substrate for these transporters. The DNP-NAC removed from perfusate was promptly excreted into bile, eliciting a dose-dependent choleresis. DNP-NAC itself constituted approximately 75% of the total dose recovered in bile, reaching a concentration of 9 mM when livers were perfused in a recirculating mode with an initial DNP-NAC concentration of 250 microM. Other biliary metabolites included DNP-SG, DNP-cysteinylglycine, and DNP-cysteine. DNP-SG was likely formed by a spontaneous retro-Michael reaction between glutathione and DNP-NAC. Subsequent degradation of DNP-SG by biliary gamma-glutamyltranspeptidase and dipeptidase activities accounts for the cysteinylglycine and cysteine conjugates, respectively. These findings indicate the presence of efficient hepatic mechanisms for sinusoidal uptake and biliary excretion of circulating mercapturic acids in rat liver and demonstrate that the liver plays a role in their whole body elimination.