In vitro mechanisms of probenecid-associated alterations in acetaminophen glucuronide hepatic disposition

The effect of probenecid (PRB) on factors regulating the hepatic disposition of acetaminophen glucuronide (AG) was investigated in vitro. Potential interactions in metabolism or binding to cytosolic proteins were examined. In the absence of PRB, AG formation in rat hepatic S9 fractions was saturable (V(max) = 2.77 +/- 0.36 nmol/ min/mg protein; K(M) = 18.0 +/- 0.92 mM). PRB significantly decreased V(max), but not K(M), for AG formation, consistent with noncompetitive inhibition. Various models were fit to the AG formation rate vs. acetaminophen (APAP) and PRB concentration data to elucidate the mechanism of inhibition by PRB. A partial noncompetitive inhibition model (K(i) = 1.10 +/- 0.01 mM) described the data best based on model selection criteria. AG did not bind to the cytosolic protein ligandin (glutathione S-transferase A1). These data indicate that PRB is a potent partial noncompetitive inhibitor of acetaminophen glucuronidation in vitro. PRB-associated alterations in AG hepatic disposition in vivo are not due to altered binding of AG to GSTA1 but may be attributed in part to impaired AG formation.