We wish to test the hypothesis that hepatic formation of metabolites occurs as a distributed-in-space phenomenon: the sojourns of a locally generated metabolite will differ from that of preformed metabolite due to their differing points of entry into the liver. These differences occur because input as generated metabolite depends on drug uptake and metabolism, and formation of any metabolite from a precursor must precede metabolite elimination. The elimination of metabolite generated from precursor and preformed metabolite will differ despite involvement of the same metabolizing enzyme(s) and processes for transfer across cells. We propose to develop an indepth theoretical treatment of a drug that forms a) a single metabolite which in turn is not further eliminated (metabolized or excreted), b) a single metabolite which is further eliminated, and c) two or more metabolites formed by competing pathways. Experimentally, we wish to study the following substrates and their metabolites in the perfused rat liver preparation: a) 3H- acetaminophen (A) and 3H-salicylamide and their 35S-sulfate conjugates; b) 14C-enalapril and 3H-enalaprilat; 3H-morphine and 14C-morphine glucuronide; 14C-acetanilide and its p-alkoxy analogs to 14C-A and 14C-AS; c) 3H-harmol and 3H-4- methylumbelliferone and their labeled (35S)-sulfates and (14C)- glucuronides (in absence and presence of sulfate inhibitor, DCNP), compounds that will be assayed by TCL and HPLC. While maintaining a single pass liver perfusion with steadystate infusions of unlabeled drug or preformed metabolite (varying levels of input concentrations), a rapid injection of radiolabeled drug and metabolite and vascular reference compounds (51Cr- RBC, 125I-albumin, and 32p-phosphate) will be used to produce a set of multiple indicator dilution curves (MID). The various techniques (normal, retrograde, hepatic artery) of oncethrough perfusion and MID will provide information on underlying processes: red cell and protein binding, influx (barrier-limited, flowlimited, and carrier- or concentrative mechanisms), efflux, and sequestration of drug and metabolite formation as well as the behaviour of preformed metabolite under both linear and nonlinear conditions. The theoretical development and the experimentally-acquired information will further our knowledge on the differences in hepatic handling between a generated vs preformed metabolite, and provide a solid basis for understanding the toxicological/pharmacological differences in generation and processing of drug metabolites.
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