HEPATIC FORMATION AND HANDLING OF DRUG METABOLITES

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The long-term goal of the research proposal is to provide a thorough understanding of the space- and time-related changes associated with drug disappearance and metabolite formation in the liver, the major drug metabolizing organ. Methods for probing zonal metabolic heterogeneities: single pass prograde [P, inflow into portal vein (PV)] and retrograde [R, inflow into hepatic vein (HV)] and hepatic artery-portal vein, hepatic artery-hepatic vein [HAPV-HAHV, dual inflow system with substrates given to HA] perfusion and the multiple indicator dilution (MID) technique will be used to examine mechanisms of elimination and transport across hepatocyte membranes for several precursor-metabolite pairs. An injection of a mixture of vascular [51Cr-labeled red blood cells, 125I-labeled albumin, 58CoEDTA (similar to [14C]sucrose)] and cellular (D2O) reference noneliminated indicators and 14C- and 3H-labeled precursor and product will be given, during steady-state bulk perfusion by PR or HAPV-HAHV. The outflow profiles thus obtained will be appropriately analyzed and referenced with respect to the noneliminated reference indicators. Modeling is able to account for the binding effects due to red cells, plasma, or tissue proteins, provide the physiological volumes and the influx, efflux, and sequestration coefficients for both precursor and metabolite, and identify the rate-controlling step. For Aim 1, the hepatic processing of benzoic acid, hippuric acid, taurolithocholic acid 3- sulfate, tracer salicylamide and phenacetin, morphine, morphine-3beta- and 6beta- glucuronides, 4-methyl-umbelliferone (4MU), 4MU glucuronide, harmol, harmol sulfate and glucuronide conjugates, estrone sulfate, estrone, and estradiol (with and without inhibitors of sulfation/desulfation) will be examined. The sulfate conjugate of the bile acid, and of estrone, 4-MU or harmol will be given simultaneously to test for interactions in transport and removal. For Aim 2, the micro-mixing of the hepatic artery and portal vein will be investigated with a full set of noneliminated references with HA or PV injections, then with HA or PV infusion of carrier-mediated [bromosulfophthalein (BSP) and its glutathione conjugate (BSP-GSH)] and flow-limited (salicylamide) substrates at varying HA:PV flow ratios. Events underlying single, parallel, and sequential pathways, futile cycling, enzyme zonation, and competition in uptake will be provided in the above studies. The permeability of the peribiliary capillary plexus to solutes and the potential reduction in liver mass with the probes infused into the HA will be delineated. These studies should lend important insight into mechanisms of drug-metabolite processing of pharmacologically important sulfate and glucuronide conjugates, and differences in preformed vs. generated metabolite removal due to the differing origins.