Competing pathways in drug metabolism. II. An identical, anterior enzymic distribution for 2- and 5-sulfoconjugation and a posterior localization for 5-glucuronidation of gentisamide in the rat liver

Abstract

Gentisamide (2,5-dihydroxybenzamide, GAM), a substrate that forms two monosulfates at the 2 and 5 positions (GAM-2S and GAM-5S) and a monoglucuronide at the 5 position (GAM-5G), was delivered at 8 or 80 microM by normal (N) and retrograde (R) flows to the once-through rat liver preparation. At the lower (8 microM) input concentration, ratios of conjugate formation rate, GAM-5S/GAM-5G and GAM-2S/GAM-5G, were decreased significantly (4.01 +/- 1.42 to 2.93 +/- 0.99, and 1.13 +/- 0.65 to 0.66 +/- 0.41, respectively) whereas a small but significant increase in the steady-state extraction ratio, E (0.89 +/- 0.029 to 0.94 +/- 0.016), was observed upon changing the flow direction from N to R. At the higher input GAM concentration (80 microM), conjugate formation rate ratios were relatively constant for GAM-5S/GAM-5G (1.18 +/- 0.08 to 1.11 +/- 0.12) and GAM-2S/GAM-5G (0.33 +/- 0.05 to 0.31 +/- 0.05) upon changing flow direction from N to R, despite a slight increase in E from 0.87 +/- 0.023 to 0.91 +/- 0.016 was observed. These results suggest that the sulfotransferase activities responsible for 2- and 5-sulfoconjugations are identically distributed and localized anterior to 5-glucuronidation activities during a normal flow of substrate into the rat liver (entering the portal vein and exiting the hepatic vein), and that the presence of uneven distribution of conjugation activities is discriminated only at the lower input drug concentration. At high concentration (greater than Km for all systems), saturation of all pathways occurs, and other anteriorly/identically distributed competing pathways would fail to perturb downstream intrahepatic drug concentrations and the resultant conjugation rates. The lack of change in metabolic profiles renders the condition unsuitable for examination of uneven distribution of enzymes in the liver. These observations were generally predicted by theoretical enzymic models of consistent distribution patterns. Because 2- and 5-sulfation were mediated by systems of similar Km but different Vmax values, two possibilities, the same isozyme of sulfotransferase being involved in the formation of two enzyme-substrate complexes to form two distinctly different products or two isozymes of sulfotransferases of identical distribution, were discussed.

Futile cycling between 4-methylumbelliferone and its conjugates in perfused rat liver

“…The shift in metabolite proportions from 0.005 to 63 pmol/L was not accompanied by changes in the hepatic extraction ratio. The observation is akin to those observed for other flow-limited substrates: harmol(49, 50), gentisamide (40,51) which are substrates that are sulfated (high affinity, low capacity pathway) predominantly in periportal hepatocytes and glucuronidated (low affinity, high capacity) virtually identically among all hepatocytes. The anterior sulfation pathway exerts an effect on glucuronidation downstream and predominates at low concentration, wherein not all downstream hepatocytes are metabolically recruited because of depletion of substrate caused by upstream metabolism.…”

Carrier-mediated entry of 4-methylumbelliferyl sulfate: Characterization by the multiple-indicator dilution technique in perfused rat liver

“…See Table 5 for the fitted parameters (K m and V max ) for the saturable and nonsaturable (P diff S) components for the influx of 4MUS into the rat liver. compounds such as harmol, 20 7-hydroxycoumarin, 38 gentisamide, 39 and salicylamide 40 in perfused rat livers. The above conclusion is based on an equal access of 4MUS with prograde and retrograde flow directions.…”