Competition between two enzymes for substrate removal in liver: Modulating effects due to substrate recruitment of hepatocyte activity

Abstract

Modulating effects of competing pathways, exemplified by sulfation (high affinity-low capacity) and glucuronidation (low affinity-high capacity), on drug disappearance and metabolite formation were investigated in a simulation study. The phenomenon of substrate recruitment of hepatocyte activity in drug removal and metabolite formation was shown with respect to inlet substrate concentration, and drug processing from inlet to outlet by enzyme systems localized differentially along the sinusoidal flow path in liver. Three enzymic distribution models: (A) sulfation and glucuronidation evenly distributed in liver, (b) sulfation occurring exclusively in the first half of the liver and glucuronidation in the second half, and (C) glucuronidation solely in the first half and sulfation in the second half, were described. The influence of K_mand V_max of the competing pathway, including enzyme induction (increase in V_max), on any given pathway was also explored. Competing pathways exert their effects on other given pathways by modulating intrahepatic drug concentration from the inlet to outlet of the liver. When a competing pathway is similarly distributed or is at an anterior location to another pathway, the former pathway effectively reduces intrahepatic drug concentrations which reach downstream hepatocytes for recruitment of activity. For example, when glucuronidation activity is anterior to sulfation activity (defined with respect to flow direction), sulfation is without an effect on glucuronidation, but glucuronidation exerts a maximal influence over sulfation rates (Model C). When glucuronidation is in direct competition with sulfation (Model A) or is posteriorly distributed to sulfation (Model B), saturation of the high-affinity sulfation pathway leads to greater fluxes of substrate available downstream for glucuronidation. This results in an apparent compensatory increase in glucuronidation with reduced sulfation capacity, which occurs at input concentrations greater than the K_m for sulfation but less than the K_m for glucuronidation. This compensation pattern is more prominent for highly extracted compounds where both sulfation and glucoronidation are effective pathways in drug removal, and where large intrahepatic drug concentration gradients are expected. Since the physiologic description of intraheptic drug concentration is often described by a concentration gradient from the inlet to outlet of the liver, the logarithmic average concentration has been used to estimate the mean liver concentration in the determination of kinetic constants for enzymic reactions. The appropriateness of the method for competing pathways is presently assessed for four phenolic substrates: 1-naphthol, acetaminophen, harmol, and salicylamide, compounds listed in order of increasing extraction ratios. By employing published values for the enzymatic constants, the simulated sulfation and glucuronidation rates, accordingly for the three cases A, B, and C, and the logarithmic concentrations were refitted to the Michaelis-Menten equation. A comparison of the assigned and fitted kinetic constants revealed that for case A, the enzymatic constants agreed well for both sulfation and glucuronidation. For cases B and C of unevenly distributed enzymes, the fitted enzymatic constants for sulfation, the high-affinity pathway, were in good agreement with assigned values. Those for the low affinity, glucuronidation pathways, however, differed for the highly cleared drugs harmol and salicylamide, and for acetaminophen, for which glucuronidation is a very poor metabolic pathway. But for 1-naphthol, where the assigned K_m's as well as the V_max's for sulfation and glucuronidation are similar, reasonably good agreement between the fitted and assigned enzymatic constants was obtained. These differential observations for competing pathways are explained by the perturbation of intrahepatic concentrations and the phenomenon of substrate recruitment of hepatocyte activities, for poorly or highly extracted compounds.