Competing pathways in drug metabolism. I. Effect of input concentration on the conjugation of gentisamide in the once-through in situ perfused rat liver preparation

Sulfation and glucuronidation are two parallel pathways for the metabolism of phenolic substrates. Gentisamide (GAM) was used as a model compound to examine the effects of parallel competing pathways on drug disappearance and metabolite formation in the once-through perfused rat liver preparation. GAM was found to form one glucuronide (GAM-5G) and two sulfate (GAM-2S and GAM-5S) conjugates. These GAM conjugates were biosynthesized in recirculating rat liver preparations, and were isolated by preparative high-performance liquid chromatography. Specific incorporation of ³⁵S-sodium sulfate and [¹⁴C]glucose into GAM sulfate and glucuronide conjugates revealed corresponding elution patterns as labeled GAM metabolites. Their identities were characterized by enzymatic and acid hydrolyses and by NMR spectroscopy. Gentisamide-5-sulfate (GAM-5S) and gentisamide-5-glucuronide (GAM-5G) are major metabolites, and gentisamide-2-sulfate (GAM-2S) is a minor metabolite. Single-pass rat liver perfusions were used to examine the effect of stepwise increases/decreases of input GAM concentration (C(ln)) on the extraction ratio (E) of GAM and formation of metabolites. The E of GAM remained constant (about 0.89) at input concentrations from 0.9 to 120 μM and decreased at C(In) > 120 μM. Metabolite patterns, however, changed with GAM C(In), even when E was constant at C(In) up to 120 μM. GAM-5S was present as the major metabolite of GAM at all GAM C(In)s in most liver preparations but the proportions of GAM-5S and GAM-2S decreased at increasing C(In); the proportion of GAM-5G, a minor metabolite at low C(In), increased with increasing C(In). Biliary excretion rates at steady state accounted for 5.3 ± 2.7% (mean ± S.D.) of the input rate: GAM-5G was the predominant metabolite found. Fitting the metabolic data (sum of the rates of efflux in bile and perfusate at steady state) and the logarithmic average drug concentration at the various C(In) to the Michaelis-Menten equation furnished parameter estimates for the three metabolic pathways. The estimated K(m) and V(max) values were quite comparable: for GAM-2S formation, 22 μM and 287 nmol/min; for GAM-5S formation 26 μM and 978 nmol/min; for GAM-5G formation, 71 μM and 1062 nmol/min, indicating that sulfation and glucuronidation are effective competing pathways of each other. In viewing the fate of GAM over the C(In) range, a changing metabolic fate of GAM over a constant E was noted at C(In) < 120 μM: the sum of the rates of metabolite formation was proportional to C(In). The ratio for formation rates of GAM-5G/GAM-2S increased with increasing C(In), whereas GAM-5S/GAM-2S remained relatively constant with C(In). The observations may be explained purely on the difference between the K(m) values for glucuronidation and sulfation in evenly distributed enzyme systems, or to an anterior sulfation, posterior glucuronidation system. The lower K(m) values for sulfation of GAM (in GAM-2S and GAM-5S formation) render the pathways very efficient at low and not high inlet substrate concentration in the liver, modulating the intrahepatic GAM concentration in the former case, either at the same or at an anterior site to glucuronidation activities.