The occurrence of two hepatic microsomal sites for amobarbital hydroxylation

Amobarbital metabolism in human liver and in rat liver, lung, kidney, and small intestine was measured in vitro using thin-layer chromatography (TLC) for separation of metabolites generated from incubation with [2-14C]amobarbital. Formation of 3'-hydroxyamobarbital (C-OH) occurred primarily in the liver. The kinetics of C-OH formation by rat liver microsomes or isolated hepatocytes could be described by a Michaelis-Menten model incorporating two metabolic sites, one characterized by high-affinity and low-velocity constants (Km = 0.054 +/- 0.012 mM, Vmax = 16.89 +/- 4.27 nmol C-OH x g liver-1 x min-1), the other by low-affinity and high-velocity (Km = 0.679 +/- 0.097 mM, Vmax = 66.0 +/- 5.41 nmol C-OH x g liver-1 x min-1). The kinetic parameters of the high-affinity site differed significantly between whole cells and homogenates. Pretreatment with phenobarbital for 3 days induced only the high-affinity site. Quantitation of C-OH formation in four human liver samples from several sources showed that metabolism may conform to the two-site model observed in rat liver.