DETERMINATION OF APPARENT KINETIC CONSTANTS OF THE MICROSOMAL HYDROXYLATION OF AMOBARBITAL, HEXOBARBITAL, AND PENTOBARBITAL

¹ Department of Pharmacology, University of Minnesota Medical School

Apparent kinetic constants for the hydroxylation of amobarbital, hexobarbital, and pentobarbital by hepatic microsomes from the rat were obtained by measuring product formation as determined by high-pressure anion exchange chromatography. Michaelis constants (millimolar) and maximum velocities (nanomoles of product formed per nmol of cytochrome P-450 per hr) for amobarbital, hexobarbital, and pentobarbital were 0.24 and 89, 0.19 and 345, and 0.25 and 100, respectively. The addition of NADH to reaction mixtures containing a NADPH-generating system increased the maximum velocities of amobarbital, hexobarbital, and pentobarbital by 77, 249, and 47%, respectively. The binding of the barbiturates to cytochrome P-450, expressed as binding constants (K₈), were about one order of magnitude lower than the Michaelis constants for the hydroxylation of the barbiturates. Amobarbital, hexobarbital, and pentobarbital stimulated NADPH-cytochrome P-450 reductase activity by 2.9, 4.6, and 2.0 nmol of cytochrome P-450 reduced per min.