Kinetics of acetyl CoA: arylamine N-acetyltransferase from rapid and slow acetylator human liver.

Michaelis-Menten kinetic constants for sulfamethazine (SMZ) and p-aminobenzoic acid (PABA) metabolism were determined in 2 very rapid, 8 intermediate, and 10 slow acetylator human livers. The mean apparent KM values for the monomorphic substrate PABA were 70 +/- 20, 180 +/- 50, and 310 +/- 30 microM for the slow, intermediate, and very rapid enzymes, respectively, whereas the polymorphic substrate SMZ exhibited little interphenotypic KM variation. Compared to the slow enzymes, the rapid acetylators exhibited mean apparent Vmax values 15- and 20-fold greater for PABA and SMZ, respectively. Furthermore, under in vitro conditions where enzyme saturation was achieved, PABA showed polymorphic acetylation characteristics, in contrast to events in vivo, where monomorphic acetylation patterns were observed. As the acetyl coenzyme A concentration in the reaction mixture was increased, the KM and Vmax for PABA increased, in accordance with "ping-pong" kinetic principles. As occurs with polymorphic substrates, a binary ping-pong mechanism appears to govern N-acetylation of monomorphic substrates in human liver, as evidenced by initial velocity patterns and limiting values for the KM and Vmax for PABA.

Human N-acetylation genotype determination with urinary caffeine metabolites.

The human acetylation genotype was determined by measuring urinary caffeine metabolites by use of a modification of a previously published HPLC method. The problem of separation of 7-methylxanthine (7X) from 1-methyluric acid (IU) in urine extracts was achieved by adding a phenyl column, in tandem with a C18 reverse-phase column, by means of a methanol:aqueous acetic acid gradient elution system. The urinary molar ratios of (AAMU)/(AAMU + 1U + 1X) and (AAMU)/(1X) were estimated in 20 subjects phenotyped with dapsone, with 100% concordance for the [AAMU]/[1X] ratio. A population study of 42 unrelated individuals exhibited trimodal distribution in acetylation capacity, consistent with the Hardy-Weinberg theory of population genetics. Definitive pedigree analysis of 16 families (75 subjects) resulted in significant similarity between the observed genotypic matings and those expected by classical Mendelian segregation. This noninvasive genotyping method promises to be useful in future investigation of the relationship between the human acetylation polymorphism and clinical disorders.