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.