Validity of an ELISA for N-acetyltransferase-2 (NAT2) phenotyping

A competitive antigen ELISA was previously developed for NAT2 phenotyping, using caffeine as the probe drug. The ELISA phenotypes by measuring the ratio of 5-acetamido-6-amino-3-methyluracil (AAMU) and 1-methylxanthine (1X) after transformation of 5-acetamido-6-formylamino-3-methyluracil (AFMU) to AAMU, in contrast to capillary electrophoresis high-pressure liquid chromatography (HPLC) which phenotype by measuring the AFMU/1X ratio. The ELISA phenotyping was previously determined in 30 samples and correlated well with phenotypes determined by capillary electrophoresis (29/30). The correlation was extended with the standard HPLC methodology by expanding the data set by 146 in order to test the validity of the ELISA methodology. The correlation with HPLC in this larger sample size was 96%; whereas the correlation between the two methods for determination of 1X was high (r(2)=0.90), that for determination of AAMU by ELISA and AFMU by HPLC was low (r(2)=0.53). The poor correlation between the two methodologies could not be attributed to the age of urine samples, nor to a significant decomposition of AFMU in the body prior to collection of the urine sample. The addition of a simple caffeine metabolite extraction method, originally developed for HPLC analysis of metabolites, to the ELISA phenotyping protocol produced a methodology with absolute correlation to the standard HPLC method.