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 rat io of 5-acetamido-6-amino-3-methyluracil (AAMU) and 1-methylxanthine (1X) a fter transformation of 5-acetamido-6-formylamino-3-methyluracil (AFMU) to A AMU, in contrast to capillary electrophoresis high-pressure liquid chromato graphy (HPLC) which phenotype by measuring the AFMU/1X ratio. The ELISA phe notyping was previously determined in 30 samples and correlated well with p henotypes determined by capillary electrophoresis (29/30). The correlation was extended with the standard HPLC methodology by expanding the data set b y 146 in order to test the validity of the ELISA methodology. The correlati on with HPLC in this larger sample size was 96%; whereas the correlation be tween 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 analys is of metabolites, to the ELISA phenotyping protocol produced a methodology with absolute correlation to the standard HPLC method. (C) 2001 Elsevier S cience B.V. All rights reserved.