Phenotyping of N-acetyltransferase type 2 by caffeine from uncontrolled dietary exposure.
OBJECTIVE
The standard approach for phenotyping of the human arylamine N-acetyltransferase 2 (NAT2) uses urinary caffeine metabolite ratios after a caffeine test dose taken in after methylxanthine abstinence. We tested whether these standardization measures were still needed when a more sensitive quantification technique was used.


METHODS
A new liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the quantification of the caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), and 1-methylurate (1U) was developed. Urine samples from 77 healthy volunteers collected before and 5-6 h after oral intake of 150-200 mg caffeine were analyzed. The lower limits of quantification were 0.1 microg/ml for caffeine, 1X, 1U, and AFMU, and 0.2 microg/ml for AAMU.


RESULTS
The urinary NAT2 ratios (AFMU+AAMU) / (AFMU+AAMU+1X+1U) before and after caffeine intake correlated well in 65 volunteers (r(2)=0.827; P< 0.0001). In 12 participants (16%), metabolite concentrations in urine before caffeine intake were below the quantification limit. NAT2 genotyping, done in 41 volunteers for four SNPs, corroborated the phenotyping results.


CONCLUSIONS
NAT2 activity can be determined from a spontaneous urine probe in most subjects by quantification of caffeine metabolites arising from non-standardized dietary caffeine exposure using LC-MS/MS. This may facilitate the phenotyping procedure.
Jetter
Alexander
A
Department of Pharmacology, Clinical Pharmacology, University of Cologne, Gleueler Strasse 24, 50931 Köln, Germany. alexander.jetter@medizin.uni-koeln.de
Kinzig-Schippers
Martina
M
Illauer
Michael
M
Hermann
Robert
R
Erb
Katharina
K
Borlak
Jürgen
J
Wolf
Helga
H
Smith
Gillian
G
Cascorbi
Ingolf
I
Sörgel
Fritz
F
Fuhr
Uwe
U
D000107:Acetylation; D000284:Administration, Oral; D000328:Adult; D001191:Arylamine N-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D002110:Caffeine / Q000008:administration & dosage / Q000378:metabolism / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid; D004032:Diet; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry / Q000652:urine; D014527:Uric Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000652:urine; D014970:Xanthines / Q000737:chemistry / Q000652:urine
D014970:Xanthines; D014527:Uric Acid; C030530:1-methyluric acid; D002110:Caffeine; D014498:Uracil; C053085:1-methylxanthine; C036692:5-acetylamino-6-formylamino-3-methyluracil; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human; C054188:5-acetylamino-6-amino-3-methyluracil