Phenotyping of N-acetyltransferase type 2 by caffeine from uncontrolled dietary exposure.

BACKGROUND AND 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. CONCLUSION: 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.

Acrylamide: increased concentrations in homemade food and first evidence of its variable absorption from food, variable metabolism and placental and breast milk transfer in humans.

We have developed a liquid chromatography/mass spectrometry (LC-MS/MS) assay to determine acrylamide in various body fluids. The assay also allows the reliable quantitation of acrylamide in food. In a total of 11 healthy male and female subjects, we were able to show that acrylamide from food given to humans is in fact absorbed from the gut. The half-lives determined in two male subjects were 2.2 and 7 h. Acrylamide was found in human breast milk and penetrated the human placenta (n = 3). The variability of acrylamide concentrations found in this investigation is most likely caused by variable intersubject bioavailability and metabolism. This may be an important indication that the assessment of the risk from acrylamide for the individual may be very difficult without knowing the concentrations of acrylamide in the body. This should be considered in the design of any risk assessment study or post hoc analysis of earlier studies. At this time, we suggest that pregnant women and breast-feeding mothers avoid acrylamide-containing food.