Hendrik Modick, André Schütze, Claudia Pälmke, Tobias Weiss
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Hendrik Modick, André Schütze, Claudia Pälmke, Tobias Weiss
Human-biomonitoring of N-acetyl-4-aminophenol (paracetamol/acetaminophen) - the major metabolite of aniline Hendrik Modick, André Schütze, Claudia Pälmke, Tobias Weiss, Thomas Brüning, Holger M. Koch Institute for Prevention and Occupational Medicine of the German Social Accident Insurance – Institute of the Ruhr - University Bochum (IPA), Bochum, Germany Methods To further investigate the quantity and the sources of internal para-NAAP exposure we continued to determine para-NAAP in urine samples from the general population, workers exposed to aniline and users of paracetamol. We determined total urinary para-NAAP, after enzymatic hydrolysis, as biomarker for internal paracetamol exposure. Cleanup and enrichment was achieved via on-line turbulent flow chromatography, chromatographic separation via RP-HPLC. Quantification was performed with tandem mass spectrometry by isotope dilution quantification with a limit of quantification (LOQ) of 0.5 µg/L[4]. References [1] Human Biomonitoring Commission of the German Ministry for Environment, Stoffmonographie und Referenzwerte für monocyklische Aminoaromaten im Urin, Bundesgesundheitsblatt 54 (2011) 650. [2] Kao, J. Faulkner J, Bridges, JW. Metabolism of aniline in pigs, rats and sheep, Drug. Metab. Dispos. 6 (1978) 549. [3]Camann, DE. Schultz, ST. Yau, AY. Heilbrun, LP. Zuniga, MM. Palmer, RF. Miller, CS. Acetaminophen, pesticide, and diethylhexyl phthalate metabolites, anandamide, and fatty acids in deciduous molars: potential biomarkers of perinatal exposure, J. Expos. Sci. Environ. Epidemiol. (2012) http://dx.doi.org/10.1038/jes.2012.71. [4]Modick, H. Schütze, A. Pälmke, C. Weiss, T. Brüning, T. Koch HM, Rapid determination of N-acetyl-4-aminophenol (paracetamol) in urine by tandem mass spectrometry coupled with on-line clean-up by two dimensional turbulent flow/reversed phase liquid chromatography J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 925 (2013) 33. [5] Regulation (EU) No. 37/2010 of the European Commission, 2010. [6]Koch, HM. Lorber, M. Christensen KLY. Pälmke, C. Koslitz, S. Brüning T. Identifying sources of phthalate exposure with human biomonitoring: Results of a 48 h fasting study with urine collection and personal activity patterns. Int. J. Hyg. Environ. Health (2013), http:// dx.doi.org/10.1016/j.ijheh.2012.12.002 So far, in our daily lab routine the analytical method was applied to over 2000 urine samples. Para-NAAP could be detected in all samples analyzed with a wide range of concentrations (table 1, figure 1), with a Table 1: Human biomonitoring data on urinary para-NAAP in the general population Smokers (n = 1,059) Non-smokers (n = 1,048) Total (n = 2,107) 10,824 4,108 7,477 Median [µg/L] 68.2 54.2 61.8 Min. [µg/L] 0. 5 0.95 0.65 Max. [µg/L] 2,274,296 580,357 2,274,296 4,464 3,292 4,047 Mean [µg/L] 95th Percentile [µg/L] relative cumulative frequency [%] However, in addition to paracetamol medication and exposure to ubiquitous aniline other sources for urinary para-NAAP in the general population cannot be excluded. Paracetamol is approved for veterinary use in the European Union[5]. Unfortunately, data on paracetamol residues in food of animal origin is limited. Hence, nutrition may also be a possible source 1,000,000 A B 100,000 10,000 1,000 100 10 100% 90% 1 80% 0 12 24 time [h] 70% 36 48 Fig. 2: Urinary excretion of para-NAAP After (A) single tablet of paracetamol (500 mg) and (B) Occupational exposure to aniline (7.7 mg/m³, 8h) 60% 50% 40% 30% 20% 10% 0% 0,1 10,000 1 10 100 1000 10000 100000 1000000 10000000 para-NAAP concentration [µg/L] Fig. 1: Human biomonitoring of N-acetyl-4-aminophenol Relative cumulative frequency of urinary para-NAAP concentrations from our daily lab routine, n > 2000 median of 61.8 µg/ L and a 95th percentile of 4,047 µg/L. Despite the fact that para-NAAP is the major metabolite of aniline and aniline is an integral part of tobacco smoke, there are no significantly higher levels of para-NAAP in smokers. This is in good accordance with HBM studies on aniline (determination of aniline in urine and hemoglobin adducts in blood) which reported no impact of tobacco smoking on urinary aniline[1]. After a single tablet of paracetamol (500 mg) maximum urinary concentrations of para-NAAP (>400 mg/L) occur ~4h – 12h after intake (figure 2). 32h – 48h after intake para-NAAP concentrations are still in the mg/L range (~5 mg/L). If we consider urinary para-NAAP concentrations above such a preliminary cut off of 5 mg/L to be due to direct intake of paracetamol, 4.7 % of the above 2000 individuals investigated would have taken paracetamol within 48h prior to sampling. In individuals with inhalation exposure to aniline slightly below the occupational threshold limit value of 7.7 mg aniline/m³ air, we observed maximum urinary concentrations of para-NAAP well in the mg/L range (figure 2). c (para-NAAP) [µg/L] Aniline is an important large-volume intermediate in several industrial processes (e.g. rubber, colorants, pesticides and pharmaceuticals). Exposure to aniline is therefore both of occupational and environmental relevance. The general population is known to be ubiquitously exposed to aniline[1]. N-acetyl-4-aminophenol (para-NAAP) is the major urinary metabolite of aniline representing 75%-86% of the dose[2]. Furthermore, para-NAAP, also known under the trade names acetaminophen/paracetamol is one of the most commonly used over-the-counter analgesics. Orally administered paracetamol is predominantly (~80%) excreted as unchanged para-NAAP in the form of glucuronide and sulphate conjugates. Recent epidemiological studies suggest that intrauterine exposure to para-NAAP is a risk factor for the development of male reproductive disorders. Human biomonitoring (HBM) data on the body burden of the general population (and potentially exposed subpopulations) to para-NAAP is sparse. In a study from 2012 para-NAAP has been detected in deciduous molars of 9 of 21 subjects[3]. In a human biomonitoring pilot study we determined urinary para-NAAP. Paracetamol users excreted urinary para-NAAP in concentrations up to the mg/L range (depending on dose and timing). However, also all non-users of paracetamol excreted urinary para-NAAP in concentrations overlapping with those of paracetamol users[4]. Results c (para-NAAP) [µg/L] Introduction 1,000 100 10 1 48h period of fasting 0 12 24 full meal 36 time [h] 48 60 Fig. 3: 48 h fasting study Creatinine adjusted urinary para-NAAP concentrations of two male volunteers during a 48h fasting period, on semi-logarithmic scale of internal body burdens to para-NAAP. To investigate food as a possible source of paracetamol exposure, we analyzed samples from a 2-day fasting study[6] for urinary para-NAAP. In all volunteers (2 male, 2 female, 25 – 40 years) para-NAAP concentrations decreased continuously and considerably during the time of fasting. Figure 3 shows the decrease in para-NAAP concentrations over the fasting time for the two male volunteers. However, in both volunteers elimination half-times were about 7h, which is slightly longer than elimination half-times after oral intake of paracetamol (~ 5h). Conclusion Within this study we present, for the first time, human biomonitoring data of the internal body burden to para-NAAP/ paracetamol in the general population. We could show that the general population is ubiquitously exposed to para-NAAP or its metabolic precursors. We investigated some possible routes of exposure (PCM medication, occupational exposure to aniline, nutrition). All of these routes might contribute to the observed para-NAAP body burden. A urinary para-NAAP level of 5mg/L seems to be a suitable cut-off to indicate a recent PCM medication in the general population. However, we could show that (occupational) exposure to aniline is also clearly related to internal body burden with para-NAAP / paracetamol. The decrease of para-NAAP excretion during a fasting experiment hints to nutrition as one important source of urinary para-NAAP in the general population.