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.