Excretion of 19‐norandrosterone after consumption of boar meat

Abstract

The consumption of the offal of noncastrated pigs can lead to the excretion of 19‐norandrosterone (NorA) in urine of humans. In doping control, GC/C/IRMS is the method of choice to differentiate between an endogenous or exogenous origin of urinary NorA. In some cases, after the consumption of wild boar offal, the δ¹³C values of urinary NorA fulfill the criteria of an adverse analytical finding due to differing food sources of boar and consumer. However, consumption of wild boar's offal is not very common in Germany, and thus, the occurrence of such an analytical finding is unlikely. In contrast, the commerce with wild boar meat has increased in Germany within the last years. Up to 20,000 tons of wild boar meat are annually consumed. In order to probe for the probability of the occurrence of urinary NorA after consumption of wild boar meat, human urine samples were tested following the ingestion of commercially available game. In approximately half of the urine samples, traces of NorA were detected postadministration of 200 to 400 g boar meat. The highest urinary concentration was 2.9 ng/ml, and significant amounts were detected up to 9 h after the meal. δ¹³C values ranged from −18.5‰ to −23.5‰, which would have led to at least two adverse analytical findings if the samples were collected in an antidoping context. IRMS analysis on German boar tissue samples showed that δ¹³C values for wild boar's steroids are unpredictable and may vary seasonally.

Full text

SPECIAL ISSUE - RESEARCH ARTICLE
Excretion of 19-norandrosterone after consumption of
boar meat
Frank Hülsemann
1
| Gregor Fußhöller
1
| Christine Lehn
2
| Mario Thevis
1
1
Institute of Biochemistry, German Sport
University Cologne, Cologne, Germany
2
Institute of Legal Medicine, University of
Munich, Munich, Germany
Correspondence
Frank Hülsemann, Institute of Biochemistry,
German Sport University Cologne, Cologne,
Germany.
Email: f.huelsemann@biochem.dshs-koeln.de
Abstract
The consumption of the offal of noncastrated pigs can lead to the excretion of
19-norandrosterone (NorA) in urine of humans. In doping control, GC/C/IRMS is the
method of choice to differentiate between an endogenous or exogenous origin of
urinary NorA. In some cases, after the consumption of wild boar offal, the δ
13
C
values of urinary NorA fulfill the criteria of an adverse analytical finding due to differ-
ing food sources of boar and consumer. However, consumption of wild boar's offal is
not very common in Germany, and thus, the occurrence of such an analytical finding
is unlikely. In contrast, the commerce with wild boar meat has increased in Germany
within the last years. Up to 20,000 tons of wild boar meat are annually consumed. In
order to probe for the probability of the occurrence of urinary NorA after consump-
tion of wild boar meat, human urine samples were tested following the ingestion of
commercially available game. In approximately half of the urine samples, traces of
NorA were detected postadministration of 200 to 400 g boar meat. The highest uri-
nary concentration was 2.9 ng/ml, and significant amounts were detected up to 9 h
after the meal. δ
13
C values ranged from −18.5‰to −23.5‰, which would have led
to at least two adverse analytical findings if the samples were collected in an
antidoping context. IRMS analysis on German boar tissue samples showed that δ
13
C
values for wild boar's steroids are unpredictable and may vary seasonally.
KEYWORDS
boar meat, carbon isotope ratios, norandrosterone
1|INTRODUCTION
In doping control analysis, isotope ratio mass spectrometry (IRMS) is
used to distinguish between an endogenous or exogenous origin of the
urinary 19-norsteroid norandrosterone (NorA).
1,2
In humans, NorA can
be naturally found in urine in small amounts (<2 ng/ml) formed, for
example, by demethylation of androsterone (A) and, at higher concen-
trations, during pregnancy.
2–4
NorA can also be formed in urine by in
situ microbial degradation of A.
4,5
In addition, NorA is the main
urinary metabolite of the therapeutic nortestosterone (NT, nandrolone)
as well as of prohormones such as 4-norandrostenediol,
5-norandrostenediol, or 4-norandrostenedione (NAED), which are all
prohibited in sports.
5,6
A source of so-called “pseudo-endogenous”uri-
nary NorA can be the consumption of the offal of noncastrated pigs.
5–8
In boars, the highest concentrations of 19-norsteroids can be
found in testicles (NAED up to 84 μg/kg, NT up to 172 μg/kg), liver,
and kidney.
9
Much lower but nevertheless significant amounts have
been found in boar meat (NAED up to 0.9 μg/kg, NT up to 3.6 μg/
kg).
9
Although the consumption of boar offal is not particularly com-
mon in Germany, the consumption of wild boar meat is increasing
Received: 30 June 2020 Revised: 28 September 2020 Accepted: 17 October 2020
DOI: 10.1002/dta.2958
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd
Drug Test Anal. 2020;1–6. wileyonlinelibrary.com/journal/dta 1

within the last years with around 600,000 wild boars harvested per
hunting season,
10
corresponding to approximately 10,000 to 20,000
tons of wild boar meat being consumed in Germany every year.
11
We have reported previously that the carbon isotopic composition
of free-ranging (“wild”) boar tissue and steroids in Germany may vary
seasonally and can result in δ
13
Cvaluesfrom−13‰to −24‰.
8
The
forage of free ranging boars is usually dominated by C
3
-plants (wheat,
barley, acorns, etc.) in winter and spring, but as soon as maize crops are
available, the boars rely elusively on this C
4
-plant as primary forage.
Thus, the consumption of offal but also potentially meat can lead to the
urinary excretion of NorA with a carbon isotopic signature different
from the consumer. This “pseudo-endogenous”origin of NorA may lead
to adverse analytical findings in doping control testing if δ
13
Cvaluesof
consumer and consumed animal differ more than 3‰.
5
It has been stated improbable that concerning antidoping tests,
significant amounts of urinary NorA in humans may originate from the
consumption of boar meat,
12
and if so, the corresponding δ
13
C values
of such urinary NorA would be “endogenous-like.”
4
As these assump-
tions are yet to be corroborated, an excretion study was conducted to
verify or falsify the possibility of an adverse analytical finding after
consumption of boar meat.
2|EXPERIMENTAL
2.1 |Test meals and participants
The meals were prepared using varying amounts of boar meat prod-
ucts (Table 1), with weights ranging from 187 to 491 g (prepared
weight). The products were randomly selected and obtained from
butcheries or online distributors. Each volunteer consumed one meal,
with time and side dishes being arbitrary. Nine male volunteers with
an average body weight of 90.4 ± 8.9 kg and three female volunteers
(67.0 ± 5.6 kg) were included in the study. The participants were
requested to collect one urine sample prior to the meal and all urine
samples for a period of 24 h after meat consumption. Samples were
stored at +4C until analysis. The participants gave written informed
consent prior to the study. Test meals were not checked for NorA
content or carbon isotope composition.
2.2 |Sample preparation of urine for GC/MS/MS
The samples were prepared according to the laboratory internal stan-
dard operating procedure for anabolic steroids.
13
Conjugated and
unconjugated steroids were extracted from urine at pH 9.6 with tert-
butylmethyl ether (TBME, in-house purified by distillation) following
enzymatic hydrolysis of the glucuronides at pH 7 (β-glucuronidase,
Roche). After centrifugation, the organic layer was transferred and
evaporated to dryness. The dry residue was derivatized with 100 μlof
N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA, Macherey-
Nagel)/ammonium iodide (Sigma-Aldrich)/ethanethiol (Merck, for syn-
thesis) (v:w:v, 1000:2:3).
2.3 |GC/MS/MS
The GC/EI-MS/MS experiments were performed in accordance to
earlier protocols
14
using a Trace 1310 gas chromatograph interfaced
to a TSQ 8000 triple quadrupole mass spectrometer (all Thermo Sci-
entific). The GC system was equipped with an Ultra1 capillary column
(length 17 m, i.d. 0.2 mm, film thickness 0.1 μm, Agilent) in split (1:10)
mode. The initial GC oven temperature was 184C, increasing at 3C/
min to 232C and at 40C/min to a final temperature of 310C.
TABLE 1 Type and amount of test meals including maximum urinary NorA concentrations and δ
13
C values after consumption for all
participants (m = male, f = female)
Type Amount/g [NorA]
max
/ng/ml NorA/NorE δ
13
C
ERC
/‰δ
13
C
NorA
/‰jΔδj/‰
1 (m) Roast
a
187 <1 3.5 −22.9 −19.7 3.2
2 (m) Roast
b
491
3 (m) Roast
a
410 2.9 7.6 −24.3 −18.5 5.8
4 (m) Roast
a
200
5 (f) Roast
a
200
6 (m) Ham
a
300 2.5 4.9 −23.3 −23.5 0.2
7 (f) Canned meat
a
220 <1 4.0 −22.8 −21.4 1.4
8 (f) Canned meat
a
215 <1 4.3 −23.8 −21.9 1.9
9 (m) Canned meat
c
235
10 (m) Canned meat
c
240
11 (m) Jerky
c
90/(315)
d
12 (m) Jerky
c
120/(420)
d
a
Obtained from a meat market, Northern Germany.
b
Local supplier, Southern Germany.
c
Online distributor, Estonian origin.
d
Number in brackets represents the corresponding raw weight of the dried meat.
2HÜLSEMANN ET AL.

Helium (4.6, Linde) was used as carrier gas (0.9 ml/min, constant pres-
sure) and argon (5.0, Linde) as collision gas. The injector and interface
temperatures were both set to 300C, and the ion source was oper-
ated at 250C. Ionization was accomplished using electron ionization
(EI) (70 eV).
2.4 |Sample preparation of urine for GC/C/IRMS
Preparation of the urine samples (20–30 ml) followed the laboratory
internal standard operating procedure
15
that comprised the following
steps: solid-phase extraction (Chromabond C18, 500 mg, 6 ml,
Macherey-Nagel) with methanol (LC grade, J.T. Baker) followed by a
liquid–liquid extraction with TBME (GC grade, Merck), an enzymatic
hydrolysis with β-glucuronidase (Roche) at pH 7, and a second liquid–
liquid extraction with n-pentane (pa, Merck) at pH 9.6. By means of
two different HPLC runs, the steroids of interest were separated and
fractionated. The first HPLC was a reversed-phase purification on a
XBridge RP18 5 μm column, followed by a second HPLC employing a
XBridge C18 column 5 μm (both columns from Waters). The fractions
were dried and acetylated for IRMS.
2.5 |GC/C/IRMS of urinary NorA
Samples were measured per GC/C/IRMS on a Trace 1310 gas
chromatograph equipped with an HP-5MS chromatographic column
and coupled via a ConFlo IV to a MAT 253 isotope ratio mass spec-
trometer (all Thermo Scientific). Carbon isotope ratios are expressed
in per mill relative to VPDB. Monitoring gas (CO
2
, purity 4.5, Linde)
was scale calibrated using acetylated steroid mixtures USADA 33-1
(Cornell University, Ithaca, NY) comprising 5α-androstan-3β-ol
acetate, 5α-androstan-3α-ol-17-one acetate, 5β-androstan-3α-ol-11,-
17-dione acetate, and 5α-cholestane
16
and CU/PCC 44-1 (Cornell
University, Ithaca, NY), which comprises 5α-androstan-3α-ol-17-one
acetate, 5β-androstan-3α,17β-diacetate, 5α-cholestane, and
5β-pregnan-3α-20α-diacetate. Accuracy of the instrument was
checked using quality control charts for all steroids analyzed in every
sequence, as well as using the internal working standard
5α-androstan-3β-ol acetate. Sample preparation was checked using
negative and positive control samples according to the WADA
technical document.
5
2.6 |EA/IRMS of bristles
Bristles were washed using distilled water and soaked for 30 min
in methanol (Roth)/chloroform (pa, Merck; v:v, 2:1) in an ultrasonic
bath (Bandolin Sonorex). After drying, they were cut into 10 mm
segments. As a multi-isotopic analysis (CHNS) was performed, and
due to the limited sample volume, only two to three segments per
animal were analyzed for δ
13
C. Between 1.8 and 2.0 mg of bristles
were weighed into tin capsules in quadruplicate. Samples were
analyzed at isolab GmbH, Schweitenkirchen, Germany, using a
Vario EL Cube elemental analyzer (Elementar Analysensysteme)
connected to a mass spectrometer (Isoprime). Internal standards
used during analysis were casein (Kremer Pigmente) and two
different horse tail hair samples (both from local suppliers). Scale
calibrations were performed with NBS 22 (−30.03‰, IAEA) and
IRMM-BCR 657 (−10.76‰, IRMM). The analytical precisions using
at least quadruplicate measurements were δ
13
C = ±0.1‰. The data
were drift corrected using repeated measurement of laboratory
standard after 30 samples.
3|RESULTS AND DISCUSSION
3.1 |Urinary concentrations of NorA
No NorA was detected in the “blank”urine samples sampled prior
to ingestion of the boar meat. Therefore, endogenous production of
NorA of the volunteers can be excluded. NorA was detected in
urine samples of five of the 12 volunteers (Table 1). Two of them
exhibited significant amounts of NorA with maximum concentrations
of 2.9 and 2.5 ng/ml (1.9 and 2.1 ng/ml after adjustment for
specific gravity),
5
whereas in the urine samples of the other three
volunteers, only traces of NorA lower than 1 ng/ml were found
(Figure 1). No NorA was detected in the urine samples of the
remaining seven volunteers. Peak NorA concentrations were
reached after 3.3 to 6.0 h after consumption of the boar meat. All
urine samples from volunteers that showed traces of urinary NorA
tested negative 24 h after the meal.
For all urine samples, also the concentration of nor-
etiocholanolone (NorE) was determined. The NorA/NorE ratio, which
is an indicator of an exogenous administration of 19-norsteroids,
5
was
increased (>3) in all urine samples with NorA concentrations greater
than 0.5 ng/ml. The results for NorA concentrations and NorA/NorE
ratios of this study were similar to those found in another studies
after ingestion of boar's offal.
7,17
FIGURE 1 Excretion profiles of urinary NorA after consumption
of boar testicles (white circles),
7
mixed meals of boar meat and offal
(grey circles),
17
and boar meat (black circles). The horizontal line
reflects the 2.5 ng/ml cut-off level for mandatory IRMS analyses
HÜLSEMANN ET AL.3

3.2 |δ
13
C of urinary NorA
For each volunteer those urine samples with the detectable NorA
concentrations were further analyzed by GC/C/IRMS. Pregnanediol
(PD) was analyzed as endogenous reference compound (ERC) with
δ
13
C values around −24.3‰and −22.8‰and, thus, in good agree-
ment with typical values observed for German inhabitants.
18
Urinary
NorA showed δ
13
C values between −23.5‰and −18.5‰(Table 1).
Accordingly, the absolute differences between ERC and NorA as
defined in the relevant technical Document of WADA ranged from
jΔδj= 0.2‰to 5.8‰for the volunteers. Hence, urine samples of two
study participants yielded jΔδj> 3, which would be considered as
adverse analytical findings (AAF) according to currently enforced
regulations.
5
All urine samples with traces of NorA resulted from the ingestion
of boar meat obtained from the same meat market in Northern Ger-
many; however, not all meals prepared from meat originating from
that market (n= 7) led to an excretion of urinary NorA. It is assumed
that the meat was derived from different animals, as the products
were either purchased at different times and/or the best-before dates
were not identical. Further, deviating δ
13
C values of the urinary NorA
indicate different sources of the metabolite's precursor.
3.3 |δ
13
C of boar's bristles
Bristles of seven wild boars hunted in Germany were analyzed per
EA/IRMS. Two wild boars originated from Northern Germany, one
boar was from in North-Rhine Westphalia (data have already been
published before
8
), and four boars were from southern Germany
(Figure 2). The wide range of δ
13
C values within the bristles of one
individual, published earlier,
7
were confirmed by the herein conducted
additional analyses, although the intraindividual variation differed
between the animals. There was neither any spatial difference in δ
13
C
detectable nor any difference according to age.
Almost all boars showed a dietary shift from C
3
to C
4
-plant based
diets (or vice versa) within their bristles. It is known that the δ
13
C
values of mammalian (and human) body protein, either hair keratin or
muscle protein, rapidly adapts toward a change in the
13
C content of
the diet.
19–22
According to these studies, δ
13
C values for mammalian
FIGURE 2 Range of δ
13
C values of wild
boar's bristles in Germany. Each circle with
corresponding δ
13
C values represents one
individual at its origin. Values for individual
marked with an asterisk (*) are from a previous
publication
8
4HÜLSEMANN ET AL.

hair of −15‰can be attributed to an almost completely corn-based
diet.
19,20,22
On the other hand, δ
13
C values of −24‰and lower are
attributed to exclusively C
3
-plants in the diet.
19,20,22
The wide range of δ
13
C values of about 10‰found in boar's
bristles correspond to values found for urinary NorA after consump-
tion of boar meat or offal, which range from −13‰to −24‰. From
diet experiments, it is known that human protein (hair keratin) and
endogenous steroids adapt concurrently to dietary changes (C
3
-to
C
4
-plant based food or vice versa).
21,23
The absolute δ
13
C values of
steroids and hair protein are comparable, although individual offsets
up to 2.5‰exist, with the urinary steroids being, in most of the cases,
more depleted in
13
C than hair protein.
3.4 |Interpretation of human urinary NorA
Our results confirm the hypothesis that low urinary NorA concentra-
tions in humans can be the result of the consumption of meat from
uncastrated male pigs. Although the concentration of 19-norsteroids
in wild boar meat is lower than in offal such as testicles or liver, the
consumption of a typical meal of wild boar meat can lead to urinary
concentrations around 2 ng/ml (after adjustment for specific gravity).
5
In order to distinguish between an endogenous or exogenous origin
according to TD2019NA for urinary concentrations of NorA greater
than 2.5 ng/ml (after adjustment for specific gravity), IRMS is manda-
tory; below 2.5 ng/ml, IRMS is optional. Due to the varying and
unpredictable diets of wild boars (in Germany) δ
13
C values of urinary
NorA can present values from −15‰to −24‰. As typical endoge-
nous values for steroids of a German population are around
−23‰,
8,18,21,24
urinary NorA deriving from wild boar meat may be
interpreted as “endogenous,”if the boar's diet was C
3
-based;
conversely, it can be identified as “exogenous”if the animal was on a
C
4
-based diet. According to TD2019NA, an absolute difference
between ERC and NorA greater than 3‰has to be reported as an
adverse analytical finding.
5
To our knowledge, synthetic pharmaceutical preparations of
19-norsteroids exhibit δ
13
C values between −33‰and −21‰.
25,26
Thus, to date, urinary NorA presenting more enriched δ
13
C values is
more likely an indication for boar meat (or offal) ingestion than for the
administration of a synthetic 19-norsteroid.
The fact of varying δ
13
C values of wild boar could also be prob-
lematic for people living in countries with a high consumption of
C
4
-plants like the United States or Southern Africa. Human endoge-
nous δ
13
C values in these countries are enriched in
13
C compared
with Germany, and for these, there is the possibility of adverse analyt-
ical findings after the consumption of the meat of 19-norsteroid
producing C
3
-fed boars.
Moreover, it appears unlikely that after the consumption of boar
meat urinary concentrations of NorA rise above 15 ng/ml, which is
the upper cut-off level for a GC/C/IRMS target analysis of suspicious
samples.
5
The highest urinary concentration of NorA after consump-
tion of boar meat in our study was 2.9 ng/ml (410 g of prepared
meat). Higher urinary concentrations of NorA may be found after con-
sumption of offal, or mixed meals of meat and offal.
7,8,17
4|CONCLUSION
The herein presented results show that detectable amounts of NorA
may occur in human urine after ingestion of wild boar meat. As it has
been shown previously, it is unpredictable which δ
13
Cvaluesof
urinary NorA can be expected after such a meal. It is still advisable to
avoid the term “endogenous”for δ
13
C values of 19-norsteroid metabo-
lites of a free ranging animal in comparison with δ
13
C values of human
urinary steroids. It has been confirmed that the δ
13
C values for animal
19-norsteroids vary between −13‰and −25‰and do not necessarily
correlate with δ
13
C values found for human individuals of the same
geographical region. Not only the consumption of wild boar's offal in
the hours preceding a doping control test but also the consumption of
wild boar meat may result in an atypical or even positive test result,
albeit the urinary NorA concentrations are expected to be lower than
after consumption of wild boar's offal. Both athletes as well as anti-
doping laboratories and authorities should still be aware of this aspect.
ACKNOWLEDGEMENTS
The authors thank the Manfred Donike Institute for Doping Analysis
(Cologne, Germany), the Doping Authority Netherlands (Capelle aan
den Ijssel, The Netherlands), and the Federal Ministry of the Interior
of the Federal Republic of Germany (Berlin, Germany) for supporting
the presented work. Open access funding enabled and organized by
Projekt DEAL.
ORCID
Frank Hülsemann https://orcid.org/0000-0001-8624-4097
Mario Thevis https://orcid.org/0000-0002-1535-6451
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How to cite this article: Hülsemann F, Fußhöller G, Lehn C,
Thevis M. Excretion of 19-norandrosterone after consumption
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