Excretion of 19-norandrosterone after consumption of boar meat

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 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. 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.


| 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 concentrations, during pregnancy. [2][3][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" urinary NorA can be the consumption of the offal of noncastrated pigs. [5][6][7][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 common in Germany, the consumption of wild boar meat is increasing 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 C values from −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 C values of 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.

| Test meals and participants
The meals were prepared using varying amounts of boar meat products (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.

| Sample preparation of urine for GC/MS/MS
The samples were prepared according to the laboratory internal standard operating procedure for anabolic steroids. 13 Conjugated and unconjugated steroids were extracted from urine at pH 9.6 with tertbutylmethyl 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 μl of   Helium (4.6, Linde) was used as carrier gas (0.9 ml/min, constant pressure) and argon (5.0, Linde) as collision gas. The injector and interface temperatures were both set to 300 C, and the ion source was operated at 250 C. Ionization was accomplished using electron ionization (EI) (70 eV).

| Sample preparation of urine for GC/C/IRMS
Preparation of the urine samples (20-30 ml) followed the laboratory internal standard operating procedure 15   F I G U R E 1 Excretion profiles of urinary NorA after consumption of boar testicles (white circles), 7 mixed meals of boar meat and offal (grey circles), 17  All urine samples with traces of NorA resulted from the ingestion of boar meat obtained from the same meat market in Northern Germany; 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.

| 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][20][21][22] According to these studies, δ 13 C values for mammalian F I G U R E 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 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  with Germany, and for these, there is the possibility of adverse analytical 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 consumption 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 consumption 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 C values of urinary NorA can be expected after such a meal. It is still advisable to avoid the term "endogenous" for δ 13