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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Sex steroid levels in urine of cattle of different ages: evaluation of abuse control procedures a
b
a
Tomaz Snoj , Jozica Dolenc & Silvestra Kobal a
Veterinary Faculty, Institute of Physiology, Pharmacology and Toxicology, University of Ljubljana, Ljubljana, Slovenia b
Veterinary Faculty, Institute for Food Hygiene, University of Ljubljana, Ljubljana, Slovenia Accepted author version posted online: 03 Jan 2014.Published online: 04 Mar 2014.
Click for updates To cite this article: Tomaz Snoj, Jozica Dolenc & Silvestra Kobal (2014) Sex steroid levels in urine of cattle of different ages: evaluation of abuse control procedures, Food Additives & Contaminants: Part A, 31:4, 614-620, DOI: 10.1080/19440049.2013.880000 To link to this article: http://dx.doi.org/10.1080/19440049.2013.880000
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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 4, 614–620, http://dx.doi.org/10.1080/19440049.2013.880000
Sex steroid levels in urine of cattle of different ages: evaluation of abuse control procedures Tomaz Snoja*, Jozica Dolencb and Silvestra Kobala a Veterinary Faculty, Institute of Physiology, Pharmacology and Toxicology, University of Ljubljana, Ljubljana, Slovenia; bVeterinary Faculty, Institute for Food Hygiene, University of Ljubljana, Ljubljana, Slovenia
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(Received 22 October 2013; accepted 27 December 2013) Levels of several natural urinary steroids have been determined in the urine of a large number of animals of different cattle categories in the context of steroid abuse in beef production. Bovine animals of different breeds, sex and age included in the Slovene national residue detection plan for steroid abuse were studied. Urine from 120 males and 174 females was analysed. Urinary boldenone, boldione, androstenedione, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, progesterone, stanozolol, trenbolone, trenbolone acetate, 17α-ethinylestradiol, 17α-methyltestosterone, epitestosterone, 17β-estradiol, testosterone, and nandrolone were determined by LC-MS/MS. Epitestosterone was found in all bulls; while the proportion of animals containing testosterone and androstenedione increased with age. Testosterone was not detected in bulls less than 5 months of age. Epitestosterone levels, however, were not age dependent. The ratio of testosterone to epitestosterone thus increased with age, from 0.13 ± 0.09 at 1–7 months to 0.42 ± 0.10 at 25–38 months. It was significantly (p < 0.01) higher in bulls above 13 months than in younger animals. In contrast to males, no urinary testosterone was found in females, whereas epitestosterone, androstenedione, progesterone and estradiol were present. The proportion of animals of various age groups in which epitestosterone was detected ranged from 68% to 100%, but the differences were not significant. The presence of both estradiol and progesterone in the same sample was not observed in any animal. The results of this study could be helpful in determining physiological urinary steroid levels in order to provide a baseline for the control of steroid abuse in beef production. Keywords: steroids; cattle; urine; steroid abuse; beef production; LC-MS/MS
Introduction Urinary sex hormones are determined in humans for doping control and in cattle as a tool for detecting the use of prohibited growth promoters. According to Council Directive 96/22/EC, the use of steroids and other growth promoters (thyreostats and β-adrenergics) is prohibited in the European Union (European Union 1996). Since compounds that improve fattening in beef production are allowed in some other parts of the world, their presence in the European Union has to be controlled. Urine is used as the matrix when screening for such illicit steroid compounds. In contrast to the well-established sample collection and steroid detection procedures, interpretation of the results is sometimes difficult, since no reference values for urinary endogenous steroids have been defined. Although the presence of several steroid compounds, such as androstenedione, epitestosterone, testosterone, progesterone, estradiol, nandrolone and boldenone, that occur normally in different matrices of various animal species has been widely reported (Scarth et al. 2009), the natural physiological levels of those compounds in body fluids are still not well established. Although control levels of plasma, muscle and liver steroids have been proposed (CRL Guidance 2007), few data and no recommended concentrations or acceptable levels of natural urinary steroids in cattle are available. *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Growth promoters are used in some parts of the world in the form of implants that contain steroid esters of progesterone, testosterone, estradiol and trenbolone in various combinations (Doyle 2000). Following their administration, free steroids are released from steroid esters and act as growth promoters. Since steroid esters are foreign to the body, confirmation of their use is not in doubt. In contrast, the presence of the free (hydrolysed) steroid is not easy to confirm. Steroid esters are present in the blood for a very short period, although they accumulate in hair, where they can be detected (Boyer et al. 2007; Stolker et al. 2009). Measurement of free steroid concentrations does not always lead to confirmation of steroid abuse (Nielen et al. 2006). Thus, measurement and interpretation of metabolic profiles have become important tools to investigate the differences between treated and untreated individuals (Gómez et al. 2012; Fabregat et al. 2013). Deviation from a normal fingerprint can trigger further investigations. Additionally, the abuse of steroids can be confirmed by stable carbon isotope analysis (Ferchaud et al. 2000; Hebestreit et al. 2006) or by determining specific steroid or non-steroid biomarkers (Gardini et al. 2006; Draisci et al. 2007; Dervilly-Pinel et al. 2011; Scarth et al. 2011; Stella et al. 2011), but these methods require special and expensive equipment, are time consuming and are presently not widely used.
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Food Additives & Contaminants: Part A Several authors have expressed concern regarding the ability to confirm the abuse of prohibited compounds in cattle fattening by using HPLC, GC or immunoassays in the absence of isotope deviation measurements (Buisson et al. 2005; Hebestreit et al. 2006; Doue et al. 2012). Some steroids that are endogenous in the organism can be used as growth promoters (Stephany 2010), thus their basic levels have to be known and taken into account in the control of growth promoter abuse. The current knowledge of urinary steroid levels is based on several studies involving only a limited numbers of animals. Furthermore, some of these studies were performed decades ago using methods that were not as accurate and precise as those available today (Angeletti et al. 2006). We have therefore determined the levels of several sex steroids endogenous to urine in a large number of cattle of different categories in order to provide a baseline for the control of steroid abuse in beef production. Materials and methods Animals and samples The study was performed on bovine animals of different breeds, sex and age that were included in the national residue monitoring programme of steroid abuse in the period January 2007–June 2013 in the Republic of Slovenia. Urine from 120 males and 174 females was analysed. Samples were collected by official veterinarians during animal urination or directly from the bladder after slaughter in the slaughterhouse. Each sample record included the date of animal’s birth. 50 ml samples were stored at –20°C until analysed by LC-MS/MS. Chemicals, reagents and materials Solvents and reagents were of analytical grade, excepting the solvent for the mobile phase, which was HPLC grade. β-Glucuronidase/aryl sulphatase stabilised water solution was from Merck (Darmstadt, Germany). The reference steroids, 17-β-testosterone (testosterone), 17-α-testosterone (epitestosterone), ethinylestradiol, 17-β-estradiol, αboldenone and β-boldenone, were from Sigma-Aldrich (Taufkirchen, Germany). Trenbolone, trenbolone acetate, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, progesterone, stanozolol, 19-nortestosterone, methyltestosterone, androstenedione, norandrostenedione and androstadienedione were from LGC Standards (Wesel, Germany). Bond Elut C18 columns 500 mg/3 ml (Varian, Mulgrave, Australia) and Isolute NH2 100 mg/3 ml (Biotage, Uppsala, Sweden) were used for SPE. The measurements were performed by Acquity UPLC Xevo TQ MS (Waters, Milford, MA, USA) using an Acquity UPLC BEH C18 1.7 µm, 50 mm × 2.1 mm (Waters) analytical column for separating substances.
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Analytical procedures Urine samples were thawed at RT. A total of 10 ml of water and 1 ml of 0.2 M sodium acetate buffer solution was added to approximately 10 g of sample. If necessary, pH was adjusted to 5.2. The samples were then submitted to an enzymatic deconjugation step using β-glucuronidase/ aryl sulphatase. After overnight incubation at 37°C, samples were purified by SPE. Samples were applied to activated and conditioned Varian C18 cartridges. The cartridges were washed with water and a mixture of methanol/water (60/40 v/v). For steroids elution 6 ml of methanol were used. Eluates were evaporated to dryness under nitrogen at 40°C. The residues were dissolved in 2 ml ethyl acetate/methanol (80/20 v/v). These samples were applied to conditioned Isolute cartridges and eluted with mixture of ethyl acetate/methanol (80/20 v/v). The solvent was evaporated to dryness under nitrogen at 40°C. The dry residues were dissolved in 1 ml of mixture acetonitrile/water. The supernatant was filtered through a 0.45 µm filter into an HPLC vial. Liquid chromatography was performed using a Waters XEVO Acquity UPLC system with a UPLC BEH C18 analytical column (Waters). A total of 5 mM formic acid in water and 50 mM formic acid in water/acetonitrile (10/ 90 v/v) were used for gradient elution. The injection volume was 10 µl. For mass spectrometric detection a XEVO TQMS (Waters) was operated in the negative ESI (ESI–). This method was developed and validated for the simultaneous quantitative determination of steroids (Table 1). The LOD for an individual steroid was 1 µg kg–1 and the LOQ was 2 µg kg–1. Recoveries ranged from 90% to 110%. Table 1.
Urinary steroids with product and precursor ions.
Steroid Androstadiendione Androstenedione α-Boldenone β-Boldenone 17-α-Testosterone 17-β-Testosterone Medroxyprogesterone Medroxyprogesterone acetate Melengesterol acetate Methyltestosterone Norandrostendione 19-Nortestosterone Progesterone Stanozolol Trenbolone Trenbolone acetate Equiline Ethinylestradiol 17β-Estradiol
Precursor ion (m/z)
Product ion (m/z)
285 287 287 287 289 289 345 387
121 97 269 121 271 97 123 327
397 303 273 275 315 329 271 313 267 295 271
279 285 109 109 97 81 199 253 143 145 145
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Data were analysed using the SPSS 17.0 (Chicago, IL, USA) commercial software. Means and standard error of the mean (mean ± SE) were calculated for urinary steroid content. A Chi-square test was used for determining the proportion of each steroid in each animal category. One way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) post-hoc test was used for testing statistically significant differences of content of the urinary steroid between the animal categories. Pearson’s correlation coefficient analysis was performed to determine whether there is a statistically significant correlation between urinary steroids. A value of p < 0.05 was considered as being statistically significant. Only measurements that exceeded the LOD were included in the ANOVA and correlation coefficient analysis.
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Results Urinary steroids in bulls In all 120 of the bull urinary samples analysed, no boldenone, boldione, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, norandrostenedione, progesterone, stanozolol, trenbolone, trenbolone acetate, 17αethinylestradiol, 17α-methyltestosterone, 17β-estradiol and nandrolone were found. With respect to animal age, androstenedione, epitestosterone and testosterone were found in various proportions, as shown in Table 2. Table 2 also shows mean, median,
T/ET ratio
Statistical analysis
* *
0.40 0.30 0.20 0.10 0.00
1–7
8–12 13–24 Age (months)
25–38
Figure 1. Urinary testosterone-to-epitestosterone ratio in bulls of different ages. T/ET ratios are means ± SE; *values differing significantly (p < 0.01) from that at 8–12 months of age.
minimal and maximal levels of androstenedione, epitestosterone and testosterone in different animal age groups. Urinary testosterone was not present earlier than in the fifth month of age. The urinary testosterone-to-epitestosterone ratio (T/ET ratio) in different age groups of bulls is shown in Figure 1. The mean T/ET ratio increased with age from 0.13 ± 0.09 at 1–7 months of age to 0.42 ± 0.10 at 25– 38 months of age. It was significantly (p < 0.01) higher in bulls after 13 months of age compared with animals at 8–12 months of age. Pearson’s correlation coefficient analysis showed significant (p < 0.001) positive correlation between urinary testosterone and epitestosterone (r2 = 0.52) when measurements of bulls of all ages were included in the
Table 2. Proportion of bulls from each age group in which urinary androstenedione, epitestosterone or testosterone were detected with mean, median, minimal and maximal detected values. Age 1–7 months, N = 17 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg 8–12 months, N = 6 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg 13–24 months, N = 78 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg 25–38 months, N = 19 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg
Androstenedione
Epitestosterone
Testosterone
kg–1)
29 2.79 ± 0.51 2.7 1.77–4.7
100 25.99 ± 4.89 26 2.2–72
12a 5.6 ± 2.40 5.6 3.2–8
kg–1)
33 1.15 ± 0.04 1.15 1.1–1.2
100 27.77 ± 10.65 16.15 3.3–69
83 4.24 ± 1.28 3.00 2–8.8
kg–1)
44 1.76 ± 0.17 1.35 1–4.7
100 22.77 ± 1.72 20.90 2.62–89
80 7.13 ± 0.83 5.60 1.2–38.7
kg–1)
42 2.16 ± 0.41 1.95 1–4.6
100 24.34 ± 4.10 20.20 2.8–75
95 9.50 ± 1.59 8.55 1.6–20.2
Note: aValue differs significantly from those for other animal ages (p < 0.001).
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analysis. In the separate animal groups, correlation coefficient analysis showed positive, but non-significant, correlations at 1–7 and 8–12 months of age (r2 = 0.37 and 0.74 respectively) and significant (p < 0.01) positive correlations at 13–24 and 25–38 months of age (r2 = 0.58 and 0.68 respectively). The urinary T/ET ratio showed significantly higher levels at 13–24 and 25–38 months of age than at 8–12 months of age.
Urinary steroids in heifers and cows In the 174 female cattle urinary samples analysed, no boldenone, boldione, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, norandrostenedione, stanozolol, trenbolone, trenbolone acetate,
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17α-ethinylestradiol, 17α-methyltestosterone, testosterone or nandrolone were found. Androstenedione, epitestosterone, progesterone and estradiol were identified in various proportions depending on age (Table 3). Values were below the LOD in three, three, one and seven animals of age groups 8–12, 13–24, 37–48 and over 60 months, respectively. Simultaneous occurrence of urinary androstenedione and estradiol or progesterone and estradiol was not found in any sample. Androstenedione and progesterone were found together in one sample in the age group 1– 7 months, once in the group 13–24 months, and twice in the group 37–48 months. Chi-square analysis showed no significant differences in proportions of different urinary steroids at different ages
Table 3. Proportion of female cattle from each age group in which urinary androstenedione, epitestosterone, progesterone or estradiol were detected with mean ± SE, median, minimal and maximal detected values. Age 1–7 months, N = 27 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) 8–12 months, N = 28 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) 13–24 months, N = 54 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) 25–36 months, N = 16 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) 37–48 months, N = 11 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum – maximum (μg kg–1) 49–60 months, N = 10 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) Over 60 months, N = 28 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1)
Androstenedione
Epitestosterone
Progesterone
11 1.13 ± 0.09 1.10 1–5.5
100 23.19 ± 4.60 18.00 2.9–117
18 2.15 ± 0.54 2.15 1–4
0 – – –
4 1.4a 1.40a 1.4a
89 20.58 ± 3.1 16.00 4.3–49
0 – – –
0 – – –
4 1.55 ± 0.15 1.55 1.4–1.7
94 26.01 ± 4.47 17.10 1–185
6 1.5 ± 0.35 1.20 1.1–2.2
2 2.4a 2.40a 2.4a
0 – – –
100 16.48 ± 3.57 9.90 2.3–56
6 4.4a 4.40a 4.4a
6 2a 2.00a 2a
27 1.4 ± 0.31 1.20 1–2
91 30.16 ± 6.16 31.80 5.8–70
27 1.57 ± 0.43 1.30 1–2.4
0 – – –
0 – – –
100 26 ± 7.42 16.85 5.4–70
10 2a 2.00a 2a
0 – – –
0 – – –
75 33.33 ± 6.05 26.80 2–86
7 1.55 ± 0.35 1.55 1.2–1.9
14 4.25 ± 0.50 4.4 2.9–5.3
Note: aOnly one sample from the group exceeded the LOD.
Estradiol
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(Table 3). Similarly, no significant differences were found between androstenedione, epitestosterone, progesterone and estradiol levels at different ages. Discussion In the European Union urine is collected for inspection purposes as a matrix for controlling steroid abuse, since the concentrations of steroids in urine are much higher than in blood. The presence of synthetic steroid compounds in urine constitutes direct proof of steroid administration to the animal (Scarth et al. 2009). In this study on cattle individuals were tested for the presence of several synthetic or bovine urine non-specific steroids, such as αand β-boldenone, boldione, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, norandrostenedione, stanozolol, trenbolone, trenbolone acetate, 17α-ethinylestradiol, 17α-methyltestosterone and nandrolone. Since none of these compounds was detected in any of the animals, we assume that steroids or other steroids that metabolise to any of the above-mentioned steroids were not introduced to the observed animals around the time of sample collection. Most of the steroids tested for do not appear naturally in cattle urine. The only exception is boldenone, which is present in faeces (Pompa et al. 2006; Arioli et al. 2008). It can be present naturally in cattle urine (Le Bizec et al. 2006; Stephany 2010) although it was reported to occur in urine for only a few hours after administration (Draisci et al. 2007). Nevertheless, we found no urinary boldenone in any epimeric form, suggesting that boldenone is present very rarely, if ever, in intact (i.e. not contaminated with faeces) bovine urine. In our study urinary epitestosterone was found in all males of all age groups (1–38 months). Statistical analysis showed no significant variation of mean epitestosterone level between age groups. Epitestosterone values in every bull age group range from low, scarcely exceeding the LOD, to high (Table 2). Interestingly, high values were found not only in mature bulls but also in calves. Epitestosterone in bulls is produced in testis and also from testosterone in liver and in blood (Starka 2003). The fact that the urinary epitestosterone level is also influenced by testosterone administration is a strong indication that, given the high individual variation, the urinary epitestosterone level is not a reliable indicator of steroid abuse. In contrast to epitestosterone, the occurrence of urinary testosterone is age dependent, being significantly lower in bulls in the range of 1–7 months (no urinary testosterone was found under the age of 5 months) than in older animals (Table 2). Its frequency of occurrence increased with age and reached 94.7% at the age of 24–38 months. Similarly, mean urinary testosterone levels increased with age, but not significantly so, probably due to high
individual variability (Table 2). Bulls reach puberty at around 5 months of age, at which time the production of testosterone increases (Renaville et al. 1996; Cestnik et al. 2001). The significant positive correlation of epitestosterone and testosterone levels confirms the conversion of testosterone to epitestosterone, as described by Starka (2003). Since urinary testosterone is age dependent and epitestosterone is not, the T/ET ratio fluctuates with age. Although more or less successful as a screening tool for doping control in athletes (WADA 2004), successful use in controlling steroid abuse in beef production is questionable. Angeletti et al. (2006) reported increased T/ET ratios in calves treated with a steroid cocktail containing testosterone and epitestosterone, although the differences from the control animal group were not significant (Angeletti et al. 2006). Our results show a significantly higher mean T/ET ratio in bulls at 13–24 months of age than at ages 8– 12 months. In bulls younger than 8 months the mean T/ET ratio is even lower (Figure 1), but the number of animals in which the urinary testosterone level was above the LOD was very small. In this age group testosterone was detected in only 12% of animals, thus similarly limiting determination of the T/ET ratio. In bulls younger than 5 months, the T/ET ratio could not be calculated since the urinary testosterone level was below the LOD. These data are especially important for assessment of testosterone abuse in animals younger than 13 months, since a high urinary T/ET ratio in such animals could be considered as ‘suspicious’. Urinary androstenedione was found in bulls in all age groups with frequencies that did not vary significantly. In the biosynthesis of steroids, androstenedione is a precursor of testosterone, since 17β-hydroxysteroid dehydrogenase converts androstenedione to testosterone. It is also a metabolite of boldenone (Merlanti et al. 2007). Most probably the observed levels of androstenedione are those that normally occur in bulls’ urine. The list of urinary steroids found in heifers and cows differs from that in bulls. Testosterone was not found in any of the 174 females, even though epitestosterone was detected in a relatively high proportion (Table 3) (but not in all animals, as was found in bulls). In contrast to bulls, the T/ET ratio in females is therefore not a significant parameter. The source of epitestosterone is probably the active ovary, since it was found in follicular fluid and is also derived from androstenedione and testosterone (Starka 2003). Urinary epitestosterone concentrations did not differ significantly between age groups, suggesting that age is not a factor in epitestosterone metabolism and excretion. In certain females urinary epitestosterone exceeded the mean value by five- to seven-fold (Table 3). These results suggest that in cows, as in humans (Dehenin et al. 1987), epitestosterone formation depends on reproductive status, as well as on reproductive disorders, resulting in high individual variability. The
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Food Additives & Contaminants: Part A proportion of females in which urinary androstenedione, progesterone and estradiol were detected is much lower than that in which urinary epitestosterone was detected and, similarly to epitestosterone, differences in mean concentrations of these steroids between the groups were not significant. Furthermore, in animals in which androstenedione was detected, its level scarcely exceeded the LOD, while progesterone and estradiol in particular animals reached 4.4 and 5.3 μg kg–1 respectively (Table 3). In this study estradiol was not detected together with androstenedione in the same urine sample, nor estradiol with progesterone. The reason why estradiol and progesterone were not observed in the same sample is that they are secreted during different phases of the oestrus cycle (Christensen et al. 1974). On the other hand, the occurrence of androstenedione and progesterone at the same time is expected since the former is involved in the biosynthetic pathway of steroids as a progesterone metabolite or an estradiol and testosterone precursor (Scarth et al. 2009). The fact that androstenedione and estradiol were not observed together in any urine sample is therefore probably a coincidence, since either androstenedione or estradiol was detected in a limited number of samples. In addition, as described by Scarth et al. (2009), mean urinary testosterone and epitestosterone levels are approximately three-fold higher in mature bulls compared with females. Regarding testosterone, this is in line with the results of this study; however, mean epitestosterone values show no differences between males and females (Tables 2 and 3). The variations in the quantitative levels of steroids found in the present study could also be the consequence of different hydration status of the animals. This could be corrected by using normalisation to urine creatinine levels. However, the role of creatinine as a marker of hydration status is somewhat unsure (Scarth et al. 2011). Furthermore, the urinary samples in this study originated from a national monitoring residue programme which did not include these parameters. Some recent studies described the exact metabolism of exogenous steroids in bovines (Ferchaud et al. 2000; Angeletti et al. 2006; Le Bizec et al. 2006; Draisci et al. 2007; Dervilly-Pinel et al. 2011; Scarth et al. 2011). The results of the present study supplement this information with urinary levels of endogenous steroids from a large number of animals of different sex and ages. It cannot be confirmed with certainty that all the urinary samples used in this study originate from animals that were not treated with illegal growth promoters. However, the fact that no indicators, such as the presence of synthetic steroids in urine or the illegal market with steroids containing implants, were observed in the period of sample collection makes the introduction of illicit compounds in the examined animals highly unlikely.
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The results of this study therefore constitute additional understanding of sex steroid excretion dynamics in cattle. In addition to the recorded physiological levels of some urinary steroids, the data presented should also be helpful in evaluating the results from screening methods for steroid abuse.
Conclusions In summary, epitestosterone, testosterone and androstenedione are normally present in the urine of bulls. Epitestosterone was found in all observed males. In contrast to epitestosterone, however, urinary testosterone occurrence is age dependent. However, since urinary testosterone is age dependent and epitestosterone is not, the T/ET ratio also fluctuates with age and is thus a good parameter for assessing animal testosterone status, especially in young animals. Urinary epitestosterone, androstenedione, progesterone and estradiol were found in heifers and cows. The proportion of animals in which urinary androstenedione, progesterone and estradiol were detected is much lower than the proportion in which epitestosterone was observed. Testosterone was not present in any of the female animals and presence of both estradiol and progesterone in the same sample was not observed. The results of this study will be helpful in evaluating the results of steroid abuse screening methods.
Acknowledgements The authors are thankful to Dr Roger H. Pain for English editing and suggestions.
Funding The authors acknowledge The Slovenian Research Agency [grant number P4-0053] and The Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant protection which enabled the study.
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Hebestreit M, Flenker U, Buisson C, Andre F, Le Bizec B, Fry H, Lang M, Presii Weigert A, Heinrich K, Hird S, Schanzer W. 2006. Application of stable carbon isotope analysis to the detection of testosterone administration to cattle. J Agric Food Chem. 54:2850–2858. Le Bizec B, Courant F, Gaudin I, Bichon E, Destrez B, Schilt R, Draisci R, Monteau F, Andre F. 2006. Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldion in cattle 1. Metabolite profiles of boldenone, boldenone esters and boldion in cattle urine. Steroids. 71:1078–1087. Merlanti R, Gallina G, Capolongo F, Contiero L, Biancotto G, Dacasto M, Montesissa C. 2007. An in vitro study on metabolism of 17beta-boldenone and boldione using cattle liver and kidney subcellular fractions. Anal Chim Acta. 586:177–183. Nielen MWF, Bovee TFH, Heskamp HH, Lasaroms JJP, Sanders MB, Van Rhijn JA, Groot MJ, Hooqenboom LA. 2006. Screening for estrogen residues in calf urine: comparition of a validated yeast estrogen bioassay and gas chromatography-tandem mass spectrometry. Food Addit Contam. 23:1123–1131. Pompa G, Arioli F, Fracchiolla ML, Sgoifo Rossi CA, Bassini AL, Stella S, Biondi PA. 2006. Neoformation of boldenone and relation steroids in faeces of veal calves. Food Addit Contam. 23:126–132. Renaville R, Massart S, Sneyers M, Falaki M, Gengier N, Burny A, Portetelle D. 1996. Dissociation of increases in plasma insulin-like growth factor I and testosterone during the onset of puberty in bulls. J Reprod Fertil. 106:79–86. Scarth J, Akre C, van Ginkel L, Le Bizec B, De Brabander H, Korth W, Points J, Teale P, Kay J. 2009. Presence and metabolism of endogenous adrogenic-anabolic steroid hormones in meat-producing animals: a review. Food Addit Contam. 26:640–671. Scarth JP, Clarke A, Teale P, Mill A, Macarthur R, Kay J. 2011. Detection of endogenous steroid abuse in cattle: results from population studies in the UK. Food Addit Contam. 28:44–61. Starka L. 2003. Epitestosterone. J Steroid Biochem Molecul Biol. 87:27–34. Stella R, Biancotto G, Krogh M, Angeletti R, Pozza G, Sorgato MC, James P, Andrighetto I. 2011. Protein expression changes in skeletal muscle in response to growth promoter abuse in beef cattle. J Proteome Res. 10:2744–2757. Stephany RW. 2010. Hormonal growth promoting agents in food producing animals. In: Thieme D, Hammersbach P, editors. Handbook of experimental pharmacology. Berlin: SpringerVerlag; p. 355–367. Stolker AAM, Groot MJ, Nijrolder AW, Lasaroms JP, Blokland MH, Riedmaier I, Becker C, Meyer HH, Nielen MW. 2009. Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment. Anal Bioanal Chem. 395:1075–1087. WADA. 2004. Reporting and evaluating guidance for testosterone, epitestosterone, T/ET ratio and other endogenous steroids [cited Aug 13]. Available from: http://www.wadaama.org/Documents/World_Anti-Doping_Program/WADPIS-Laboratories/Technical_Documents/WADA_TD2004 EAAS_Reporting_Evaluation_Testosterone_Epitestosterone_TE_Ratio_EN.pdf
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Sex steroid levels in urine of cattle of different ages: evaluation of abuse control procedures a
b
a
Tomaz Snoj , Jozica Dolenc & Silvestra Kobal a
Veterinary Faculty, Institute of Physiology, Pharmacology and Toxicology, University of Ljubljana, Ljubljana, Slovenia b
Veterinary Faculty, Institute for Food Hygiene, University of Ljubljana, Ljubljana, Slovenia Accepted author version posted online: 03 Jan 2014.Published online: 04 Mar 2014.
Click for updates To cite this article: Tomaz Snoj, Jozica Dolenc & Silvestra Kobal (2014) Sex steroid levels in urine of cattle of different ages: evaluation of abuse control procedures, Food Additives & Contaminants: Part A, 31:4, 614-620, DOI: 10.1080/19440049.2013.880000 To link to this article: http://dx.doi.org/10.1080/19440049.2013.880000
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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 4, 614–620, http://dx.doi.org/10.1080/19440049.2013.880000
Sex steroid levels in urine of cattle of different ages: evaluation of abuse control procedures Tomaz Snoja*, Jozica Dolencb and Silvestra Kobala a Veterinary Faculty, Institute of Physiology, Pharmacology and Toxicology, University of Ljubljana, Ljubljana, Slovenia; bVeterinary Faculty, Institute for Food Hygiene, University of Ljubljana, Ljubljana, Slovenia
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(Received 22 October 2013; accepted 27 December 2013) Levels of several natural urinary steroids have been determined in the urine of a large number of animals of different cattle categories in the context of steroid abuse in beef production. Bovine animals of different breeds, sex and age included in the Slovene national residue detection plan for steroid abuse were studied. Urine from 120 males and 174 females was analysed. Urinary boldenone, boldione, androstenedione, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, progesterone, stanozolol, trenbolone, trenbolone acetate, 17α-ethinylestradiol, 17α-methyltestosterone, epitestosterone, 17β-estradiol, testosterone, and nandrolone were determined by LC-MS/MS. Epitestosterone was found in all bulls; while the proportion of animals containing testosterone and androstenedione increased with age. Testosterone was not detected in bulls less than 5 months of age. Epitestosterone levels, however, were not age dependent. The ratio of testosterone to epitestosterone thus increased with age, from 0.13 ± 0.09 at 1–7 months to 0.42 ± 0.10 at 25–38 months. It was significantly (p < 0.01) higher in bulls above 13 months than in younger animals. In contrast to males, no urinary testosterone was found in females, whereas epitestosterone, androstenedione, progesterone and estradiol were present. The proportion of animals of various age groups in which epitestosterone was detected ranged from 68% to 100%, but the differences were not significant. The presence of both estradiol and progesterone in the same sample was not observed in any animal. The results of this study could be helpful in determining physiological urinary steroid levels in order to provide a baseline for the control of steroid abuse in beef production. Keywords: steroids; cattle; urine; steroid abuse; beef production; LC-MS/MS
Introduction Urinary sex hormones are determined in humans for doping control and in cattle as a tool for detecting the use of prohibited growth promoters. According to Council Directive 96/22/EC, the use of steroids and other growth promoters (thyreostats and β-adrenergics) is prohibited in the European Union (European Union 1996). Since compounds that improve fattening in beef production are allowed in some other parts of the world, their presence in the European Union has to be controlled. Urine is used as the matrix when screening for such illicit steroid compounds. In contrast to the well-established sample collection and steroid detection procedures, interpretation of the results is sometimes difficult, since no reference values for urinary endogenous steroids have been defined. Although the presence of several steroid compounds, such as androstenedione, epitestosterone, testosterone, progesterone, estradiol, nandrolone and boldenone, that occur normally in different matrices of various animal species has been widely reported (Scarth et al. 2009), the natural physiological levels of those compounds in body fluids are still not well established. Although control levels of plasma, muscle and liver steroids have been proposed (CRL Guidance 2007), few data and no recommended concentrations or acceptable levels of natural urinary steroids in cattle are available. *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Growth promoters are used in some parts of the world in the form of implants that contain steroid esters of progesterone, testosterone, estradiol and trenbolone in various combinations (Doyle 2000). Following their administration, free steroids are released from steroid esters and act as growth promoters. Since steroid esters are foreign to the body, confirmation of their use is not in doubt. In contrast, the presence of the free (hydrolysed) steroid is not easy to confirm. Steroid esters are present in the blood for a very short period, although they accumulate in hair, where they can be detected (Boyer et al. 2007; Stolker et al. 2009). Measurement of free steroid concentrations does not always lead to confirmation of steroid abuse (Nielen et al. 2006). Thus, measurement and interpretation of metabolic profiles have become important tools to investigate the differences between treated and untreated individuals (Gómez et al. 2012; Fabregat et al. 2013). Deviation from a normal fingerprint can trigger further investigations. Additionally, the abuse of steroids can be confirmed by stable carbon isotope analysis (Ferchaud et al. 2000; Hebestreit et al. 2006) or by determining specific steroid or non-steroid biomarkers (Gardini et al. 2006; Draisci et al. 2007; Dervilly-Pinel et al. 2011; Scarth et al. 2011; Stella et al. 2011), but these methods require special and expensive equipment, are time consuming and are presently not widely used.
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Food Additives & Contaminants: Part A Several authors have expressed concern regarding the ability to confirm the abuse of prohibited compounds in cattle fattening by using HPLC, GC or immunoassays in the absence of isotope deviation measurements (Buisson et al. 2005; Hebestreit et al. 2006; Doue et al. 2012). Some steroids that are endogenous in the organism can be used as growth promoters (Stephany 2010), thus their basic levels have to be known and taken into account in the control of growth promoter abuse. The current knowledge of urinary steroid levels is based on several studies involving only a limited numbers of animals. Furthermore, some of these studies were performed decades ago using methods that were not as accurate and precise as those available today (Angeletti et al. 2006). We have therefore determined the levels of several sex steroids endogenous to urine in a large number of cattle of different categories in order to provide a baseline for the control of steroid abuse in beef production. Materials and methods Animals and samples The study was performed on bovine animals of different breeds, sex and age that were included in the national residue monitoring programme of steroid abuse in the period January 2007–June 2013 in the Republic of Slovenia. Urine from 120 males and 174 females was analysed. Samples were collected by official veterinarians during animal urination or directly from the bladder after slaughter in the slaughterhouse. Each sample record included the date of animal’s birth. 50 ml samples were stored at –20°C until analysed by LC-MS/MS. Chemicals, reagents and materials Solvents and reagents were of analytical grade, excepting the solvent for the mobile phase, which was HPLC grade. β-Glucuronidase/aryl sulphatase stabilised water solution was from Merck (Darmstadt, Germany). The reference steroids, 17-β-testosterone (testosterone), 17-α-testosterone (epitestosterone), ethinylestradiol, 17-β-estradiol, αboldenone and β-boldenone, were from Sigma-Aldrich (Taufkirchen, Germany). Trenbolone, trenbolone acetate, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, progesterone, stanozolol, 19-nortestosterone, methyltestosterone, androstenedione, norandrostenedione and androstadienedione were from LGC Standards (Wesel, Germany). Bond Elut C18 columns 500 mg/3 ml (Varian, Mulgrave, Australia) and Isolute NH2 100 mg/3 ml (Biotage, Uppsala, Sweden) were used for SPE. The measurements were performed by Acquity UPLC Xevo TQ MS (Waters, Milford, MA, USA) using an Acquity UPLC BEH C18 1.7 µm, 50 mm × 2.1 mm (Waters) analytical column for separating substances.
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Analytical procedures Urine samples were thawed at RT. A total of 10 ml of water and 1 ml of 0.2 M sodium acetate buffer solution was added to approximately 10 g of sample. If necessary, pH was adjusted to 5.2. The samples were then submitted to an enzymatic deconjugation step using β-glucuronidase/ aryl sulphatase. After overnight incubation at 37°C, samples were purified by SPE. Samples were applied to activated and conditioned Varian C18 cartridges. The cartridges were washed with water and a mixture of methanol/water (60/40 v/v). For steroids elution 6 ml of methanol were used. Eluates were evaporated to dryness under nitrogen at 40°C. The residues were dissolved in 2 ml ethyl acetate/methanol (80/20 v/v). These samples were applied to conditioned Isolute cartridges and eluted with mixture of ethyl acetate/methanol (80/20 v/v). The solvent was evaporated to dryness under nitrogen at 40°C. The dry residues were dissolved in 1 ml of mixture acetonitrile/water. The supernatant was filtered through a 0.45 µm filter into an HPLC vial. Liquid chromatography was performed using a Waters XEVO Acquity UPLC system with a UPLC BEH C18 analytical column (Waters). A total of 5 mM formic acid in water and 50 mM formic acid in water/acetonitrile (10/ 90 v/v) were used for gradient elution. The injection volume was 10 µl. For mass spectrometric detection a XEVO TQMS (Waters) was operated in the negative ESI (ESI–). This method was developed and validated for the simultaneous quantitative determination of steroids (Table 1). The LOD for an individual steroid was 1 µg kg–1 and the LOQ was 2 µg kg–1. Recoveries ranged from 90% to 110%. Table 1.
Urinary steroids with product and precursor ions.
Steroid Androstadiendione Androstenedione α-Boldenone β-Boldenone 17-α-Testosterone 17-β-Testosterone Medroxyprogesterone Medroxyprogesterone acetate Melengesterol acetate Methyltestosterone Norandrostendione 19-Nortestosterone Progesterone Stanozolol Trenbolone Trenbolone acetate Equiline Ethinylestradiol 17β-Estradiol
Precursor ion (m/z)
Product ion (m/z)
285 287 287 287 289 289 345 387
121 97 269 121 271 97 123 327
397 303 273 275 315 329 271 313 267 295 271
279 285 109 109 97 81 199 253 143 145 145
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Data were analysed using the SPSS 17.0 (Chicago, IL, USA) commercial software. Means and standard error of the mean (mean ± SE) were calculated for urinary steroid content. A Chi-square test was used for determining the proportion of each steroid in each animal category. One way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) post-hoc test was used for testing statistically significant differences of content of the urinary steroid between the animal categories. Pearson’s correlation coefficient analysis was performed to determine whether there is a statistically significant correlation between urinary steroids. A value of p < 0.05 was considered as being statistically significant. Only measurements that exceeded the LOD were included in the ANOVA and correlation coefficient analysis.
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Results Urinary steroids in bulls In all 120 of the bull urinary samples analysed, no boldenone, boldione, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, norandrostenedione, progesterone, stanozolol, trenbolone, trenbolone acetate, 17αethinylestradiol, 17α-methyltestosterone, 17β-estradiol and nandrolone were found. With respect to animal age, androstenedione, epitestosterone and testosterone were found in various proportions, as shown in Table 2. Table 2 also shows mean, median,
T/ET ratio
Statistical analysis
* *
0.40 0.30 0.20 0.10 0.00
1–7
8–12 13–24 Age (months)
25–38
Figure 1. Urinary testosterone-to-epitestosterone ratio in bulls of different ages. T/ET ratios are means ± SE; *values differing significantly (p < 0.01) from that at 8–12 months of age.
minimal and maximal levels of androstenedione, epitestosterone and testosterone in different animal age groups. Urinary testosterone was not present earlier than in the fifth month of age. The urinary testosterone-to-epitestosterone ratio (T/ET ratio) in different age groups of bulls is shown in Figure 1. The mean T/ET ratio increased with age from 0.13 ± 0.09 at 1–7 months of age to 0.42 ± 0.10 at 25– 38 months of age. It was significantly (p < 0.01) higher in bulls after 13 months of age compared with animals at 8–12 months of age. Pearson’s correlation coefficient analysis showed significant (p < 0.001) positive correlation between urinary testosterone and epitestosterone (r2 = 0.52) when measurements of bulls of all ages were included in the
Table 2. Proportion of bulls from each age group in which urinary androstenedione, epitestosterone or testosterone were detected with mean, median, minimal and maximal detected values. Age 1–7 months, N = 17 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg 8–12 months, N = 6 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg 13–24 months, N = 78 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg 25–38 months, N = 19 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg
Androstenedione
Epitestosterone
Testosterone
kg–1)
29 2.79 ± 0.51 2.7 1.77–4.7
100 25.99 ± 4.89 26 2.2–72
12a 5.6 ± 2.40 5.6 3.2–8
kg–1)
33 1.15 ± 0.04 1.15 1.1–1.2
100 27.77 ± 10.65 16.15 3.3–69
83 4.24 ± 1.28 3.00 2–8.8
kg–1)
44 1.76 ± 0.17 1.35 1–4.7
100 22.77 ± 1.72 20.90 2.62–89
80 7.13 ± 0.83 5.60 1.2–38.7
kg–1)
42 2.16 ± 0.41 1.95 1–4.6
100 24.34 ± 4.10 20.20 2.8–75
95 9.50 ± 1.59 8.55 1.6–20.2
Note: aValue differs significantly from those for other animal ages (p < 0.001).
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analysis. In the separate animal groups, correlation coefficient analysis showed positive, but non-significant, correlations at 1–7 and 8–12 months of age (r2 = 0.37 and 0.74 respectively) and significant (p < 0.01) positive correlations at 13–24 and 25–38 months of age (r2 = 0.58 and 0.68 respectively). The urinary T/ET ratio showed significantly higher levels at 13–24 and 25–38 months of age than at 8–12 months of age.
Urinary steroids in heifers and cows In the 174 female cattle urinary samples analysed, no boldenone, boldione, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, norandrostenedione, stanozolol, trenbolone, trenbolone acetate,
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17α-ethinylestradiol, 17α-methyltestosterone, testosterone or nandrolone were found. Androstenedione, epitestosterone, progesterone and estradiol were identified in various proportions depending on age (Table 3). Values were below the LOD in three, three, one and seven animals of age groups 8–12, 13–24, 37–48 and over 60 months, respectively. Simultaneous occurrence of urinary androstenedione and estradiol or progesterone and estradiol was not found in any sample. Androstenedione and progesterone were found together in one sample in the age group 1– 7 months, once in the group 13–24 months, and twice in the group 37–48 months. Chi-square analysis showed no significant differences in proportions of different urinary steroids at different ages
Table 3. Proportion of female cattle from each age group in which urinary androstenedione, epitestosterone, progesterone or estradiol were detected with mean ± SE, median, minimal and maximal detected values. Age 1–7 months, N = 27 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) 8–12 months, N = 28 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) 13–24 months, N = 54 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) 25–36 months, N = 16 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) 37–48 months, N = 11 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum – maximum (μg kg–1) 49–60 months, N = 10 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1) Over 60 months, N = 28 Proportion (%) Mean (μg kg–1) ± SE Median (μg kg–1) Minimum–maximum (μg kg–1)
Androstenedione
Epitestosterone
Progesterone
11 1.13 ± 0.09 1.10 1–5.5
100 23.19 ± 4.60 18.00 2.9–117
18 2.15 ± 0.54 2.15 1–4
0 – – –
4 1.4a 1.40a 1.4a
89 20.58 ± 3.1 16.00 4.3–49
0 – – –
0 – – –
4 1.55 ± 0.15 1.55 1.4–1.7
94 26.01 ± 4.47 17.10 1–185
6 1.5 ± 0.35 1.20 1.1–2.2
2 2.4a 2.40a 2.4a
0 – – –
100 16.48 ± 3.57 9.90 2.3–56
6 4.4a 4.40a 4.4a
6 2a 2.00a 2a
27 1.4 ± 0.31 1.20 1–2
91 30.16 ± 6.16 31.80 5.8–70
27 1.57 ± 0.43 1.30 1–2.4
0 – – –
0 – – –
100 26 ± 7.42 16.85 5.4–70
10 2a 2.00a 2a
0 – – –
0 – – –
75 33.33 ± 6.05 26.80 2–86
7 1.55 ± 0.35 1.55 1.2–1.9
14 4.25 ± 0.50 4.4 2.9–5.3
Note: aOnly one sample from the group exceeded the LOD.
Estradiol
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(Table 3). Similarly, no significant differences were found between androstenedione, epitestosterone, progesterone and estradiol levels at different ages. Discussion In the European Union urine is collected for inspection purposes as a matrix for controlling steroid abuse, since the concentrations of steroids in urine are much higher than in blood. The presence of synthetic steroid compounds in urine constitutes direct proof of steroid administration to the animal (Scarth et al. 2009). In this study on cattle individuals were tested for the presence of several synthetic or bovine urine non-specific steroids, such as αand β-boldenone, boldione, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, norandrostenedione, stanozolol, trenbolone, trenbolone acetate, 17α-ethinylestradiol, 17α-methyltestosterone and nandrolone. Since none of these compounds was detected in any of the animals, we assume that steroids or other steroids that metabolise to any of the above-mentioned steroids were not introduced to the observed animals around the time of sample collection. Most of the steroids tested for do not appear naturally in cattle urine. The only exception is boldenone, which is present in faeces (Pompa et al. 2006; Arioli et al. 2008). It can be present naturally in cattle urine (Le Bizec et al. 2006; Stephany 2010) although it was reported to occur in urine for only a few hours after administration (Draisci et al. 2007). Nevertheless, we found no urinary boldenone in any epimeric form, suggesting that boldenone is present very rarely, if ever, in intact (i.e. not contaminated with faeces) bovine urine. In our study urinary epitestosterone was found in all males of all age groups (1–38 months). Statistical analysis showed no significant variation of mean epitestosterone level between age groups. Epitestosterone values in every bull age group range from low, scarcely exceeding the LOD, to high (Table 2). Interestingly, high values were found not only in mature bulls but also in calves. Epitestosterone in bulls is produced in testis and also from testosterone in liver and in blood (Starka 2003). The fact that the urinary epitestosterone level is also influenced by testosterone administration is a strong indication that, given the high individual variation, the urinary epitestosterone level is not a reliable indicator of steroid abuse. In contrast to epitestosterone, the occurrence of urinary testosterone is age dependent, being significantly lower in bulls in the range of 1–7 months (no urinary testosterone was found under the age of 5 months) than in older animals (Table 2). Its frequency of occurrence increased with age and reached 94.7% at the age of 24–38 months. Similarly, mean urinary testosterone levels increased with age, but not significantly so, probably due to high
individual variability (Table 2). Bulls reach puberty at around 5 months of age, at which time the production of testosterone increases (Renaville et al. 1996; Cestnik et al. 2001). The significant positive correlation of epitestosterone and testosterone levels confirms the conversion of testosterone to epitestosterone, as described by Starka (2003). Since urinary testosterone is age dependent and epitestosterone is not, the T/ET ratio fluctuates with age. Although more or less successful as a screening tool for doping control in athletes (WADA 2004), successful use in controlling steroid abuse in beef production is questionable. Angeletti et al. (2006) reported increased T/ET ratios in calves treated with a steroid cocktail containing testosterone and epitestosterone, although the differences from the control animal group were not significant (Angeletti et al. 2006). Our results show a significantly higher mean T/ET ratio in bulls at 13–24 months of age than at ages 8– 12 months. In bulls younger than 8 months the mean T/ET ratio is even lower (Figure 1), but the number of animals in which the urinary testosterone level was above the LOD was very small. In this age group testosterone was detected in only 12% of animals, thus similarly limiting determination of the T/ET ratio. In bulls younger than 5 months, the T/ET ratio could not be calculated since the urinary testosterone level was below the LOD. These data are especially important for assessment of testosterone abuse in animals younger than 13 months, since a high urinary T/ET ratio in such animals could be considered as ‘suspicious’. Urinary androstenedione was found in bulls in all age groups with frequencies that did not vary significantly. In the biosynthesis of steroids, androstenedione is a precursor of testosterone, since 17β-hydroxysteroid dehydrogenase converts androstenedione to testosterone. It is also a metabolite of boldenone (Merlanti et al. 2007). Most probably the observed levels of androstenedione are those that normally occur in bulls’ urine. The list of urinary steroids found in heifers and cows differs from that in bulls. Testosterone was not found in any of the 174 females, even though epitestosterone was detected in a relatively high proportion (Table 3) (but not in all animals, as was found in bulls). In contrast to bulls, the T/ET ratio in females is therefore not a significant parameter. The source of epitestosterone is probably the active ovary, since it was found in follicular fluid and is also derived from androstenedione and testosterone (Starka 2003). Urinary epitestosterone concentrations did not differ significantly between age groups, suggesting that age is not a factor in epitestosterone metabolism and excretion. In certain females urinary epitestosterone exceeded the mean value by five- to seven-fold (Table 3). These results suggest that in cows, as in humans (Dehenin et al. 1987), epitestosterone formation depends on reproductive status, as well as on reproductive disorders, resulting in high individual variability. The
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Food Additives & Contaminants: Part A proportion of females in which urinary androstenedione, progesterone and estradiol were detected is much lower than that in which urinary epitestosterone was detected and, similarly to epitestosterone, differences in mean concentrations of these steroids between the groups were not significant. Furthermore, in animals in which androstenedione was detected, its level scarcely exceeded the LOD, while progesterone and estradiol in particular animals reached 4.4 and 5.3 μg kg–1 respectively (Table 3). In this study estradiol was not detected together with androstenedione in the same urine sample, nor estradiol with progesterone. The reason why estradiol and progesterone were not observed in the same sample is that they are secreted during different phases of the oestrus cycle (Christensen et al. 1974). On the other hand, the occurrence of androstenedione and progesterone at the same time is expected since the former is involved in the biosynthetic pathway of steroids as a progesterone metabolite or an estradiol and testosterone precursor (Scarth et al. 2009). The fact that androstenedione and estradiol were not observed together in any urine sample is therefore probably a coincidence, since either androstenedione or estradiol was detected in a limited number of samples. In addition, as described by Scarth et al. (2009), mean urinary testosterone and epitestosterone levels are approximately three-fold higher in mature bulls compared with females. Regarding testosterone, this is in line with the results of this study; however, mean epitestosterone values show no differences between males and females (Tables 2 and 3). The variations in the quantitative levels of steroids found in the present study could also be the consequence of different hydration status of the animals. This could be corrected by using normalisation to urine creatinine levels. However, the role of creatinine as a marker of hydration status is somewhat unsure (Scarth et al. 2011). Furthermore, the urinary samples in this study originated from a national monitoring residue programme which did not include these parameters. Some recent studies described the exact metabolism of exogenous steroids in bovines (Ferchaud et al. 2000; Angeletti et al. 2006; Le Bizec et al. 2006; Draisci et al. 2007; Dervilly-Pinel et al. 2011; Scarth et al. 2011). The results of the present study supplement this information with urinary levels of endogenous steroids from a large number of animals of different sex and ages. It cannot be confirmed with certainty that all the urinary samples used in this study originate from animals that were not treated with illegal growth promoters. However, the fact that no indicators, such as the presence of synthetic steroids in urine or the illegal market with steroids containing implants, were observed in the period of sample collection makes the introduction of illicit compounds in the examined animals highly unlikely.
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The results of this study therefore constitute additional understanding of sex steroid excretion dynamics in cattle. In addition to the recorded physiological levels of some urinary steroids, the data presented should also be helpful in evaluating the results from screening methods for steroid abuse.
Conclusions In summary, epitestosterone, testosterone and androstenedione are normally present in the urine of bulls. Epitestosterone was found in all observed males. In contrast to epitestosterone, however, urinary testosterone occurrence is age dependent. However, since urinary testosterone is age dependent and epitestosterone is not, the T/ET ratio also fluctuates with age and is thus a good parameter for assessing animal testosterone status, especially in young animals. Urinary epitestosterone, androstenedione, progesterone and estradiol were found in heifers and cows. The proportion of animals in which urinary androstenedione, progesterone and estradiol were detected is much lower than the proportion in which epitestosterone was observed. Testosterone was not present in any of the female animals and presence of both estradiol and progesterone in the same sample was not observed. The results of this study will be helpful in evaluating the results of steroid abuse screening methods.
Acknowledgements The authors are thankful to Dr Roger H. Pain for English editing and suggestions.
Funding The authors acknowledge The Slovenian Research Agency [grant number P4-0053] and The Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant protection which enabled the study.
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