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Evolution of the δ13C values of testosterone metabolites (epitestosterone and etiocholanolone) and precursor (DHEA) over a 35 day period. Results for a Normande cow M (15 years old) treated (at J0) with testosterone enanthate and subjected to a feed based on maize silage and hay.
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The detection of exogenous testosterone in bovine urine was investigated by using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The carbon isotopic ratio measurement of epitestosterone, etiocholanolone (testosterone metabolite) and DHEA (testosterone precursor) in female bovine urines after testosterone enanthate admini...
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Context 1
... the end of the first experiment, the cattle feed was replaced by mainly maize silage and a small percentage of hay. Two weeks later, the Normande cow received a second intramuscular dose of testosterone enanthate. The results of GC/C/IRMS analysis of all urine samples are shown in Fig. 5. The mean d 13 C values of DHEA became progressively richer in 13 C content (from À24 to À19.6%) until the end of the experimental study. This minor variation can be explained by the steroid biosynthesis from maize silage; one can observe that the value stabilised after J29 (d 13 C = À19.6%). Likewise, after testosterone ...
Context 2
... as a result of maize feeding. After testosterone administration, the d 13 C value of the Rapid Commun. Mass Spectrom. 14, 652-656 (2000) metabolites changed rapidly to reach a maximum difference between epitestosterone, etiocholanolone and DHEA of 7.5%. Comparison of this large difference with that obtained for an adult animal (maximum = 4%) ( Fig. 5) led us to conclude that the lower is the endogenous biosynthesis of testosterone metabolites (low ppb level in heifers), the more important is the difference between the 13 C/ 12 C ratios of metabolites and precursor. Indeed, the relative metabolite concentration due to exogenous administration is significantly more important in young ...
Context 3
... animal could also be a potential factor influencing the 13 C/ 12 C ratio of the endogenous steroid, but the comparison of Fig 3 to 5, for the same cattle feeding, shows no difference in the d 13 C values of the precursor. Indeed, the Holstein cow (Fig. 3) and the Normande cow (Fig. 4) showed similar DHEA d 13 C values, whereas the Normande cow ( Fig. 5) stabilised to À19.6% at the end of maize treatment compared with the Blonde d'Aquitaine heifer d 13 C value, which is of À20%. Even if this phenomenon was predictable (i.e. independence between breed and 13 C content), we should verify it was ...
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Citations
... The small differences between endogenous and exogenous anabolic steroids isotopic deviations are one of the main difficulties encountered when applying the GC-Isotope Ratio Mass Spectrometry (IRMS) technique. In fact, the endogenous isotopic deviation of anabolic steroids depends mainly on the diet [83,84]. Depending on the type of food consumed, the isotopic deviation of ingested cholesterol, and hence deriving anabolic steroids, will not be the same. ...
Testosterone is a key compound of the anabolic androgenic steroids (AAS) family. It has largely been misused in human and animal doping targeting a muscle tissue growth and an enhancement of performances. Such practices constitute a violation against ethical values, food safety, and animal welfare. Consequently, the use of such substance is regulated by WADA and International committees for some animal species such as equine and bovine. Although efficient, the detection of testosterone misuse remains challenging in some cases due to its endogenous origin and its inter- and intra-individual level fluctuation in biological fluids. Novel analytical strategies have been developed and are continuously evolving in order to tackle this issue and to provide a better control of testosterone misuse.
... The average 13 C VPDB values for AEdiol, from T 0 until the end of the experiment, were −26.71‰ and −25.69‰ for cow A and cow B, respectively, representative for a feeding regime very low in C4-plant material content [33], while for cow C, this was −20.91‰. Even though the last sample was taken 3 weeks after the feed change, the data suggest that a steady state has not yet been reached at the end of the experiment, which is in line with previous findings, indicating adjustment periods close to 30 days [33,34]. More remarkable is the observed difference between AEdiol and BAA-PD in response to the feeding change. ...
... More remarkable is the observed difference between AEdiol and BAA-PD in response to the feeding change. While the values for AEdiol changed gradually over the total time, as previously reported for androgenic compounds [15,33,34], rising 8.84‰ in total, they already augmented 5.74‰ within the first 3 days for BAA-PD, resulting in a significant offset up to 5.04‰ between AEdiol and BAA-PD before the treatment. This offset could also explain the large impact of the endogenous dilution observed through the relatively small 13 C VPDB values obtained after the treatment. ...
Currently, no analytical method is available to demonstrate progesterone administration in biological samples collected in rearing animals, and therefore, tracking the abuse of this popular growth promoter is arduous. In this study, a method is presented to reveal progesterone (PG) treatment on the basis of carbon isotope measurement of 5β-pregnane-3α, 20α-diol (BAA-PD), a major PG metabolite excreted in bovine urine, by gas chromatography-mass spectrometry/combustion/isotope ratio mass spectrometry (GC-MS/C/IRMS). 5-Androstene-3β,17α-diol (AEdiol) is used as endogenous reference compound. Intermediate precisions (n=11) of 0.56‰ and 0.68‰ have been determined for AEdiol and BAA-PD, respectively. The analytical method was used for the very first time to successfully differentiate urine samples collected in treated and untreated animals.
... 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. ...
... 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. ...
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.
... This methodology has been applied successfully in urine to trace the administration of testosterone and estradiol esters in cattle (Buisson et al., 2005; Hebestreit et al., 2006). However, no thresholds of reference for these changes in the 13C/12C ratios of steroid hormones have been established yet, since their variations depend largely on the diet of the animal and also other factors such as age, sex or breed (Cawley et al., 2009; Ferchaud et al., 2000). In addition, GC-C-IRMS methods imply long and complicated steps of extraction and purification of the sample and also the use of semi-preparation HPLC and derivatization procedures (Buisson et al., 2005; Hebestreit et al., 2006). ...
... 17␣-boldenone). As exogenous sources of boldenone are characterised by depleted composition values in 13 C [24][25][26][27], it would be feasible to demonstrate its administration so far the concentration of target metabolites exceeded 10 g L −1 . Finally, the diagnostic character of the 6-hydroxymetabolite of boldenone as a potential marker of boldenone administration would merit to be definitively investigated. ...
Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17beta-boldenone-sulphate. The analytical procedure included direct extraction-purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap). Decision limit (CCalpha) and detection capability (CCbeta) were respectively 0.2 microg L(-1) and 0.4 microg L(-1) on triple quadrupole and 0.1 microg L(-1) and 0.2 microg L(-1) on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17beta-Boldenone-sulphate was measurable up to 36h after oral administration of boldione, and 30 days after 17beta-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.
... It is generally conceded that there are no recognized and formally validated limits which can be used in a regulatory context to distinguish between natural concentrations of endogenous hormones, such as testosterone, which may be found in tissues, from enhanced concentrations of the free hormone which might result from administration of products containing synthetic versions of the hormone (Ferchaud et al. 2000;Pre´vost et al. 2001). There is considerable variability in the normal concentrations of endogenous hormones which may be influenced by various factors, including gender, age, environment, diet and health status. ...
... Research has been conducted to develop methods which can distinguish between endogenous and exogenous-source natural hormones. While it has been demonstrated that isotope ratio mass spectrometry may be used to distinguish between natural endogenous hormones and their synthetic exogenous versions based on the ratio of 13 C/ 12 C, this requires highly specialized equipment not currently found in most residue control laboratories and is not in widespread use (Ferchaud et al. 2000;Pre´vost et al. 2001). Since the information on natural concentrations of hormones found in normal tissues has not provided a basis for identification of exogenous treatment and the isotope ratio approach is not readily accessible to most residue laboratories, a simpler approach was investigated. ...
The detection of hormone abuse for growth promotion in food animal production is a global concern. Initial testing for hormones in Canada was directed at the compounds approved for use in beef cattle, melengestrol acetate, trenbolone acetate and zeranol, and the banned compound diethylstilbestrol (DES). No hormonal growth promoters are approved for use in veal production in Canada. However, instances of use of trenbolone and clenbuterol were detected in Canada in the 1990s. During the development of a new analytical method for testosterone and progesterone, there were reports of suspicious injection sites being found in veal calves. Upon implementation of the method, analysis of investigative samples revealed significant residues of testosterone in some injection sites. To prove that the source of these residues was exogenous, a fully validated method for hormone esters was developed to confirm the presence of exogenous hormones in these injection sites. The QUECHERS model was employed in methods development and resulted in a simple, effective extraction technique that consisted of sample pre-homogenization, liquid/liquid partitioning, extract dilution, filtration and use of LC/MS/MS to provide detection selectivity. The result was an adaptable MS/MS confirmation technique that meets the needs of Canadian regulatory authorities to confirm the misuse of injectable testosterone, and potentially other hormones, in food animal production.
... In a study involving one participant who received 50 mg of epitestosterone, the ␦ 13 C value of a diol metabolite (measured as the diacetate) of epitestosterone was highly negative (15 ). In addition, after the administration of testosterone enanthate to cattle, the ␦ 13 C value of diacetates of urine epitestosterone fell from approximately Ϫ25‰ to Ϫ30‰ and remained low for more than 16 days (unlike humans, cattle metabolize testosterone to epitestosterone) (25 ). The time course of urinary epitestosterone ␦ 13 C values after epitestosterone administration in humans has not been reported. ...
Epitestosterone is prohibited by sport authorities because its administration will lower the urinary testosterone/epitestosterone ratio, a marker of testosterone administration. A definitive method for detecting epitestosterone administration is needed.
We developed a gas chromatography-combustion-isotope ratio mass spectrometry method for measuring the delta(13)C values for urinary epitestosterone. Sample preparation included deconjugation with beta-glucuronidase, solid-phase extraction, and semipreparative HPLC. Epitestosterone concentrations were determined by gas chromatography-mass spectrometry for urines obtained from a control group of 456 healthy males. Epitestosterone delta(13)C values were determined for 43 control urines with epitestosterone concentrations > or =40 microg/L (139 nmol/L) and 10 athletes' urines with epitestosterone concentrations > or =180 microg/L (624 nmol/L), respectively.
The log epitestosterone concentration distribution was gaussian [mean, 3.30; SD, 0.706; geometric mean, 27.0 microg/L (93.6 nmol/L)]. The delta(13)C values for four synthetic epitestosterones were low (less than or equal to -30.3 per thousand) and differed significantly (P <0.0001). The SDs of between-assay precision studies were low (< or =0.73 per thousand). The mean delta(13)C values for urine samples obtained from 43 healthy males was -23.8 per thousand (SD, 0.93 per thousand). Nine of 10 athletes' urine samples with epitestosterone concentrations >180 microg/L (624 nmol/L) had delta(13)C values within +/- 3 SD of the control group. The delta(13)C value of epitestosterone in one sample was -32.6 per thousand (z-score, 9.4), suggesting that epitestosterone was administered. In addition, the likelihood of simultaneous testosterone administration was supported by low delta(13)C values for androsterone and etiocholanolone.
Determining delta(13)C values for urinary epitestosterone is useful for detecting cases of epitestosterone administration because the mean delta(13)C values for a control group is high (-23.8 per thousand) compared with the delta(13)C values for synthetic epitestosterones.
... Cette méthode indirecte consiste non pas à rechercher des substances anormales, toxiques pour le consommateur ou interdites, mais à considérer l'effet de "perturbation" de ces substances sur le métabolisme par rapport à une situation métabolique de référence. Habituellement, le traitement est mis en évidence par le spectre de masse de la molécule suspectée (1,8,16). La nouvelle technique permet de rechercher dans les urines de bovins, une signature biologique trahissant l'utilisation d'anabolisant. ...
Cellular and Molecular Biology TM 47 (Supplément), 93-108 0145-5680/01 Imprimé en France. 2001 Cell. mol. Biol. TM 93 Gérard PASCAL Ingénieur biochimiste de l'Institut National des Sciences Appliquées de Lyon, Gérard PASCAL est depuis Janvier 1998 directeur scientifique à l'INRA responsable de la nutrition humaine et de la sécurité sanitaire des aliments. Il y a créé le département de recherche en Nutrition (NASA) qu'il a dirigé de 1989 à 1992 puis de 1996 à 1998. Il est membre de la CGB depuis sa création en 1986. Il a présidé la section de nutrition du CSHPF de 1988 à 1992 et le Comité scientifique de l'alimentation humaine à Bruxelles de 1992 à 1997. Expert de l'OMS en sécurité des aliments depuis 1993, il préside actuellement le Conseil scientifique de l'AFSSA et le Comité Scientifique Directeur de l'Union Européenne. Sylvain MAHE Docteur en Sciences, spécialité sciences alimentaires (ENSIA, Universités Paris VII et XI, 1987) et diplômé de l'Institut Pasteur de Paris (Microbiologie Générale, Bactériologie Systématique 1984-1985), Sylvain MAHÉ est Chargé de Recherches à l'Institut National de Recherche Agronomique (INRA) depuis 1988. De 1990 à 2000 il a animé une équipe de recherche INRA qui étudie le métabolisme des protéines alimentaires durant la phase postprandiale et les répercussions physiologiques et fonctionnelles, chez l'Homme. En septembre 2000 il a rejoint la Direction Scientifique de Nutrition Humaine et Sécurité des Aliments (INRA, DS-NHSA) où il est chargé de la veille et prospective scientifique.
This unique and definitive reference on hormone abuse in food producing animals is for scientists, regulators and consumers. It contains the results of a meeting held in November 2006 to discuss the progress made by the Veterinary Medicines Directorate (VMD) supported programme on steroid abuse detection. When the "hormone ban" first arose in the EU, there were only 15 Member States. There are now 27 and some newer members lack the background knowledge of this issue. This book sets out to summarise the history and show the significant progress that has been, and continues to be, made in this area. It is the only comprehensive review of this subject available and contains input from leading researchers from around the world. The initial chapters provide valuable background information. For example, the chapter on toxicology and risk covers the controversies arising from the interpretation of the effects of artificial hormones in meat-producing animals. The book then goes on to deal with how the issue has been managed via national and international detection programmes. It finishes by covering the resulting cutting edge analytical science including current research using "omics"/ profiling for "natural hormones" and novel detection techniques such as IRMS. This book offers readers an insight into the risk management of an important food related issue and how current analytical analyses can assist evidence-based risk assessments. There is comprehensive coverage of all past and current issues relating to growth promoting hormone abuse in animals.
