Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites : II. Identification and quantification of 19-norsteroids responsible for 19-norandrosterone and 19-noretiocholanolone excretion in human urine / De Wasch K, Le Bizec B, De Brabander H, André F, Impens S. Rapid Commun Mass Spectrom. 2001;15(16):1442-7.
Laboratory of Chemical Analysis, Ghent University, Faculty of Veterinary Medicine, Salisburylaan 133, B-9820 Merelbeke, Belgium.

In previous work (Le Bizec et al., Rapid Commun. Mass Spectrom. 2000; 14: 1058), it was demonstrated that a boar meal intake could lead to possible false accusations of abuse of 17beta-nortestosterone in antidoping control. The aim of the present study was to identify and quantify endogenous 19-norsteroids in boar edible tissue at concentrations that can alter the steroid urinary profile in humans, and lead to excretion of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). The samples were analysed in two laboratories. The methodologies used for extraction and detection (GC/MS(EI) and LC/MS/MS(APCI+)) are compared and discussed. 19-Norandrostenedione (NAED), 17beta- and 17alpha-nortestosterone (bNT, aNT), and 17beta- and 17alpha-testosterone (bT, aT) were quantified. The largest concentrations of NAED and bNT were observed in testicles (83 and 172 microg/kg), liver (17 and 63 microg/kg) and kidney (45 and 38 microg/kg). A correlation between the bNT and NAED content of a typical meal prepared with boar parts and the excreted concentrations of 19-NA and 19-NE in human urine was demonstrated.

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19-norandrosterone and 19-noretiocholanolone in human urine / B. Le Bizec, I. Gaudin, F. Monteau, F. Andre, S. Impens, K. De Wasch, H. De Brabander

• Rapid Communications in Mass Spectrometry 14 (2000) 12 (30 June), p. 1058-1065
• PMID: 10861987
• DOI: 10.1002/1097-0231(20000630)14:12<1058::AID-RCM991>3.0.CO;2-7

Abstract

For the first time in the field of steroid residues in humans, demonstration of 19-norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone (19-NE: 3alpha-hydroxy-5beta-estran-17-one) excretion in urine subsequent to boar consumption is reported. Three male volunteers agreed to consume 310 g of tissues from the edible parts (meat, liver, heart and kidney) of a boar. The three individuals delivered urine samples before and during 24 h after meal intake. After deconjugation of phase II metabolites, purification and specific derivatisation of target metabolites, the urinary extracts were analysed by mass spectrometry. Identification was carried out using measurements obtained by gas chromatography/high resolution mass spectrometry (GC/HRMS) (R = 7000) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (positive electrospray ionisation (ESI+)). Quantification was realised using a quadrupole mass filter. 19-NA and 19-NE concentrations in urine reached 3.1 to 7.5 microg/L nearby 10 hours after boar tissue consumption. Levels returned to endogenous values 24 hours after. These two steroids are usually exploited to confirm the exogenous administration of 19-nortestosterone (19-NT: 17beta-hydroxyestr-4-en-3-one), especially in the antidoping field. We have thus proved that eating tissues of non-castrated male pork (in which 17beta-nandrolone is present) might induce some false accusations of the abuse of nandrolone in antidoping.

Segura J, Monfort N, Ventura R. Drug Test Anal. 2012 Nov;4(11):876-81. doi: 10.1002/dta.405. Epub 2012 Mar 9.
Bioanalysis Research Group, IMIM Hospital del Mar Research Institute, Barcelona, Spain.

The use of blood doping is forbidden by the World Anti-Doping Agency. Several practices, such as blood transfusions are used to increase oxygen delivery to muscles and all of them are highly pursued. In this regard, the development of accurate methodologies for detecting these prohibited practices is one of the current aims of the anti-doping control laboratories. Flow cytometry methods are able to detect allogeneic blood transfusions but there is no official methodology available to detect autologous blood transfusions. This paper reviews protocols, including the Athlete Biological Passport, that use indirect markers to detect misuse of blood transfusions, especially autologous blood transfusions. The methods of total haemoglobin mass measurements and the detection of metabolites of blood bags plasticizers in urine are reviewed. The latter seems to be an important step forward because it is a fast screening method and it is based on urine, a fluid widely available for doping control. Other innovative approaches to blood transfusion detection are also mentioned. A combination of the reported methodologies and the implementation of the Athlete Biological Passport is becoming a promising approach.

Sanchis-Gomar F, Banfi G, Lippi G.
Transfusion. 2012 Mar;52(3):680; author reply 680-1. doi: 10.1111/j.1537-2995.2011.03433.x.
Comment on Plasticizers excreted in urine: indication of autologous blood transfusion in sports. [Transfusion. 2012]

Monfort N, Ventura R, Platen P, Hinrichs T, Brixius K, Schänzer W, Thevis M, Geyer H, Segura J.Transfusion. 2012 Mar;52(3):647-57. doi: 10.1111/j.1537-2995.2011.03331.x. Epub 2011 Sep 2.
Bioanalysis Research Group, IMIM Hospital del Mar Research Institute, the Universitat Pompeu Fabra, Barcelona, Spain.

BACKGROUND:
Misuse of autologous blood transfusions in sports remains undetectable. The metabolites of the plasticizer di-(2-ethylhexyl)phthalate (DEHP) were recently proposed as markers of blood transfusion, based on high urinary concentrations of these compounds observed in patients subjected to blood transfusion. This study evaluates DEHP metabolites in urine for detecting autologous blood transfusion.

STUDY DESIGN AND METHODS:
One blood bag was drawn from moderately trained subjects and the red blood cells (RBCs) were reinfused after different storage periods. Group 1 (12 subjects) was reinfused after 14 days, and Group 2 (13 subjects), after 28 days of storage. Urine samples were collected before and after reinfusion for determination of the concentrations of three DEHP metabolites, mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate, and mono-(2-ethyl-5-oxohexyl)phthalate.

RESULTS:
Concentrations of DEHP metabolites on the days before reinfusion were in agreement with those described after common environmental exposure. A few hours after the reinfusion a significant increase was observed for all metabolites in all volunteers. Concentrations 1 day later were still higher (p < 0.05) than before reinfusion. Variations in urine dilution supported normalization by specific gravity. Concentrations of DEHP metabolites tended to be higher after longer storage times of RBCs.

CONCLUSION:
Autologous transfusion with RBCs stored in plastic bags provokes an acute increase in the urinary concentrations of DEHP metabolites, allowing the detection of this doping malpractice. The window of detection is approximately 2 days. The method might be applied to urine samples submitted for antidoping testing.

Evaluation and analysis of exposure levels of di(2-ethylhexyl) phthalate from blood bags / Inoue K, Kawaguchi M, Yamanaka R, Higuchi T, Ito R, Saito K, Nakazawa H. - (Clin Chim Acta. 2005 Aug;358(1-2):159-66)

Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Tokyo 142-8501, Japan

BACKGROUND:

The US FDA and The Ministry of Health, Labor and Welfare of Japan have indicated that the risk assessment of di(2-ethylhexyl) phthalate (DEHP) released from polyvinyl chloride (PVC) medical devices requires immediate attention. In particular, the analysis of the exposure to DEHP from blood bags is very important for medical treatment. However, human exposure to DEHP via blood transfusion remains poorly understood. We evaluated DEHP and mono(2-ethylhexyl) phthalate (MEHP) levels, migration patterns, and metabolism in blood products for the detailed assessment of exposure to DEHP.

METHODS:
A method that is based on column-switching liquid chromatography-electrospray mass spectrometry (LC-MS) coupled with on-line extraction was used for the direct analysis of DEHP and MEHP in the blood products. From the Japanese Red Cross Society, 78 blood products (red blood cell concentrate: n=18, irradiated red blood cell concentrate: n=18, whole blood: n=18, blood platelet: n=18, and frozen plasma: n=6) were sampled in January 2003 for use in this study.

RESULTS:
The detection levels of DEHP and MEHP ranged from 1.8 to 83.2 microg/ml and from 0.1 to 9.7 microg/ml, respectively. The levels of MEHP and DEHP in the blood products were increased with increasing storage time. In addition, whole blood products in PVC bags had the highest DEHP levels compared to the other blood products. Our results indicate that the maximum level of human exposure to DEHP released from blood bags is 0.7 mg/kg weight/time.

CONCLUSION:
This first quantitative evidence may be useful for the risk assessment of DEHP released from blood bags.

Backhouse SH, Whitaker L, Petróczi A.
Scand J Med Sci Sports. 2011 Aug 9. doi: 10.1111/j.1600-0838.2011.01374

Abstract

Nutritional supplement (NS) use is widespread in sport. This study applied an integrated social cognitive approach to examine doping attitudes, beliefs, and self-reported doping use behavior across NS users (n = 96) and nonusers (n = 116). Following ethical approval, 212 competitive athletes (age mean = 21.4, s = 4.5; 137 males) completed self-reported measures of doping-related social cognitions and behaviors, presented in an online format where completion implied consent. Significantly more NS users (22.9%) reported doping compared with nonusers (6.0%; U = 4628.0, P < 0.05). NS users presented significantly more positive attitudes toward doping (U = 3152.0, P < 0.05) and expressed a significantly greater belief that doping is effective (U = 3152.0, P < 0.05). When presented with the scenario that performance-enhancing substances are effective and increase the possibility of winning, NS users were significantly more in favor of competing in situations that allow doping (U = 3504.5, P < 0.05). In sum, doping use is three-and-a-half times more prevalent in NS users compared with nonusers. This finding is accompanied by significant differences in doping attitudes, norms, and beliefs. Thus, this article offers support for the gateway hypothesis; athletes who engage in legal performance enhancement practices appear to embody an "at-risk" group for transition toward doping. Education should be appropriately targeted.

CAS 2010/A/2307 World Anti-Doping Agency (WADA) v. Jobson Leandro Pereira de Oliveira, Confederação Brasileira de Futebol (CBF) & Superior Tribunal de Justiça Desportiva (STJD)

• Football
• Doping (cocaine)
• CAS Jurisdiction ratione personae over the STJD
• CAS Jurisdiction ratione personae over any athlete registered with a Brazilian federation
• Impossibility to re-classify a substance
• Standard of utmost care
• Player’s age and degree of experience
• Commencement of the suspension period

1. The STJD is a justice body which is an integral part of the organizational structure of the CBF, with no legal personality of its own. At least for international purposes the decisions of the STJD, although independently reached, must be considered to be the decisions of the CBF. As a result, the STJD has no autonomous legal personality and may not be considered as Respondent on its own in a CAS appeal arbitration concerning one of its rulings. Consequently, the CAS does not have jurisdiction ratione personae over the STJD.

2. According to Brazilian law, official sports practice in Brazil is governed by national and international rules and by sporting practice rules of each type of sport, accepted by the respective national federations. In particular, athletes practicing professional sport have the duty to abide by international sports rules. As a result of these provisions and in accordance with CAS jurisprudence, international sports rules are directly applicable to Brazilian sport. Hence, any athlete registered with a Brazilian federation is directly bound by the international rules accepted by that federation, including any provision therein giving jurisdiction to the CAS.

3. Under the FIFA Anti-Doping Regulations (ADR), the inclusion of a substance in the Prohibited List and its classification is final. Thus, a panel is bound by the fact that cocaine was classified as a prohibited substance and it cannot re-classify it as a specified substance.

4. In accordance with the applicable strict standard of utmost care, except only in the most “truly exceptional cases”, the presence of prohibited substances in an athlete’s system constitutes a failure in fulfilling that duty. A cocaine-dependency syndrome is not specific and decisive, and cannot be considered an exceptional circumstance which is so “truly exceptional” as to explain a player’s departure from the expected standard of behavior.

5. The age and experience of an athlete must be considered in the context of all the relevant circumstances in order to determine whether they might mitigate the athlete’s fault or negligence.

6. Four months after the decision was rendered appears to be a reasonable span of time to hear a doping case. In case of an adjudicating process of almost twenty months since the date of the sample collection, with this duration not attributable to the player, it is fair to apply the principle set forth in Article 53 (2) of the FIFA ADR and to start the period of ineligibility at an earlier date than the day of notification of the award.

On 6 May 2010 the Brazilian football Superior Court of Sport Justice (STJD) decided by majority to impose a reduced 6 month period of ineligibility on the football player Jobson Leandro Pereira de Oliveira after he twice had tested positive for the prohibited substance Cocaine in December 2009.

Hereafter in December 2010 the World Anti-Doping Agency (WADA) appealed the STJD Decision with the Court of Arbitration for Sport (CAS). WADA requested the Panel to set aside the Appealed Decision and to impose a 2 year period of inelgibility on the Athlete.

The Athlete asserted with evidence that he bore no significant fault or negligence because, at the relevant time, he was allegedly suffering from an irresistible coercion caused by his Cocaine-dependence syndrome that did not allow him to control the use of Cocaine:

Preliminary the Panel establishes that it does have jurisdiction ratione personae over WADA, the Athlete, the CBS and the Appealed Decision. The Panel holds that it does not have jurisdiction personae over the STJD.

In this case the Panel assessed and addressed the following issues:

• a.) Has the Athlete committed an anti-doping rule violation?
• b.) If the answer to question a) is affirmative, what would be the appropriate sanction to be imposed on the Athlete?
• c.) What would be the legal consequences of the Panel’s findings?

The Panel finds that the elements offered by the Athlete are not sufficient to establish, on the balance of probability, that he bore no ‘significant fault or negligence’. The Panel does not find the evidence presented by the Athlete to be specific and decisive to explain the Athlete’s departure from the expected standard of behavior.

Moreover, the Panel is not convinced that the circumstances of the present case are ‘truly exceptional’ so as to reduce the Athlete’s responsibility. In fact, the Panel finds that the Athlete’s degree of fault or negligence, viewed in the totality of the circumstances, is clearly ‘significant’ in relation to the anti-doping rule violation.

The Panel has found that article 47 (1) FIFA ADR is not applicable to the case at hand, as Cocaine is not considered a ‘specified substance’ under the FIFA ADR. Furthermore, the Panel has found that the Athlete's degree of fault or negligence, viewed in the totality of the circumstances, was clearly ‘significant’ in relation to the anti-doping rule violation.

In addition, the Panel has found that imposing a two-year period of ineligibility on the Athlete is compatible with international law and human rights requirements.

Therefore the Court of Arbitration for Sport decides on 14 September 2011:

1.) The CAS has jurisdiction both ratione materiae and ratione personae to entertain the appeal of the World Anti-Doping Agency (WADA) against the Confederação Brasileira de Futebol (CBF) and Mr Jobson Leandro Pereira de Oliveira, while it has no jurisdiction ratione personae in respect of the Superior Tribunal de Justiça Desportiva (STJD).

2.) The Appeal of WADA against the decision of the STJD dated 6 May 2010 of the STJD is upheld.

3.) The decision dated 6 May 2010 of the STJD is set aside.

4.) Mr Jobson Leandro Pereira de Oliveira is suspended from 6 September 2010 for a period of two years, less the period of suspension of six months already served.

(…)

7.) All other prayers for relief are dismissed.

Haff, G. Gregory PhD, CSCS, FNSCA
Strength & Conditioning Journal: 29(1):50-57, February 2007.

summary: This is the second part of a 2-part roundtable series that presents current information and opinions about anabolic androgenic steroids.

(C) 2007 National Strength and Conditioning Association

CAS 2002/A/374 M. / International Olympic Committee (IOC)

Related cases:

• CAS 2002_A_400 Johann Muehlegg vs FIS
  January 24, 2003
• IOC 2002 IOC vs Johann Muehlegg
  February 24, 2002
  
  

• Cross Country Skiing
• Doping
• Accreditation of the laboratory to conduct EPO testing
• Nature of Aranesp
• Validity of the testing procedure to detect Aranesp Sanction in case of out-of-competition doping control

1. The fact that an accreditation for the isoelectric focusing test was not granted to the laboratory at the time when the samples were tested does not mean that this laboratory was not capable of conducting the r-EPO test. The Olympic Movement Antidoping Code (OMAC) specifically provides for the evolution of scientific knowledge and testing procedures. What must be established to the comfortable satisfaction of the tribunal is that the testing procedure as carried out was in accordance with the prevailing standards and practices of the scientific community.

2. Aranesp is a substance, which has the effect of artificially boosting the oxygen in the blood by the introduction of a greater number of red blood cells, and for an elite performance athlete these additional red blood cells translate into enhanced stamina. The natural hormone EPO and r-EPO have precisely the same physiological effects. Aranesp is an analogue and mimetic of the Prohibited Substance r-EPO.

3. Aranesp is a Prohibited Substance and can not be produced naturally unlike r-EPO that has an overlapping fingerprint with EPO and can cause doubts as to whether the isoform is natural or artificial in nature. Therefore, it does not matter that there may be overlap with the natural bands of EPO as there can be no doubt that there was use of Aranesp and its source can not possibly be that of the human body. Therefore, the direct urine test employed to detect r-EPO can also be applied to detect Aranesp. The notable difference between the two applications is that Aranesp does not require a threshold safety margin to protect against false positives because of overlap, as does r-EPO.

4. The closing words of Article 3.5 OMAC indicate that where an athlete commits an out-of-competition doping offence, at least all the results obtained after the date the sample was taken shall be invalidated. The proper interpretation of this Article could be construed as limiting the invalidation of results to those results that were achieved after the later of the date the positive result was recorded or the date final judgment on the issue is rendered. This interpretation would result in the absurdity that an athlete could compete up until the final adjudication of a doping infraction and not have any results obtained in the interim period invalidated. This is contrary to the purpose of the OMAC and such an interpretation cannot be accepted. This article operates not to determine what results will be invalidated, but the date on which the invalidation of results is effectively imposed.

The Spanish Athlete Johann Muehlegg competed in the Men's 30 km Free Mass Start (February 9, 2002), the Men's 10 km Free Pursuit (February 14, 2002) and the Men's 50 km Classical (February 23, 2002). Muehlegg placed first in all three events acquiring Spain's only medals of the Games.

In February 2002 the International Olympic Committee (IOC) reported an anti-doping rule violation againt the Athlete after his A and B samples tested positive for the prohibited substance Darbepoetin (dEPO).

Consequently on 24 February 2002 the IOC decided that the Athlete was disqualified and excluded from the 2002 Salt Lake City Olympic Winter Games.

Hereafter in March 2002 the Athlete appealed the IOC Decision with the Court of Arbitration for Sport (CAS 2002/A/374). Also in June 2002 the Athlete appealed with CAS the decision of the International Ski Federation (FIS) to sanction the Athlete for 2 years after he tested positive for dEPO) (CAS 2002/A/400).

The Athlete requested the Panel to set aside the IOC decision and he disputed the reliability of the testing method and the  testing result.

Following assessment of the case the Panel concludes that the IOC Executive Board properly found Muehlegg to have committed a doping infraction and hereby upholds that decision. Also the Panel finds that the IOC Executive Board properly exercised its authority under the Olympic Charter and the OMAC to invalidate Muehlegg’s results in the 50km classical cross-country event, withdraw the gold medal obtained, and exclude him from the Games.

Therefore on 24 January 2003 the Court of Arbitration for Sport decides:

1.) The appeal filed by Johann Muehlegg on 16 March 2002 is dismissed.

2.) The decision of the Executive Board of the International Olympic Committee of 24 February 2002 is upheld.

3.) The award is pronounced without costs, except for the court office fee of CHF 500 (five hundred Swiss Francs) paid by Johann Muehlegg which is kept by the CAS.

4.) Johann Muehlegg is ordered to pay the sum of CHF 12’000.- (Twelve thousand Swiss Francs), to the IOC in contribution towards its legal costs.