Doping Test Results Dependent on Genotype of Uridine Diphospho-Glucuronosyl Transferase 2B17, the Major Enzyme for Testosterone Glucuronidation

1 Jul 2008

Doping Test Results Dependent on Genotype of Uridine Diphospho-Glucuronosyl Transferase 2B17, the Major Enzyme for Testosterone Glucuronidation / Jenny Jakobsson Schulze, Jonas Lundmark, Mats Garle, Ilona Skilving, Lena Ekström, Anders Rane. - (The Journal of Clinical Endocrinology & Metabolism 93 (2008) 7 (1 July)p. p. 2500-2506)

  • DOI: 10.1210/jc.2008-0218


Abstract

Context: Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. The large variation in testosterone glucuronide (TG) excretion and its strong association with a deletion polymorphism in the uridine diphospho-glucuronosyl transferase (UGT) 2B17 gene challenge the accuracy of the T/E ratio test.

Objective: Our objective was to investigate whether genotype-based cutoff values will improve the sensitivity and specificity of the test.

Design: This was an open three-armed comparative study.

Participants: A total of 55 healthy male volunteers with either two, one, or no allele [insertion/insertion, insertion/deletion, or deletion/deletion (del/del)] of the UGT2B17 gene was included in the study.

Intervention: A single im dose of 500 mg testosterone enanthate was administered.

Main Outcome Measures: Urinary excretion of TG after dose and the T/E ratio during 15 d were calculated.

Results: The degree and rate of increase in the TG excretion rate were highly dependent on the UGT2B17 genotype with a 20-fold higher average maximum increase in the insertion/insertion group compared with the del/del group. Of the del/del subjects, 40% never reached the T/E ratio of 4.0 on any of the 15 d after the dose. When differentiated cutoff levels for the del/del (1.0) and the other genotypes (6.0) were applied, the sensitivity increased substantially for the del/del group, and false positives in the other genotypes were eliminated.

Conclusions: Consideration of the genetic variation in disposition of androgens will improve the sensitivity and specificity of the testosterone doping test. This is of interest not only for combating androgen doping in sports, but also for detecting and preventing androgen abuse in society.

The impact of genetics and hormonal contraceptives on the steroid profile in female athletes

10 Apr 2014

The impact of genetics and hormonal contraceptives on the steroid profile in female athletes / Jenny J. Schulze, Jenny E. Mullen, Emma Berglund Lindgren, Magnus Ericsson, Lena Ekström, Angelica Lindén Hirschberg. - (Frontiers in Endocrinology (2014, 10 April); p. 1-6)

  • PMID: 24782830
  • PMCID: PMC3989562
  • DOI: 10.3389/fendo.2014.00050


Abstract

The steroid module of the Athlete Biological Passport, the newest innovation in doping testing, is currently being finalized for implementation. Several factors, other than doping, can affect the longitudinal steroid profile. In this study, we investigated the effect of hormonal contraceptives (HC) as well as the effect of three polymorphisms on female steroid profiles in relation to doping controls. The study population consisted of 79 female elite athletes between the ages of 18 and 45. HC were used by 32% of the subjects. A full urinary steroid profile was obtained using World Anti-Doping Agency accredited methods. In addition all subjects were genotyped for copy number variation of UGT2B17 and SNPs in UGT2B7 and CYP17. Subjects using HC excreted 40% less epitestosterone as compared to non-users (p = 0.005) but showed no difference in testosterone excretion. When removing individuals homozygous for the deletion in UGT2B17, the testosterone to epitestosterone (T/E) ratio was 29% higher in the HC group (p = 0.016). In agreement with previous findings in men, copy number variation of UGT2B17 had significant effect on female urinary testosterone excretion and therefore also the T/E ratio. Subjects homozygous for the T allele of CYP17 showed a lower urinary epitestosterone concentration than the other CYP17 genotypes. It is of great importance that the athlete's steroidal passport can compensate for all possible normal variability in steroid profiles from women. Therefore, considering the large impact of HC on female steroid profiles, we suggest that the use of HC should be a mandatory question on the doping control form.

Sudden cardiac death in sports: could we save Pheidippides?

8 Jan 2021

Sudden cardiac death in sports: could we save Pheidippides? / Asterios Deligiannis, Evangelia Kouidi. - (Acta Cardiologica (2021, 8 January))

  • PMID: 33416030
  • DOI: 10.1080/00015385.2020.1867388


Abstract

Hereditary diseases under the age of 35 are the most common underlying heart disease, leading to sudden cardiac death (SCD) in competitive sports, while in older people, atherosclerotic coronary artery disease (CAD) is the main cause. The following preventive measures are recommended: (a) The pre-participation cardiovascular screening, (b) the genetic testing, (c) the use of implantable cardioverter-defibrillator (ICD), (d) the prohibition of doping in sports, (e) the prevention of 'exercise-induced' cardiac complications, (f) the reduction of high-risk factors for CAD, and (g) the use of cardiopulmonary resuscitation. The cost-effectiveness of the electrocardiograms in the pre-participation screening programs remains questionable. Genetic testing is recommended in borderline cases and positive family history. Athletes with ICD can, under certain conditions, participate in competitive sports. Excessive endurance exercise appears to harm the endothelium, promotes inflammatory processes and leads to fibrosis in the myocardium, and calcium deposition in the coronary vessels. Cardiac arrest may be reversed if cardiopulmonary resuscitation is performed and a defibrillator is immediately used. Thus, equipping all fields with automatic external defibrillators are recommended.

Nine prohibited stimulants found in sports and weight loss supplements

23 Mar 2021

Nine prohibited stimulants found in sports and weight loss supplements: deterenol, phenpromethamine (Vonedrine), oxilofrine, octodrine, beta-methylphenylethylamine (BMPEA), 1,3-dimethylamylamine (1,3-DMAA), 1,4-dimethylamylamine (1,4-DMAA), 1,3-dimethylbutylamine (1,3-DMBA) and higenamine /  Pieter A. Cohen, John C. Travis, Céline Vanhee, Dana Ohana, Bastiaan J. Venhuis. - (Clinical Toxicology (2021, 23 March; p. 1-8)

  • PMID: 33755516
  • DOI: 10.1080/15563650.2021.1894333


Abstract

Background: Weight loss and sports supplements containing deterenol have been associated with serious adverse events including cardiac arrest.

Objective: To determine the presence and quantity of experimental stimulants in dietary supplements labeled as containing deterenol sold in the United States.

Methods: Dietary supplements available for sale in the US and labeled as containing deterenol or one of its synonyms (e.g., isopropylnorsynephrine and isopropyloctopamine) were purchased online. For each brand, one container or subsample was analyzed by NSF International (Ann Arbor, MI) and one container or subsample by the Netherland's National Institute for Public Health and the Environment (RIVM, Bilthoven, The Netherlands). When differences existed between the two containers or subsamples of the same brand, both products were reanalyzed by Sciensano (Brussels, Belgium). NSF International carried out qualitative and quantitative analyses using ultra-high-performance liquid chromatography (UHPLC) quadrupole-Orbitrap mass spectrometry. RIVM performed qualitative and quantitative analysis using UHPLC quadrupole time-of-flight mass spectrometry. Sciensano carried out qualitative analysis using UHPLC quadrupole-Orbitrap mass spectrometry.

Results: Seventeen brands of supplements were analyzed. Many brands included more than one prohibited stimulant in the same product: 4 brands (24%, 4/17) included 2 stimulants, 2 (12%, 2/17) combined 3 stimulants, and 2 (12%, 2/17) combined 4 stimulants. The range of quantities per recommended serving size of the 9 stimulants detected were 2.7 mg to 17 mg of deterenol; 1.3 mg to 20 mg of phenpromethamine (Vonedrine); 5.7 mg to 92 mg of beta-methylphenylethylamine (BMPEA); 18 mg to 73 mg of octodrine; 18 mg to 55 mg of oxilofrine; 48 mg of higenamine; 17 mg of 1,3-dimethylamylamine (1,3-DMAA); 1.8 mg to 6.6 mg of 1,3-dimethylbutylamine (1,3-DMBA); and 5.3 mg of 1,4-dimethylamylamine (1,4-DMAA).

Conclusion: Weight loss and sports supplements listing deterenol as an ingredient contained 9 prohibited stimulants and 8 different mixtures of stimulants, with as many as 4 experimental stimulants per product. These cocktails of stimulants have never been tested in humans and their safety is unknown.

Overview of Prohibited Substances and Prohibited Methods from the IOC Lists & WADA Prohibited Lists (1968-2021)

30 Mar 2021

Complete Overview of Prohibited Substances and Prohibited Methods from the IOC Lists (1968-2003) & WADA Prohibited Lists (2004- ) / ed. M.C. Tuk, Olivier de Hon. - Doping Authority Netherlands (Dopingautoriteit); Anti-Doping Knowledge Center (ADKC), 2021

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Metabolic studies of hypoxia-inducible factor stabilisers IOX2, IOX3 and IOX4 (in vitro) for doping control

17 Jan 2021

Metabolic studies of hypoxia-inducible factor stabilisers IOX2, IOX3 and IOX4 (in vitro) for doping control / Moses Philip, Binoy Mathew, Tajudheen K. Karatt, Zubair Perwad, Michael Benedict Subhahar, Abdul Khader Karakka Kal. - (Drug Testing and Analysis (2021) 17 January)

  • PMID: 33458935
  • DOI: 10.1002/dta.3000


Abstract

The transcriptional activator hypoxia-inducible factor (HIF) is a vital arbitrator in the performance of cellular responses lacking oxygen supply in aerobic organisms. Because these compounds are capable of enhancing the organism's capacity for molecular oxygen transport, they possess great potential for abuse as a performance-enhancing agent in sports. A comprehensive study of the metabolic conversion of the most popular HIF stabilisers such as IOX2, IOX3 and IOX4 using equine liver microsomes (in vitro) is reported. The parents and their metabolites were identified and characterised by liquid chromatography-mass spectrometry in negative ionisation mode using a QExactive high-resolution mass spectrometer. Under the current experimental condition, a total of 10 metabolites for IOX2 (three phase I and seven phase II), nine metabolites for IOX3 (four phase I and five phase II) and five metabolites for IOX4 (three phase I and two phase II) were detected. The outcome of the present study is as follows: (1) all the three IOX candidates are prone to oxidation, results in subsequent monohydroxylated, and some dihydroxylated metabolites. (2) Besides oxidation, there is a possibility of hydrolysis and de-alkylation, which results in corresponding carboxylic acid and amide, respectively. (3) The glucuronide and sulphate conjugate of the parent drugs as well as the monohydroxylated analogues were observed in this study. The characterised in vitro metabolites can potentially serve as target analytes for doping control analysis.

Measurement of urinary cobalt as its complex with 2-(5-chloro-2-pyridylazo)-5-diethylaminophenol by liquid chromatography-tandem mass spectrometry for the purpose of anti-doping control

22 Jan 2021

Measurement of urinary cobalt as its complex with 2-(5-chloro-2-pyridylazo)-5-diethylaminophenol by liquid chromatography-tandem mass spectrometry for the purpose of anti-doping control / Tim Sobolevsky, Brian Ahrens. - (Drug Testing and Analysis (2021) 22 January)

  • PMID: 33484083
  • DOI: 10.1002/dta.3004


Abstract

Cobalt is well known for its ability to stimulate erythropoiesis via stabilization of hypoxia-inducible factors. In sports, this can provide a competitive benefit to athletes, so the World Anti-Doping Agency prohibits the use of cobalt in any form except its cobalamin vitamers. As of now, cobalt in biological fluids is detected by inductively coupled plasma mass spectrometry (ICP-MS), a technique which has very limited availability in anti-doping laboratories. Therefore, a quantitative method based on liquid chromatography-tandem mass spectrometry capable of measuring urinary cobalt in the form of its complex with 2-(5-chloro-2-pyridylazo)-5-diethylaminophenol (5-Cl-PADAP) has been developed and validated. A cobalt complex with deuterium-labeled 5-Cl-PADAP was used as internal standard. The method was found linear over the concentration range of 5-500 ng/ml with a combined standard uncertainty less than 10% at 15, 200, and 450 ng/ml. Stability of cobalt ions in urine was investigated over the course of 2 months; the concentration of free Co2+ was observed to decline by approximately 50% but restored upon hydrolysis with hydrochloric acid. Unlike ICP-MS, this method is practically unaffected by the presence of cyanocobalamin as the latter is resistant to acid hydrolysis. Notwithstanding the lack of formalized threshold concentration of cobalt in urine, it is highly desirable that more anti-doping laboratories engage in testing for cobalt levels to better understand the prevalence of cobalt misuse in athletes. Given that cobalt salts are inexpensive and easily obtainable, the risk of such abuse should not be underestimated.

A validated method for the quantification of IOX-2, a potent prolyl hydroxylase inhibitor in equine urine and plasma using liquid chromatography-high-resolution mass spectrometry

2 Feb 2021

A validated method for the quantification of IOX-2, a potent prolyl hydroxylase inhibitor in equine urine and plasma using liquid chromatography-high-resolution mass spectrometry / Ezra Mikhail, Erik Siccardi, Ali Bawazir, Ambika Rajesh, Seetharani Prathyush, Duaa Mohammad Kamal Al Wazani, Muhammed Sabeek, Thomas John. - (Drug Testing and Analysis (2021) 2 February)

  • PMID: 33533201
  • DOI: 10.1002/dta.3010

Abstract

IOX-2 is a potent inhibitor of enzyme prolyl hydroxylases-2 (PHD) that plays a critical role in regulating hypoxia inducible factor (HIF) abundance and oxygen homeostasis. Federation for Equestrian Sports has listed HIF activators as prohibited substances to prevent their usage in doping. Consequently, it became essential to develop adequate knowledge and testing methods to detect it in equine sports drug testing samples. The validated method utilizes ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry in order to detect extremely low concentration of the analyte present in both matrices. Confirmation for the presence of the analyte was achieved by comparing ion ratios, retention time, and accurate mass. Method linear range for plasma was between 0.25 to 100 ng/ml, and limit of detection (LOD) was 0.075 ng/ml. The linear range for urine was 0.125 to 100 ng/ml, and LOD was 0.025 ng/ml. Intraday precision at 0.5, 10, and 50 ng/ml was between 4.0% and 9.7% for plasma and 4.2% and 10.4% for urine. Accuracy at 0.5, 10, and 50 ng/ml was between 91% and 94% for plasma and 99% and 103% for urine. Elimination profile of IOX-2 in equine plasma and urine was carried out using the developed method in which two horses were intravenously administered IOX-2 and samples were collected. Metabolic profile in plasma and urine was investigated. IOX-2 was detected for a minimum of 54 and 151 h of post administration in plasma and urine, respectively, thereby providing a valuable tool for evaluating its misuse in equine racing.

Keywords: IOX-2; PHD inhibitor; equine doping; plasma; urine.

Anabolic steroid use and ischaemic stroke in a young fitness enthusiast

4 Feb 2021

Anabolic steroid use and ischaemic stroke in a young fitness enthusiast / James Choulerton, Nishan Guha, Rebecca Squires. - (BMJ Case Reports 14 (2021) 2 (4 February))

  • PMID: 33542023
  • PMCID: PMC7868212 (available on 2023-02-04)
  • DOI: 10.1136/bcr-2020-234241


Abstract

We report a case of ischaemic stroke in a 34-year-old male recreational bodybuilder following a 3-month period of anabolic androgenic steroid (AAS) use and 1-month period of 'post-cycle therapy' (tamoxifen and clomiphene citrate), the latter treatments aimed at restoring normal endogenous testosterone production after initial AAS use. We hypothesise a transient drug-related prothrombotic state with paradoxical embolisation via an atrial septal defect which was later found on bubble echocardiogram. We highlight a rare but important cause of stroke in younger patients which is relevant given the increasing use of AAS misuse among casual fitness enthusiasts. We explore the various possible mechanisms by which AAS use can increase ischaemic stroke risk in such patients.

Effects of hypoxic preconditioning combined with altitude training on CD55, CD59 and the immune function of swimmers

17 Jan 2021

Effects of hypoxic preconditioning combined with altitude training on CD55, CD59 and the immune function of swimmers / Xi Wang, Lin Huang, Huan Gao. - (Annals of Palliative Medicine 10 (2021) 1 (January); p. 509-517)

  • PMID: 33545782
  • DOI: 10.21037/apm-20-2379


Abstract

Background: Hypoxic preconditioning refers to a certain intensity and time of hypoxic exposure before hypoxic stress, which makes the body produce endogenous protection to enhance the body's tolerance to subsequent more severe hypoxia. However, there are few studies on the effects of hypoxic preconditioning combined with altitude training on the immune system of athletes.

Methods: Nine swimmers from Shanghai underwent 3-week hypoxic preconditioning [living high-training low (HiLo)] combined with 3-week altitude training. CD55 and CD59 expression in red blood cells (RBCs), CD55 and CD59 expression in white blood cells (WBCs), RBC count, WBC count, T lymphocyte CD3, CD4, CD8 expression, and immunoglobulins IgG, IgM, and IgA were measured 4 times: before the start of hypoxic preconditioning, in the first week of hypoxic preconditioning, at the end of hypoxic preconditioning (i.e., before the start of altitude training), and at the end of altitude training.

Results: CD55 and CD59 expression in RBCs significantly increased in the first week of hypoxic preconditioning (P<0.05), returned to baseline levels at the end of preconditioning, and significantly increased again during altitude training (P&lt0.05). CD55 and CD59 expression in WBCs decreased significantly during hypoxic preconditioning (P&lt0.05) and increased significantly during altitude training (P&lt0.05). CD3 expression first decreased and then increased in the hypoxic preconditioning phase, then decreased again in the altitude training phase. However, there was no significant difference in each phase. CD4/CD8 expression after altitude training was significantly lower than that before altitude training (P&lt0.05), but was not significantly different from that before the start of hypoxic preconditioning. IgG, IgM, and IgA did not fluctuate significantly throughout the experimental phase.

Conclusions: After hypoxic preconditioning combined with altitude training, the expression of CD55 and CD59 on the surface of RBCs and WBCs increased significantly, and T lymphocyte CD4/CD8 expression also increased. These results suggest an improvement in the complement regulation system and RBC immune function. Hypoxic preconditioning can therefore improve immunity and enhance the physical function of athletes during altitude training.

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