Evaluation of testosterone/epitestosterone ratio influential factors as determined in doping analysis.
The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in doping analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair analysis, and high-performance liquid chromatography-mass spectrometry, are discussed. (+info)
A novel method utilising markers of altered erythropoiesis for the detection of recombinant human erythropoietin abuse in athletes.
BACKGROUND AND OBJECTIVE: The use of recombinant human erythropoietin (r-HuEPO) to enhance athletic performance is prohibited. Existing tests cannot readily differentiate between exogenous and endogenous EPO. Therefore the aim of our study was to investigate possible indirect detection of r-HuEPO use via blood markers of altered erythropoiesis. DESIGN AND METHODS: Twenty-seven recreational athletes were assigned to three groups prior to a 25 day drug administration phase, with the following protocols: EPO+IM group (n = 10), 50 Ukg(-1) r-HuEPO at a frequency of 3wk(-1), 100 mg intramuscular (IM) iron 1wk(-1) and a sham iron tablet daily; EPO+OR group (n = 8), 50 U.kg(-1) r-HuEPO 3wk(-1), sham iron injection 1wk(-1) and 105 mg of oral elemental iron daily; placebo group (n = 9), sham r-HuEPO injections 3wk(-1), sham iron injections 1wk(-1) and sham iron tablets daily. Each group was monitored during and for 4 weeks after drug administration. RESULTS: Models incorporating combinations of the variables reticulocyte hematocrit (RetHct), serum EPO, soluble transferrin receptor, hematocrit (Hct) and % macrocytes were analyzed by logistic regression. One model (ON-model) repeatedly identified 94-100% of r-HuEPO group members during the final 2 wk of the r-HuEPO administration phase. One false positive was recorded from a possible 189. Another model (OFF-model) incorporating RetHct, EPO and Hct was applied during the wash-out phase and, during the period of 12-21 days after the last r-HuEPO injection, it repeatedly identified 67-72% of recent users with no false positives. INTERPRETATION AND CONCLUSIONS: Multiple indirect hematologic and biochemical markers used simultaneously are potentially effective for identifying current or recent users of r-HuEPO. (+info)
Discrimination of prohibited oral use of salbutamol from authorized inhaled asthma treatment.
BACKGROUND: The administration of salbutamol is permitted only by inhalation by the International Olympic Committee (IOC) for the management of asthma and exercise-induced asthma in athletes. The establishment of criteria to distinguish between the (IOC) authorized use (inhaled) and the (IOC) prohibited use (oral) of salbutamol appeared possible using simultaneous evaluation of variables based on the concentration of nonconjugated enantiomers of salbutamol excreted in urine. METHODS: Urine was collected from asthmatic and nonasthmatic swimmers who had received various preexercise doses of oral (five doses of 4 mg) or inhaled (two doses of 100 microgram) salbutamol. Urine was also obtained from subjects who had received the maximum dosage of inhaled salbutamol advisable for competing athletes to provide protection from exercise-induced asthma and treatment of asthma (1600 microgram in 24 h, 800 microgram being in the last 4 h). All samples were analyzed to determine the total amount of unchanged salbutamol excreted in urine and the ratio between the S: and R: enantiomers. RESULTS: The discriminant function D = -3.776 + 1.46 x 10(-3) ([S:(+)] + [R:(-)]) + 1.012 ([S:(+)]/[R(-)]) can be used to classify data into two groups, inhaled and oral. The confirmatory criterion suggested (cutoff at D = 1.06, 4 SD from the mean D value of the inhaled distribution) has been verified in different sets of samples showing suspicious concentrations by conventional screening procedures in doping control. An 11.8% false-negative (oral classified as inhaled) rate is assumed with the confirmatory criterion proposed, but virtually no false positives (inhaled classified as oral) are obtained (<1 in 33 000). CONCLUSIONS: The overall procedure recommended is to screen all samples and to apply the confirmation criterion proposed to samples showing free racemic salbutamol concentrations >500 microgram/L by gas chromatography-mass spectrometry or free + conjugated racemic salbutamol concentrations >1400 microgram/L by ELISA. (+info)
Performance characteristics of a carbon isotope ratio method for detecting doping with testosterone based on urine diols: controls and athletes with elevated testosterone/epitestosterone ratios.
BACKGROUND: Carbon isotope ratio methods are used in doping control to determine whether urinary steroids are endogenous or pharmaceutical. METHODS: Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was used to determine the delta(13)C values for 5 beta-androstane-3 alpha,17 beta-diyl diacetate (5 beta A), 5 alpha-androstane-3 alpha,17 beta-diyl diacetate (5 alpha A), and 5 beta-pregnane-3 alpha,20 alpha-diyl diacetate (5 beta P) in a control group of 73 healthy males and 6 athletes with testosterone/epitestosterone ratios (T/E) >6. RESULTS: The within-assay precision SDs for 5 beta A, 5 alpha A, and 5 beta P were +/- 0.27 per thousand, +/- 0.38 per thousand, and +/- 0.28 per thousand, respectively. The between-assay precision SDs ranged from +/- 0.40 per thousand to +/- 0.52 per thousand. The system suitability and batch acceptance scheme is based on SDs. For the control group, the mean delta(13)C (SD) values were -25.69 per thousand (+/- 0.92 per thousand), -26.35 per thousand (+/- 0.68 per thousand), and -24.26 per thousand (+/- 0.70 per thousand), for 5 beta A, 5 alpha A, and 5 beta P, respectively. 5 beta P was greater than 5 beta A and 5 alpha A (P <0.01), and 5 beta A was greater than 5 alpha A (P <0.01). The means - 3 SD were -28.46 per thousand, -28.39 per thousand, and -26.37 per thousand for 5 beta A, 5 alpha A, and 5 beta P, respectively. The maximum difference between 5 beta P and 5 beta A was 3.2 per thousand, and the maximum 5 beta A/5 beta P was 1.13. Three athletes with chronically elevated T/Es had delta(13)C values consistent with testosterone administration and three did not. CONCLUSIONS: This GC-C-IRMS assay of urine diols has low within- and between-assay SDs; therefore, analysis of one urine sample suffices for doping control. The means, SDs, +/-3 SDs, and ranges of delta(13)C values in a control group are established. In comparison, testosterone users have low 5 beta A and 5 alpha A, large differences between 5 beta A or 5 alpha A and 5 beta P, and high 5 beta A/5 beta P and 5 alpha A/5 beta P ratios. (+info)
Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis.
BACKGROUND AND OBJECTIVES: The detection of recombinant human erythropoietin (r-HuEPO) abuse by athletes remains problematic. The main aim of this study was to demonstrate that the five indirect markers of altered erythropoiesis identified in our earlier work were reliable evidence of current or recently discontinued r-HuEPO use. A subsidiary aim was to refine weightings of the five markers in the initial model using a much larger data set than in the pilot study. A final aim was to verify that the hematologic response to r-HuEPO did not differ between Caucasian and Asiatic subjects. DESIGN AND METHODS: Recreational athletes resident in Sydney, Australia (Sydney, n = 49; 16 women, 33 men) or Beijing, China (Beijing, n=24; 12 women, 12 men) were randomly assigned to r-HuEPO or placebo groups prior to a 25 day administration phase. Injections of r-HuEPO (or saline) were administered double-blind at a dose of 50 IU/kg three times per week, with oral iron (105 mg) or placebo supplements taken daily by all subjects. Blood profiles were monitored during and for 4 weeks after drug administration for hematocrit (Hct), reticulocyte hematocrit (RetHct), percent macrocytes (%Macro), serum erythropoietin (EPO) and soluble transferrin receptor (sTfr), since we had previously shown that these five variables were indicative of r-HuEPO use. RESULTS: The changes in Hct, RetHct, %Macro, EPO and sTfr in the Sydney trial were qualitatively very similar to the changes noted in our previous administration trial involving recreational athletes of similar genetic origin. Statistical models developed from Fisher's discriminant analysis were able to categorize the user and placebo groups correctly. The same hematologic response was demonstrated in Beijing athletes also administered r-HuEPO. INTERPRETATION AND CONCLUSIONS: This paper confirms that r-HuEPO administration causes a predictable and reproducible hematologic response. These markers are disturbed both during and for several weeks following r-HuEPO administration. This work establishes an indirect blood test which offers a useful means of detecting and deterring r-HuEPO abuse. Ethnicity did not influence the markers identified as being able to detect athletes who abuse r-HuEPO. (+info)
Clenbuterol in the horse: confirmation and quantitation of serum clenbuterol by LC-MS-MS after oral and intratracheal administration.
Clenbuterol is a beta2 agonist/antagonist bronchodilator, and its identification in post-race samples may lead to sanctions. The objective of this study was to develop a specific and highly sensitive serum quantitation method for clenbuterol that would allow effective regulatory control of this agent in horses. Therefore, clenbuterol-d9 was synthesized for use as an internal standard, an automated solid-phase extraction method was developed, and both were used in conjunction with a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to allow unequivocal identification and quantitation of clenbuterol in 2 mL of serum at concentrations as low as 10 pg/mL. Five horses were dosed with oral clenbuterol (0.8 microg/kg, BID) for 10 days, and serum was collected for 14 days thereafter. Serum clenbuterol showed mean trough concentrations of approximately 150 pg/mL. After the last dose on day 10, serum clenbuterol reached a peak of approximately 500 pg/mL and then declined with a half-life of approximately 7 h. Serum clenbuterol declined to 30 and 10 pg/mL at 48 and 72 h after dosing, respectively. By 96 h after dosing, the concentration was below 4 pg/mL, the limit of detection for this method. Compared with previous results obtained in parallel urinary experiments, the serum-based approach was more reliable and satisfactory for regulation of the use of clenbuterol. Clenbuterol (90 microg) was also administered intratracheally to five horses. Peak serum concentrations of approximately 230 pg/mL were detected 10 min after administration, dropping to approximately 50 pg/mL within 30 min and declining much more slowly thereafter. These observations suggest that intratracheal administration of clenbuterol shortly before race time can be detected with this serum test. Traditionally, equine drug testing has been dependent on urine testing because of the small volume of serum samples and the low concentrations of drugs found therein. Using LC-MS-MS testing, it is now possible to unequivocally identify and quantitate low concentrations (10 pg/mL) of drugs in serum. Based on the utility of this approach, the speed with which new tests can be developed, and the confidence with which the findings can be applied in the forensic situation, this approach offers considerable scientific and regulatory advantages over more traditional urine testing approaches. (+info)
The prevalence of the use of androgenic anabolic steroids by adolescents in a county of Sweden.
BACKGROUND: The prevalence of the use of androgenic anabolic steroids has been poorly studied in Europe. This study was undertaken to examine the prevalence of the misuse--the non-medical use--of androgenic anabolic steroids among adolescents in a county of Sweden. METHODS: The total population of 16 and 17 year old male and female adolescents in a county on the south-west coast of Sweden was studied. The investigation was done by an anonymous multiple-choice questionnaire. The questionnaire was completed by 5,827 pupils and statistically analysed. The participation rate was 95%. RESULTS: Among male adolescents 16 and 17 years old, 3.6% and 2.8% had misused androgenic anabolic steroids, respectively. These male adolescents had also misused alcohol, growth hormones and narcotic drugs more than the steroid hormone non-users. Among female adolescents there was no recorded misuse of these drugs (0.0%). CONCLUSIONS: The misuse of androgenic anabolic steroids is a reality in both small and large municipalities in Sweden. The prevalence figures are higher among 16 year old compared to 17 year old male adolescents. There is an association between this drug misuse and other substance misuse such as narcotic drugs. Female adolescents do not misuse steroid hormones. The findings indicate the need for preventive work among male adolescents in order to induce adolescents not to start misusing androgenic anabolic steroids. (+info)
Insulin, growth hormone and sport.
This review examines some interesting 'new' histories of insulin and reviews our current understanding of its physiological actions and synergy with GH in the regulation of metabolism and body composition. It reviews the history of GH abuse that antedates by many years the awareness of endocrinologists to its potent anabolic actions. Promising methods for detection of GH abuse have been developed but have yet to be sufficiently well validated to be ready for introduction into competitive sport. So far, there are two promising avenues for detecting GH abuse. The first uses immunoassays that can distinguish the isomers of pituitary-derived GH from the monomer of recombinant human GH. The second works through demonstrating circulating concentrations of one or more GH-sensitive substances that exceed the extremes of normal physiological variability. Both methods require blood rather than urine samples. The first method has a window of opportunity lasting about 24 h after an injection and is most suitable for 'out of competition' testing. The second method has reasonable sensitivity for as long as 2 weeks after the last injection of GH and is uninfluenced by extreme exercise and suitable for post-competition samples. This method has a greater sensitivity in men than in women. The specificity of both methods seems acceptably high but lawyers need to decide what level of scientific probability is needed to obtain a conviction. Both methods need further validation before implementation. Research work carried out as part of the fight against doping in sport has opened up a new and exciting area of endocrinology. (+info)