The effect of athletes` hyperhydration on the urinary 'steroid profile' markers in doping control analysis.

The urinary 'steroid profile' in doping control analysis is a powerful tool aimed at detecting intra-individual deviations related to the abuse of endogenous steroids. Factors altering the steroid profile include, among others, the excessive fluid intake leading to low endogenous steroids concentrations compared to an individual's normal values. Cases report the use of hyperhydration by athletes as a masking method during anti-doping urine sample collection. Seven healthy physically active non-smoking Caucasian males were examined for a 72-hour period using water and a commercial sports drink as hyperhydration agents (20 mL/kg body weight). Urine samples were collected and analyzed according to World Anti-Doping Agency (WADA) technical documents. Although, significant differences were observed on the endogenous steroid concentrations under the studied hyperhydration conditions, specific gravity adjustment based on a reference value of 1.020 can eliminate the dilution induced effect. Adjustment methods based on creatinine and urinary flow rate were also examined; however, specific gravity was the optimum method in terms of effectiveness to adjust concentrations close to the baseline steroid profile and practicability. No significant effect on the urinary steroid ratios was observed with variability values within 30% of the mean for the majority of data. Furthermore, no masking on the detection ability of endogenous steroids was observed due to hyperhydration. It can be concluded that any deviation on the endogenous steroid concentrations due to excessive fluid intake can be compensated by the specific gravity adjustment and therefore, hyperhydration is not effective as a masking method on the detection of the abuse of endogenous steroids.

The effect of cumulative endurance exercise on leptin and adiponectin and their role as markers to monitor training load.

Leptin and adiponectin play an essential role in energy metabolism. Leptin has also been proposed as a marker for monitoring training load. So far, no studies have investigated the variability of these hormones in athletes and how they are regulated during cumulative exercise. This study monitored leptin and adiponectin in 15 endurance athletes twice daily in the days before, during and after a 9-day simulated cycling stage race. Adiponectin significantly increased during the race (p = 0.001) and recovery periods (p = 0.002) when compared to the baseline, while leptin decreased significantly during the race (p < 0.0001) and returned to baseline levels during the recovery period. Intra-individual variability was substantially lower than inter-individual variability for both hormones (leptin 34.1 vs. 53.5%, adiponectin 19% vs. 37.2%). With regards to exercise, this study demonstrated that with sufficient, sustained energy expenditure, leptin concentrations can decrease within the first 24 hours. Under the investigated conditions there also appears to be an optimal leptin concentration which ensures stable energy homeostasis, as there was no significant decrease over the subsequent race days. In healthy endurance athletes the recovery of leptin takes 48-72 hours and may even show a supercompensation-like effect. For adiponectin, significant increases were observed within 5 days of commencing racing, with these elevated values failing to return to baseline levels after 3 days of recovery. Additionally, when using leptin and adiponectin to monitor training loads, establishing individual threshold values improves their sensitivity.

The effect of fasting during Ramadan on parameters of the haematological and steroidal modules of the athletes biological passport - a pilot study.

This study investigated the effect of Ramadan on the haematological and steroid module of the Athletes Biological Passport (ABP) of the World Anti-Doping Agency (WADA). Nine healthy physically active subjects were tested in the morning and afternoon for two days before and three days during Ramadan. Sample collection and all analyses were performed according to WADA technical documents. Although there were significant changes in the haemoglobin concentration during Ramadan, especially during the first fasting week, none of the subjects in this study exceeded the individually calculated thresholds of the ABP. No significant effects on testosterone/epitestosterone (T/E) ratio were observed but only the afternoon specific gravity (SG) of the urine was elevated. Thus, when urinary steroid concentrations are required, SG corrections need to be performed. The haematological and the steroid module of the ABP can be reliably applied during Ramadan as the observed changes are only marginal.

Markers of mesterolone abuse in sulfate fraction for doping control in human urine.

This manuscript describes the direct detection of mesteroloe sulfo-conjugated metabolites by liquid chromatography/quadrupole/time of flight mass spectrometry (LC/Q/TOFMS) with special focus on evaluation of their retrospective detectability and their structure elucidation. A comparison of their long-term detectability, with the mesterolone main metabolite (1α-methyl-5α-androstan-3α-ol-17-one) excreted in glucuronide fraction and detected by gas chromatography/high resolution mass spectrometry (GC/HRMS), is also presented. Studies on mesterolone were performed with samples obtained from two excretion studies after single oral administration of Proviron© by healthy volunteers. Potential sulfate metabolites were detected in post administration samples after liquid-liquid extraction (LLE) with ethyl acetate and LC/TOFMS analysis, in negative mode. Twelve mesterolone sulfate metabolites from the first excretion study and nine from the second were subsequently confirmed by LC/Q/TOFMS. Finally, six mesterolone sulfate metabolites were considered important taking into account their abundance and long-term detectability, encoded as M1, M2, M4, M5, M6 and M7. The proposed mesterolone sulfate metabolites M1, M2, M4 and M5 (excreted as sulfates) have the same retrospectivity with the main mesterolone metabolite, excreted in glucuronide fraction. For metabolite characterization, LC fractionation was performed. The metabolites were identified and characterized by GC/MS, after solvolysis and derivatization. Combined mass spectra data from trimethyl-silyl (TMS), TMS-enolTMS and methoxime-TMS derivatives were taken into account for the characterization of these metabolites. It was concluded that M1 is 1α-methyl-5α-androstan-3ß-ol-17 one, M2 is 1α-methyl-5α-androstan-3α-ol-17 one, M4 is 1α-methyl-5a-androstan-3ß, 16z-diol-17-one, M5 is 1α-methyl-5α-androstan-17z,4ξ-diol-3one, M6 is 1α-methyl-5α-androstan-3z,6z-diol-17-one and M7 is 4z-hydroxy-1α-methyl-5α-androstan-3,17-dione.

Variability of serum markers of erythropoiesis during 6 days of racing in highly trained cyclists.

The athlete biological passport for the fight against doping is currently based on longitudinal monitoring for abnormal changes in cellular blood parameters. Serum parameters related to altered erythropoiesis could be considered for inclusion in the passport. The aim of this study was to quantify the changes in such parameters in athletes during a period of intense exercise.12 highly trained cyclists tapered for 3 days before 6 days of simulated intense stage racing. Morning and afternoon blood samples were taken on most days and analysed for total protein, albumin, soluble transferrin receptor and ferritin concentrations. Plasma volume was determined via total haemoglobin mass measured by carbon-monoxide rebreathing. Percent changes in means from baseline and percent standard errors of measurement (analytical error plus intra-athlete variation) on each measurement occasion were estimated with mixed linear modelling of log-transformed measures. Means of all variables changed substantially in the days following the onset of racing, ranging from -13% (haemoglobin concentration) to +27% (ferritin). After the second day, errors of measurement were generally twice those at baseline. Plasma variables were affected by heavy exercise, either because of changes in plasma volume (total protein, albumin, haemoglobin), acute phase/inflammatory reactions (ferritin) or both (soluble transferrin receptor). These effects need to be taken into consideration when integrating a plasma parameter into the biological passport model for athletes.

The effect of a period of intensive exercise on the isoform test to detect growth hormone doping in sports.

OBJECTIVE: The major objective of this study was to investigate the effects of several days of intense exercise on growth hormone (hGH) testing using the World Anti-Doping Agencies hGH isoform differential immunoassays. Additionally the effects of circadian variation and exercise type on the isoform ratios were also investigated. STUDY DESIGN: 15 male athletes performed a simulated nine day cycling stage race. Blood samples were collected twice daily over a period of 15 days (stage race+three days before and after). hGH isoforms were analysed by the official WADA immunoassays (CMZ Assay GmbH). RESULTS: All measured isoform ratios were far below the WADA decision limits for an adverse analytical finding. Changes in the isoform ratios could not be clearly connected to circadian variation, exercise duration or intensity. CONCLUSIONS: The present study demonstrates that the hGH isoform ratios are not significantly affected by exercise or circadian variation. We demonstrated that heavy, long term exercise does not interfere with the decision limits for an adverse analytical finding.

The impact of long-haul air travel on variables of the athlete's biological passport.

INTRODUCTION: Dehydration, fluid shifts or changes in coagulation occurring during air travel can trigger distinct reactions in the haematological system. Athletes are concerned that these effects might impair sporting performance, increase the risk of thrombosis or cause abnormalities in blood values that might be mistaken for doping in the 'Athlete's biological passport' (ABP) a longitudinal monitoring of haematological variables in antidoping. The aim of the study was to investigate key variables of the ABP before and after a long-haul flight in athletes. METHODS: Fifteen endurance athletes were submitted to ABP blood samples in the morning before and after arrival of an 8 h flight. Two additional samples were obtained in the morning and the evening 3 days after the travel. Twelve nontravelling subjects served as controls. RESULTS: Haemoglobin concentration was higher before than after travel in athletes (+0.5 g/dL, P = 0.038), a similar pattern was observed 3 days after the travel. No difference was observed in the control group. Reticulocyte% did not show any significant changes in neither of the groups. CONCLUSION: The observed changes are in line with normal diurnal variations. There is no indication that travel will affect haematological variables in way that might be mistaken for blood doping.