Abstract
Doping is a multifaceted phenomenon, officially defined as the occurrence of one or more of the antidoping rule violations as stated in the World Anti-Doping Code: the presence of a prohibited substance (or its metabolites), the misuse or attempted use or possession or trafficking of a prohibited substance or method, the refusal or failure for testing, the (attempted) administration to any athlete of any prohibited method or substance and tampering or any attempt to tamper collected samples are all considered as doping offences.Excessive fluid intake, i.e., hyperhydration is not prohibited as a practice according to the World Anti-Doping Agency`s (WADA) current regulations. However, hyperhydration may be adopted by athletes as a masking method during anti-doping sample collection to influence the excretion patterns of doping agents and, therefore, manipulate their detection. The aim of the present study was to evaluate the role of hyperhydration as a potential doping masking method, by ...
Doping is a multifaceted phenomenon, officially defined as the occurrence of one or more of the antidoping rule violations as stated in the World Anti-Doping Code: the presence of a prohibited substance (or its metabolites), the misuse or attempted use or possession or trafficking of a prohibited substance or method, the refusal or failure for testing, the (attempted) administration to any athlete of any prohibited method or substance and tampering or any attempt to tamper collected samples are all considered as doping offences.Excessive fluid intake, i.e., hyperhydration is not prohibited as a practice according to the World Anti-Doping Agency`s (WADA) current regulations. However, hyperhydration may be adopted by athletes as a masking method during anti-doping sample collection to influence the excretion patterns of doping agents and, therefore, manipulate their detection. The aim of the present study was to evaluate the role of hyperhydration as a potential doping masking method, by assessing the effect of excessive fluid ingestion on the urinary steroidal and haematological Athlete Biological Passport (ABP), on the urinary endogenous luteinizing hormone (LH) excretion patterns and on the blood (plasma/serum) and urinary pharmacokinetic (PK) characteristics and detection of two representative prohibited substances, namely, Epoetin beta (rHuEPO) and budesonide (BDS) after a single subcutaneous and oral dose administration, respectively. The presentation of the study results is structured into three Parts (A-C). In the first part, “Part A: Introduction & Study design”, the rational of the study and a description of the study protocol and methodology is given together with the study aim and objectives, as “Chapter 1: Introduction” and “Chapter 2: Study design and methodology”. The full study protocol, as reviewed by the Health, Medical and Research Committee of WADA (10D21CG) and approved by the Ethics Committee of the Anti-Doping Lab Qatar, Doha, Qatar (F2013000006) is presented in Annex C. In brief, seven healthy, Caucasian, recreationally active non-smoking male volunteers were enrolled in the study and participated in all the study phases. The study comprised the following phases: a baseline phase, i.e., no drug administration, with and without hyperhydration and two drug phases, i.e., drug administration, with and without hyperhydration. The aim of the baseline phase was to examine the hyperhydration effect on the blood and urinary profiles of endogenous substances, i.e., endogenous anabolic androgenic steroids, endogenous erythropoietin, endogenous luteinizing hormone and on the haemodynamic markers included in the ABP. The aim of the drug phase was to perform a PK study of the selected doping substances, i.e., rHuEPO and BDS, administered as a single dose, in order to examine the hyperhydration effect on the serum and urinary concentration-time profiles and subsequent detection of each substance during doping control analysis. Hyperhydration was induced by either water or a commercial sports drink within 30 minutes by the ingestion of a bolus of 20 mL hyperhydration agent/kg body weight (i.e., 1400 mL for a 70-kg male). The collection and storage of urine and blood samples were performed according to the WADA`s official guidelines, following the sampling schedule described in the study protocol. All the serum and urine sample analysis were performed by validated analytical methods according to the WADA`s official guidelines and technical documents. Statistical data analysis was performed by SPSS version 25.0 (IBM SPSS Statistics for Windows, Version 25.0, IBM Corporation, Armonk, NY, USA) software package. Phoenix® version 8.0 PK/PD software package (Certara, Princeton, NJ, USA) was used for the PK data analysis. Non-compartmental analysis (NCA) was performed in all the cases while PK modeling was performed for rHuEPO.The second and third part of the study presentation (Parts B & C) are structured into chapters and each chapter is organized to be read independently. More specifically:“Part B: The effect of athletes` hyperhydration on haematological parameters and urinary profiles of endogenous biomarkers in doping control analysis”, consists of three chapters describing the effect of hyperhydration on the haematological parameters of the ABP (Chapter 3), on the urinary “steroid profile” markers (Chapter 4) and on the urinary endogenous LH concentrations (Chapter 5), respectively, as follows:Chapter 3: Effect of hyperhydration on the haematological parameters of the Athlete Biological Passport in doping control analysisABP is an indirect approach, implemented by WADA, aimed at detecting blood manipulation based on abnormal changes in haematological markers. Cases report the use of hyperhydration by athletes as a masking method during anti-doping urine sample collection which could also potentially mask suspicious fluctuations on ABP profiles. This study investigated the hyperhydration effect of different agents on the ABP haematological markers, i.e., haemoglobin concentration, reticulocyte percentage and calculated OFF-hr score, with and without rHuEPO administration. Seven healthy, physically active volunteers were recruited for a five-week period; Baseline Phase (Weeks 1 to 2) and rHuEPO Phase (Weeks 3 to 5). Water and a commercial sports drink were used as hyperhydration agents at a volume of 20 mL/kg body weight. According to the study protocol, to examine the hyperhydration effect on the normal ABP profile of each volunteer, only hyperhydration was implemented at 0, 24 and 48 hours during the baseline phase by either water or sports drink. During the rHuEPO phase, volunteers received a single 3000 IU dose of Epoetin beta with hyperhydration to be implemented at 0, 24 and 48 hours after drug administration. Blood and urine samples were collected and analyzed according to the WADA guidelines and technical documents. No significant effect (P > 0.05, 95% CI) on ABP markers was observed due to hyperhydration at any time during the study. Pre- and post-hyperhydration data were not significant different independently of the hyperhydration agent compared to individual baseline data with and without EPO administration. In conclusion, hyperhydration does not affect ABP haematological markers.Chapter 4: Effect of hyperhydration on the urinary ‘steroid profile’ markers in doping control analysisThe 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. The 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 WADA`s technical documents. Although, significant differences were observed on the endogenous steroid concentrations under the studied hyperhydration conditions, SG-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, SG 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 SG-adjustment and, therefore, hyperhydration is not effective as a masking method on the detection of the abuse of endogenous steroids.Chapter 5: Effect of hyperhydration on the urinary luteinizing hormone concentrations in doping control analysisLow urinary LH values have been discussed as a marker to detect steroid abuse. However, suppressed LH concentrations related to highly diluted urine samples could be a misleading indication of anabolic steroid abuse. One aim of the present study was to examine the effect of hyperhydration on the interpretation of LH findings during doping control analysis and to investigate different possibilities to correct volume related changes in urinary LH concentrations. The 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 WADA`s technical documents. Baseline urinary LH concentrations for each individual were within the acceptable physiological range (7.11 ± 5.42 IU/L). Comparison of the measured LH values for both hyperhydration phases (Phase A: 4.24 ± 5.60 IU/L, Phase B: 4.74 ± 4.72 IU/L) with the baseline (‘normal’) values showed significant differences (Phase A: P < 0.001, Phase B: P < 0.001) suggesting the clear effect of the urine dilution due to hyperhydration. However, adjustment of urinary LH concentrations by specific gravity based on a reference value of 1.020 seems to adequately correct the hyperhydration induced decrease on LH levels. “Part C: The effect of athletes’ hyperhydration on the detection sensitivity and PK profiles in both urine and blood of exogenously administered prohibited substances in doping control analysis” consists of two chapters describing the effect of hyperhydration on the serum and urinary concentration-time profiles of Epoetin beta (Chapter 6) and BDS (Chapter 7) after subcutaneous and oral single dose administration, respectively, as follows:Chapter 6: Effect of hyperhydration on detection sensitivity and pharmacokinetic parameters of recombinant human erythropoietin in urine and serum doping control analysisThe aim of the present study was to assess the hyperhydration effect on the detection sensitivity of rHuEPO by Sodium N-Lauroylsarcosinate (‘Sarcosyl’) Polyacrylamide Gel Electrophoresis (SAR-PAGE) analysis. The influence of hyperhydration on the serum and urinary PK profiles of rHuEPO was also investigated. The seven healthy physically active non-smoking Caucasian males were participated in a 31-day clinical study comprised a baseline (Days 0, 1‒3, 8‒10) and a drug phase (Days 15‒17, 22‒24, 29‒31). Epoetin beta was administered subcutaneously at a single dose of 3000 IU on Days 15, 22 and 29. Hyperhydration was applied in the morning on three consecutive days (Days 1‒3, 8‒10, 22‒24, 29‒31), i.e., 0, 24 and 48 hours after first fluid ingestion. Water and a commercial sports drink were used as hyperhydration agents (20 mL/kg body weight). Serum and urinary concentration-time profiles were best described by a one compartment PK model with first order absorption. Results showed no significant difference (P > 0.05, 95% CI) on serum or urinary EPO concentrations under hyperhydration conditions. No significant differences (P > 0.05, 95% CI) due to hyperhydration for any of the serum PK parameters were also observed. Renal excretion of endogenous and rHuEPO, as reflected on the urinary cumulative amount, was increased approximately twice after hyperhydration and this supports the non-significant difference on the urinary concentrations. Analysis of serum and urine samples was able to detect rHuEPO up to 72 hours after drug administration. The detection window of rHuEPO remained unaffected after water or sports drink ingestion. Hyperhydration had no effect on the detection sensitivity of EPO either in serum or urine samples. Chapter 7: Effect of hyperhydration on the detection sensitivity and pharmacokinetic parameters of BDS and its metabolites in urine and plasma doping control analysisThe aim of the present study was to investigate if hyperhydration could influence the excretion and subsequent detection of BDS and its main metabolites (6β-hydroxy-budesonide and 16α-hydroxy-prednisolone) during doping control analysis by leading to concentrations below the WADA reporting level (30 ng/mL). The influence of hyperhydration on the plasma and urinary PK profiles of BDS and metabolites was also examined. The seven healthy physically active non-smoking Caucasian males were participated in a 15-day clinical study. BDS was administered orally at a single dose of 9 mg on Days 1, 7 and 13. Hyperhydration was applied in the morning on two consecutive days, i.e., 0 and 24 hours after first fluid ingestion. Water and a commercial sports drink were used as hyperhydration agents (20 mL/kg body weight). Results showed no significant difference (P > 0.05, 95% CI) on plasma or urinary PK parameters under hyperhydration conditions for all the analytes. However, significant differences (P < 0.05, 95% CI) due to hyperhydration were observed on the urinary concentrations of BDS and metabolites. To compensate the dilution effect due to hyperhydration, different adjustment methods were applied based on specific gravity, urinary flow rate and creatinine. All the applied methods were able to adjust the concentration values close to the baseline ones for each analyte, however, specific gravity was the optimum method in terms of effectiveness and practicability. Furthermore, no masking on the detection sensitivity of BDS and its metabolites was observed due to hyperhydration either in plasma or urine samples. In conclusion, the present study revealed a clear effect of hyperhydration on the urinary profiles of endogenous biomarkers under the applied experimental conditions. However, concentration adjustment according to the WADA official SG-adjustment method proved to adequately compensate the dilution effect, while the dilution did not affect the detection sensitivity of the applied analytical methods. Based on the results of the present study, SG-adjustment of urinary concentrations of the studied biomarkers independent of the initial SG of the urine sample could be useful. No hyperhydration effect was observed on either serum or urine EPO concentration-time profiles or the reliability, robustness and detection sensitivity of the routine anti-doping analysis of rHuEPO. On the contrary, urinary concentrations of BDS and its metabolites namely, 6OH-BDS and 16OH-PREDN, were affected due to dilution after hyperhydration leading to increased percentage of samples below the reporting level (MRPL) of 30 ng/mL. Nevertheless, the WADA official SG-adjustment method was able to compensate the dilution effect by adjusting the concentration values closed to the baseline and, therefore, decreasing the percentage of samples below the MRPL.Despite the small number of volunteers enrolled, the results of the study are quite clear and promising regarding the effect of hyperhydration on the urine profiles of endogenous biomarkers, as well as on the serum/plasma and urinary profiles of exogenously administered prohibited doping substances as per WADA`s regulations. However, to explore the results of the present study and to further investigate their applicability for doping control purposes, multiple dose administration of the WADA prohibited substances, as well as different scenarios of hyperhydration implementation (e.g., twice a day), would be interesting.
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