RESUMEN
BACKGROUND: Despite implementation of the Athlete Biological Passport 10 years ago, blood doping remains difficult to detect. Thus, there is a need for new biomarkers to increase the sensitivity of the adaptive model. Transcriptomic biomarkers originating from immature reticulocytes may be reliable indicators of blood manipulations. Furthermore, the use of dried blood spots (DBSs) for antidoping purposes constitutes a complementary approach to venous blood collection. Here, we developed a method of quantifying the RNA-based 5'-aminolevulinate synthase 2 (ALAS2) biomarker in DBS. MATERIALS: The technical, interindividual, and intraindividual variabilities of the method, and the effects of storage conditions on the production levels of ALAS2 RNA were assessed. The method was used to monitor erythropoiesis stimulated endogenously (blood withdrawal) or exogenously (injection of recombinant human erythropoietin). RESULTS: When measured over a 7-week period, the intra- and interindividual variabilities of ALAS2 expression in DBS were 12.5%-42.4% and 49%, respectively. Following withdrawal of 1 unit of blood, the ALAS2 RNA in DBS increased significantly for up to 15 days. Variations in the expression level of this biomarker in DBS samples were more marked than those of the conventional hematological parameters, reticulocyte percentage and immature reticulocyte fraction. After exogenous stimulation of erythropoiesis via recombinant human erythropoietin injection, ALAS2 expression in DBS increased by a mean 8-fold. CONCLUSIONS: Monitoring of transcriptomic biomarkers in DBS could complement the measurement of hematological parameters in the Athlete Biological Passport and aid the detection of blood manipulations.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Pruebas con Sangre Seca/métodos , Eritropoyesis/genética , 5-Aminolevulinato Sintetasa/sangre , 5-Aminolevulinato Sintetasa/metabolismo , Adulto , Biomarcadores/sangre , Doping en los Deportes/métodos , Eritropoyetina , Femenino , Voluntarios Sanos , Humanos , Masculino , ARN , TranscriptomaRESUMEN
In the light of recurring anti-doping news, this article discusses some special cases and extends to the future prospects of developing effective anti-doping strategies. After recalling the current principles of the fight against doping, the use of substances tole-rated at a certain threshold, and the therapeutic use exemptions (TUE) are discussed. Authorized substances with a health risk for athletes are discussed before concluding on the development of the athlete's biological passport as a future common ground for anti-doping and medical follow-up of athletes. In conclusion, this approach emphasizes the imperative dialogue between anti-doping organizations and sports medicine in order to defend good practices preserving the intrinsic value of sport.
En réponse aux actualités antidopage, cet article traite quelques cas particuliers et s'étend aux perspectives futures de développement de stratégies antidopage efficaces. Après avoir rappelé les principes actuels de la lutte antidopage, il aborde l'utilisation de substances tolérées à un certain seuil et évoque les autorisations à usage thérapeutique (AUT). Les substances autorisées mais pouvant présenter un risque sanitaire pour les athlètes sont discutées avant de conclure sur le développement du passeport biologique de l'athlète comme futur terrain commun pour la lutte antidopage et le suivi médical des sportifs. En conclusion, cette approche souligne le dialogue impératif entre organisations antidopage et médecine du sport afin de défendre de bonnes pratiques à même de préserver la valeur intrinsèque du sport.
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Doping en los Deportes , Deportes , Atletas , Doping en los Deportes/prevención & control , Humanos , Organizaciones , Detección de Abuso de SustanciasRESUMEN
Sodium-glucose cotransporter (SGLT) 2 inhibition is a well-known target for the treatment of type 2 diabetes, renal disease and chronic heart failure. The protein SGLT2 is encoded by SLC5A2 (Solute Carrier Family 5 Member 2), which is highly expressed in renal cortex, but also in the testes where glucose uptake may be essential for spermatogenesis and androgen synthesis. We postulated that in healthy males, SGLT2 inhibitor therapy may affect gonadal function. We examined the impact on gonadal and steroid hormones in a post-hoc analysis of a double-blind, randomized, placebo-controlled research including 26 healthy males who were given either placebo or empagliflozin 10 mg once daily for four weeks. After one month of empagliflozin, there were no discernible changes in androgen, pituitary gonadotropin hormones, or inhibin B. Regardless of BMI category, the administration of empagliflozin, a highly selective SGLT2 inhibitor, did not alter serum androgen levels in men without diabetes. While SGLT2 is present in the testes, its inhibition does not seem to affect testosterone production in Leydig cells nor inhibin B secretion by the Sertoli cells.
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Compuestos de Bencidrilo , Glucósidos , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Masculino , Humanos , Compuestos de Bencidrilo/farmacología , Glucósidos/farmacología , Adulto , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Método Doble Ciego , Testículo/metabolismo , Testículo/efectos de los fármacos , Testosterona/sangre , Inhibinas/sangre , Inhibinas/metabolismo , Persona de Mediana Edad , Transportador 2 de Sodio-Glucosa/metabolismo , Andrógenos/metabolismo , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Células de Sertoli/metabolismo , Células de Sertoli/efectos de los fármacosRESUMEN
CONTEXT: Detection of endogenous anabolic androgenic steroids (EAAS), like testosterone (T), as doping agents has been improved with the launch of the Steroidal Module of the Athlete Biological Passport (ABP) in urine samples. OBJECTIVE: To target doping practices with EAAS, particularly in individuals with low level of biomarkers excreted in urine, by including new target compounds measured in blood. DESIGN: T and T/androstenedione (T/A4) distributions were obtained from 4 years of anti-doping data and applied as priors to analyze individual profiles from 2 T administration studies in female and male subjects. SETTING: Anti-doping laboratory. Elite athletes (n = 823) and male and female clinical trials subjects (n = 19 and 14, respectively). INTERVENTION(S): Two open-label administration studies were carried out. One involved a control phase period followed by patch and then oral T administration in male volunteers and the other followed female volunteers during 3 menstrual cycles with 28 days of daily transdermal T application during the second month. MAIN OUTCOME MEASURE(S): Serum samples were analyzed for T and A4 and the performance of a longitudinal ABP-based approach was evaluated for T and T/A4. RESULTS: An ABP-based approach set at a 99% specificity flagged all female subjects during the transdermal T application period and 44% of subjects 3 days after the treatment. T showed the best sensitivity (74%) in response to transdermal T application in males. CONCLUSIONS: Inclusion of T and T/A4 as markers in the Steroidal Module can improve the performance of the ABP to identify T transdermal application, particularly in females.
Asunto(s)
Doping en los Deportes , Detección de Abuso de Sustancias , Femenino , Humanos , Masculino , Esteroides Anabólicos Androgénicos , Androstenodiona , Atletas , Esteroides , TestosteronaRESUMEN
The steroidal module of the athlete biological passport (ABP) targets the use of pseudo-endogenous androgenous anabolic steroids in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive oral contraceptive pill (OCP) cycles in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and epitestosterone (E) were below the limit of quantification of 1 ng/ml in 62% of the samples. Biomarkers' variability ranged between 31% and 41%, with a significantly lesser variability for ratios (except for T/E [41%]): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5α-androstane-3α,17ß-diol/5ß-androstane-3α,17ß-diol (p < 0.001). In serum, markers' variability (testosterone: 24%, androstenedione: 23%, dihydrotestosterone: 19%, and T/A4: 16%) was significantly lower than in urine (p < 0.001). Urinary A/Etio increased by >18% after the first 2 weeks (p < 0.05) following withdrawal blood loss. In contrast, serum T (0.98 nmol/l during the first week) and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p < 0.05), respectively, in the following weeks. Our results outline steroidal variations during the OCP cycle, highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear negative impact on the subsequent interpretation of steroid profile of the ABP. With a greater analytical sensitivity and lesser variability for steroids in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module.
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Doping en los Deportes , Humanos , Femenino , Esteroides/orina , Testosterona/orina , Dihidrotestosterona/orina , AnticoncepciónRESUMEN
Different calibration strategies are used in liquid chromatography hyphenated to mass spectrometry (LC-MS) bioanalysis. Currently, the surrogate matrix and surrogate analyte represent the most widely used approaches to compensate for the lack of analyte-free matrices in endogenous compounds quantification. In this context, there is a growing interest in rationalizing and simplifying quantitative analysis using a one-point concentration level of stable isotope-labeled (SIL) standards as surrogate calibrants. Accordingly, an internal calibration (IC) can be applied when the instrument response is translated into analyte concentration via the analyte-to-SIL ratio performed directly in the study sample. Since SILs are generally used as internal standards to normalize variability between authentic study sample matrix and surrogate matrix used for the calibration, IC can be calculated even if the calibration protocol was achieved for an external calibration (EC). In this study, a complete dataset of a published and fully validated method to quantify an extended steroid profile in serum was recomputed by adapting the role of SIL internal standards as surrogate calibrants. Using the validation samples, the quantitative performances for IC were comparable with the original method, showing acceptable trueness (79%-115%) and precision (0.8%-11.8%) for the 21 detected steroids. The IC methodology was then applied to human serum samples (n = 51) from healthy women and women diagnosed with mild hyperandrogenism, showing high agreement (R2 > 0.98) with the concentrations obtained using the conventional quantification based on EC. For IC, Passing-Bablok regression showed proportional biases between -15.0% and 11.3% for all quantified steroids, with an average difference of -5.8% compared to EC. These results highlight the reliability and the advantages of implementing IC in clinical laboratories routine to simplify quantification in LC-MS bioanalysis, especially when a large panel of analytes is monitored.
Asunto(s)
Esteroides , Espectrometría de Masas en Tándem , Femenino , Humanos , Espectrometría de Masas en Tándem/métodos , Calibración , Reproducibilidad de los Resultados , Cromatografía Liquida/métodosRESUMEN
BACKGROUND: Most current state-of-the-art strategies to generate individual adaptive reference ranges are designed to monitor one clinical parameter at a time. An innovative methodology is proposed for the simultaneous longitudinal monitoring of multiple biomarkers. The estimation of individual thresholds is performed by applying a Bayesian modeling strategy to a multivariate score integrating several biomarkers (compound concentration and/or ratio). This multimodal monitoring was applied to data from a clinical study involving 14 female volunteers with normal menstrual cycles receiving testosterone via transdermal route, as to test its ability to detect testosterone administration. The study samples consisted of urine and blood collected during 4 weeks of a control phase and 4 weeks with a daily testosterone gel application. RESULTS: Integrating multiple biomarkers improved the detection of testosterone gel administration with substantially higher sensitivity compared with the distinct follow-up of each biomarker, when applied to selected urine and serum steroid biomarkers, as well as the combination of both. Among the 175 known positive samples, 38% were identified by the multimodal approach using urine biomarkers, 79% using serum biomarkers and 83% by combining biomarkers from both biological matrices, whereas 10%, 67% and 64% were respectively detected using standard unimodal monitoring. SIGNIFICANCE AND NOVELTY: The detection of abnormal patterns can be improved using multimodal approaches. The combination of urine and serum biomarkers reduced the overall number of false-negatives, thus evidencing promising complementarity between urine and blood sampling for doping control, as highlighted in the case of the use of transdermal testosterone preparations. The generation in a multimodal setting of adaptive and personalized reference ranges opens up new opportunities in clinical and anti-doping profiling. The integration of multiple parameters in a longitudinal monitoring is expected to provide a more complete evaluation of individual profiles generating actionable intelligence to further guide sample collection, analysis protocols and decision-making in clinics and anti-doping.
Asunto(s)
Doping en los Deportes , Detección de Abuso de Sustancias , Humanos , Femenino , Teorema de Bayes , Detección de Abuso de Sustancias/métodos , Testosterona/orina , Esteroides/orina , BiomarcadoresRESUMEN
The haematological module of the athlete biological passport (ABP) monitors longitudinal haematological variations that could be indicative of blood manipulation. This study applied a multi-parametric model previously validated in elite cyclists to compare inferred and actual PV variations, whereas the potential influence of the oral contraceptive pill (OCP) cycle on the ABP blood biomarkers and plasma volume (PV) in 14 physically active women taking OCPs was also investigated. Blood and serum samples were collected each week for 8 weeks, and the ABP haematological variables were determined according to the World Anti-Doping Agency guidelines. Transferrin (sTFN), ferritin (FERR), albumin (ALB), calcium (Ca), creatinine (CRE), total protein (TP) and low-density lipoprotein (LDL) were additionally computed as 'volume-sensitive' variables in a multivariate analysis to determine individual estimations of PV variations. Actual PV variations were indirectly measured using a validated carbon monoxide rebreathing method. We hypothesised ABP markers to be stable during a standard OCP cycle and estimated PV variations similar to measured PV variations. Measured PV variations were in good agreement with the predictions and allowed to explain an atypical passport finding (ATPF). The ABP biomarkers, Hbmass and PV were stable over 8 weeks. Significant differences occurred only between Week 7 and Week 1, with lower levels of haemoglobin concentration ([Hb]), haematocrit (HCT) and red blood cell count (RBC)(-4.4%, p < 0.01; -5.1%, p < 0.01; -5.2%, p < 0.01) and higher levels of PV at week 7 (+9%, p = 0.05). We thus concluded that estimating PV variations may help interpret individual ABP haematological profiles in women.
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Doping en los Deportes , Volumen Plasmático , Atletas , Biomarcadores , Anticoncepción , Femenino , HumanosRESUMEN
In the context of steroid analyses, the use of blood could represent a valuable complement to urine. While the blood steroid profile is currently being established to aid unveiling testosterone (T) doping, this matrix is also well suited for detection of exogenous anabolic steroids and steroid esters. In this study, a method to determine a simplified blood steroid profile in combination with the direct detection of exogenous anabolic steroids and steroid esters using just one serum aliquot was developed to obtain a comprehensive analytical workflow. Following the first chromatographic analysis of endogenous and exogenous steroids, samples were derivatised with Girard's reagent T (GT) to improve the ionisation of steroid esters and re-injected. The quantitative performance for T, androstenedione (A4) and 5α-dihydrotestosterone (DHT) was evaluated and the method was validated for qualitative analysis of exogenous analogues with estimated limits of detection (LOD) between 50 and 500 pg/ml. To demonstrate the applicability of the method, samples collected from a clinical study with an oral administration of testosterone undecanoate (TU) to 19 male volunteers were then analysed. The individual serum steroid profiles with the endogenous markers T, A4 and DHT were established as well as the concentrations of TU. TU was detected in all 19 volunteers up to 24 h, while DHT represented the most promising biomarker in endogenous steroid profile for the detection of oral TU administration. These results showed that the selected approach to combine exogenous and endogenous steroid analysis has the potential to strengthen T doping detection in the future.
Asunto(s)
Doping en los Deportes , Espectrometría de Masas en Tándem , Masculino , Humanos , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión/métodos , Esteroides/análisis , Testosterona , Dihidrotestosterona , Androstenodiona , ÉsteresRESUMEN
Urine is currently the matrix of choice for the detection of exogenous substances but also for the application of the steroidal module of the Athlete Biological Passport (ABP) consisting in a longitudinal monitoring of steroid biomarkers. To fill the limitations related to urine, the longitudinal monitoring of serum steroids concentration in the so-called 'blood steroid profile' has recently been proposed. Although serum samples are collected much less than urine samples, plasma derived from ABP whole blood samples used for the full blood count could be exploited for the quantification of endogenous steroids. Alternatively, dried blood spots (DBS) that are much easier to collect could also serve as matrix for the steroid profile. In this study, we compared the concentration levels of several endogenous steroids measured in three different blood matrices (serum, plasma and DBS) collected from 100 elite athletes participating in the 2019 Doha World Athletics Championships using UHPLC-MS/MS. Plasma and serum samples were collected by venipuncture, whereas DBS were generated from whole blood samples. Although steroids demonstrated a good agreement between the three matrices, a slight but acceptable underestimation (10%-20%) was observed in plasma compared with serum. The difference between DBS and the two other matrices was dependent of the bias between serum and plasma. We also showed that a generic HCT correction for DBS could be a valuable approach for quantitative measurements. This study demonstrates the possibility to use three different matrices for the quantification of endogenous steroids although the slight discrepancies should be considered for longitudinal evaluation.
Asunto(s)
Doping en los Deportes , Espectrometría de Masas en Tándem , Humanos , Cromatografía Líquida de Alta Presión , Esteroides/orina , Atletas , Pruebas con Sangre SecaRESUMEN
In women, hormonal fluctuations related to the menstrual cycle may impose a great source of variability for some biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. In this study, the sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers, was investigated for the monitoring of a 28-day T gel treatment combined with endogenous fluctuation of the menstrual cycle in 14 healthy female subjects. Additionally, the analysis of urinary target compounds was performed on a subset of samples for endogenous/exogenous origin via isotope ratio mass spectrometry (IRMS). In serum, concentrations of T and dihydrotestosterone (DHT) increased significantly during the treatment, whereas in urine matrix the most affected biomarkers were found to be the ratios of testosterone/epitestosterone (T/E) and 5α-androstane-3α,17ß-diol/epitestosterone (5αAdiol/E). The detection capability of both urinary biomarkers was heavily influenced by [E], which fluctuated depending on the menstrual cycle, and resulted in low sensitivity of the urinary steroidal ABP module. On the contrary, an alternative approach by the longitudinal monitoring of serum T and DHT concentrations with the newly proposed T/androstenedione ratio showed higher sensitivity. The confirmatory IRMS results demonstrated that less than one third of the tested urine samples fulfilled the criteria for positivity. Results from this study demonstrated that the 'blood steroid profile' represents a powerful complementary approach to the 'urinary module' and underlines the importance of gathering bundle of evidence to support the scenario of an endogenous prohibited substance administration.
Asunto(s)
Doping en los Deportes , Epitestosterona , Biomarcadores/orina , Dihidrotestosterona , Femenino , Humanos , Ciclo Menstrual , Esteroides/orina , Detección de Abuso de Sustancias/métodos , Testosterona/orina , Congéneres de la TestosteronaRESUMEN
The quantification of a large panel of endogenous steroids in serum by LC-MS/MS represents a powerful clinical tool for the screening or diagnosis of diverse endocrine disorders. This approach has also demonstrated excellent sensitivity for the detection of testosterone misuse in the anti-doping field, especially in female athlete population. In both situations, the use of dried blood spots (DBS) could provide a viable alternative to invasive venous blood collection. Here, the evaluation of DBS sampling for the quantification of a panel of endogenous steroids using UHPLC-MS/MS is described. The UHPLC-MS/MS method was validated for quantitative analysis of eleven free and eight conjugated steroids and was then used for the analysis of DBS samples collected in 14 healthy women during a normal menstrual cycle (control phase) followed by a 28-days testosterone gel treatment (treatment phase). Results were compared with those obtained from serum matrix. Satisfactory performance was obtained for all compounds in terms of selectivity, linearity, accuracy, precision, combined uncertainty, stability as well as extraction recovery and matrix effects. In control phase, high correlation was observed between DBS and serum concentrations for most compounds. In treatment phase, higher testosterone concentrations were observed in capillary than in venous DBS, suggesting a possible interference resulting from testosterone contamination on finger(s) used for gel application. Steroid profiling in capillary DBS represents a simple and efficient strategy for monitoring endogenous steroid concentrations and their fluctuation in clinical context of steroid-related disorders, or for the detection of testosterone abuse in anti-doping.
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Esteroides , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Pruebas con Sangre Seca , Femenino , Humanos , Reproducibilidad de los Resultados , TestosteronaRESUMEN
Tramadol is a synthetic opioid drug used in the treatment of chronic and acute pain. An abnormal prevalence of its misuse in elite sport to overcome pain resulting from prolonged physical effort was recently reported. However, besides its antinociceptive effects, tramadol consumption is associated with negative effects such as numbness, confusion, and reduced alertness. This fact prompted the Union Cycliste Internationale to ban the use of tramadol in cycling competitions. Herein, we present the development of a dried blood spot (DBS) sample collection and preparation method followed by a liquid-chromatography mass spectrometry (LC-MS) analysis to rapidly determine the presence of tramadol and its two main metabolites in blood samples. The detection window of each analyte was evaluated and the analysis of performance on various MS platforms (HRMS and MS/MS) was assessed. Tramadol and its two main metabolites were detected up to 12 h after the intake of a single dose of 50 mg of tramadol in positive controls. In professional cycling competitions, 711 DBS samples collected from 361 different riders were analysed using the developed methodology, but all returned negative results (absence of parent and both metabolite compounds). In the context of professional cycling, we illustrate a valid method bringing together the easiness of collection and minimal sample preparation required by DBS, yet affording the performance standards of MS determination. The proposed method to detect tramadol and its metabolites was successfully implemented in cycling races with a probable strong deterrent effect.
Asunto(s)
Analgésicos Opioides/sangre , Ciclismo/fisiología , Doping en los Deportes/prevención & control , Pruebas con Sangre Seca/métodos , Dolor/prevención & control , Detección de Abuso de Sustancias/métodos , Tramadol/sangre , Adulto , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Doping en los Deportes/métodos , Pruebas con Sangre Seca/normas , Hematócrito/métodos , Hematócrito/normas , Humanos , Límite de Detección , Masculino , Detección de Abuso de Sustancias/normas , Factores de TiempoRESUMEN
Aim: Quantitative endogenous steroid profiling in blood appears as a complementary approach to the urinary module of the World Anti-Doping Agency's Athlete Biological Passport Steroidal Module for the detection of testosterone doping. To refine this approach further, a UHPLC-MS/MS method was developed for the simultaneous determination of 14 free and 14 conjugated steroids in serum. Results: The method was validated for quantitative purposes with satisfactory results in terms of selectivity, linearity range, trueness, precision and combined uncertainty (<20%). The validated method was then applied to serum samples from both healthy women and women diagnosed with mild hyperandrogenism. Conclusion: The UHPLC-MS/MS method showed promising capability in quantifying free and conjugated steroids in serum and determining variations of their concentration/distribution within serum samples from different populations.
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Doping en los Deportes , Esteroides/sangre , Detección de Abuso de Sustancias , Atletas , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Hiperandrogenismo/sangre , Espectrometría de Masas en TándemRESUMEN
Erythropoietin (EPO) is the main hormone regulating red blood cell (RBC) production. The large-scale production of a recombinant human erythropoietin (rHuEPO) by biotechnological methods has made possible its widespread therapeutic use as well as its misuse in sports. Since the marketing of the first epoetin in 1989, the development has progressed to the third-generation analogs. However, the production of rHuEPO is costly, and the frequent administration of an injectable formula is not optimal for compliance of therapeutic patients. Hence, pharmaceutical industries are currently developing alternative approaches to stimulate erythropoiesis, which might offer new candidates for doping purposes. The hypoxia inducible factors (HIF) pathway is of particular interest. The introduction of new erythropoiesis-stimulating agents (ESAs) for clinical use requires subsequent development of anti-doping methods for detecting the abuse of these substances. The detection of ESAs is based on two different approaches, namely, the direct detection of exogenous substances and the indirect detection, for which the effects of the substances on specific biomarkers are monitored. Omics technologies, such as ironomics or transcriptomics, are useful for the development of new promising biomarkers for the detection of ESAs. Finally, the illicit use of ESAs associates with multiple health risks that can be irreversible, and an essential facet of anti-doping work is to educate athletes of these risks. The aim of this review is to provide an overview of the evolution of ESAs, the research and implementation of the available detection methods, and the side effects associated with the misuse of ESAs.
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Eritropoyetina/efectos adversos , Eritropoyetina/análisis , Sustancias para Mejorar el Rendimiento/efectos adversos , Sustancias para Mejorar el Rendimiento/análisis , Doping en los Deportes , Eritropoyetina/farmacología , Humanos , Sustancias para Mejorar el Rendimiento/farmacología , Transcriptoma/genéticaRESUMEN
Autologous blood transfusion is a powerful means of improving performance and remains one of the most challenging methods to detect. Recent investigations have identified 3 candidate reticulocytes genes whose expression was significantly influenced by blood transfusion. Using quantitative reverse transcription polymerase chain reaction as an alternative quantitative method, the present study supports that delta-aminolevulinate synthase 2 (ALAS2), carbonic anhydrase (CA1), and solute carrier family 4 member 1 (SLC4A1) genes are down-regulated post-transfusion. The expression of these genes exhibited stronger correlation with immature reticulocyte fraction than with reticulocytes percentage. Moreover, the repression of reticulocytes' gene expression was more pronounced than the diminution of immature reticulocyte fraction and reticulocyte percentage following blood transfusion. It suggests that the 3 candidate genes are reliable predictors of bone marrow's response to blood transfusion and that they represent potential biomarkers for the detection of this method prohibited in sports.
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Transfusión de Sangre Autóloga , Doping en los Deportes , Eritropoyesis , Transcriptoma , 5-Aminolevulinato Sintetasa/genética , Adulto , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Transfusión de Sangre Autóloga/métodos , Anhidrasa Carbónica I/genética , Doping en los Deportes/métodos , Regulación hacia Abajo , Humanos , Masculino , Reticulocitos/citología , Reticulocitos/metabolismoRESUMEN
Innovation in medical research has been diverted at multiple occasions to enhance human performance. The predicted great progress in gene therapy has raised some concerns regarding its misuse in the world of sports (gene doping) for several years now. Even though there is no evidence that gene doping has ever been used in sports, the continuous improvement of gene therapy techniques increases the likelihood of abuse. Therefore, since 2004, efforts have been invested by the anti-doping community and WADA for the development of detection methods. Several nested PCR and qPCR-based strategies exploiting the absence of introns in the transgenic DNA have been proposed for the long-term detection of transgene in blood. Despite their great sensitivity, those protocols are hampered by limitations of the techniques that can be cumbersome and costly. The purpose of this perspective is to describe a new approach based on loop-mediated isothermal amplification (LAMP) for the detection of gene doping. This protocol enables a rapid and simple method to amplify nucleic acids with a high sensitivity and specificity and with a simple visual detection of the results. LAMP is already being used in clinical application for the detection of viruses or mutations. Therefore, this technique has the potential to be further developed for the detection of foreign genetic material in elite athletes. Copyright © 2017 John Wiley & Sons, Ltd.
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Doping en los Deportes , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transgenes/genética , Humanos , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , Transgenes/fisiologíaRESUMEN
The concentration of hepcidin, a key regulator of iron metabolism, is suppressed during periods of increased erythropoietic activity. The present study obtained blood samples from 109 elite athletes and examined the correlations between hepcidin and markers of erythropoiesis and iron metabolism (i.e., haemoglobin, erythropoietin (EPO), ferritin, erythroferrone (ERFE), and iron concentration). Furthermore, an administration study was undertaken to examine the effect of recombinant human EPO (rhEPO) delta (Dynepo™) on hepcidin concentrations in healthy male volunteers. The effects on hepcidin were then compared with those on reticulocyte percentage (Ret%) and ferritin concentration. There was a significant positive correlation between hepcidin and ferritin, iron, and haemoglobin levels in athletes, whereas hepcidin showed an inverse correlation with ERFE. Administration of rhEPO delta reduced hepcidin levels, suggesting that monitoring hepcidin may increase the sensitivity of the Athlete Biological Passport (ABP) for detecting rhEPO abuse. Copyright © 2016 John Wiley & Sons, Ltd.
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Eritropoyetina/administración & dosificación , Eritropoyetina/sangre , Hepcidinas/sangre , Detección de Abuso de Sustancias/métodos , Adulto , Biomarcadores/sangre , Doping en los Deportes , Eritropoyesis/efectos de los fármacos , Ferritinas/sangre , Hemoglobinas/análisis , Humanos , Hierro/sangre , Masculino , Recuento de Reticulocitos , Reticulocitos/citología , Reticulocitos/efectos de los fármacos , Adulto JovenRESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and thus influence many cellular and physiological processes. miRNAs are also present in cell-free body fluids such as plasma or serum, and these circulating miRNAs are very stable, sensitive, and specific biomarkers of pathophysiological states. In this study, we investigated whether circulating miRNAs could serve as biomarkers of exogenous testosterone administration. Misuse of testosterone as a performance-enhancing drug is thought to be widespread in sports. Detection of testosterone through the urinary steroid profile of the Athlete Biological Passport faces several obstacles, indicating that new biomarkers are required. To this end, we analyzed plasma miRNA levels by high-throughput quantitative real-time PCR. Plasma samples were obtained before and at several time points after transdermal and oral testosterone administration. Screening identified three potential candidate miRNAs that were altered by both routes of testosterone administration. Longitudinal monitoring of these candidates revealed that variation in two of them (miR-150 and miR-342), relative to the corresponding levels in control samples, was testosterone-independent. However, levels of the liver-specific miR-122 increased 3.5-fold 1 day after drug intake. Given that testosterone is metabolized by the liver, this observation suggests that miR-122 in cell-free fluids may be used as a sensitive biomarker of testosterone misuse via multiple dosing routes and could therefore be integrated into a blood-based multiparametric follow-up.