New analytical methodologies for doping control – detection of anabolic androgenic steroids in human urine

Dissertation submitted to Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa in fulfilment of the requirements for the degree of Doctor of Philosophy (Biochemistry - Biotechnology)

Anti-doping testing at the 2008 European football championship.

Big sports events like the 2008 European Football Championship are a challenge for anti-doping activities, particularly when the sports event is hosted by two different countries and there are two laboratories accredited by the World Anti-Doping Agency. This challenges the logistics of sample collection as well as the chemical analyses, which must be carried out timeously. The following paper discusses the handling of whereabouts information for each athlete and the therapeutic use exemption system, experiences in sample collection and transportation of blood and urine samples, and the results of the chemical analysis in two different accredited laboratories. An overview of the analytical results of blood profiling and growth hormone testing in comparison with the distribution of the normal population is also presented.

Time and temperature dependant changes in red blood cell analytes used for testing recombinant erythropoietin abuse in sports.

There has been a long debate since the introduction of blood analysis prior to major sports events, to find out whether blood samples should be analysed right away on the site of competition or whether they should be transported and analysed in an anti-doping laboratory. Therefore, it was necessary to measure blood samples and compare the results obtained right after the blood withdrawal with those obtained after a few hours delay. Furthermore, it was interesting to determine the effect of temperature on the possible deterioration of red blood cell analytes used for testing recombinant erythropoietin abuse. Healthy volunteers were asked to give two blood samples and one of these was kept at room temperature whereas the second one was put into a refrigerator. On a regular basis, the samples were rolled for homogenisation and temperature stabilisation and were analysed with the same haematological apparatus. The results confirmed that blood controls prior to competition should be performed as soon as possible with standardised pre-analytical conditions to avoid too many variations notably on the haematocrit and the reticulocyte count. These recommendations should ideally also be applied to the all the blood controls compulsory for the medical follow up, otherwise unexplainable values could be misinterpreted and could for instance lead to a period of incapacity.

Methods for detection and confirmation of Hematide?/peginesatide in anti-doping samples.

Since the 1990's, cheating athletes have abused substances to increase their oxygen transport capabilities; among these substances, recombinant EPO is the most well known. Currently, other investigational pharmaceutical products are able to produce an effect similar to EPO but without having chemical structures related to EPO; these are the synthetic erythropoiesis stimulating agents (ESAs). Peginesatide (also known as Hematide?) is being developed by Affymax and Takeda and, if approved by regulatory authorities, could soon be released on the international market. To detect potential athletic abuse of this product and deter athletes who consider cheating, we initiated a collaboration to implement a detection test for anti-doping purposes. Peginesatide is a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. It is undetectable using current anti-doping tests due to its lack of sequence homology to EPO. To detect and deter potential abuse of peginesatide, we initiated an industry/antidoping laboratory collaboration to develop and validate screening and confirmation assays so that they would be available before peginesatide reaches the market. We describe a screening ELISA and a confirmation assay consisting of immune-purification followed by separation with SDS-PAGE and revelation with Western double blotting. Both assays can detect 0.5 ng/mL concentrations of peginesatide in blood samples, enabling detection for several days after administration of a physiologically relevant dose. This initial report describes experimental characterization of these assays, including testing with a blinded set of samples from a clinical study conducted in healthy volunteers.

Detection of human growth hormone doping in urine: out of competition tests are necessary.

The misuse of human growth hormone (hGH) in sport is deemed to be unethical and dangerous because of various adverse effects. Thus, it has been added to the International Olympic Committee list of banned substances. Until now, the very low concentration of hGH in the urine made its measurement difficult using classical methodology. Indeed, for routine diagnosis, only plasma measurements were available. However, unlike blood samples, urine is generally provided in abundant quantities and is, at present, the only body fluid allowed to be analysed in sport doping controls. A recently developed enzyme-linked immunosorbent assay (Norditest) makes it now possible, without any extraction, to measure urinary hGH (u-hGH) in a dynamic range of 2-50 ng hGH/l. In our protocol, untreated and treated non-athlete volunteers were followed. Some of them received therapeutical doses of recombinant hGH (Norditropin) for one week either intramuscularly (three increasing doses) or subcutaneously (12 i.u. every day). The u-hGH excretion after treatment showed dramatic increases of 50-100 times the basal values and returned to almost the mean normal level after 24 h. u-hGH was also measured in samples provided by the anti-doping controls at major and minor competitions. Depending on the type of efforts made during the competition, the hGH concentration in urine was dramatically increased. Insulin-like growth factor binding proteins and beta 2-microglobulins in urine and/or in blood could be necessary for the correct investigation of any hGH doping test procedure.

Anti-doping testing at the 2008 European football championship.

Big sports events like the 2008 European Football Championship are a challenge for anti-doping activities, particularly when the sports event is hosted by two different countries and there are two laboratories accredited by the World Anti-Doping Agency. This challenges the logistics of sample collection as well as the chemical analyses, which must be carried out timeously. The following paper discusses the handling of whereabouts information for each athlete and the therapeutic use exemption system, experiences in sample collection and transportation of blood and urine samples, and the results of the chemical analysis in two different accredited laboratories. An overview of the analytical results of blood profiling and growth hormone testing in comparison with the distribution of the normal population is also presented.

Determination of Vasopressin and Desmopressin in urine by means of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry for doping control purposes.

The anti-diuretic neurohypophysial hormone Vasopressin (Vp) and its synthetic analogue Desmopressin (Dp, 1-desamino-vasopressin) have received considerable attention from doping control authorities due to their impact on physiological blood parameters. Accordingly, the illicit use of Desmopressin in elite sport is sanctioned by the World Anti-Doping Agency (WADA) and the drug is classified as masking agent. Vp and Dp are small (8-9 amino acids) peptides administered orally as well as intranasally. Within the present study a method to determine Dp and Vp in urinary doping control samples by means of liquid chromatography coupled to quadrupole high resolution time-of-flight mass spectrometry was developed. After addition of Lys-Vasopressin as internal standard and efficient sample clean up with a mixed mode solid phase extraction (weak cation exchange), the samples were directly injected into the LC-MS system. The method was validated considering the parameters specificity, linearity, recovery (80-100%), accuracy, robustness, limit of detection/quantification (20/50 pg mL(-1)), precision (inter/intra-day<10%), ion suppression and stability. The analysis of administration study urine samples collected after a single intranasal or oral application of Dp yielded in detection windows for the unchanged target analyte for up to 20 h at concentrations between 50 and 600 pg mL(-1). Endogenous Vp was detected in concentrations of approximately 20-200 pg mL(-1) in spontaneous urine samples obtained from healthy volunteers. The general requirements of the developed method provide the characteristics for an easy transfer to other anti-doping laboratories and support closing another potential gap for cheating athletes.

Circulating microRNAs as biomarkers for detection of autologous blood transfusion.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate various biological processes. Cell-free miRNAs measured in blood plasma have emerged as specific and sensitive markers of physiological processes and disease. In this study, we investigated whether circulating miRNAs can serve as biomarkers for the detection of autologous blood transfusion, a major doping technique that is still undetectable. Plasma miRNA levels were analyzed using high-throughput quantitative real-time PCR. Plasma samples were obtained before and at several time points after autologous blood transfusion (blood bag storage time 42 days) in 10 healthy subjects and 10 controls without transfusion. Other serum markers of erythropoiesis were determined in the same samples. Our results revealed a distinct change in the pattern of circulating miRNAs. Ten miRNAs were upregulated in transfusion samples compared with control samples. Among these, miR-30b, miR-30c, and miR-26b increased significantly and showed a 3.9-, 4.0-, and 3.0-fold change, respectively. The origin of these miRNAs was related to pulmonary and liver tissues. Erythropoietin (EPO) concentration decreased after blood reinfusion. A combination of miRNAs and EPO measurement in a mathematical model enhanced the efficiency of autologous transfusion detection through miRNA analysis. Therefore, our results lay the foundation for the development of miRNAs as novel blood-based biomarkers to detect autologous transfusion.

Use of forensic investigations in anti-doping

The fight against doping is mainly focused on direct detection, using analytical methods for the detection of doping agents in biological samples. However, the World Anti-Doping Code also defines doping as possession, administration or attempted administration of prohibited substances or methods, trafficking or attempted trafficking in any prohibited substance or methods. As these issues correspond to criminal investigation, a forensic approach can help assessing potential violation of these rules.In the context of a rowing competition, genetic analyses were conducted on biological samples collected in infusion apparatus, bags and tubing in order to obtain DNA profiles. As no database of athletes' DNA profiles was available, the use of information from the location detection as well as contextual information were key to determine a population of suspected athletes and to obtain reference DNA profiles for comparison.Analysis of samples from infusion systems provided 8 different DNA profiles. The comparison between these profiles and 8 reference profiles from suspected athletes could not be distinguished.This case-study is one of the first where a forensic approach was applied for anti-doping purposes. Based on this investigation, the International Rowing Federation authorities decided to ban not only the incriminated athletes, but also the coaches and officials for 2 years.

The effect of a period of intense exercise on the marker approach to detect growth hormone doping in sports.

The major objective of this study was to investigate the effects of several days of intense exercise on the growth hormone marker approach to detect doping with human growth hormone (hGH). In addition we investigated the effect of changes in plasma volume on the test. Fifteen 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). Plasma volumes were estimated by the optimized CO Rebreathing method. IGF-1 and P-III-NP were analyzed by Siemens Immulite and Cisbio Assays, respectively. All measured GH 2000 scores were far below the published decision limits for an adverse analytical finding. The period of exercise did not increase the GH-scores; however the accompanying effect of the increase in Plasma Volume yielded in essentially lower GH-scores. We could demonstrate that a period of heavy, long-term exercise with changes in plasma volume does not interfere with the decision limits for an adverse analytical finding. Copyright © 2014 John Wiley & Sons, Ltd.

Monitoring of biological markers indicative of doping: the athlete biological passport

The athlete biological passport (ABP) was recently implemented in anti-doping work and is based on the individual and longitudinal monitoring of haematological or urine markers. These may be influenced by illicit procedures performed by some athletes with the intent to improve exercise performance. Hence the ABP is a valuable tool in the fight against doping. Actually, the passport has been defined as an individual and longitudinal observation of markers. These markers need to belong to the biological cascade influenced by the application of forbidden hormones or more generally, affected by biological manipulations which can improve the performance of the athlete. So far, the haematological and steroid profile modules of the ABP have been implemented in major sport organisations, and a further module is under development. The individual and longitudinal monitoring of some blood and urine markers are of interest, because the intraindividual variability is lower than the corresponding interindividual variability. Among the key prerequisites for the implementation of the ABP is its prospect to resist to the legal and scientific challenges. The ABP should be implemented in the most transparent way and with the necessary independence between planning, interpretation and result management of the passport. To ensure this, the Athlete Passport Management Unit (APMU) was developed and the WADA implemented different technical documents associated to the passport. This was carried out to ensure the correct implementation of a profile which can also stand the challenge of any scientific or legal criticism. This goal can be reached only by following strictly important steps in the chain of production of the results and in the management of the interpretation of the passport. Various technical documents have been then associated to the guidelines which correspond to the requirements for passport operation. The ABP has been completed very recently by the steroid profile module. As for the haematological module, individual and longitudinal monitoring have been applied and the interpretation cascade is also managed by a specific APMU in a similar way as applied in the haematological module. Thus, after exclusion of any possible pathology, specific variation from the individual norms will be then considered as a potential misuse of hormones or other modulators to enhance performance.

Stability and robustness of blood variables in an antidoping context.

Introduction:  With the setting up of the newly Athlete's Biological Passport antidoping programme, novel guidelines have been introduced to guarantee results beyond reproach. We investigated in this context, the effect of storage time on the variables commonly measured for the haematological passport. We also wanted to assess for these variables, the within and between analyzer variations. Methods:  Blood samples were obtained from top level male professional cyclists (27 samples for the first part of the study and 102 for the second part) taking part to major stage races. After collection, they were transported under refrigerated conditions (2 °C < T < 12 °C), delivered to the antidoping laboratory, analysed and then stored at approximately 4 °C to conduct analysis at different time points up to 72 h after delivery. A mixed-model procedure was used to determine the stability of the different variables. Results:  As expected haemoglobin concentration was not affected by storage and showed stability for at least 72 h. Under the conditions of our investigation, the reticulocytes percentage showed a much better stability than previous published data (> 48 h) and the technical comparison of the haematology analyzer demonstrated excellent results. Conclusion:  In conclusion, our data clearly demonstrate that as long as the World Anti-Doping Agency's guidelines are followed rigorously, all blood results reach the quality level required in the antidoping context.

Circulating miRNAs: a new generation of anti-doping biomarkers

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. Cell-free miRNAs detected in blood plasma are used as specific and sensitive markers of physiological processes and some diseases. Circulating miRNAs are highly stable in body fluids, for example plasma. Therefore, profiles of circulating miRNAs have been investigated for potential use as novel, non-invasive anti-doping biomarkers. This review describes the biological mechanisms underlying the variation of circulating miRNAs, revealing that they have great potential as a new class of biomarker for detection of doping substances. The latest developments in extraction and profiling technology, and the technical design of experiments useful for anti-doping, are also discussed. Longitudinal measurements of circulating miRNAs in the context of the athlete biological passport are proposed as an efficient strategy for the use of these new markers. The review also emphasizes potential challenges for the translation of circulating miRNAs from research into practical anti-doping applications.