Application of the Athlete Biological Passport Approach to the Detection of Growth Hormone Doping.

CONTEXT: Because of its anabolic and lipolytic properties, growth hormone (GH) use is prohibited in sport. Two methods based on population-derived decision limits are currently used to detect human GH (hGH) abuse: the hGH Biomarkers Test and the Isoforms Differential Immunoassay. OBJECTIVE: We tested the hypothesis that longitudinal profiling of hGH biomarkers through application of the Athlete Biological Passport (ABP) has the potential to flag hGH abuse. METHODS: Insulin-like growth factor 1 (IGF-1) and procollagen III peptide (P-III-NP) distributions were obtained from 7 years of anti-doping data in elite athletes (n = 11 455) and applied as priors to analyze individual profiles from an hGH administration study in recreational athletes (n = 35). An open-label, randomized, single-site, placebo-controlled administration study was carried out with individuals randomly assigned to 4 arms: placebo, or 3 different doses of recombinant hGH. Serum samples were analyzed for IGF-1, P-III-NP, and hGH isoforms and the performance of a longitudinal, ABP-based approach was evaluated. RESULTS: An ABP-based approach set at a 99% specificity level flagged 20/27 individuals receiving hGH treatment, including 17/27 individuals after cessation of the treatment. ABP sensitivity ranged from 12.5% to 71.4% across the hGH concentrations tested following 7 days of treatment, peaking at 57.1% to 100% after 21 days of treatment, and was maintained between 37.5% and 71.4% for the low and high dose groups 1 week after cessation of treatment. CONCLUSION: These findings demonstrate that longitudinal profiling of hGH biomarkers can provide suitable performance characteristics for use in anti-doping programs.

The Proteomic Signature of Recombinant Growth Hormone in Recreational Athletes.

OBJECTIVE: Administration of human growth hormone (hGH) is prohibited in competitive sport and its detection in an athlete's sample triggers an adverse analytical finding. However, the biological processes that are modulated by recombinant hGH are not well characterized and associated blood serum proteins may constitute new biomarkers for hGH misuse. METHODS: Thirty-five recreational athletes were enrolled in a study to investigate the time- and dose-dependent response of serum protein levels to recombinant hGH administration. Participants were randomly assigned to 4 groups, receiving 1 of 3 different doses of recombinant hGH or a placebo. Bio samples were collected at 22 time points over a period of 13 weeks, starting 4 weeks before treatment, during 3 weeks of treatment, and at 6 weeks' follow-up. A total of 749 serum samples were analyzed for 1305 protein markers using the SOMAscan proteomics platform. RESULTS: We identified 66 proteins that significantly associated with recombinant hGH administration and dosage, including well known hGH targets, such as IGF1, but also previously unknown hGH-related proteins (eg, protease inhibitors, WFIKKN1, and chemokines, CCL2). Network analysis revealed changes in specific biological pathways, mainly related to the immune system and glucose metabolism. CONCLUSION: Our analysis suggests that hGH administration affects biological processes more strongly than previously acknowledged. Some of the proteins were dysregulated even after hGH treatment and could potentially be developed into biomarkers for hGH misuse. Moreover, our findings suggest new roles for hGH-associated proteins in the etiology of hGH-related diseases and may indicate new risks that may be associated with hGH misuse.

Analytical challenges in the detection of peptide hormones for anti-doping purposes.

Although significant progress has been achieved during the past few years with the introduction of new assays and analytical methodologies, the detection and quantification of protein analytes, in particular of peptide hormones, continues to pose analytical challenges for the World Anti-Doping Agency-accredited anti-doping laboratories. In this article, the latest achievements in the application of MS-based methodologies and specific biochemical and immunological assays to detect some of the prohibited substances listed in section S2 of the World Anti-Doping Agency List of Prohibited Substances and Methods are reviewed. In addition, we look towards the future by focusing on some of the most promising analytical approaches under development for the detection of so-called 'biomarkers of doping'.

Ensuring high quality in anti-doping laboratories.

The worldwide network of World Anti-Doping Agency (WADA)-accredited anti-doping laboratories plays a fundamental role in supporting the global fight against doping in sport. This role is dependent on the ability to provide accurate, reliable and comparable data in identifying and measuring the presence of prohibited substances and methods. The accredited laboratories participate in WADA's External Quality Assessment Scheme (EQAS) program, which provides the structure to continuously assess and improve laboratory performance in compliance to the requirements of the International Standard for Laboratories and related Technical Documents. The WADA EQAS is comprised of various programs, including a blind EQAS, a double-blind EQAS and an educational EQAS, each with specific goals with regard to monitoring and improving laboratory competence. In this article, the anti-doping rules and processes that govern granting and maintenance of WADA laboratory accreditation, aimed at ensuring a high-quality of laboratory operations within the framework of the global fight against doping in sport, are reviewed.

Measurement uncertainty in anti-doping quantitative analysis for prohibited threshold substances.

The standards of laboratory performance of the World Anti-Doping Agency (WADA)-accredited laboratories are defined in the WADA International Standard for Laboratories and its associated Technical Documents. These sets of rules aim to harmonize the production of valid laboratory test results and evidentiary data as well as the reporting of laboratory analytical findings. The determination of anti-doping rule violations in sport made on the basis of analytical quantitative confirmatory analyses for the presence of prohibited threshold substances, in particular, requires the application of specific compliance decision rules, which are established in the WADA Technical Document on Decision Limits. In this article, the use of measurement uncertainty information in the establishment of compliance Decision Limits and in evaluating the performance of a laboratory's quantitative analytical procedures over time and in relation to other laboratories through WADA's External Quality Assessment Scheme program is reviewed and discussed. Furthermore, a perspective is provided on the emerging challenges associated with the harmonization of the quantitative measurement of large-molecular weight biomolecules.

Determination of urinary concentrations of pseudoephedrine and cathine after therapeutic administration of pseudoephedrine-containing medications to healthy subjects: implications for doping control analysis of these stimulants banned in sport.

Due to its stimulatory effects on the central nervous system, and its structural similarity to banned stimulants such as ephedrine and methamphetamine, pseudoephedrine (PSE) at high doses is considered as an ergogenic aid for boosting athletic performance. However, the status of PSE in the International Standard of the Prohibited List as established under the World Anti-Doping Code has changed over the years, being prohibited until 2003 at a urinary cut-off value of 25 µg/ml, and then subsequently removed from the Prohibited List during the period 2004-2009. The re-consideration of this position by the World Anti-Doping Agency (WADA) List Expert Group has led to the reintroduction of PSE in the Prohibited List in 2010. In this manuscript, we present the results of two WADA-sponsored clinical studies on the urinary excretion of PSE and its metabolite cathine (CATH) following the oral administration of different PSE formulations to healthy individuals at therapeutic regimes. On this basis, the current analytical urinary threshold for the detection of PSE as a doping agent in sport has been conservatively established at 150 µg/ml

The list of prohibited substances and methods in sport: structure and review process by the world anti-doping agency.

The List of Prohibited Substances and Methods (the List) is the International Standard that determines what is prohibited in sport in- and out-of-competition. The official text of the List is produced by the World Anti-Doping Agency (WADA), the international independent organization responsible for promoting, coordinating and monitoring the fight against doping in sport. The drafting of the annual List is a highly interactive and consultative process involving scientific and medical experts in anti-doping, sport federations and governments. In this article, the elements that compose the List as well as the process behind its annual revision and update are presented.

Investigating the use of stimulants in out-of-competition sport samples.

The List of Prohibited Substances and Methods (the List), an International Standard published yearly by the World Anti-Doping Agency (WADA), determines which substances and methods are prohibited in sport in- and out-of-competition. Stimulants are included within drug class S.6 under the in-competition testing section of the List. Athletes may be tempted to use stimulants as ergogenic aids in-competition in order to temporarily improve their mental and/or physical functions by increasing alertness, aggressiveness, motivation, locomotion, heart rate, and reducing fatigue. The Prohibited List Expert Group, responsible for the maintenance of the List, approved WADA funding for a two-year study to determine whether athletes were also using stimulants to benefit from their performance-enhancing effects during the training phase between competitions (i.e., out-of-competition). This study, involving 11 WADA-accredited laboratories, found that the use of stimulants by athletes during training was not significantly prevalent (0.36% of positive findings), suggesting that this issue does not, at the moment, pose a further challenge to the fight against doping in sport. In addition, the study supports the current structure in the Prohibited List that differentiates banned substances into the in- and out-of-competition classifications.

Detection of GH abuse in sport: Past, present and future.

Due to its considered performance enhancing effects, human growth hormone (hGH) is abused as a doping agent in sport. Its misuse also carries potentially serious side effects to a person's health. Consequently, hGH and its releasing factors are prohibited in sport, as established in the Prohibited List which is updated and published yearly by the World Anti-Doping Agency (WADA). In order to fight the menace that hGH doping poses to the spirit of sport and to the health of athletes, the sport movement and the anti-doping authorities, initially led by the International Olympic Committee (IOC) and later by WADA, have put substantial efforts into developing tests for its detection. Currently, a primary analytical approach, the isoform differential immunoassay, has been implemented in WADA-accredited laboratories. In parallel, a second, indirect approach for the detection of hGH abuse, based on the quantification of hGH-associated biological markers, has been developed. The final aim is to combine both methodologies to improve the sensitivity and expand the time window to detect doping with hGH. In addition, novel analytical procedures, based on proteomic and genomic technologies as well as the use of mass spectrometry-based methods of detection, are being investigated for future application in hGH anti-doping tests.

Hormone abuse in sports: the antidoping perspective.

Since ancient times, unethical athletes have attempted to gain an unfair competitive advantage through the use of doping substances. A list of doping substances and methods banned in sports is published yearly by the World Anti-Doping Agency (WADA). A substance or method might be included in the List if it fulfills at least two of the following criteria: enhances sports performance; represents a risk to the athlete's health; or violates the spirit of sports. This list, constantly updated to reflect new developments in the pharmaceutical industry as well as doping trends, enumerates the drug types and methods prohibited in and out of competition. Among the substances included are steroidal and peptide hormones and their modulators, stimulants, glucocorticosteroids, beta2-agonists, diuretics and masking agents, narcotics, and cannabinoids. Blood doping, tampering, infusions, and gene doping are examples of prohibited methods indicated on the List. From all these, hormones constitute by far the highest number of adverse analytical findings reported by antidoping laboratories. Although to date most are due to anabolic steroids, the advent of molecular biology techniques has made recombinant peptide hormones readily available. These substances are gradually changing the landscape of doping trends. Peptide hormones like erythropoietin (EPO), human growth hormone (hGH), insulin, and insulin-like growth factor I (IGF-I) are presumed to be widely abused for performance enhancement. Furthermore, as there is a paucity of techniques suitable for their detection, peptide hormones are all the more attractive to dishonest athletes. This article will overview the use of hormones as doping substances in sports, focusing mainly on peptide hormones as they represent a pressing challenge to the current fight against doping. Hormones and hormones modulators being developed by the pharmaceutical industry, which could emerge as new doping substances, are also discussed.