Abuse of growth hormone among young athletes

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Pediatr Clin North Am. 2007 Aug;54(4):823-43, xiii
Buzzini SR
Department of Pediatrics, Geisinger Medical Center, 100 North Academy Avenue, Danville, PA 17822, USA

The underground abuse of growth hormone (GH) among young athletes presents a challenge to medical professionals. Health care professionals providing knowledgeable guidance regarding healthy ways to improve performance and appearance, as well as accurate information regarding substances' perceived benefits, risks, and unknown qualities, is invaluable to the young athlete. Further research focused on the profile and motivation of young people who use GH is essential to understanding and intervening better with those who use these substances.

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Sex, drugs and sports: prostaglandins, epitestosterone and sexual development

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Med Hypotheses. 2007;69(4):829-35. Epub 2007 Mar 23
Sanders BK
College of Letters and Science, University of California, Berkeley, CA 94720, USA

Amateau and McCarthy's findings published in Nature Neuroscience (June 2004) are noteworthy for suggesting a role for prostaglandins in sexual development. However, evidence suggests that in manipulating PGE2, they unknowingly implicated 3alpha-hydroxysteroid dehydrogenase [E.C. 1.1.1.50], 3(or 17)alpha-hydroxysteroid dehydrogenase [E.C. 1.1.1.209] and their respective products, androsterone (ADT) and epitestosterone (EpiT), in the developmental masculinization of sex behavior. EpiT is generally regarded as a hormonally inactive 17alpha-epimer of testosterone (T). In rats, the kidney is the primary site of EpiT formation, whereas in humans it originates from the gonads, with only a small contribution secreted by the adrenals. Because the ratio of T to EpiT is nearly constant, it is presently used for assessing steroid abuse in competitive sports, where the World Anti-Doping Agency (WADA) considers a T/EpiT ratio >4 evidence of T doping. Despite its central role in the detection of illict anabolic steroid use, our knowledge of factors effecting EpiT production is poor. Clues in the literature, however, reveal that prostaglandin-mediated processes, such as LHRH release, may influence its production. Antimycotics, NSAIDs, and opioid analgesics used in sports medicine are all known to effect prostaglandin E2 synthesis. Primary PGs are potent inhibitors of ADT oxidation, while indomethacin, a prostaglandin blocker, powerfully inhibits 3alpha-HSD reduction and ADT oxidation. This is significant because ADT inhibits the oxidation of EpiT, and may modulate its antiandrogenic and neuroprotective effects. It is hypothesized that the T/EpiT ratio is increased by COX-2 inhibitors and opiod analgesics, and decreased by antimycotics that do not impair testosterone biosynthesis. Given the devastating personal and career consequences that may result from false positive drug tests, substantive research on the effects of PGE2 manipulations on EpiT is warranted.

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Testosterone precursors: use and abuse in pediatric athletes

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Pediatr Clin North Am. 2007 Aug;54(4):787-96, xii
Smurawa TM, Congeni JA
Northeastern Ohio Universities College of Medicine, 4209 State Route 44, PO Box 95, Rootstown, OH 44272, USA

The dietary supplements androstenedione, dehydroepiandrosterone, and androstenediol are precursors in the endogenous production of testosterone. The efficacy and safety of these prohormones are not well established but are promoted to have the same androgenic effects on building muscle mass and strength as anabolic-androgenic steroids. Studies have demonstrated repeatedly that acute and long-term administration of these oral testosterone precursors does not effectively increase serum testosterone levels and fails to produce any significant changes in lean body mass, muscle strength, or performance improvement compared with placebo. The Anabolic Steroid Control Act of 2004 lists androstenedione as a schedule III controlled substance, and it is regulated by the U.S. Food and Drug Administration. Testosterone precursors are banned by most major sports organizations.

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The continuing story of nutritional supplements and doping infractions

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Br J Sports Med. 2007 Nov;41(11):800-5; discussion 805
de Hon O, Coumans B.
Anti-Doping Authority of The Netherlands

Nutritional supplements can be a source of positive doping cases as some supplements contain prohibited substances without showing this on their label. This problem has existed for some time and has been extensively studied in the past 8 years. The sport of tennis has played a particular role in this problem because of some peculiar doping cases within its community. This article focuses on this particular doping problem, explaining the background and reviewing the available literature. It presents the first 3 years of experience within the Netherlands Security System Nutritional Supplements Elite Sports ("Nederlands Zekerheidssysteem Voedingssupplementen Topsport" or NZVT) and explains the most extensive system established to combat this particular doping problem. The NZVT experience has shown that paper-based quality systems are still prone to possible contaminations, which leads to the conclusion that the best possible solution for athletes who wish to use nutritional supplements must include laboratory-based analysis for doping substances, preferably repeated for every new batch. The most important educational message, however, is to use a nutritional supplement only if it is deemed of benefit by a nutritional expert.

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Gene doping: a review of performance-enhancing genetics

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Pediatr Clin North Am. 2007 Aug;54(4):807-22, xii-xiii
Gaffney GR, Parisotto R
Division of Children and Adolescent Psychiatry, Department of Psychiatry, University of Iowa College of Medicine, 200 Hawkins Road, Iowa City, IA 52242, USA

Unethical athletes and their mentors have long arrogated scientific and medical advances to enhance athletic performance, thus gaining a dishonest competitive advantage. Building on advances in genetics, a new threat arises from athletes using gene therapy techniques in the same manner that some abused performance-enhancing drugs were used. Gene doping, as this is known, may produce spectacular physiologic alterations to dramatically enhance athletic abilities or physical appearance. Furthermore, gene doping may present pernicious problems for the regulatory agencies and investigatory laboratories that are entrusted to keep sporting events fair and ethical. Performance-enhanced genetics will likewise present unique challenges to physicians in many spheres of their practice.

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Short-term recombinant human growth hormone administration improves respira tory function in abstinent anabolic-androgenic steroid users

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Growth Horm IGF Res. 2007 Aug;17(4):328-35. Epub 2007 May 23
Graham MR, Baker JS, Evans P, Kicman A, Cowan D, Hullin D, Davies B.
Health and Exercise Science Research Unit, Faculty of Health Sport and Science, University of Glamorgan, Pontypridd, Wales, UЛ

OBJECTIVES: To determine whether 6 days recombinant human growth hormone (rhGH) administration, in an abstinent anabolic-androgenic steroid (AAS) using group had any respiratory, endurance exercise and biochemical effects compared with an abstinent AAS control group. METHODS: Male subjects (n=48) were randomly divided, using a single blind procedure into two groups: (1) control group (C) n=24, means+/-SD, age 32+/-11 years; height 1.8+/-0.06 m; (2) rhGH using group (0.019 mg kg(-1) day(-1)) (GH) n=24, means+/-SD, age 32+/-9 years; height 1.8+/-0.07 m. Anthropometry, respiratory muscle function and endurance exercise were investigated. Respiratory measurements examined, were forced expiratory volume in one second, forced vital capacity, maximum inspiratory pressure and maximum expiratory pressure. Endurance exercise was assessed by measuring peak oxygen uptake (VO(2)peak). Biochemical analysis included; haemoglobin, packed cell volume, glucose, sodium, urea, creatinine, total protein, albumin, testosterone and insulin like growth factor-I (IGF-I). RESULTS: Forced expiratory volume in one second/forced vital capacity, maximum inspiratory pressure, maximum expiratory pressure, and IGF-I significantly increased compared with the control group (all P<0.05).

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Technology Insight: detecting growth hormone abuse in athletes

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Nature Clin Pract Endocrinol Metab. 2007 Nov;3(11):769-77
Bidlingmaier M, Strasburger CJ.
M Bidlingmaier is Head of the Endocrine Research Laboratories, Medizinische Klinik - Innenstadt (Medical Department), Klinikum der Universitat, Ludwig-Maximilians University, Munich, Germany

Athletes recognized the performance-enhancing potential of human growth hormone when it became available for treatment of short stature in growth-retarded children. Although no controlled clinical studies have demonstrated a significant benefit in highly trained adults with normal pituitary function, the practice of doping increased with the introduction of recombinant human growth hormone. Evidence of widespread abuse has been gathered by police and customs authorities or provided by former athletes. It has been difficult to develop a test to prove the administration of exogenous growth hormone in athletes because of its specific physiological and biochemical properties. Significant progress has only recently been made, particularly via two differing approaches. The 'marker approach' utilizes characteristic changes in concentrations of pharmacodynamic end points of growth hormone action, for example serum concentrations of insulin-like growth factor I and factors related to bone and soft tissue turnover. The 'isoform approach' detects changes in the molecular isoform composition of circulating growth hormone evoked by the administration of exogenous recombinant growth hormone. The isoform approach was applied at the Olympic Games in Athens in 2004 and in Turin in 2006. Used in a complementary way in an out-of-competition setting, these methods are a powerful tool with which to detect growth hormone abuse in sports.

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"Because I know it will!": placebo effects of an ergogenic aid on athletic performance

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J Sport Exerc Psychol. 2007 Jun;29(3):382-94
McClung M, Collins D.
Scottish Institute of Sport, Blackburn, West Lothian, Scotland

In the perpetual quest for better performance, athletes are using an increasingly diverse range of ergogenic aids. Some are permitted; however, this "drug" use is often seen as an ethically questionable behavior. A variety of research suggests that much of the impact of such aids may be due to expectancy-the belief that the substance will aid performance. It would be useful to demonstrate this to athletes considering such usage, especially as a pillar of antidrug education. Accordingly, this investigation used sodium bicarbonate and placebo additives in a double disassociation design, with athletes completing a series of 1,000-m time trials. Results showed that believing one had taken the substance resulted in times almost as fast as those associated with consuming the drug itself. In contrast, taking the drug without knowledge yielded no significant performance increment. Results are discussed against the backdrop of applying expectancy effects in high-performance sport, including dissuading athletes from using illegal aids.

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LC-MS/MS method for confirmation of recombinant human erythropoietin and darbepoetin alpha in equine plasma

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Anal Chem. 2007 Jun 15;79(12):4627-35. Epub 2007 May 15
Guan F, Uboh CE, Soma LR, Birks E, Chen J, Mitchell J, You Y, Rudy J, Xu F, Li X, Mbuy G.
University of Pennsylvania School of Veterinary Medicine, New Bolton Center Campus, 382 West Street Road, Kennett Square, Pennsylvania 19348, USA

Recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production. Consequently, they are abused in human and equine sports. To deter their abuse in the horse racing industry, a sensitive and reliable method for confirmation of these agents in equine plasma has been in urgent need. Such a method by LC-MS/MS is described in this paper. The method involved analyte enrichment by immunoaffinity separation using anti-rhEPO antibody linked to magnetic beads, digestion by trypsin, and analysis by LC-MS/MS. Two specific proteotypic peptides, 46VNFYAWK52 and 144VYSNFLR150 from rhEPO and DPO were employed for confirmation of the analytes based on chromatographic retention times and major product ions. The limit of confirmation of this method was 0.2 ng/mL, and the limit of detection was 0.1 ng/mL for rhEPO and DPO in equine plasma. This method was successful in confirming the presence of rhEPO and DPO in plasma samples collected from research horses to which rhEPO or DPO was administered and from racehorses following competition and in noncompetition samples in North America. To our knowledge, this is the first LC-MS method with adequate sensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples. This method provides a powerful enforcement tool that was lacking in the fight against the abuse of rhEPO and DPO in the horse racing industry.

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Clenbuterol marketed as dietary supplement

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Biomed Chromatogr. 2007 Oct 15; [Epub ahead of print]
Parr MK, Koehler K, Geyer H, Guddat S, Schanzer W.
Centre for Preventive Doping Research, German Sport University Cologne, Carl-Diem-Weg 6, 50933 Cologne, Germany

In several studies it has been demonstrated that products containing pharmaceutically active ingredients are marketed as dietary supplements. Most of these products contain anabolic steroids. Recently products for weight loss containing active drugs have also appeared on the market. In the present case a healthy male ordered the product 'Anabolic burner' via the Internet. The product was received from a German dispatcher and paid by bank transfer to a German bank account. After ingesting one tablet he reported tremor and delivered a urine sample. This urine was found to contain 2 ng/mL of clenbuterol utilizing LC-MS/MS analysis. Additionally the product itself was analyzed with GC-MS for clenbuterol, yielding a content of about 30 microg per tablet. The beta-2 agonist clenbuterol is only legally available on prescription and is classified as prohibited doping substance in sports. The present case for the first time confirms the presence of clenbuterol in a dietary supplement. It again demonstrates the common problem with products on the supplement market, where non-licensed pharmaceuticals and doping substances are easily available. The ingestion of these products containing additions of therapeutic drugs can lead to side effects and/or interactions with conventional medicines.

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Intermittent hypoxia exposure in a hypobaric chamber and erythropoietin abuse interpretation

J Sports Sci. 2007 Sep;25(11):1241-50
Abellan R, Ventura R, Remacha AF, Rodriguez FA, Pascual JA, Segura J.
Pharmacology Research Unit, Institut Municipal d'Investigacio Medica, IMIM-UPF, Barcelona, Spain

The aim of this study was to assess the effect of intermittent hypoxia exposure on direct and indirect methods used to evaluate recombinant human erythropoietin (rhEPO) misuse. Sixteen male triathletes were randomly assigned to either the intermittent hypoxia exposure group (experimental group) or the control normoxic group (control group). The members of the experimental group were exposed to simulated altitude (from 4000 to 5500 m) in a hypobaric chamber for 3 h per day, 5 days a week, for 4 weeks. Blood and urine samples were collected before and after the first and the final exposures, and again 2 weeks after the final exposure. While serum EPO significantly increased after the first [from a mean 8.3 IU x l(-1) (s = 3.2) to 16.6 IU x l(-1) (s = 4.7)] and final exposures [from 4.6 IU x l(-1) (s = 1.4) to 24.8 IU x l(-1) (s = 9.3)], haemoglobin, percentage of reticulocytes, and soluble transferrin receptor were not elevated. Second-generation ON/OFF models (indirect rhEPO misuse detection) were insensitive to intermittent hypoxia exposure. The distribution of the urinary EPO isoelectric profiles (direct rhEPO misuse detection) was altered after intermittent hypoxia exposure with a slight shift towards more basic isoforms. However, those shifts never resulted in misinterpretation of results. The intermittent hypoxia exposure protocol studied did not produce any false-positive result for indirect or direct detection of rhEPO misuse in spite of the changes in EPO serum concentrations and urinary EPO isoelectric profiles, respectively.

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Blood-borne infections

Clin Sports Med. 2007 Jul;26(3):425-31
Pirozzolo JJ, LeMay DC.
CentraCare Hospital, Orlando, FL 32819, USA

Blood-borne infections are transmitted by way of direct blood contact from one individual to another from injured skin or a mucous membrane. Blood-borne infections can also be transmitted through blood doping and drug abuse and through sexual contact. Risk factors for hepatitis B virus (HBV) HBV infection include travel to regions with endemic hepatitis. Prevention of blood-borne pathogens in the student-athlete should focus on traditional transmission routes and off-the-field behavior because experts believe that field transmission of blood-borne pathogens is minimal. Worldwide, HBV, hepatitis C virus (HCV), and HIV are the most common pathogens encountered. This article focuses on HBV and HCV as being the most prevalent in athletics.

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Proteases in doping control analysis

Int J Sports Med. 2007 Jul;28(7):545-9. Epub 2007 May 24
Thevis M, Maurer J, Kohler M, Geyer H, Schanzer W.
Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Cologne, Germany

Urine manipulation in sports drug testing has become a serious problem for doping control laboratories, and recent scandals in elite endurance sports have revealed the problem of urine manipulation presumably using proteases, which will impede the detection of drugs such as erythropoietin (EPO) or other peptide hormones. Using commonly accepted analytical strategies, a protocol was developed enabling the determination of elevated protease activities in doping control specimens followed by the visualization of protein degradation and identification of proteases such as chymotrypsin, trypsin and papain. Therefore, protease detection kits based on fluorescein isothiocyanate-labeled casein were employed, and protease concentrations greater than 15 microg/mL of urine entailed subsequent 1-dimensional gel electrophoretic visualization of urinary proteins. The presence of 20 microg of proteases per mL of urine caused a complete degradation of proteins usually observed in urinary matrices ("trace of burning"), while respective proteases were still detected in spiked urine samples after 10 days of storage at + 4 and - 20 degrees C. Identification of target proteases at respective molecular weights was accomplished using bottom-up sequencing approaches based on in-gel digestion of separated enzymes followed by capillary liquid chromatography--Orbitrap tandem mass spectrometry.

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Detection of recombinant human erythropoietin in urine for doping analysis: interpretation of isoelectric profiles by discriminant analysis

Also see:
Delanghe JR, Bollen M, Beullens M. Testing for recombinant erythropoietin. Am J Hematol. 2007 Oct 4 [Doping J Record]
Koudinov AR. WADA, IOC Testing For Erythropoietin Is Faked. The Doping Journal Vol. 3, 1 (2006) Published online September 24, 2006 [
FullText]

Electrophoresis. 2007 Jun;28(12):1875-81
Lasne F, Thioulouse J, Martin L, de Ceaurriz J.
Agence francaise de lutte contre le dopage, Department des analyses, Chatenay-Malabry, France.
PubMed ID & Record: 17503402

The detection in urine of recombinant human erythropoietin (rHuEPO), a hormone misused by endurance athletes as a doping agent, is based on the differentiation of its isoelectric pattern from that of the corresponding natural hormone. Different empirical criteria have been proposed for discriminating the images of the patterns but none of them have been elaborated from a rational statistical approach. Discriminant analysis was applied to a dataset of profiles defined as positive (116 profiles from 26 subjects) (presence of rHuEPO and possibly residual natural endogenous hormone) and negative (131 profiles from 131 subjects) (presence of natural endogenous hormone only). The different bands were numbered according to a template of 16 possible positions and their relative intensities constituted the 16 variables of the statistical analysis. This method was then tested with data from an administration trial of low doses (6.7-10 IU/kg) following high-dose (265 IU/kg) injections (71 profiles from one subject). The analysis of the dataset clearly separated the negative and positive profiles. A cross-validation procedure confirmed that the analysis was extremely stable: with ten-fold cross-validation, no false positives were observed even with 100,000 simulations. Furthermore, the detection of rHuEPO in the profiles from the low-dose trial was greatly improved in comparison with a previously validated empirical criterion.

Testing for recombinant erythropoietin

Am J Hematol. 2007 Oct 4
Delanghe JR, Bollen M, Beullens M.
Department of Clinical Chemistry, University Hospital, Ghent, Belgium.
PubMed ID & record: 17918255

Also see: Koudinov AR. WADA, IOC Testing For Erythropoietin Is Faked. The Doping Journal Vol. 3, 1 (2006) Published online September 24, 2006 [FullText]

Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEpo. Both direct tests (indicating the presence of exogeneous Epo isoforms) and indirect tests (indicating hematological changes induced by exogenous Epo administration) can be used for Epo detection. At present, the test adopted by the World Anti Doping Agency is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and rhEpo. However, the adopted monoclonal anti-Epo antibodies are not monospecific. Therefore, the test can occasionally lead to the false-positive detection of rhEpo (epoetin-beta) in post-exercise, protein-rich urine, or in case of contamination of the sample with microorganisms. An improved preanalytical care may counteract a lot of these problems. Adaptation of the criteria may be helpful to further refine direct Epo testing. Indirect tests have the disadvantage that they require blood instead of urine samples, but they can be applied to detect a broader range of performance improving techniques which are illicitly used in sports.

Hb mass measurement suitable to screen for illicit autologous blood transfusions

Med Sci Sports Exerc. 2007 Oct;39(10):1748-1756
Pottgiesser T, Umhau M, Ahlgrim C, Ruthardt S, Roecker K, Schumacher YO.
Medizinische Universitatsklinik, Abteilung Rehabilitative und Praventive Sportmedizin, Freiburg, GERMANY; and Zentrale Einrichtung Transfusionsmedizin, Universitatsklinikum Freiburg, Freiburg, GERMANY
PubMed ID & Record: 17909402

PURPOSE: An increase of hemoglobin (Hb) mass is the key target of blood doping practices to enhance performance as it is a main determinant of maximal oxygen uptake. Although detection methods exist for doping with recombinant EPO and homologous blood transfusions, autologous transfusions remain virtually undetectable. In this context, the most sensitive parameter would be a determination of Hb mass itself. The purpose therefore was to establish whether Hb mass measurements by the optimized CO-rebreathing method allow screening for the withdrawal and reinfusion of autologous red blood cells. METHODS:: The optimized CO-rebreathing method was used for evaluation of Hb mass in two groups at three time points (duplicate measurements: 1) baseline, 2) after donation, and 3) after reinfusion). Group I (N = 6) was to donate and receive 1 unit of packed red cells (PRC) in contrast to two PRC in group II (N = 4). The time span between withdrawal and reinfusion was 2 d. RESULTS:: The mean Hb content of the blood units was 59.0 +/- 3.9 g (group I) and 108.3 +/- 1.3 g (group II). Hb mass decreased significantly after blood withdrawal (-89 +/- 16 g in group I and -120 +/- 14 g in group II) and increased significantly after reinfusion (group I: 70 +/- 16 g; group II: 90 +/- 9 g) but was lower than at baseline (group I: -19 +/- 17 g; group II: -30 +/- 14 g). The total error of measurements for the duplicate measures ranged between 0.8 and 3.1% (Hb mass: 6.4-22.1 g). CONCLUSION:: Hb mass determination with the optimized CO-rebreathing method has sufficient precision to detect the absolute differences in Hb mass induced by blood withdrawal and autologous reinfusion. Thus, it may be suited to screen for artificially induced alterations in Hb mass.

Prevalence and risk factors of anabolic-androgenic steroids (AAS) abuse among adolescents and young adults in Poland

Soz Praventivmed. 2006;51(6):392-398.
Rachon D, Pokrywka L, Suchecka-Rachon K.
Department of Immunology, Medical University of Gdansk, Poland.
PubMed ID & Record: 17658145

OBJECTIVE: To estimate the prevalence of anabolic-androgenic steroids (AAS) abuse among adolescent and young adults in Poland. METHOD: 3,687 men (48.2%) and women (51.8%), median age 23 (interquartile range 19-30 years) participated in a survey via a "pop-up window" which appeared on two popular Polish internet portals during one month. Questions concerning their body image, exercise behaviour, education level and use of anabolic-androgenic steroids were asked. RESULTS: The prevalence of anabolic-androgenic steroids use was 6.2% among males and 2.9% among females. Male AAS users, compared to non-users, were more often concerned about their physical appearance, were less educated and often engaged in some sport activity. Among female AAS users, no significant differences concerning self-body image satisfaction or participation in sports were found. However, compared to non-users, female AAS users were less educated. CONCLUSION: The abuse of AAS is a reality in Poland and may become a serious health concern among adolescents and young adults.

Rapid screening of doping agents in human urine by vacuum MALDI-linear ion trap mass spectrometry

Anal Chem. 2007 Aug 1;79(15):6020-6. Epub 2007 Jun 30
Kosanam H, Prakash PK, Yates CR, Miller DD, Ramagiri S.
Department of Chemistry, University of Memphis, Memphis, Tennessee 38152, USA
PubMed ID & Record: 17602668

Detection of doping agents in urine frequently requires extensive separation prior to chemical analyses. Gas or liquid chromatography coupled to mass spectrometry has produced accurate and sensitive assays, but chromatographic separations require time and, sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique. Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and betamethasone were chosen as study compounds. Different MALDI matrixes like alpha-cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl) porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range (m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit of detection and lower limit of quantification for all five analytes have been determined to be 0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal standard, and the quantitative measurements were achieved by the selective reaction monitoring (SRM) mode. The method was validated and showed consistency in the results. Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and quantification of doping agents in urine.

Medical report from the 2006 FIFA World Cup Germany

Br J Sports Med. 2007 Sep;41(9):578-81; discussion 581. Epub 2007 May 17
Dvorak J, Junge A, Grimm K, Kirkendall D.
FIFA Medical Assessment and Research Centre, Schulthess Clinic, Zurich, Switzerland
PMID: 17510228

OBJECTIVE: To continue the injury surveillance of FIFA-sponsored football tournaments and report on other medical aspects of the 2006 FIFA World Cup. DESIGN: Prospective epidemiological injury surveillance and descriptive summary of additional medical aspects. SETTING: Major international football tournament. PARTICIPANTS: National team players, doctors and referees at the 2006 FIFA World Cup Germany. MAIN OUTCOME MEASURES: Injury type, location and rate. RESULTS: 145 injuries were reported for the 64 matches of the 2006 FIFA World Cup Germany-an overall injury rate of 68.7 per 1000 match hours (95% CI 57.5 to 79.9) or 2.3 injuries per match, in comparison with 2.7 injuries per match in the 2002 FIFA World Cup (p = NS). Physical examinations before participation uncovered no hidden cardiovascular problems. Once the tournament started, no referees were unable to complete their duties. There were no positive doping tests. CONCLUSIONS: The injury rate for this World Cup was below that of 2002, but consistent with the overall injury rate per match since data collection began in 1998. There continues to be no evidence of systematic doping in international football.

Doping and respiratory system

Monaldi Arch Chest Dis. 2007 Mar;67(1):53-62
Casali L, Pinchi G, Puxeddu E.
Department of Internal Medicine, Section of Respiratory Disease, University of Perugia, Italy
PubMed ID & Record: 17564285

Historically many different drugs have been used to enhance sporting performances. The magic elixir is still elusive and the drugs are still used despite the heavy adverse effects. The respiratory system is regularly involved in this research probably because of its central location in the body with several connections to the cardiovascular system. Moreover people are aware that O2 consumption and its delivery to mitochondria firstly depend on ventilation and on the respiratory exchanges. The second step consists in the tendency to increase V'O2 max and to prolong its availability with the aim of improving the endurance time and to relieve the fatigue. Many methods and substances had been used in order to gain an artificial success. Additional oxygen, autologous and homologous transfusion and erythropoietin, mainly the synthetic type, have been administered with the aim of increasing the amount of oxygen being delivered to the tissues. Some compounds like stimulants and caffeine are endowed of excitatory activity on the CNS and stimulate pulmonary ventilation. They did not prove to have any real activity in supporting the athletic performances. Beta-adrenergic drugs, particularly clenbuterol, when administered orally or parenterally develop a clear illicit activity on the myosin fibres and on the muscles as a whole. Salbutamol, terbutaline, salmeterol and formoterol are legally admitted when administrated by MDI in the treatment of asthma. The prevalence of asthma and bronchial hyperactivity is higher in athletes than amongst the general population. This implies that clear rules must be provided to set a correct diagnosis of asthma in the athletes and a correct therapy to align with the actual guidelines according to the same rights of the "other" asthmatic patients.

Similarity of polygenic profiles limits the potential for elite human physical performance

J Physiol. ePub 2007 Sep 27
Williams AG, Folland JP.
Manchester Metropolitan University.
PubMed ID & Record: 17901117

Human physical capability is influenced by many environmental and genetic factors, and it is generally accepted that physical capability phenotypes are highly polygenic. However, the ways relevant polymorphisms combine to influence physical capability of individuals and populations are unknown. Initially, the literature was searched to identify associations between 23 genetic polymorphisms and human endurance phenotypes. Next, typical genotype frequencies of those polymorphisms in the general population were obtained from suitable literature. Using probability calculations, we found only a 0.0005% chance of a single individual in the world having the 'preferable' form of all 23 polymorphisms. As the number of DNA variants shown to be associated with human endurance phenotypes continues to increase, the probability of any single individual possessing the 'preferable' form of each polymorphism will become even lower. However, with population turnover, the chance of such genetically gifted individuals existing increases. To examine the polygenic endurance potential of a human population, a 'total genotype score' (for the 23 polymorphisms) was calculated for each individual within a hypothetical population of 1,000,000. There was considerable homogeneity in terms of genetic predisposition to high endurance potential, with 99% of people differing by no more than seven genotypes from the typical profile. Consequently, with population turnover world and Olympic records should improve even without further enhancement of environmental factors, as more 'advantageous' polygenic profiles occasionally, though rarely, emerge. More broadly, human potential appears limited by the similarity of polygenic profiles at both the 'elite sport' and 'chronic disorder' ends of the performance continuum.

Monitoring Gene Therapy by External Imaging of mRNA: Pilot Study on Murine Erythropoietin

Ther Drug Monit. 2007 Oct;29(5):612-618.
Segura J, Fillat C, Andreu D, Llop J, Millan O, de la Torre BG, Nikolovski Z, Gomez V, Andreu N, Pinyot A, Castelo R, Gispert JD, Pascual JA.
From the *Institut Municipal d'Investigacio Medica IMIM-Hospital del Mar; daggerCentre de Regulacio Genomica; double daggerUniversitat Pompeu Fabra; and section signInstitut d'Alta Tecnologia, Barcelona, Spain.
PubMed ID & record: 17898652

Gene therapy is anticipated as being an important medical development. Essential to its effectiveness is the appropriate activity (protein expression) in the expected target cells. A noninvasive diagnostic procedure of successful gene expression will be of paramount importance to validate its use or its misuse (eg, sports gene doping). Externally detectable labeled oligonucleotide hybridizing with the messenger RNA generated by the transferred gene has been proposed as a possibility to monitor successful gene therapy. The authors selected the erythropoietin gene (Epo) for a pilot study on erythropoietin protein expression in mouse muscle. Oligonucleotides of peptide nucleic acid (PNA) type capable of antisense binding to unique murine Epo-mRNA sequences were synthesized by solid phase methods, and elongated at the N-terminus with the HIV Tat (48-60) cell penetrating peptide. They were labeled with fluorescence and radioactive tags to verify penetration and longer half-life properties in Epo gene transfected C2C12 mouse muscle cells as compared with corresponding wild-type cells. Downregulation of newly expressed erythropoietin protein in such cells additionally confirmed the penetration and hybridizing properties of the selected labeled oligonucleotide. I-labeled Tat-PNAs were intravenously injected into mice that had previously received the Epo gene into the right tibialis muscle by DNA electrotransfer. Preferential accumulation of radioactivity in the transferred limb as compared with the contralateral limb was ascertained, especially for I-Tat-CTA CGT AGA CCA CT (labeled Tat-PNA 1). This study provides experimental data to support the potential use of external noninvasive image detection to monitor gene therapy. The extension of the approach to more sensitive methods for whole-body external detection such as positron emission tomography appears feasible.

Determination of tuaminoheptane in doping control urine samples

Eur J Mass Spectrom (Chichester, Eng). 2007;13(3):213-21. LinkOut
Thevis M, Sigmund G, Koch A, Schanzer W.
Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Carl-Diem Weg 6, 50933 Cologne, Germany.
PubMed ID & record: 17881789

Since January 2007, the list of prohibited substances established by the World Anti-Doping Agency includes the sympathomimetic compound tuaminoheptane (1-methyl-hexylamine, 2-heptylamine). Primarily used as nasal decongestant drug it has been considered relevant for sports drug testing due to its stimulating properties. A confirmatory gas chromatographic-mass spectrometric procedure was developed including liquidliquid extraction and imine formation of tuaminoheptane employing various aldehydes and ketones such as formaldehyde, acetaldehyde, benzaldehyde and acetone. Extraction and derivatisation conditions were optimised for utmost efficiency, and characteristic fragment ions obtained after electron ionisation allowed for a sensitive and selective analytical assay, which was validated with regard to recovery (50%), lower limit of detection (20 ng mL(1)) as well as interday- and intraday precision (<15%). The applicability to authentic urine samples was demonstrated using administration study specimens obtained from two male persons using Rhinofluimucil (tuaminoheptane hemisulfate) for intranasal application. The administered drug was detected up to 46 h after repeated topical instillation of a total of approximately 3 mg.