Problems with Growth Hormone Doping in Sports: Isoform Methods

  • Martin Bidlingmaier
  • Zida Wu
  • Christian J. Strasburger
Chapter
Part of the Endocrine Updates book series (ENDO, volume 29)

Abstract

Human growth hormone (GH), a 191 amino acid proteohormone, is abused in sports. Although there were only few positive test result reported from accredited antidoping laboratories until today, there is enough evidence to assume its wider abuse for performance enhancement: Former athletes have confessed to have abused GH, and ampoules containing recombinant GH have been found in the possession of active athletes or trainers by police and customs officials. For a long time, the detection of GH abuse by testing laboratories was not possible. The main reason why it was difficult to develop an appropriate test was the identity in amino acid sequence between pituitary and recombinant GH, making a distinction by means of physicochemical characterization impossible. Over the last decade, two approaches have been followed to detect abuse of GH. One approach, based on characterization of changes in concentrations of proteins which are produced in response to GH (the “marker approach”), is described elsewhere in this book. Here, we are describing an alternative strategy, called the “differential immunoassay” or “isoform approach.” Pituitary GH consists of a spectrum of molecular isoforms, whereas recombinant hGH preparations are made of the 22 kD isoform only. After injection, secretion of the pituitary-derived isoforms is blunted by negative feedback, and the 22 kD isoform becomes predominant in circulation. Specific immunoassays employing monoclonal antibodies with a preference for the one or the other isoform can be used to detect these changes. Two sets of specific assays have been validated for this purpose and are currently implemented in antidoping laboratories. Future directions for developing the isoform approach include the specific measurement of the 20 kD isoform, which is reduced after application of 22 kD GH, and also the identification of GH isoforms by methods other than immunoassays, including plasmon surface resonance, two dimensional gel electrophoresis, and mass spectrometry.

Keywords

Glutathione Electrophoresis Half Life 

References

  1. 1.
    Baumann G. Growth hormone heterogeneity: Genes, isohormones, variants, and binding ­proteins. Endocr Rev. 1991;12:424–49CrossRefPubMedGoogle Scholar
  2. 2.
    Hirt H, Kimelman J, Birnbaum MJ, Chen EY, Seeburg PH, Eberhardt NL, Barta A. The human growth hormone gene locus: Structure, evolution, and allelic variations. DNA. 1987;6:59–70CrossRefPubMedGoogle Scholar
  3. 3.
    Igout A, Van Beeumen J, Frankenne F, Scippo ML, Devreese B, Hennen G. Purification and biochemical characterization of recombinant human placental growth hormone produced in Escherichia coli. Biochem J. 1993;295:719–24PubMedGoogle Scholar
  4. 4.
    Cooke NE, Ray J, Watson MA, Estes PA, Kuo BA, Liebhaber SA. Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns. J Clin Invest. 1988;82:270–5CrossRefPubMedGoogle Scholar
  5. 5.
    Wallace JD, Cuneo RC, Bidlingmaier M, Lundberg PA, Carlsson L, Boguszewski CL, Hay J, Healy ML, Napoli R, Dall R, Rosen T, Strasburger CJ. The response of molecular isoforms of growth hormone to acute exercise in trained adult males. J Clin Endocrinol Metab. 2001;86:200–6CrossRefPubMedGoogle Scholar
  6. 6.
    Nindl BC. Exercise modulation of growth hormone isoforms: Current knowledge and future directions for the exercise endocrinologist. Br J Sports Med. 2007;41:346–8; discussion 348Google Scholar
  7. 7.
    Baumann G. Growth hormone heterogeneity in human pituitary and plasma. Horm Res. 1999;51:2–6CrossRefPubMedGoogle Scholar
  8. 8.
    Popii V, Baumann G. Laboratory measurement of growth hormone. Clin Chim Acta. 2004; 350:1–16CrossRefPubMedGoogle Scholar
  9. 9.
    Baumann G, Stolar MW, Buchanan TA. The metabolic clearance, distribution, and degradation of dimeric and monomeric growth hormone (GH): Implications for the pattern of circulating GH forms. Endocrinology. 1986;119:1497–501CrossRefPubMedGoogle Scholar
  10. 10.
    Baumann G, Amburn K, Shaw MA. The circulating growth hormone (GH)-binding protein complex: A major constituent of plasma GH in man. Endocrinology. 1988;122:976–84CrossRefPubMedGoogle Scholar
  11. 11.
    Kratzsch J, Selisko T, Birkenmeier G. Identification of transformed alpha 2-macroglobulin as a growth hormone-binding protein in human blood. J Clin Endocrinol Metab. 1995;80: 585–90CrossRefPubMedGoogle Scholar
  12. 12.
    Bidlingmaier M, Freda PU. Measurement of human growth hormone by immunoassays: Current status, unsolved problems and clinical consequences. Growth Horm IGF Res. 2010; 20:19–25Google Scholar
  13. 13.
    Pokrajac A, Wark G, Ellis AR, Wear J, Wieringa GE, Trainer PJ. Variation in GH and IGF-I assays limits the applicability of international consensus criteria to local practice. Clin Endocrinol (Oxf). 2007;67:65–70CrossRefGoogle Scholar
  14. 14.
    Wieringa GE, Trainer PJ. Commentary: Harmonizing growth hormone measurements: Learning lessons for the future. J Clin Endocrinol Metab. 2007;92:2874–5CrossRefPubMedGoogle Scholar
  15. 15.
    Boguszewski CL, Hynsjo L, Johannsson G, Bengtsson BA, Carlsson LM. 22-kD growth hormone exclusion assay: A new approach to measurement of non-22-kD growth hormone isoforms in human blood. Eur J Endocrinol. 1996;135:573–82CrossRefPubMedGoogle Scholar
  16. 16.
    Wada M, Uchida H, Ikeda M, Tsunekawa B, Naito N, Banba S, Tanaka E, Hashimoto Y, Honjo M. The 20-kilodalton (kDa) human growth hormone (hGH) differs from the 22-kDa hGH in the complex formation with cell surface hGH receptor and hGH-binding protein circulating in human plasma. Mol Endocrinol. 1998;12:146–56CrossRefPubMedGoogle Scholar
  17. 17.
    Keller A, Wu Z, Kratzsch J, Keller E, Blum WF, Kniess A, Preiss R, Teichert J, Strasburger CJ, Bidlingmaier M. Pharmacokinetics and pharmacodynamics of GH: Dependence on route and dosage of administration. Eur J Endocrinol. 2007;156:647–53CrossRefPubMedGoogle Scholar
  18. 18.
    Ishikawa M, Yokoya S, Tachibana K, Hasegawa Y, Yasuda T, Tokuhiro E, Hashimoto Y, Tanaka T. Serum levels of 20-kilodalton human growth hormone (GH) are parallel those of 22-kilodalton human GH in normal and short children. J Clin Endocrinol Metab. 1999;84:98–104CrossRefPubMedGoogle Scholar
  19. 19.
    Leung KC, Howe C, Gui LY, Trout G, Veldhuis JD, Ho KK. Physiological and pharmacological regulation of 20-kDa growth hormone. Am J Physiol Endocrinol Metab. 2002;283:E836–43PubMedGoogle Scholar
  20. 20.
    Tsushima T, Katoh Y, Miyachi Y, Chihara K, Teramoto A, Irie M, Hashimoto Y. Serum ­concentration of 20K human growth hormone (20K hGH) measured by a specific enzyme-linked immunosorbent assay. Study Group of 20K hGH. J Clin Endocrinol Metab. 1999;84:317–22CrossRefPubMedGoogle Scholar
  21. 21.
    Sinha YN, Jacobsen BP. Human growth hormone (hGH)-(44-191), a reportedly diabetogenic fragment of hGH, circulates in human blood: Measurement by radioimmunoassay. J Clin Endocrinol Metab. 1994;78:1411–8CrossRefPubMedGoogle Scholar
  22. 22.
    Wu Z, Bidlingmaier M, Friess SC, Kirk SE, Buchinger P, Schiessl B, Strasburger CJ. A new nonisotopic, highly sensitive assay for the measurement of human placental growth hormone: Development and clinical implications. J Clin Endocrinol Metab. 2003;88:804–11CrossRefPubMedGoogle Scholar
  23. 23.
    Celniker AC, Chen AB, Wert RM, Jr., Sherman BM. Variability in the quantitation of circulating growth hormone using commercial immunoassays. J Clin Endocrinol Metab. 1989;68:469–76CrossRefPubMedGoogle Scholar
  24. 24.
    Bidlingmaier M, Suhr J, Ernst A, Wu Z, Keller A, Strasburger CJ, Bergmann A. High-sensitivity chemiluminescence immunoassays for detection of growth hormone doping in sports. Clin Chem. 2009;55:445–53CrossRefPubMedGoogle Scholar
  25. 25.
    Hayakawa M, Shimazaki Y, Tsushima T, Kato Y, Takano K, Chihara K, Shimatsu A, Irie M. Metabolic effects of 20-kilodalton human growth hormone (20K-hGH) for adults with growth hormone deficiency: Results of an exploratory uncontrolled multicenter clinical trial of 20K-hGH. J Clin Endocrinol Metab. 2004;89:1562–71CrossRefPubMedGoogle Scholar
  26. 26.
    Bidlingmaier M, Strasburger CJ. Technology insight: Detecting growth hormone abuse in athletes. Nat Clin Pract Endocrinol Metab. 2007;3:769–77CrossRefPubMedGoogle Scholar
  27. 27.
    Bidlingmaier M, Wu Z, Strasburger CJ. Test method: GH. Baillieres Best Pract Res Clin Endocrinol Metab. 2000;14:99–109CrossRefPubMedGoogle Scholar
  28. 28.
    Wu Z, Bidlingmaier M, Dall R, Strasburger CJ. Detection of doping with human growth hormone. Lancet. 1999;353:895CrossRefPubMedGoogle Scholar
  29. 29.
    Wallace JD, Cuneo RC, Bidlingmaier M, Lundberg PA, Carlsson L, Boguszewski CL, Hay J, Boroujerdi M, Cittadini A, Dall R, Rosen T, Strasburger CJ. Changes in non-22-kilodalton (kDa) isoforms of growth hormone (GH) after administration of 22-kDa recombinant human GH in trained adult males. J Clin Endocrinol Metab. 2001;86:1731–7CrossRefPubMedGoogle Scholar
  30. 30.
    Saugy M, Robinson N, Saudan C, Baume N, Avois L, Mangin P. Human growth hormone doping in sport. Br J Sports Med. 2006;40 Suppl 1:i35–9CrossRefPubMedGoogle Scholar
  31. 31.
    Gutierrez-Gallego R, Bosch J, Such-Sanmartin G, Segura J. Surface plasmon resonance immuno assays – A perspective. Growth Horm IGF Res. 2009;19:388–98CrossRefPubMedGoogle Scholar
  32. 32.
    Kohler M, Puschel K, Sakharov D, Tonevitskiy A, Schanzer W, Thevis M. Detection of recombinant growth hormone in human plasma by a 2-D PAGE method. Electrophoresis. 2008;29:4495–502CrossRefPubMedGoogle Scholar
  33. 33.
    Kohler M, Thomas A, Puschel K, Schanzer W, Thevis M. Identification of human pituitary growth hormone variants by mass spectrometry. J Proteome Res. 2009;8:1071–6CrossRefPubMedGoogle Scholar
  34. 34.
    Segura J, Gutierrez-Gallego R, Ventura R, Pascual JA, Bosch J, Such-Sanmartin G, Nikolovski Z, Pinyot A, Pichini S. Growth hormone in sport: Beyond Beijing 2008. Ther Drug Monit. 2009;31:3–13CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Martin Bidlingmaier
    • 1
  • Zida Wu
  • Christian J. Strasburger
  1. 1.Endocrine Laboratory, Medizinische Klinik – InnenstadtLudwig-Maximilians-UniversityMunichGermany

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