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“Beyond Reasonable Doubt - an Analysis of the Uncertainty Behind a Positive Test For UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE HEALTH AND LIFE SCIENCES School of Medicine “Beyond Reasonable Doubt - An Analysis of the Uncertainty behind a Positive Test for Growth Hormone Abuse in Sport” by Ioulietta Erotokritou-Mulligan MSc, BSc(Hons) Thesis for the degree of Doctor of Philosophy June 2008 2 | P a g e ABSTRACT Growth hormone (GH) is a protein hormone which occurs naturally in the body and it is produced by the pituitary gland. The anabolic properties of GH have made this an attractive substance to be used by patients with catabolic medical problems and athletes that wish to enhance their athletic performance. However, to date, there is no scientific evidence to confirm that its use will enhance physical performance in sport. Aside from the morality issues regarding GH abuse in sports, the use of GH has been associated with a number of potential health risks. GH is on the International Olympic Committee (IOC) Prohibited Substance list. To date however, there is no established method available for detecting its abuse reliably. In 1999 the GH-2000 research group proposed a GH detection methodology to the IOC. The method uses the concentration values of two GH-dependent markers; Insulin Growth Factor-I (IGF-I) and Procollagen type III peptide (P-III-P). Concerns were raised by the IOC regarding the uncertainty involved in this methodology on areas such as the effect of ethnicity, injuries and the physiological variability on its ability to find an athlete guilty beyond reasonable doubt. The GH-2004 project was set up at the University of Southampton in order to address these issues by undertaking several studies to assess the effect of these potential confounding factors. This thesis used data collected by the GH-2004 project to examine the issues raised by the IOC. Furthermore, work was undertaken to validate the performance of the proposed method on independent data which will provide further evidence of the reliability of the GH-2000 detection method. An examination into the uncertainty surrounding the use of immunoassay methodology in this method was also carried out. This thesis demonstrates that the GH-2000 detection method is robust when used for individuals of any ethnic group regardless of any sport injuries experienced by athletes. Furthermore, it is shown that the physiological variability of the concentration of the two GH- dependent markers will not adversely affect the performance of this method. The validity of this method is also successfully demonstrated using independently collected data. Before this method is officially implemented, there is important laboratory analysis which needs to be undertaken, which will enable us to determine reliable cut-off limits, which will both maximise the sensitivity of the test but also reduce the rate of false positive results. It has been shown that this methodology is a powerful method and should help to cut down the abuse of GH. Keywords: Growth Hormone, Doping, Sports, IGF-I, Injury, Ethnicity, Physiological variability, Immunoassays 3 | P a g e ABBREVIATIONS ALS Acid-labile subunit BMI Body mass index CV Coefficient of variation EF3 Discriminant function for females EF3b Age-adjusted Discriminant function for females EM1 Discriminant function for males EM1b Age-adjusted discriminant function for males FDA Food and Drug Administration GH Growth hormone hGH Human growth hormone ICTP Carboxyl-terminal cross-linked telopeptide of type I collagen IGFBP IGF-binding protein IGF-I Insulin growth factor – I IOC International Olympic Committee OLR Ordinary Least-Squares Regression Osteo Osteocalcin PICP Carboxyl-terminal propeptide of type I procollagen 4 | P a g e P-III-P Procollagen type III peptide rhGH Recombinant human growth hormone Te Testosterone USADA United States Anti-Doping Agency WADA World Anti-Doping Agency 5 | P a g e DECLARATION OF AUTHORSHIP I, IOULIETTA EROTOKRITOU-MULLIGAN, declare that the thesis entitled “Beyond Reasonable Doubt - an Analysis of the Uncertainty behind a Positive Test for Growth Hormone Abuse” and the work presented in it are my own. I confirm that: this work was done wholly or mainly while in candidature for a research degree at this University; where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated; where I have consulted the published work of others, this is always clearly attributed; where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work; I have acknowledged all main sources of help; where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself; none of this work has been published before submission Signed: ……………………………………………………………………….. Date:……………………………………………………………………………. 6 | P a g e ACKNOWLEDGEMENTS The principal investigators of the GH-2004 project were Dr. Richard I.G. Holt, Reader of Endocrinology and Metabolism at the University of Southampton and Professor Peter Sönksen, Emeritus Professor at the University of Southampton. Dr. Richard I.G. Holt was responsible for designing all studies covered in this project. Statistical input on the study design was made by Dr. Eryl Bassett, Senior Lecturer in Statistics at the University of Kent. The initial Ethics application for approval of these studies was made by Dr. Richard I.G. Holt. Subsequent revisions were made by the GH-2004 Research Fellow; Dr Cathy McHugh. Recruitment for study participants was undertaken by members of the GH-2004 project team; myself, Dr Richard I.G. Holt, Dr Cathy McHugh, Ms Clare Hartley (Project Manager 2002-2004), Mr Rod Park (Project Manager 2004-2006) and the medical students: Jenny Bacon, Kate Harrison, Ben Curtis, Luke Smith, Victoria Wells, Barnaby Fontaine and Polly Milward. Furthermore, Dr. Richard Seah recruited athletes with a sporting injury for the GH- 2004 Injury Study. Compliance assessments, sample collection and anthropological measurements were undertaken by Dr Cathy McHugh or under the supervision of Dr McHugh by one of the medical students involved in the GH-2004 project. Data quality assessment was a collective effort by the whole of the GH-2004 project team. Laboratory Analysis of samples collected by the GH-2004 project was undertaken by the WADA accredited laboratory at King’s College in London by Professor David Cowan and Mr Christiaan Bartlett. I would like to thank the nurses of the Wellcome Trust Clinical Research Facility at the University of Southampton for their assistance in recruitment and follow-up of the subjects. 7 | P a g e Furthermore, I would also like to thank Dr. Astrid Kniess and Professor Alessandro Sartorio for sharing the results of their studies for the purposes of the analysis presented in this thesis. In addition, I would like to acknowledge the support of Dr Richard Budgett, Director of Medical Services for the British Olympic Association. I would also like to acknowledge the legacy of the GH-2000 Project (a European Union ‘Biomed 2’ Project Contract umber: BMH4 CT950678). The GH-2004 study was funded by the World Anti-Doping Agency and US Anti-Doping Agency without which the studies described in this thesis would not have been possible. With the oversight of my supervisors, editorial advice has been sought. No changes of intellectual content were made as a result of this advice. 8 | P a g e SPECIAL ACKNOWLEDGEMENTS I would like to genuinely thank my supervisors (in no particular order) Dr Richard Holt, Dr Eryl Bassett and Professor Peter Sönksen for being a true inspiration and for looking after me so well the last few years. I feel incredibly blessed for having such an amazing supervisory team. I could not have done this without their support and help. I would also like to thank my parents, Erotokritos and Eve for believing in me and supporting me emotionally & financially all these years. Without their love, I would have probably given up a long time ago. Last, but definitely not least, the person I owe all of this to, is my beloved husband Stewart. I would like to thank him for looking after me better than an angel could, with infinite patience and love. I hope I can now finally return some favours. Thank you. 9 | P a g e To my angel, Stewart 10 | P a g e TABLE OF CONTENTS ABSTRACT ..................................................................................................................................................... 2 ACKNOWLEDGEMENTS ............................................................................................................................ 6 SPECIAL ACKNOWLEDGEMENTS ........................................................................................................... 8 LIST OF FIGURES ...................................................................................................................................... 18 CHAPTER 1: INTRODUCTION ........................................................................................................... 23 1.1 BACKGROUND TO THE RESEARCH .......................................................................................................... 24 1.2 JUSTIFICATION FOR THIS RESEARCH ..................................................................................................... 28 1.3 METHODOLOGY .......................................................................................................................................
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