BritisM MtdUal Bulliltn (1992) Vol 48, No. 3, pp. 496-517 O The Brituh Council 1992
Peptide hormones and sport: Misuse and detection A T Kicman and D A Cowan Drug Control and Teaching Centre, King's College, Manresa Road, London, UK.
In 1989 the Medical Commission of the International Olympic Committee (IOC) introduced the new doping class of 'peptide hormones and analogues,' which include human chorionic gonadotrophin (hCG) and related compounds, adrenocorticotrophic hormone (ACTH), human growth hormone (hGH), all the releasing factors of these listed hormones, and erythropoietin (Epo). Currently there are no IOC approved definitive tests for these hormones but highly specific immunoassays combined with suitable purification techniques may be sufficient to warrant IOC approval. The importance of measuring hCG and luteinizing hormone (LH) in the control of testosterone misuse is discussed and strategies for the detection of hGH, ACTH and Epo administration are suggested.
In 1967, the International Olympic Committee (IOC) established a Medical Commission to deal with the widespread misuse of drugs in top-level sport. The Medical Commission banned the practice of doping the following year and up to the early 1980's the IOC accredited laboratories were dedicated to the testing of banned foreign substances or their metabolites in athletes' urine, e.g. anabolic-androgenic steroids. Since then, drug testing has evolved from the comparatively simple task of detecting these synthetic compounds. For example, the naturally occurring androgen testosterone was added to the IOC Medical Commission list of banned substances in 1983. In 1989 the Commission introduced the new doping class of 'peptide hormones and analogues', which included human chorionic gonadotrophin (hCG) and related com-
PEPTIDE HORMONES AND SPORT: MISUSE AND DETECTION
497
pounds, adrenocorticotrophic hormone (ACTH), human growth hormone (hGH), all the releasing factors of these listed hormones, and erythropoietin (Epo). Currently there are no IOC approved definitive tests for these hormones and hence no confirmatory methods of analysis. Nevertheless, at the Drug Control Centre, which is the IOC accredited laboratory in the UK, all urine samples obtained from male athletes are screened for abnormal concentrations of hCG. This review will describe the biological action of these hormones, the reasons why they may be misused in sport and possible means of detection. In addition to the banned peptide hormones, the importance of measuring urinary luteinizing hormone (LH) as an additional criterion in determining testosterone administration will be presented. BANNED DRUGS AND PEPTIDE HORMONE MEASUREMENT Only untimed urine samples are available from athletes as blood collection is currently considered to be too invasive to the individual. The presence of banned drugs or their metabolites are identified unequivocally by gas chromatography coupled to a mass spectrometer (GC-MS). With GC-MS, the compound of interest is separated from other potentially interfering substances by high resolution chromatography before being ionised by the mass spectrometer. The ions generated are separated according to their mass-to-charge ratio and a characteristic mass spectrum of that compound is generated. The quantification of 'peptide hormones' by GC-MS is not possible due to the unsuitable conditions of gas chromatography. However, analysis by mass spectrometry may be possible in the future by use of the recent interfacing of alternative chromatographic systems to mass spectrometers (e.g. high performance liquid chromatography) and the introduction of new ionization methods (e.g. fast atom bombardment and electrospray) for polar, charged, high mass molecules, such as hCG and LH. 1 Currently, these methods are not sufficiently sensitive and the only means of measuring small quantities of these hormones is by immunoassay. There are a number of commercially supplied immunoassay kits available for hCG, LH, hGH, ACTH and Epo, which incorporate the use of highly specific antibodies. Nevertheless the IOC does not accept the singular use of immunoassay as a definitive method of analysis and thus there are no IOC approved
498
SPORTS MEDICINE
tests for 'peptide' hormone administration. The rationale for this unacceptability is that immunoassay does not have a discriminating power equivalent to GC-MS. This problem could be overcome if potentially interfering substances in the urine were removed prior to confirmation by immunoassay. At the Drug Control Centre, ultrafiltration procedures have been introduced for this purpose in the measurement of LH and hCG. Ultrafiltration may also be of potential use for the measurement of other 'peptide' hormones. LUTEINIZING HORMONE AND HUMAN CHORIONIC GONADOTROPHIN With the knowledge of an efficient GC-MS detection system for synthetic steroids, it was anticipated that some athletes may switch to using the natural androgen testosterone to evade detection and also that male athletes could use hCG to stimulate their endogenous testosterone production.2 Human CG could also be used by male athletes to prevent testicular atrophy during and after prolonged courses of androgen administration. In 1987, 3 different commercially available immunoassay kits were used to evaluate the incidence of hCG administration in selected sport events. Out of 740 samples, 21 samples were found to have unnaturally large concentrations of urinary hCG, 3 and such a large incidence of misuse led to the IOC ban on hCG. The biochemistry, endocrinology and immunoassay of hCG has been reviewed by Kicman, Brooks and Cowan.4 Human CG and LH will be considered together, as these hormones have fundamentally the same biological action at cellular level, and both are essential components to be considered in the control of testosterone misuse in sport. The gonadotrope cells of the anterior pituitary secrete the two gonadotrophins, LH and follicle stimulating hormone (FSH). In men, LH stimulates steroidogenesis in the Leydig cells of the testes, resulting in the synthesis and release of testosterone. Both gonadotrophins, together with testosterone are important in spermatogenesis. In women, LH and FSH interact with ovarian theca and granulosa cells to stimulate steroidogenesis, follicular maturation and ovulation. Human CG is secreted in large amounts by the developing placenta or chorion during pregnancy. During the first trimester of pregnancy hCG is considered to play an important role in the maintenance of the corpus luteum for the production of progesterone and to a lesser extent oestradiol. Human
PEPTIDE HORMONES AND SPORT: MISUSE AND DETECTION
499
CG may also be necessary for male sexual development by possibly stimulating the transient secretion of testosterone by the fetal Leydig cells during the second and third trimester. Very small concentrations of hCG or hCG-like material have been measured in the serum of normal men (less than 60 years, < 1.3 IU/1; after 60 years,
Peptide hormones and sport: Misuse and detection A T Kicman and D A Cowan Drug Control and Teaching Centre, King's College, Manresa Road, London, UK.
In 1989 the Medical Commission of the International Olympic Committee (IOC) introduced the new doping class of 'peptide hormones and analogues,' which include human chorionic gonadotrophin (hCG) and related compounds, adrenocorticotrophic hormone (ACTH), human growth hormone (hGH), all the releasing factors of these listed hormones, and erythropoietin (Epo). Currently there are no IOC approved definitive tests for these hormones but highly specific immunoassays combined with suitable purification techniques may be sufficient to warrant IOC approval. The importance of measuring hCG and luteinizing hormone (LH) in the control of testosterone misuse is discussed and strategies for the detection of hGH, ACTH and Epo administration are suggested.
In 1967, the International Olympic Committee (IOC) established a Medical Commission to deal with the widespread misuse of drugs in top-level sport. The Medical Commission banned the practice of doping the following year and up to the early 1980's the IOC accredited laboratories were dedicated to the testing of banned foreign substances or their metabolites in athletes' urine, e.g. anabolic-androgenic steroids. Since then, drug testing has evolved from the comparatively simple task of detecting these synthetic compounds. For example, the naturally occurring androgen testosterone was added to the IOC Medical Commission list of banned substances in 1983. In 1989 the Commission introduced the new doping class of 'peptide hormones and analogues', which included human chorionic gonadotrophin (hCG) and related com-
PEPTIDE HORMONES AND SPORT: MISUSE AND DETECTION
497
pounds, adrenocorticotrophic hormone (ACTH), human growth hormone (hGH), all the releasing factors of these listed hormones, and erythropoietin (Epo). Currently there are no IOC approved definitive tests for these hormones and hence no confirmatory methods of analysis. Nevertheless, at the Drug Control Centre, which is the IOC accredited laboratory in the UK, all urine samples obtained from male athletes are screened for abnormal concentrations of hCG. This review will describe the biological action of these hormones, the reasons why they may be misused in sport and possible means of detection. In addition to the banned peptide hormones, the importance of measuring urinary luteinizing hormone (LH) as an additional criterion in determining testosterone administration will be presented. BANNED DRUGS AND PEPTIDE HORMONE MEASUREMENT Only untimed urine samples are available from athletes as blood collection is currently considered to be too invasive to the individual. The presence of banned drugs or their metabolites are identified unequivocally by gas chromatography coupled to a mass spectrometer (GC-MS). With GC-MS, the compound of interest is separated from other potentially interfering substances by high resolution chromatography before being ionised by the mass spectrometer. The ions generated are separated according to their mass-to-charge ratio and a characteristic mass spectrum of that compound is generated. The quantification of 'peptide hormones' by GC-MS is not possible due to the unsuitable conditions of gas chromatography. However, analysis by mass spectrometry may be possible in the future by use of the recent interfacing of alternative chromatographic systems to mass spectrometers (e.g. high performance liquid chromatography) and the introduction of new ionization methods (e.g. fast atom bombardment and electrospray) for polar, charged, high mass molecules, such as hCG and LH. 1 Currently, these methods are not sufficiently sensitive and the only means of measuring small quantities of these hormones is by immunoassay. There are a number of commercially supplied immunoassay kits available for hCG, LH, hGH, ACTH and Epo, which incorporate the use of highly specific antibodies. Nevertheless the IOC does not accept the singular use of immunoassay as a definitive method of analysis and thus there are no IOC approved
498
SPORTS MEDICINE
tests for 'peptide' hormone administration. The rationale for this unacceptability is that immunoassay does not have a discriminating power equivalent to GC-MS. This problem could be overcome if potentially interfering substances in the urine were removed prior to confirmation by immunoassay. At the Drug Control Centre, ultrafiltration procedures have been introduced for this purpose in the measurement of LH and hCG. Ultrafiltration may also be of potential use for the measurement of other 'peptide' hormones. LUTEINIZING HORMONE AND HUMAN CHORIONIC GONADOTROPHIN With the knowledge of an efficient GC-MS detection system for synthetic steroids, it was anticipated that some athletes may switch to using the natural androgen testosterone to evade detection and also that male athletes could use hCG to stimulate their endogenous testosterone production.2 Human CG could also be used by male athletes to prevent testicular atrophy during and after prolonged courses of androgen administration. In 1987, 3 different commercially available immunoassay kits were used to evaluate the incidence of hCG administration in selected sport events. Out of 740 samples, 21 samples were found to have unnaturally large concentrations of urinary hCG, 3 and such a large incidence of misuse led to the IOC ban on hCG. The biochemistry, endocrinology and immunoassay of hCG has been reviewed by Kicman, Brooks and Cowan.4 Human CG and LH will be considered together, as these hormones have fundamentally the same biological action at cellular level, and both are essential components to be considered in the control of testosterone misuse in sport. The gonadotrope cells of the anterior pituitary secrete the two gonadotrophins, LH and follicle stimulating hormone (FSH). In men, LH stimulates steroidogenesis in the Leydig cells of the testes, resulting in the synthesis and release of testosterone. Both gonadotrophins, together with testosterone are important in spermatogenesis. In women, LH and FSH interact with ovarian theca and granulosa cells to stimulate steroidogenesis, follicular maturation and ovulation. Human CG is secreted in large amounts by the developing placenta or chorion during pregnancy. During the first trimester of pregnancy hCG is considered to play an important role in the maintenance of the corpus luteum for the production of progesterone and to a lesser extent oestradiol. Human
PEPTIDE HORMONES AND SPORT: MISUSE AND DETECTION
499
CG may also be necessary for male sexual development by possibly stimulating the transient secretion of testosterone by the fetal Leydig cells during the second and third trimester. Very small concentrations of hCG or hCG-like material have been measured in the serum of normal men (less than 60 years, < 1.3 IU/1; after 60 years,
