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Adult Workshop 1

Horm Res Paediatr 2011;76(suppl 1):84–90 DOI: 10.1159/000329185

Growth Hormone in Sports:Detecting the Doped or Duped

Ken K.Y. Ho   a, b Anne E. Nelson   a

a   Pituitary Research Unit, Garvan Institute of Medical Research, and b   Department of Endocrinology,St. Vincent’s Hospital, Sydney, N.S.W. , Australia

test based on these markers must take these factors into ac-count. Extensive data now validate the GH-responsive mark-er approach, and implementation is largely dependent on establishing an assured supply of standardized assays. Con-clusions: Robust tests are available to detect GH and enforce the ban on its abuse in sports. Novel approaches that include gene expression and proteomic profiling must continue to be pursued to expand the repertoire of testing approaches available and to maintain deterrence of GH doping.

Copyright © 2011 S. Karger AG, Basel

Introduction

Although doping with growth hormone (GH) is banned by the World Anti-Doping Agency, it is widely believed to be abused in sports, often together with other banned substances such as anabolic steroids [1] . A reliable test is needed to detect the presence of these products and to enforce the ban; however, developing a test for a natu-rally occurring polypeptide such as GH has been a chal-lenge. The two main current approaches for GH detec-tion, based on isoforms of GH and on GH-responsive markers in blood, are reviewed herein, with an emphasis on the basis of each approach and its current status as a doping test.

Key Words Doping � Growth hormone � IGF-I � Procollagen I � Procollagen III

Abstract Background: Doping with growth hormone (GH) is banned; however, there is anecdotal evidence that it is widely abused. GH is reportedly often used in combination with anabolic steroids at high doses for several months. Development of a robust test for detecting GH has been challenging since re-combinant human 22-kDa GH used in doping is indistin-guishable analytically from endogenous GH and there are wide physiological fluctuations in circulating GH concentra-tions. Discussion: One approach to GH testing is based on measurement of different circulating GH isoforms using im-munoassays that differentiate between 22-kDa and other GH isoforms. Administration of 22-kDa GH results in a change in its abundance relative to other endogenous pituitary GH isoforms. The differential isoform method is, however, lim-ited by its short time window of detection. A second ap-proach that extends the time window of detection is based on detection of increased levels of circulating GH-responsive proteins, such as the insulin-like growth factor (IGF) axis and collagen peptides. As age and gender are the major deter-minants of variability for IGF-I and the collagen markers, a

Published online: July 21, 2011 HORMONERESEARCH IN PÆDIATRICS

Ken Ho, MD, FRACP Princess Alexandra Hospital, Centres for Health Research, Level 2, Building 35 Ipswich Road Woolloongabba, QLD 4102 (Australia) Tel. +61 7 3176 7667, E-Mail Ken_Ho   @   health.qld.gov.au

© 2011 S. Karger AG, Basel1663–2818/11/0767–0084$38.00/0

Accessible online at:www.karger.com/hrp

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Horm Res Paediatr 2011;76(suppl 1):84–90 85

Abuse of GH in Sports

Evidence that GH is widely abused is indicated by the number of website hits for GH supply, by customs and police drug seizures and by frequent press reports of prosecution of high-profile athletes [1] . The abuse of GH in sports is probably due to the immense pressure to per-form. When athletes were asked in a survey if they would take GH if there was a guarantee they would not get caught and they won every competition for the next 5 years – even if they later died from adverse effects related to the drug – 50% replied ‘yes’ [2] .

Abuse of GH may start at a young age. An early survey of 10th grade boys in the US indicated that 5% had taken GH, with more than half using GH in conjunction with steroids [3] . Large surveys of US secondary school stu-dents reported an increase in the use of anabolic steroids in the late 1990s, followed by a subsequent decline in prevalence [4] . A survey of college athletes in the US found that 1.2% used GH in the past 12 months [5] . Dos-es used by athletes are estimated to range from 3 to 8 mg/day for 3 to 4 days per week, often in combination with other doping agents [6] , resulting in average daily doses of 1–2 mg of GH, which is approximately 2–3 times the level of daily endogenous pituitary secretion. ‘Polyphar-macy’ is widely practiced with GH most often used in conjunction with anabolic steroids. A web-based survey reported that 25% of anabolic androgenic steroid users also used GH (1–10 mg/day) and insulin [7] .

Evidence that GH actually improves performance in athletes is lacking. Although beneficial effects on endur-ance and strength have been demonstrated in adults with GH deficiency [8] , the balance of evidence in healthy, young adults does not indicate a beneficial ef-fect of GH on these performance measures. While GH has been shown to induce a measurable protein anabol-ic effect in athletes [9] , there has been no evidence from double-blind, placebo-controlled studies that GH en-hances muscle strength or performance in trained adult athletes [10] . A recent systematic review has highlighted a paucity of published evaluations, and it remains incon-clusive as to whether GH enhances sporting perfor-mance [10] .

Due to its health risks to athletes and its potential to enhance sports performance – in addition to violating the spirit of sport – GH is listed in the 2008 List of Prohibited Substances (http://www.wada-ama.org/rtecontent/docu-ment/2008_List_En.pdf) by the World Anti-Doping Code at all times, both in- and out-of-competition.

Challenges in Developing a Robust Test

Until recently, urine was the only body fluid available for sports doping testing. Compared with a blood sample, urine is easily obtained and available in relatively large volumes. However, the concentration of GH in urine is very low (approximately 0.1–1% that in blood) and vari-able, with much of the variability not associated with fluctuations in daily serum GH levels [11, 12] . Although urinary GH concentration increases after administration of exogenous GH, it can also increase after exercise [13] . For these reasons, urinary testing for GH is unlikely to be successful.

Detection of GH is challenging since recombinant hu-man 22-kDa GH (22K), which is available commercially and used in doping, has an identical amino acid sequence to the 22K GH isoform secreted endogenously by the pi-tuitary gland; as such, the two substances are indistin-guishable using current analytical methods. Differences in glycosylation patterns have been used to distinguish between exogenous recombinant hormone and endoge-nously secreted hormone as the basis for a test for eryth-ropoietin [14] ; however, this is not currently feasible with GH since it does not have N-linked glycosylation sites in the 1–191 sequence.

GH has a short half-life of 15–20 min in the circula-tion, and exogenous GH administered by subcutaneous injection disappears rapidly from the circulation [15, 16] . The circulating concentrations of GH also vary widely since it is secreted from the pituitary in a pulsatile man-ner and is regulated by several factors, including sleep, exercise and stress [17] . With exercise, a major stimulus to GH secretion, plasma concentrations can increase up to 10-fold, dependent on the duration, intensity and na-ture of the exercise (reviewed by Gibney et al. [18] ). Be-cause of the widely fluctuating physiological GH concen-trations particularly in response to exercise, and the fact that increases in circulating GH are not specific for exog-enous GH administration, direct measurement of total circulating GH is not a viable option.

Physiological Basis for GH Tests

GH is secreted by the pituitary and circulates as a number of different isoforms. The heterogeneity of se-creted and circulating forms in humans occurs for sev-eral reasons. GH is the product of the GH-N gene and is subject to alternate splicing into different isoforms, post-translational modifications, proteolysis and binding to

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GH-binding proteins (reviewed by Baumann [19] ). The 191 amino acid, 22K GH isoform is the most abundant form of GH comprising approximately 50% of circulating GH in the monomeric form. The 20 kDa (20K) GH iso-form lacks 15 amino acids (corresponding to residues 32–46 of the 1–191 sequence) and results from alternative splicing of the GH-N gene. The 20K GH isoform is the second most abundant monomer in the circulation (ap-proximately 10–15%) and has a longer half-life in the cir-culation compared with 22K GH [16] . Proteomic analysis has identified 17 kDa splice variants in the human pitu-itary at low abundance ( ! 4% of the total pituitary GH) [20] . In addition to splice variants, GH may undergo post-translational modifications including acetylation, deam-idation and phosphorylation [20] . The different isoforms of GH also form oligomers (dimers, trimers, tetramers, pentamers and possibly higher), which are both disul-fide-linked and noncovalently associated, and comprise approximately 30% of circulating GH. Furthermore, complexes can form between GH and GH-binding pro-teins in the circulation [21] . Proteolytic fragments of GH have also been described, including 5 kDa and 17 kDa human GH [22] .

Negative feedback regulation by circulating GH and insulin-like growth factor I (IGF-I) inhibits the secretion of pituitary GH [23] ; therefore, administration of exoge-nous GH reduces the concentrations of endogenous GH isoforms secreted by the pituitary [24] . Injection of exog-enous recombinant 22K GH results in increased circulat-ing concentrations of 22K GH and a decrease in other endogenous pituitary isoforms. The change in the ratio between serum concentrations of 22K GH and other pi-tuitary-derived isoforms of GH forms the basis of one ap-proach to testing for GH [16, 24, 25] .

The second current approach to a GH doping test is based on the physiological effects of GH that result in increased circulating concentrations of proteins that have a longer half-life and a more stable serum concen-tration than GH. GH stimulates production of IGF-I, which mediates many of the anabolic actions of GH, by the liver, the main source of circulating IGF-I, and by extrahepatic tissues. GH also stimulates the hepatic pro-duction of IGF binding protein-3 (IGFBP-3) and the acid labile subunit (ALS) which, together with IGF-I, form the circulating ternary complex [26] . Therefore, in response to GH the serum concentrations of these IGF axis pro-teins increase. GH also stimulates bone and connective tissue turnover probably via IGF-I, resulting in increased concentrations of specific collagen peptides related to collagen synthesis and degradation [27] . These include

the marker of bone formation, N-terminal propeptide of type I procollagen (PINP); the marker of bone resorp-tion, C-terminal telopeptide of type I collagen (ICTP); and the marker of connective tissue synthesis, N-termi-nal propeptide of type III procollagen (abbreviated as PIIINP or PIIIP, referring to measurements by different assays) [28] . The half-lives of the IGF axis proteins and collagen markers, which range from 90 to 1 500 h [29] , are considerably longer than that of GH. The increases in the serum concentrations of these GH-responsive markers form the basis of the second approach to GH testing [30, 31] .

The GH Isoform Approach

Application of the GH isoform approach to doping testing has been made possible by the development of im-munoassays that differentiate between the isoforms of GH, in particular between 22K GH and other GH iso-forms [24] . The group led by Strasburger has developed a method based on two immunoassays that distinguish be-tween recombinant 22K GH and all endogenous GH iso-forms using specific monoclonal antibodies (MAbs) [24, 32] . One of the assays uses a MAb that preferentially rec-ognizes recombinant 22K GH (Rec-GH) and the other uses a MAb that is permissive and recognizes all pituitary isoforms (Pit-GH). The ratio of the measurements from the Rec-GH and Pit-GH assays (Rec:Pit) indicates the rel-ative abundance of 22K GH. Good separation of the Rec:Pit ratio for GH-treated versus control samples has been reported [32] . Using a highly sensitive chemilumi-nescence assay, Bidlingmaier et al. [33] have recently re-ported the detection of recombinant human GH for up to 36 h after injection.

An alternate isoform-based method to detect exoge-nous GH measures the specific 20K GH isoform together with measurement of 22K GH, using highly sensitive and selective MAbs for each of the two GH isoforms in an enzyme-linked immunosorbent assay [34, 35] . Cosecre-tion of 20K GH with 22K GH results in peaks of secretion that coincide during the day, with the circulating concen-tration of 20K in a constant proportion to 22K GH [16, 35] . Administration of exogenous GH, however, results in rapid reduction of 20K GH concentration due to negative feedback inhibition of pituitary secretion of 20K GH. Fol-lowing injection of exogenous 22K GH, the increase in circulating 22K and the reduction in 20K GH result in a rapid increase in the ratio of 22K to 20K GH [16] . Current studies from our group following daily injections of 2 mg

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GH for 8 weeks suggest that the time window for detec-tion is within 24 h of the last injection.

The ratio between 22K and 20K GH is relatively stable, with little effect of age, gender, body weight or height in the general population [35] . Using a study group of near-ly 1,000 elite athletes from four major ethnic groups, we have shown that the effects of age, gender, body mass in-dex (BMI), ethnicity and sports type on the 22K:20K ratio are minimal [36] . The stability of the ratio to the effects of demographic factors and sports types renders it a promising measure of exogenous GH abuse. All molecu-lar isoforms of GH increase with acute exercise; however, supraphysiological doses of GH suppress exercise-stimu-lated endogenous GH isoforms, further supporting the use of the isoform approach [25] .

A major limitation of the isoform approach is the short time window of detection of possibly 24–36 h after injec-tion, thereby restricting its utility primarily to no-ad-vance-notice, out-of-competition testing. The isoform approach can only detect 22K GH and does not detect administration of pituitary-derived GH or GH secreta-gogues. The differential isoform method was used in the 2004 (Athens), 2006 (Turin) and 2008 (Beijing) Olympic games. There have been no irregular findings from sports samples tested using this method to date; however, the short time window of detection makes detection highly unlikely during competition periods.

The GH-Responsive Marker Approach

The GH-responsive marker approach, based on de-tecting increased levels of GH-responsive proteins in blood, has the advantage of a longer time window of de-tection than the isoform-based approach. The collabora-tive GH-2000 group pioneered the evaluation of serum IGF axis markers: IGF-I, IGFBP-1, IGFBP-2, IGFBP-3 and ALS, and serum markers of bone and connective tissue turnover: osteocalcin, bone-specific alkaline phospha-tase, C-terminal propeptide of type I collagen (PICP), ICTP and PIIIP [37] . Acute exercise transiently increased IGF-I, IGFBP-3 and ALS levels; however, the increases were much smaller than those in response to GH admin-istration alone [38] . The same was true for osteocalcin, PICP, ICTP and PIIIP [39] .

The effect of administration of GH for 4 weeks on these GH-responsive markers was examined in a ran-domized, double-blind, placebo-controlled study in 99 young athletically trained men and women using two doses of GH: 0.33 and 0.67 mg/kg/day. The IGF axis pro-

teins IGF-I, IGFBP-3 and ALS all increased in response to GH, with the greatest response observed in IGF-I lev-el. Men were substantially more responsive than wom-en. All IGF proteins returned to baseline within a few days of cessation of treatment, except for IGF-I level, which remained elevated for a longer period of time in men [30] . All markers of bone and connective tissue turnover also increased with ICTP and PIIIP exhibiting the greatest responses, and peak increments were great-er in men than in women. Osteocalcin, ICTP and PIIIP remained significantly elevated for up to 8 weeks after cessation of treatment [40] . Other placebo-controlled studies support the potential for IGF axis and collagen peptides to be used as markers of GH abuse [41, 42] . A recent evaluation of IGFBP-4 and -5 has indicated that they will not be useful as IGF-I-independent markers [43] .

We recently evaluated the diagnostic potential of the IGF axis and collagen markers in a double-blind, place-bo-controlled study in male and female recreational ath-letes [44] . This study also evaluated the effects of con-comitant testosterone administration in men. All mark-ers increased substantially in response to GH, and the responses of all markers were greater in men than in women. IGF-I showed the greatest increase compared with IGFBP-3 and ALS in response to GH. The increases in collagen markers were greater than those of the IGF axis markers, especially for PIIINP where the magnitude was nearly 2-fold greater than that of IGF-I. The collagen marker levels not only increased more slowly than the IGF axis markers, they also decreased more slowly. Most remained elevated 6 weeks after withdrawal; in contrast, IGF markers returned rapidly to baseline within 1 week. The administration of testosterone in addition to GH did not change the time course of any marker; however, it no-tably enhanced the response of PIIINP. Testosterone alone did not affect the concentrations of IGF axis mark-ers, but modestly increased all the collagen markers. This study revealed differences in the pharmacodynamics of the IGF axis markers and collagen markers, a gender dif-ference in the responses of all the markers and an ampli-fying effect of testosterone on the collagen marker PIIINP [44] . This suggests that using both IGF-I and a collagen marker would provide the greatest discriminatory power for a doping test both during GH administration and withdrawal.

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Normative Data in Elite Athletes

The application of a method using GH-responsive markers requires extensive normative data in elite ath-letes to identify the factors influencing their levels in blood and establish normal reference ranges. We have undertaken a large cross-sectional study of IGF-I, IGFBP-3, ALS, PINP, ICTP and PIIINP in over 1,000 elite athletes from 12 countries representing four major ethnic groups. We found that age and gender were the major de-terminants of variability for IGF-I and the collagen pep-tides, whereas ethnicity accounted for a minor propor-tion ( ! 6%) of the attributable variation, except for IGFBP-3 and ALS [45] . Levels of all GH-responsive mark-ers decreased substantially with age, similar to that ob-served in the general population [46, 47] , and age was the major contributor to variability, especially for the colla-gen peptides. There were notable differences between men and women; however, the contribution of gender was smaller than that of age except for IGFBP-3 and ALS lev-els. The contributions of BMI and sports type were mod-est compared with those of age and gender. Therefore, our study of elite athletes indicated that a test based on IGF-I and the collagen markers must take age into ac-count for men and women, and that ethnicity is unlikely to be a confounder [45] . The findings on the influence of age, gender, BMI and sports type have also been con-firmed in a study of mostly Caucasian elite athletes [48] , which also concluded that sports category was not an in-dependent predictor compared with age and gender.

Marker Variability

The successful application of the markers for testing also requires data on within-subject variability of the IGF axis and collagen peptides over time. All biological mea-surements are subject to within-subject random variation and estimation of this variation has important clinical and scientific ramifications. We conducted an evaluation of short-term variability of IGF axis and collagen peptides in 1,103 elite athletes from whom multiple samples were ob-tained over a 2-week period [49] . Total variability was rep-resented by the sum of variance between and within indi-viduals and the analytical variance. Between-subject vari-ance formed a major component of the variance of the markers, accounting for 64–68% of the total variance of IGF axis markers and for 87–96% of total variance of col-lagen markers. The within-subject variance was greater for IGF axis markers (32–36%) than for collagen markers

(4–13%). Most of the within-subject variance was attribut-able to biological variability accounting for 80–95% of the within-subject variance. The coefficient of reliability, de-fined as the ratio of the between-subject variance to the total variance (the sum of between- and within-subject variances), was computed for each marker. This coeffi-cient represents the degree to which an individual’s mark-er values remain relatively consistent over repeated mea-surements, or the measure of the correlation between ‘probable’ values and ‘measured’ values. The coefficient of reliability was higher for the collagen markers than for IGF axis markers, indicating that the within-subject variabil-ity was smaller relative to between-subject variability for the collagen markers. Regression toward the mean effects was observed, and ‘probable’ values were estimated for in-dividuals based on a Bayesian model. This modeling strat-egy not only enhances the reliability but reduces the cost of GH doping tests based on the use of these markers [49] .

The effect of injury on the collagen peptides in athletes also warrants investigation. Distinct changes in serum bio-chemical bone markers, both in the early stage after frac-ture and up to several weeks later, have been described fol-lowing lower-limb fractures due to bone remodeling and collagen III synthesis in fracture healing [50, 51] . A recent study has shown that the rise in collagen peptides after soft-tissue trauma and fractures is modest compared with that observed after supraphysiological doses of GH [52] .

Algorithms Based on GH-Responsive Markers

A GH doping test based on the GH-responsive mark-ers should not rely on a single marker, but rather a com-bination of markers. The pharmacodynamic profiles of IGF axis and collagen markers are different, as shown by studies with extended washout periods that reveal a pro-longed elevation of collagen markers after cessation of GH administration [30, 40, 44] . These findings highlight the benefits of using a combination of several markers (i.e. IGF-I, IGFBP-3, PIIINP and ICTP) to detect GH dop-ing during both active administration and washout. Al-gorithms based on IGF-I and PIIINP show promise in differentiating GH- from placebo-treated subjects when compared with a single marker [31, 41, 53] . Our recent GH administration study has highlighted the potential of IGF-I, PIIINP and ICTP in combination as promising discriminators of GH administration against a reference population of elite athletes, both during and up to sev-eral weeks following treatment, due to the longer time course of the collagen marker responses [44] .

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Summary and Conclusions

Two approaches to testing for GH based on measure-ment of GH isoforms and GH-responsive markers have been extensively developed. These tests will likely be used in a complementary manner due to their different time windows of detection [54] . Their availability will enable the ban on the use of GH to be enforced. Commercial as-says are now available that will enable wide implementa-tion of the isoform-based approach. The GH-responsive marker approach will extend the time window of detec-tion of GH and the technical hurdles to its implementa-tion are currently being addressed by the anti-doping au-thorities. Additional investigations are required to ex-pand the repertoire of testing approaches available and to maintain deterrence of GH doping.

Acknowledgments

The authors’ research has been supported by the World Anti-Doping Agency and by the Australian Government through the Anti-Doping Research Program (ADRP) of the Department of Communications, Information Technology and the Arts. We thank Professors Robert Baxter, David Handelsman, Markus Sei-bel, Minoru Irie and Dr. Ray Kazlauskas for contributions to the research work.

Disclosure Statement

K.K.Y.H. is a consultant to Ipsen and Novartis and an advi-sory committee/review panel member for Lilly, Merck-Serono and Pfizer. He conducts contracted or funded research for Novar-tis and has received consulting fees from Ipsen, Novartis and Lil-ly. He received an honorarium from Pfizer in association with his presentation and resulting manuscript for the proceedings for the 41st International Symposium sponsored by Pfizer.

A.E.N. declares no conflict of interest. Production logistics including collection of manuscripts, as-

sistance to editors, obtaining reprint permissions, graphic design and layout were provided by CMM Global.

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