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By John M. MacKnight, MD
Synopsis: Markers of bone metabolism may be useful in the detection of growth hormone abuse in sport.
Source: Longobardi S, et al. Growth hormone (GH) effects on bone and collagen turnover in healthy adults and its potential as a marker of GH abuse in sports: A double blind, placebo-controlled study. J Clin Endocrinol Metab 2000;85(4): 1505-1512.
As a greater premium is placed on athletic achievement, sports medicine physicians are increasingly faced with the prospect of athletes using performance-enhancing substances. In recent years, the practice of "doping" has risen dramatically. As the means for detecting previous doping techniques, most notably the use of anabolic steroids, have improved, athletes have embraced a number of alternatives such as recombinant human growth hormone (rhGH). This study seeks to elucidate the effect of rhGH on several serum markers of bone and collagen metabolism and, in so doing, lays the foundation for their potential use in detecting growth hormone abuse in competitive sports.
This double-blind, placebo-controlled study was carried out in 99 subjects between 18-35 years of age who had been training twice weekly for at least a year. Males and females were equally represented. The subjects were randomized to receive 28 days of low-dose (0.1 IU/kg/d) subcutaneous rhGH, high-dose (0.2 IU/kg/d) subcutaneous rhGH, or placebo followed by a wash-out period of 56 days. These doses were felt to simulate those of GH doping in sport. Serial assessments were made of bone and collagen markers including serum osteocalcin, C-terminal propeptide of type I procollagen (PICP), C-terminal cross-linked telopeptide of collagen type I (ICTP), and procollagen type III N terminal extension peptide (PIIIP).
During this study, serum levels of all of the studied markers increased significantly with both low and high-dose rhGH administration. This is in keeping with previous data on the influence of GH on bone remodeling and formation. No change in these parameters was seen in the placebo group, nor was any menstrual or diurnal variation found. The most remarkable increases were seen with ICTP and PIIIP, and osteocalcin, and PIIIP remained elevated throughout the 84 days of the study. These findings support a general acceleration of bone turnover and bone formation.
From a standpoint of doping control, this study has promising implications. There is presently no approved method to detect GH doping, largely because of its short half-life, unpredictable serum levels, and minimal excretion in urine. This study shows that the studied markers are significantly elevated in both rhGH treatment groups, creating the possibility that one or more of them could be used as a surrogate marker for GH doping in sport. Moreover, these changes persisted many weeks into the wash-out period, establishing their usefulness even at times far removed from the doping incident itself, a major advance over many present screening measures.
This is also an important study in the field of GH use. Longobardi and colleagues were able to demonstrate the strong influence that exogenous GH has on the bone and collagen metabolism of exercising individuals as compared to placebo-treated controls. This may give some insight into future uses for GH administration in a therapeutic role, most notably in osteoporosis where the risk for fracture may be reduced.
The study is somewhat limited by evaluating a predominantly caucasian population exercising at levels likely well below those of athletes who are likely to be involved in doping activities. Both flaws will need to be addressed in future studies before bone metabolism markers can be confidently used for GH doping detection in the athletic population.
Use of recombinant growth hormone (somatotropin, rhGH) as a doping agent in sport is on the rise. And although rhGH has been shown to increase muscle mass, muscle strength, and exercise capacity in GH-deficient patients, the data to date do not support the notion that rhGH administration to healthy athletes increases muscle strength or aerobic power. Typically administered daily as a subcutaneous injection, rhGH stimulates production of insulin-like growth factor I (IGF-I) through which GH exerts its anabolic effects in the body. Recombinant IGF-I is now commercially available (as somatomedin-1), as well as several low molecular weight, orally active growth hormone secretagogues.
Measurements of bone and collagen metabolism in the face of rhGH administration seem to be the most promising markers for rhGH doping in athletes. Furthermore, IGF-I levels and a number of IGF-I binding proteins may remain persistently elevated after rhGH administration. Changes in the relative concentrations of a number of these markers may also eventually serve as a useful means of indirect detection of rhGH use.
Recent reports have raised the possibility of distinguishing between recombinant and endogenous GH by simultaneous radioimmunoassay of the two GH isoforms (20 and 22 kDa). Endogenous GH is composed of 22 kDa and 20 kDa isoforms while rhGH contains only the 22 kDa isoform. Thus, alterations in the normal 22:20 kDa isoform ratio could be used as a means of detecting exogenous rhGH administration. This may be a powerful direct means of testing for GH doping, but again, due to the short half-lives of the involved peptides, testing would need to be carried out within days of GH use. For now, the study by Longobardi et al raises the most promising possibility for GH doping detection.
Dating back nearly four decades, the Council of Europe in 1963, in response to rising concerns about the use of doping substances for unfair athletic advantage, established a definition for athletic doping practices: "The administering or use of substances in any form alien to the body or of physiological substances in abnormal amounts and with abnormal methods by healthy persons with the exclusive aim of attaining an artificial and unfair increase in performance in competition."
Such doping substances are generally grouped into two major categories: those used acutely for benefit during a competition and those used to enhance the effectiveness of a training regimen leading up to competition. The use of "traditional" doping agents, such as anabolic steroids and stimulants, has been tempered by their ease of detection in urine via gas chromatography and mass spectrometry. Newer performance-enhancing substances, however, create unique challenges because of the difficulty in detecting them. Newer methods of doping detection often take advantage of unique physiologic or biochemical properties of the involved agents. The following is a brief review of the most common of these newer doping measures and the current trends toward increased detection.
This is the practice of intravenously infusing autologous (reinfusion of athlete’s own blood) or nonautologous blood in order to create supraphysiologic erythrocytosis. The practice dates back to the 1960s, with its most notable incident involving cyclists on the 1984 U.S. Olympic team. Blood doping results in an increase in total aerobic power by increasing the transport of oxygen to working muscles. Evidence would also suggest that not only is the volume of oxygen delivered to the muscle increased by blood doping, but the volume of oxygen used during intense exercise is increased significantly as well. The common practice is to infuse 2-3 units of blood 1-7 days prior to competition. High intensity aerobic sports such as cycling, cross country skiing, and long-distance running are the target sport groups.
Detecting nonautologous (allogeneic) blood depends upon the demonstration of blood group differences between the athlete’s own cells and those of the transfused red cells. Autologous doping can be detected by simultaneously measuring the levels of erythropoietin, hemoglobin, bilirubin, and iron. As doping results in elevated hemoglobin levels, the secretion of endogenous erythropoietin displays a compensatory decrease. The retransfused red cells also are fragile and susceptible to hemolysis, resulting in elevations in both bilirubin and iron levels. Although there remain potential errors with this method, it has been found to detect 50% of positive cases and is at present among the best approaches.
Erythropoietin is a glycoprotein hormone secreted by renal cells to stimulate erythroid progenitor cells in the bone marrow. Repeated injections of recombinant erythropoietin (EPO) will increase hemoglobin concentration and hematocrit in a dose- and time-dependent fashion. Studies have revealed a significant increase in hemoglobin concentration and up to an 8% increase in maximal aerobic power with as few as six weeks of subcutaneous EPO administration.
Indirect methods of EPO detection center around the body’s response to its administration. Reticulocyte counts, hemoglobin, hematocrit, and red blood cell numbers all rise. Then there is a rise in large erythrocytes with low hemoglobin content (hypochromic macrocytes) and an increase in the level of soluble transferrin receptors in plasma. ELISA testing for these transferrin receptors at present is the most reliable, indirect means of screening for doping with EPO.
Direct detection of EPO is at present too costly and laborious to be of practical use. Future methods will likely take advantage of the ability to detect differences between the isoforms of exogenously administered EPO and natural endogenous erythropoietin.
Anabolic steroids and testosterone have been used for athlete doping since the 1950s. They increase muscle mass, strength, speed, and mental aggressiveness. They also decrease catabolism which allows for improved recovery from vigorous training. Because of these properties, their use is greatest among strength athletes in such sports as football, wrestling, powerlifting, sprinting, and field events. All anabolic steroids are structurally derived from testosterone, and those used in doping are generally found as an ester derivative administered in an injectable form. The most common preparations contain testosterone enanthate, cypionate, and propionate. The International Olympic Committee (IOC) formally banned anabolic steroids in 1975 with urine testing programs initiated in 1976. The first widespread testing for anabolic steroids came at the 1983 Pan American games.
Indirect screening for testosterone abuse has relied upon the urinary detection of an increase in the testosterone glucuronide: epitestosterone glucuronide ratio. Exogenously administered testosterone increases the urinary excretion of testosterone but also decreases epitestosterone excretion secondary to negative feedback on the pituitary with resultant decreased epitestosterone production. The normal mean ratio is one. A ratio value of six or greater is considered positive evidence for testosterone doping.
Additional indirect measures that take advantage of the negative feedback effect on endogenous testosterone synthesis are an increased urinary ratio of testosterone: luteinizing hormone (LH) or elevated serum ratio of testosterone: 17a-hydroxyprogesterone. The administration of one or two doses of ketoconazole, which decreases endogenous testosterone production, may also prove useful in evaluating an elevated urinary testosterone/epitestosterone ratio.
Direct measurement of testosterone esters uses serum mass spectrometry and gas chromatography to determine the 12C:13C isotope ratio of testosterone in urine. Synthetic testosterone has a much higher level of 13C than does endogenous testosterone. Presently considered a confirmatory test for those with elevated urinary testosterone ratios, it could also be used to detect doping with testosterone precursors such as dehydroepiandrosterone (DHEA), androstenedione (Andro), and dihydrotesterone (DHT). Screening for the remainder of the anabolic steroid agents at present is accomplished through the use of high-resolution mass spectrometry, first mandated at the 1996 Atlanta Olympic games.
Future trends in athletic doping will likely include the direct use of IGF-1, insulin, oral growth hormone secretagogues, and red cell substitute oxygen carriers such as stroma-free hemoglobin solutions or perfluorocarbon-based substitutes. Some are being used already to augment sport, with little if any ability to detect their use at present.
The future trends in doping control are in blood sample analysis. This creates a number of logistical and ethical concerns that will need to be resolved before blood testing becomes a standard part of the screening for performance-aiding agents in athletes. An additional technique still in its infancy for doping detection is the use of hair samples. The analysis of chemically-digested hair using gas chromatography and tandem mass spectrometry has promising implications for use in the detection of anabolic steroids and their esters, amphetamines, and corticosteroids. Though far from mainstream utilization at present, these techniques may provide the ability to take a "snapshot" of banned substance use that covers a period of time far exceeding that of today’s methods. Much additional study is required, but novel approaches must constantly be sought if the medical community is to keep pace with the ever-advancing scourge of athletic doping activity.
Physicians who care for athletes must be aware of the increased doping behaviors and must have a working knowledge of the involved agents and their means of detection. Only through the vigilance of sports medicine practitioners and the governing bodies of sport can we hope to preserve the purity of athletic competition.
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2. Bowers LD. Athletic drug testing. Clin Sport Med 1998;17(2):299-318.