University of Alberta, Edmonton, Alberta, Canada
Publication Date: July 21, 2015
Doping has a long and eventful history in athletics, and the use of performance enhancing drugs (PEDs) has become even more prevalent as technology improves. The use of drugs such as methamphetamine and methods such as blood transfusions and erythropoietin injections have led to serious side effects, including death1,2 . These PEDs pose serious risks to elite athletes, creating an expectation of use in order to reach peak performance levels, and setting an example for up and coming athletes. For these reasons, it is becoming increasingly important to develop methods for robust and reliable detection of performance enhancement. This has been particularly difficult to do in the case of growth hormone (GH), mainly because it is produced endogenously. Despite minimal evidence supporting its athletic benefit3,4 , GH use by itself and in combination with products such as erythropoietin and androgens appears to be increasingly popular4. However, significant progress in terms of detection has been made in recent years, and there are many hopeful paths to be pursued with current technology.
Development of Detection Methods
i) Levels of Growth Hormone
As GH is released in a pulsatile fashion, levels of hGH in the human body are highly variable and prone to large fluctuations. On a subject-to-subject basis secretion can be influenced by age, sex, body composition, time of day, and nutritional status5,6 . This makes it difficult to determine individual baseline levels, resulting in the suggestion of an “athlete’s passport” to track levels of different biological markers in an athlete over time7. While this accounts for many individual differences, it is costly and puts large pressure on elite athletes to take numerous drug tests throughout the year. There are also many other influences on GH levels that cannot necessarily be accounted for during normal testing, such as stimulation of GH due to psychological stress and physical exercise8,9. These are particularly important to take into account when testing GH levels in elite athletes, as major sporting events can elicit both these conditions.
ii) Previous Methods
Though urine tests have been widely used in drug detection, levels of hGH in urine are only around 0.1%-1.0% of levels found in blood7, making detection impossible with current immunoassay sensitivity and time-sensitive. GH has a very short half-life, about 15-25 minutes, with exogenously applied GH degrading even more quickly10. One solution to this may be to use hydrogel nanoparticles functionalized with Cibacron Blue F3G-A (CB), which has been shown to capture, concentrate, and preserve hGH for detection in urine11.
Difficulties with urine test concentrations have lead researchers to look towards blood analysis as a potential method for GH detection. The very short half-life of GH still has an effect here, and blood testing is a more costly and invasive method to administer. In addition to this, GH is difficult to detect due to the identical amino acid structure of the most prevalent endogenous form, 22-kilo Daltons (kD), and the most popular form of GH used in doping, recombinant human 22K GH7. This makes differentiating between endogenous and exogenous GH very difficult, and has resulted in two main types of tests involving blood sampling being developed in recent years.
i) Marker approach
The marker approach is an indirect test of recombinant growth hormone (rGH) use, which attempts to measure changes in levels of proteins produced by GH rather than directly measuring rGH. A specific point of focus has been insulin-like growth factor-I (IGF-I) system, which is believed to be affected by GH (Figure 1)(Roith, D., Bondy, C., Yakar, S. et al., 2001). However, it is believed that GH is not the only hormone that can affect IGF-I, and that IGF-I is produced in many tissues and actually affects GH12 (Figure 1). This complicates the creation of a direct link between growth hormone and IGF-I axis protein levels. One way to handle this is to show that levels of IGF-I do not exceed a certain limit when exposed to only endogenous levels of GH. This was relationship was tested by the GH-2000 committee, a GH detection-focussed group funded jointly by the International Olympic Committee and the European Union, who showed that supraphysiological doses of GH resulted in abnormal levels of IGF-I, which could therefore be a marker for its abuse13.
Figure 1: Physiological basis for the isoform and growth hormone (GH)-marker approaches to GH tests. GH is secreted from the pituitary and circulates as a number of different isoforms. The isoform approach to GH testing is based on the negative feedback that inhibits pituitary secretion of endogenous GH following GH administration. This results in a change in the ratio between serum concentrations of 22 kDa (22K) GH and other pituitary-derived isoforms of GH. GH stimulates the production of IGF-I together with its circulating binding partners insulin-like growth factor binding protein-3 (IGFBP-3) and the acid labile subunit (ALS), and also stimulates bone and connective tissue turnover, resulting in increased levels of specific collagen peptides (PINP, PIIINP, ICTP) related to collagen synthesis and degradation. The GH marker approach is based on increases in response to GH in the circulating concentrations of IGF axis and collagen peptides that have relatively long half-lives and stable concentrations (Figure from Nelson, A. etal., 2007).
Another focus of the GH-2000 committee was the measurement of bone and collagen turnover markers to determine rGH use, which was met with significant success. Specifically, it was found that GH administration in healthy, fit subjects resulted in much larger changes to levels of amino-terminal extension peptide of type III procollagen (PIIIC) and carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) compared to levels resulting from acute exercise14. The combination of IGF-I and bone turnover markers was put forward by the GH-2000 group as a potential robust method for the detection of GH abuse14 , and was further supported by data found by the GH-2004 group. This research looked more closely at confounding factors such as gender and ethnicity, and found that variations between males and females were predictable, while ethnicity did not appear to play a significant role in the test proposed by the GH-200015.
ii) Isoform Approach
This method uses the direct analysis of GH isoforms circulating within the body16, and is based on current knowledge that an increase in GH results in a subsequent negative feedback of pituitary GH release (Figure 1). While rGH is identical 22-kD GH down to a single base pair level, it can be differentiated from other circulating isoforms using two different monoclonal antibodies, one of which recognizes the 22-kD isoform and the other which recognizes a much wider variety16 (Figure 2).
Figure 2: Molecular basis of the immunoassay approach. The concentration of GH in a sample is determined by two GH assays of different specificity. After injection of recombinant human GH, the relative abundance of the 22kDa isoform is increased, leading to an increase in the ratio of the concentrations measured by assay 1 (which preferentially measures the 22 kDa GH isoform) divided by that measured by assay 2 (which measures many GH isoforms). (Figure from Bidlingmaier, M., and Strasburger, C., 2007).
The basis of this approach is that the injection of rGH will inhibit pituitary secretion of GH, resulting in an abnormally high proportion of rGH (interchangeable in this case with 22-kD) and therefore an abnormally low proportion of other isoforms16. The monoclonal antibody used to detect the 22-kD isoform is termed the “rec assay”, while the second monoclonal antibody, used to detect other isoforms, is the “pit assay”. Using these two assays allows the development of a “rec/pit ratio”, which theoretically allows for the clear determination of athletes who have an abnormally high ratio of 22-kD17. This method also helps avoid the confounding influence of exercise in the detection of rGH use following athletic competition as the ratio of 22-kD to other isoforms has been shown to decrease following exercise18, meaning that it is unlikely to give us a false positive.
The isoform approach is not without challenges, however. The issue of the short half-life of GH has an influence here, as the 20-kD isoform is most likely to be detected within 24 hours of the last rGH injection16. An athlete could theoretically avoid detection quite easily by ceasing drug injections prior to the competition, and therefore random testing would likely need to be performed throughout the competitive season to ensure thoroughness.
As methods of doping have become more advanced, the technology for detecting these drugs has as well. Both urine tests and mass spectroscopy, have proved relatively ineffective based on their current levels of accuracy. Prohibiting rGH in athletics has created unique challenges due to its structure as well as GH’s pulsatile release, very short half-life, and the influence that many factors such as sleep, stress, and exercise can have on its release. In order for both consistent and thorough testing to be done future research will need to be conducted, potentially focusing on the amplification of GH, and substances affected by GH, present in very small amounts in human urine or blood samples. However, despite these numerous challenges, significant progress has been made in recent years, proven by the successful detection of doping athletes at an international level.
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18) Wallace, J., Cuneo, R., Bidlingmaier, M., Lundberg, P., Carlsson, L., Boguszewski, C., … Strasburger, C. (2001). The Response of Molecular Isoforms of Growth Hormone to Acute Exercise in Trained Adult Males. The Journal of Clinical Endocrinology & Metabolism, 86, 200-206.
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