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This may lead to establishment of cut?off levels and description of so?called abnormal values outside the normal constellation of measures. This approach, proposed in the mid?1990s, was investigated by an international panel of endocrinologists, but did not lead to a final solution for detection of hGH doping. A main objective of the study was to investigate the variation of these secondary variables during or after exercise. IGF?1 and IGFBP?3 in the hGH biological cascade as well as selected peptides involved in bone metabolism (for example the N?terminal peptide from the pro?collagen named PIIIP) or osteocalcin were considered as good biological markers of GH doping. These variables showed slight but significant changes after acute exercise. Moreover, the interindividual variability in the reaction to GH administration makes the use of indirect measurements almost impossible in a forensic description of GH misuse. It is obvious that rather than depending on the observation of a single value, a solution may be found in an algorithm combining all the biological variables from the cascade. Nevertheless, all these investigations clearly show that the indirect approach can certainly be used for screening and targeting purposes when a biological follow up of athletes will be acceptable in the sport community. But it cannot stand in front of a court as an absolute proof of doping. The regular evaluation of individual normal ranges in sportspeople could in fact lead to, as is currently done with haematological substances, better screening and targeting of the athletes and direct detection of hGH misuse as proposed in the following section. What this study adds. The direct method of detection, based on double immunological tests needs to be well evaluated and validated. This review has described the difficulty sports authorities will face to prove hGH doping. At present, the short time window of detection of any method and the effect of exercise on natural hGH secretion still make any approach quite risky. The Strasburger’Bidlingmaier group in Munich developed a so?called direct method for the detection of hGH doping. Recombinant hGH is exclusively represented by the native 22 kDa form whereas the circulating hGH in the human blood is present in several forms (table 1 ? 1). When the recombinant form is injected in the body, this increases, for a period of time, the proportion of the 22 kDa form in comparison with all other circulating forms. Moreover, with long term use, classic back regulation of the endogenous secretion of natural hGH occurs, which favours the proportion of the major 22 kDa sequence. Isoforms Per cent 22 kDa monomer 48 20 kDa monomer 9 Modified hGH (dimers and oligomers) 30 Acidic hGH (desaminated and acylated forms) 7 Fragmented hGH (17, 12, 5, 30 kDa) Variable, buy trenbolone hexy 100 mg injectable steroids $84.00 parabolan. The proposed test was used during the Olympics in Athens (2004) and in Torino (2006). To fulfil the requirements of the World Anti?Doping Agency (WADA) code and the standards for laboratories, two double tests were applied to serum samples: the first test quantified specifically the 22 kDa form and the second test was a comprehensive assay measuring all forms present in the serum (see fig 1 ? 1). The ratio was established and a cut?off defined to differentiate normal subjects (negative samples) from those having a significant higher proportion of 22 kDa hGH (positive samples). A second double sample test was used for confirmation purposes. The time window of detection for these tests is claimed to be between 24 hours and 36 hours after the last injection, depending on the dosage used. It is thought that hGH doping, to be efficient, needs multiple injections.
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