Growth hormone: drug test

Carrying out a drug test on recombinant somatotropin hormone (STG) of the person for many years was considered as an impracticable task. However recently it has been shown that at least two independent approaches allow to establish the athlete applied recombinant STG or not. The developed analysis methods undergo testing now and it will be possible to hope to be applied as official doping test already in the near future.

The main problem is that unlike erythropoietin any of the offered methods doesn't allow to define STG in urine. It is caused first of all by extremely low concentration of hormone in urine which don't allow to find it by the existing methods. Besides, allocation of somatotropin with urine represents difficult process which is characterized by considerable variability and still remains badly studied (Saugy et al., 1996). The IEF method applied in case of ERO in urine tests is unsuitable for the analysis of STG as the last has no glycosylation websites. According to modern data, molecules STG of a recombinant origin are almost completely identical to the main fraction of molecules STG, hypophysis, and any physical and chemical differences between them isn't revealed.

Nevertheless, even despite the lack of the websites of glycosylation, STG in the blood circulatory system it is provided by mix of molecular isoforms (Baumann, 1999). The research of these isoforms progressed not so far as in case of gonadotrophin, however for the last years became possible to identify some main components. Along with the main isoform having molecular weight 22 kd and consisting of 191 amino-acid remaining balance in blood the second for quantitative representation is found, shorter isoform with a molecular weight of 20 kd in which sequence there are no amino acids 32 — 46 (Hashimoto et al., 1998; Tsushima et al., 1999; Leung et al., 2002). There are also even more STG short forms, however they come to light changeably and completely aren't analysed yet. Some of them represent products of hydrolysis or degradation of molecules STG. The STG isoforms can exist in the form of monomers, dimer and multimer consisting from identical (gomodimer) or various (heterodimeasure) isoforms.

In many cases impact of STG on a human body mediates the factor which received the name the insulin like factor of growth of I (IFR-1) produced mainly in a liver, and also locally in cartilaginous bone and many other fabrics, IFR-1 in the blood circulatory system where it contacts the specific connecting proteins (Le Roith ct al., 2001). The greatest value from them protein 3 (IGFBP-3), and also the acid and labile subjedinitsa (ALS) which products also are under control STG has IFR. At least, for IGFBP-3 it is shown that this protein can make own impact irrespective of linkng with IFR-1 and therefore can be considered as independent peptide hormone. Together IFR-1, IGFBP-3 and ALS create a threefold complex which has bigger life expectancy, in comparison with the molecules creating it separately. Do not forget take Hepatamin for better results.

One of the strategy of search of the suitable test for detection of application of STG as a stimulator consisted in assessment of quantitative changes of products of pharm dinamic impact of STG, in particular possible increase in quantity of components of the threefold complex exceeding the range of natural variability on level (Dali et al., 2000). One of advantages of such test would consist that time of life of products of pharm dinamic impact of STG exceeds time of life of the hormone that allows to increase a time span during which there is a possibility of detection of abuse of somatotropny hormone. The international scientific consortium carried out a series large-scale a research, the products of pharm dinamic impact of STG directed to studying of command depending on various factors, including sharp and chronic physical activities, age, sex, an ethnic origin and injuries (Wallace ct al., t999, 2000; Longobardi ct al., 2000; Ehmborg ct al., 2003). Confirmation of the fact of induction of the changes in structure of pharm dinamic impact happening in case of long regular application of STG and having the characteristics allowing to distinguish them from the changes induced by training occupations or other stimulators became the main achievement of the conducted researches. In particular, the statistical model describing command of a number of products of pharm dinamic impact of hormone and considering sexual distinctions was created. Also the fact that during determination of the determining factors assessment of the methods of the immune analysis used for this purpose was made and for each of them is important the specific ranges of sensitivity were established.Not all available commercial methods meet the requirements established during the researches that cause need of carrying out careful selection of systems of the immune analysis which are supposed to be used further. Besides, as very difficult statistical model is the basis for this method of testing, it is necessary to imagine limits of variability of results of the used techniques and to guarantee fixed use in tests of identical antibodies. It can represent a problem as many types of immune analysis are based on use the policlon now, but not monoclonal antibodies. However policlon antibodies can't be received in large numbers and after the termination of one batch it is impossible to guarantee that the following batch will be identical to the first. All this allows understanding why the international anti-doping organizations pay so considerable attention to development of technologies of receipt of monoclonal antibodies, suitable for use. After refining of all methodical details, this method of testing can be very effective but to comparison with the blood test applied to a drug test on ERO.

One more approach is directed to the analysis directly of STG. In difference of a range of isoforms of hormone, hypophysis, recombinant hormone is always provided by the unique form with a molecular weight of 22 kd. Also production of a recombinant form with a molecular weight of 20kg is described, but so far this protein was used only in several clinical testing. Recombinant somatropin, applied to treatment of deficit of hormone of growth at children, teenagers and persons of mature age, has molecular weight 22kg, it is obvious that the same medicines use as stimulators in sport. Such homogeneity or "lack of heterogeneity" at recombinant forms of hormone distinguishing it from a natural variety of the STG isoforms hypophysis, also makes a basis for so-called "metol of the differential immune analysis" applied to a drug test on recombinant STG (Z. Wu et al., 1999): introduction in an organism of recombinant monomeric STG with a molecular weight of 22kg leads to increase in relative content of this isoform in blood. This change in a range of isoforms of hormone becomes further even more noticeable in case of long application of STG as turning on of the mechanism of negative return regulation in this situation leads to decrease in secretion of endogenous hormone hypophysis (Wallace et al., 2001a). Screening of the monoclonal antibodies received when using various medicines of endogenous STG of the person allowed to develop two versions of the immunoanalysis. In the first option the immobilized antibodies connect mainly 22kg growth hormone isoform, and in the second is mainly somatropin "a hypophysial origin", provided various but to the size by isoforms (Bidlingmaier et al., 2000). The analysis of samples of serum by means of both versions of the immune analysis allows determining relative content of an isoform of hormone with a molecular weight of 22kg, but to comparison with the others ("total STG") and thanks to it to reveal samples with abnormally high content of 22 g of STG.It has been confirmed that changes in a range of isoforms of hormone of growth are caused only by application of recombinant STG while at stimulation of secretion after physical activity increase in quantity of all forms of hormone is observed (Wallace et al., 2001b). In addition, sensitivity of an initial method has significantly been increased (Bidlingmaicr et al., 2003). The ego has become possible thanks to receiving new monoclonal antibodies. Along with it the complex of the independent confirming tests which are also based on application of the new monoclonal antibodies having affinity to the differentiated epitopa has been developed. The last is an indispensable condition of the acceptability of the immune analysis for carrying out a drug test: each type of the analysis has to be confirmed with other analysis directed to an alternative epitop of the interesting nasmolecule that will allow to obtain the additional data necessary for identification of a molecule.

Features of a method of the differential immune analysis limit its application only to conducting doping tests he doesn't allow to distinguish the natural range of the STG isoforms and medicines of hormone extracted from hypophysis of dead people on structure. Besides, owing to extremely short half-life period of STG in the blood circulatory system (about 15 min.) the possibility of detection of use of hormone of growth as a stimulator remains limited 24 — 36 hours. It is obvious that even development of more sensitive methods won't allow to overcome this restriction as it has been shown that after disintegration of recombinant protein and the termination of the negative answer of system of feedback, hypophysis starts over again usual range of isoforms of hormone. On the other hand, the fact that for achievement of the stimulating influence hormone needs to be accepted daily increases probability of identification of the athlete applying dope during the unplanned tests which aren't connected with participation in competitions.

Application of a method of the differential immune analysis demands also unconditional justification of competency of the used types of the immune analysis. Besides, as calculation of a ratio is carried out, it is necessary to define precisely degree of reproducibility of results of separate methods and the relation taking into account potential influence of variability of results on size of the counted ratio. Essential decrease in variability of results can achieve if to use the same micro titrating plate with the immobilized antibodies for both analyses: one half of a plate is covered with monoclonal antibodies for the STG isoform with a molecular weight of 22 kd, and other half — monoclonal antibodies for total hormone of human height. After addition of calibrators, kontroly it is also model to each half of a plate, all ρε is covered with the same detecting monoclonal antibodies. Such order of carrying out the immune analysis allows to reduce significantly variability which will surely arise at uneven distribution of material of a sample between two various plates (Bidlingmaicr et al., 2000).