Growth hormone testing: Gene and protein markers

Human growth hormone testing

Recombinant human growth hormone (rhGH) is on the official banned list of substances for athletes that is maintained by the World Anti-Doping Agency due to its ability to reduce body fat, increase muscle mass and strength, and aid tissue repair. It is also used in conjunction with other performance-enhancing drugs to give athletes the edge during competition.

These properties alone are sufficient for WADA to instigate a ban but the side effects of rhGH offer a second reason. Regular users can experience pain in the muscles and joints, are more susceptible to cardiovascular problems and are at greater risk of osteoarthritis and abnormal organ growth.

RhGH doping is currently monitored using one of two tests. The direct method uses an immunoassay that measures variations in the levels of the rhGH variants that are altered after taking the hormone, but has a relatively short detection window. The indirect method targets insulin-like growth factor 1 (IGF-1) and N-terminal propeptide of type III collagen (P-III-NP), which are also raised. However, this method has limitations too as it can only be applied to serum and not plasma and IGF-1 is metabolised more quickly than P-III-NP, limiting the time for retrospective testing. Despite this, it provides a longer testing window than the direct method.

Now, a third set of biomarkers for rhGH abuse has been proposed by researchers in Italy, which they hope will be developed into an official test. The approach has been described by Jordi Segura from IMIM (Hospital del Mar Medical Research Institute) and Pompeu Fabra University, Barcelona, who worked with colleagues to identify two genes and a protein as candidates.

Genetic and proteomic biomarker candidates

The genes FN1 and RAB31 have previously been suggested as biomarkers of IGF-1 abuse following in vitro studies. This proposal is supported by the fact that patients suffering from hypertension and various kidney and bone diseases show increased levels of FN1 and its corresponding protein FN1 after IGF-1 treatment. Since rhGH increases the concentration of IGF-1, it could be assumed that it also increases the genes and the protein.

This prediction was tested on ten healthy males who were administered low doses of rhGH over three days with four other men being used as drug-free controls. The levels of the three targets were measured in serum, plasma and peripheral blood lymphocytes (PBL) by quantitative real-time PCR and immunoassays and the results confirmed the suspicion that the substances are raised by taking rhGH.

The level of the gene FN1 began to increase in PBL within 24 hours, maximising after 72 hours but remaining elevated after 216 hours. It was a similar story for RAB31, which peaked after 48 hours and was still raised after 168 hours. The concentration profile of the protein FN1 was similar to that of the gene FN1.

In contrast, the levels of IGF-1 rose rapidly following rhGH administration but fell to baseline after 168 hours. The picture for P-III-NP was even less satisfactory, as the baseline was variable and the detectability had low statistical significance.

Better testing candidates

The suitability of the protein FN1 was examined further and the preliminary results were encouraging. Acute sports activities carried out by cyclists, tennis players, swimmers and triathletes, as well as age and gender, had no effect on the basal FN1 concentrations compared with the control group.

In addition, administration of the rhGH-releasing peptide GHRP-2, also known as palmorelin, had no effect of the levels of IGF-1 or FN-1 in serum. Nor did the use of serum or plasma for measuring FN-1, both matrices yielding similar concentrations over the range 220-565 µg/mL.

The results require confirmation of a greater number of subjects over a wider age range but the preliminary data are promising. They would provide wider testing windows for the administration of rhGH than tests for IGF-1, giving the potential to catch more drug cheats. They are also better than P-III-NP, giving steadier baseline levels.

In the long-term, a routine testing procedure for FN-1 would be required with immunoassay and mass spectrometry two obvious alternatives. Standard tests for the genes FN1 and RAB31 would also be necessary.

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