Markers of erythropoiesis stimulating agents use and hypoxia

Code: ISF17R02NN

This project is aimed at identifying markers of rHuEPO misuse and separate these from markers of natural hypoxic exposure by metabolomics. Male (n=20) and female (n=20) non-competing athletes are exposed to 4 weeks sea-level investigation and after a full washout period, participants are exposed to hypobaric hypoxia at 2.320 m for four weeks. Four groups are formed in a blinded randomized and gender balanced fashion: One group (n=20) receives EPO during the sea-level period and placebo during the stay
at altitude. Another group (n=8) receives EPO both at sea-level and altitude. A third group (n=8) receives placebo both at sea-level and altitude and the fourth group (n=4) receives placebo at sea-level and EPO at altitude. The design facilitates identification of markers of EPO misuse at sea-level (n=28) as well as markers of natural altitude exposure (n=28). Each participant’s treatment response is analyzed in relation to a 4 week baseline period. Moreover, the design includes an intra-individual possibility to verify identified targets expected to be unique to rHuEPO misuse, since 8 participants are treated with rHuEPO both at sea-level and altitude. Runners are enrolled due to the good possibility of recruitment and conduction of training at altitude. The project results in a highly valuable biobank for identification of markers sensitive to initiation and termination of rHuEPO injections. Metabolomics discovery is carried out in collaboration with a world-leading research group as well as the experts at University of Cologne, Germany. Importantly, the project is a joint effort between world-leading University research, Anti-doping organizations and WADA laboratories.

Main Findings

With the present study we were able to demonstrate that recombinant human erythropoietin (rhEpo) treatment combined with altitude exposure provide an additive erythropoietic response compared to rhEpo treatment or altitude exposure alone. Accordingly, analysing the results in the Athlete

Biological Passport revealed that the passport is more sensitive to rhEpo treatment at altitude than at sea level, but a concurrent compromised specificity exist with altitude exposure.

We also investigated the potential of immature reticulocyte fraction (IRF) and the ratio between immature reticulocyte and red blood cells (IR/RBC) as novel biomarkers for rhEpo treatment. With this study we demonstrate that a low-dose rhEpo treatment alters IRF and IR/RBC compared with placebo, which also exceeds the fluctuations induced by altitude exposure in the post-treatment period. Similarly, during rhEpo treatment at altitude, both biomarkers exceeded the fluctuations induced by placebo injections at altitude. When individual thresholds were calculated, we were able to identify ~90% of the rhEpo treated participants at sea level with each variable, whereas we were able to identify ~30% and ~65% of the rhEpo treated participants at altitude for IRF and IR/RBC, respectively. Importantly, these sensitivities were obtained with a specificity >99%.

Plasma samples have been subjected to untagerted metabolomics followed and bioinformatic analyses. No biomarkers with sufficient sensitivity and specificity to discriminate between sea-level or altitude exposure could be identified. Likewise, no biomarkers able to discriminate rhEpo and placebo treatment could be identified.

Finally, the present study demonstrated that the novel markers of iron homeostasis, hepcidin and erythroferrone, are more sensitive markers of altered iron homeostasis within one week of changes in iron demands than routine iron markers. When individual thresholds were calculated, 30-40% of the rhEpo treated participants exceeded the thresholds. Our results demonstrate that erythroferrone, and to some extent hepcidin, may hold promise as novel biomarkers for detecting rhEpo misuse at altitude, but intra-individual variability is of concern and additional studies are required