Principal Investigator:
Dr. Laura Garvican-Lewis; Australian Institute of Sport

Research Summary:

Introduction: The Athlete’s Biological Passport (ABP) is a valuable tool in the fight against doping; its integrity is underpinned by understanding the magnitude of normal fluctuations of its biomarkers in response to various environmental stimuli or medical conditions e.g. altitude training or iron deficiency. The combination of intravenous iron supplementation and altitude training has a number of potentially important ramifications for the anti-doping field. Intravenous iron supplementation, via bolus injection, is currently not banned under WADA guidelines. However its potential to enhance performance via facilitating erythropoiesis warrants further investigation. Secondly, since altitude training and the treatment of iron deficiency present two potentially confounding factors to the passport, a thorough understanding of the magnitude by which the ABP can be altered when these are combined is warranted. Lastly, the combination of intravenous iron supplementation and altitude training creates an opportunity to model the end result of erythropoietic doping practices within natural and legal constraints. This allows relevant data to be collected from high-level athletes, which can be used to validate a number of novel and emerging analysis methods for doping detection.

Hypothesis: The erythropoietic response to 3 weeks of altitude training was assessed in endurance trained athletes supplemented with either oral or intravenous (IV) iron. We hypothesized that the increased bioavailability of iron via intravenous supplementation would accelerate the time-course of adaptation; therein creating a model for maximal hematological perturbations within WADA regulations, with potential for significant performance enhancement. Further, we hypothesized that the IV treated group would exhibit greater erythropoietic gains in hemoglobin mass (Hb mass) following altitude exposure, resulting in greater increases in maximal aerobic power (VO2max). Hb mass and ABP hematological parameters were measured during both the “ON” and “OFF phase” of altitude training. The sensitivity and specificity of the current ABP algorithms during these time periods was then assessed using the ABP profiles created for each athlete. We hypothesized that the expected greater enhancements in the IV treated group would result in red flags in the ABP profiles of these athletes.

Methods: The combined impact of altitude and iron supplementation on the ABP was evaluated in endurance-trained athletes (n=34) undertaking 3-weeks of simulated live-high: train-low (14 h.d-1,3000m). Athletes received either oral (ferrous sulphate), intravenous (IV, ferric carboxymaltose) or placebo iron supplementation, commencing two weeks prior and continuing throughout hypoxic exposure. Hb mass, ABP parameters and markers of iron regulation were assessed at baseline (day−14), immediately prior to (day 0), weekly during (days 8, 15), and immediately, 1, 3 and 6 weeks after the completion of altitude exposure (days 22, 28, 42 and 63). VO2max was assessed in the week prior to and week post altitude exposure. Individual ABP thresholds for hemoglobin concentration ([Hb]), reticulocyte percentage (%retic), and OFF score were calculated using the adaptive model and assessed at 99% and 99.9% specificity. Finally, a newly developed plasma volume (PV) blood test was applied to assess the possible efficacy of reducing the influence of PV contractions on the volumetric ABP markers; hemoglobin concentration ([Hb]) and the OFF-score. Serum was analyzed for Transferrin, Albumin, Calcium, Creatinine, Total Protein, and Low-Density Lipoprotein. The PV blood test (consisting of the serum markers, [Hb] and platelets) was then applied to the ABP adaptive model and new reference predictions were calculated for [Hb] and the OFF-score, thereby reducing the PV variance component.

Results: Hb mass significantly increased following altitude in IV (Mean%, [90% CI]: 3.7%, [2.8, 4.7]) and oral (3.2%, [2.2, 4.2]), and remained elevated at 7 days post-altitude in oral (2.9%, [1.5, 4.3]) and 21 days post in IV (3.0%, [1.5, 4.6]). Hb mass was not significantly higher than baseline at any timepoint in placebo. Eleven athletes returned values outside of the calculated reference ranges at 99%, with 8 at 99.9%. The percentage of athletes exceeding the thresholds in each group was similar, but IV returned the most individual occurrences. A similar frequency of abnormalities occurred across the three biomarkers, with abnormal [Hb] and OFF score values arising mainly during-, and %retic values mainly post-altitude. Removing samples collected during altitude from the model resulted in ten athletes returning abnormal values at 99% specificity, two of whom had not triggered the model previously. The PV correction refined the ABP reference predictions. The number of atypical passport findings (ATPFs) for [Hb] was reduced from 7 ATPFs of 5 subjects to 6 of 3 subjects. The OFF-score ATPFs frequency increased with the PV correction (from 9 to 13, 99% specificity); most likely the result of more specific reference limit predictions combined with the altitude-induced increase in red cell production. Importantly, all abnormal biomarker values were flagged identified by a low confidence value by the PV model’s weighting function.

Conclusions: Iron supplementation appears necessary for optimal erythropoietic adaptation to altitude exposure. However, intravenous iron supplementation during three weeks of simulated LHTL altitude training offered no additional benefit in terms of the magnitude of the erythropoietic response for non-anemic endurance athletes compared to oral supplementation. The abnormalities observed in the ABP, in response to iron supplementation and hypoxia, were not systematic and mostly in line with expected physiological adaptations. They do not represent a uniform weakness in the ABP. Nevertheless, altitude training and iron supplementation should be carefully considered by experts evaluating abnormal ABP profiles. Finally, although the multifaceted, individual physiological response to altitude confounded some results, the PV model appears capable of reducing the impact of PV fluctuations on [Hb].