Serum proteomic changes after randomized prolonged erythropoietin treatment and/or endurance training: detection of novel biomarkers

Abstract

Introduction: Despite implementation of the biological passport to detect erythropoietin abuse, a need for additional biomarkers remains. We used a proteomic approach to identify novel serum biomarkers of prolonged erythropoiesis-stimulating agent (ESA) exposure (Darbepoietin-α) and/or aerobic training.

Trial design: Thirty-six healthy young males were randomly assigned to the following groups: Sedentary-placebo (n = 9), Sedentary-ESA (n = 9), Training-placebo (n = 10), or Training-ESA (n = 8). They were treated with placebo/Darbepoietin-α subcutaneously once/week for 10 weeks followed by a 3-week washout period. Training consisted of supervised biking 3/week for 13 weeks at the highest possible intensity. Serum was collected at baseline, week 3 (high dose Darbepoietin-α), week 10 (reduced dose Darbepoietin-α), and after a 3-week washout period.

Methods: Serum proteins were separated according to charge and molecular mass (2D-gel electrophoresis). The identity of proteins from spots exhibiting altered intensity was determined by mass spectrometry.

Results: Six protein spots changed in response to Darbepoietin-α treatment. Comparing all 4 experimental groups, two protein spots (serotransferrin and haptoglobin/haptoglobin related protein) showed a significant response to Darbepoietin-α treatment. The haptoglobin/haptoglobin related protein spot showed a significantly lower intensity in all subjects in the training-ESA group during the treatment period and increased during the washout period.

Conclusion: An isoform of haptoglobin/haptoglobin related protein could be a new anti-doping marker and merits further research.

Trial registration: ClinicalTrials.gov NCT01320449.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. CONSORT flow diagram.

Fig 2. Representative 2DE gel with significant spots.

Representative 2-dimensional electrophoresis (2DE) gel of human serum. Protein spots that changed significantly (p

Fig 3. Spots affected by Darbepoietin-α treatment alone.

A total of six spots changed significantly in relation to Darbepoietin-α treatment alone, two graphs for each of the spots A-F are shown. Left panel show the average change in spot intensity for the sedentary-ESA group (n = 9). The dark grey box at the bottom indicates the boosting period (first 3 weeks) and the light grey box the maintenance period (weeks 4–10), subjects were not treated during weeks 11–13 (wash-out phase). Data are expressed as mean ± SE, level of significance are p

Fig 4. Spots with an overall Darbepoietin-α effect.

For the spots G and F that exhibited a significant Darbepoietin-α effect are shown average change in spot intensity. SP (sedentary-placebo), SE (sedentary-ESA), TP (training-placebo), and TE (training-ESA). The dark grey box at the bottom indicates the boosting period (first 3 weeks) and the light grey box the maintenance period (weeks 4–10), subjects were not treated during weeks 11–13 (wash-out phase). Individual changes (%) from baseline in all subjects in the TE and SE group are also shown. Data are expressed as mean ± SE, level of significance are p

Fig 5. Spots with overall training effect.

Average spot intensity for spots B, D, and H, which showed an overall training effect. SP (sedentary-placebo), SE (sedentary-ESA), TP (training-placebo), and TE (training-ESA). The dark grey box at the bottom indicates the boosting period (first 3 weeks) and the light grey box the maintenance period (weeks 4–10), subjects were not treated during weeks 11–13 (wash-out phase). Data are expressed as mean ± SE, level of significance are p

Fig 6. Total serum haptoglobin levels.

The individual total serum haptoglobin levels in the sedentary-ESA and training-ESA groups, respectively, throughout the study period. The dark grey box at the bottom indicates the boosting period (first 3 weeks) and the light grey box the maintenance period (weeks 4–10), subjects were not treated during weeks 11–13 (wash-out phase).