Abstract
The hematological module of the Athlete Biological Passport (ABP) is used for indirect detection of blood manipulations; however, the use of this method to detect doping, such as with microdoses of recombinant human erythropoietin (rhEPO), is problematic. For this reason, the sensitivity of ABP must be enhanced by implementing novel biomarkers. Here, we show that 5'‐aminolevulinate synthase 2 (ALAS2) mRNAs are useful transcriptomic biomarkers to improve the indirect detection of rhEPO microdosing. Moreover, the sensitivity was sufficient to distinguish rhEPO administration from exposure to hypoxic conditions. Levels of mRNAs encoding carbonate anhydrase 1 (CA1) and solute carrier family 4 member 1 (SLC4A1) RNA, as well as the linear (L) and linear + circular (LC) forms of ALAS2 mRNA, were monitored for 16 days after rhEPO microdosing and during exposure to hypoxic conditions. ALAS2 mRNAs increased by 300% compared with the baseline values after rhEPO microdosing. Moreover, ALAS2 mRNAs were not significantly increased under hypoxic conditions. By contrast, CA1 mRNA was increased after both rhEPO microdosing and hypoxia, whereas SLC4A1 mRNA did not significantly increase under either condition. Furthermore, the analyses described here were performed using dried blood spots (DBSs), which provide advantages in terms of the sample collection, transport, and storage logistics. This study demonstrates that ALAS2 mRNA levels are sensitive and specific transcriptomic biomarkers for the detection of rhEPO microdosing using the hematological module of the ABP, and this method is compatible with the use of DBSs for anti‐doping analyses.
Highlights
This study demonstrates that aminolevulinate synthase 2 (ALAS2) mRNA levels are sensitive and specific transcriptomic biomarkers for the detection of recombinant human erythropoietin (rhEPO) microdosing using the hematological module of the Athlete Biological Passport (ABP), and this method is compatible with the use of dried blood spots (DBSs) for anti-doping analyses
Indirect detection of blood manipulations using the hematological module of the Athlete Biological Passport (ABP) can be challenging, for example, in unmasking doping with microdoses of recombinant human erythropoietin.[1,2]
The levels of ALAS2 linear + circular (LC), ALAS2 L, and carbonate anhydrase 1 (CA1) mRNAs were significantly higher after rhEPO microdosing (Figure 1)
Summary
Indirect detection of blood manipulations using the hematological module of the Athlete Biological Passport (ABP) can be challenging, for example, in unmasking doping with microdoses of recombinant human erythropoietin (rhEPO).[1,2] Developing transcriptomics methods is a promising approach to improve the sensitivity of the hematological module of ABP. A number of studies have attempted to characterize the transcriptomic blood signature after rhEPO administration, and several genes have been identified as potential RNA-based biomarkers.[3,4] Among these, mRNAs encoding 5'-aminolevulinate synthase 2 (ALAS2), carbonic anhydrase 1 (CA1), and solute carrier family 4 member 1 (SLC4A1) have been proposed as candidates for the detection of blood manipulations. Salamin et al and Loria et al demonstrated that the expression of these mRNAs increased following the administration of therapeutic doses of rhEPO.[5,6,7] Both the linear and the linear + circular forms of ALAS2 mRNA (ALAS2 L and ALAS2 LC, respectively) may be useful as potential biomarkers, as both were found to increase after therapeutic injection of rhEPO.[5] Circular mRNAs have a head and tail joined at the splice site, rendering them more stable than linear mRNAs.[8] This propriety could be beneficial for monitoring RNA-based biomarkers in the anti-doping context
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