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.

中文翻译：

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在干血斑中使用基于 RNA 的 5'-氨基乙酰丙酸合酶 2 生物标志物检测重组人促红细胞生成素微剂量

运动员生物护照 (ABP) 的血液模块用于间接检测血液操作；然而，使用这种方法来检测兴奋剂，例如用微剂量的重组人促红细胞生成素 (rhEPO) 是有问题的。因此，必须通过实施新的生物标志物来提高 ABP 的敏感性。在这里，我们展示了 5'-氨基乙酰丙酸合酶 2 ( ALAS2 ) mRNA 是有用的转录组生物标志物，可用于改善 rhEPO 微量给药的间接检测。此外，灵敏度足以区分 rhEPO 给药与暴露于缺氧条件。编码碳酸酐酶 1 ( CA1 ) 和溶质载体家族 4 成员 1 ( SLC4A1 ) 的 mRNA 水平) RNA，以及线性 (L) 和线性 + 环状 (LC) 形式的ALAS2 mRNA，在 rhEPO 微剂量给药后和暴露于缺氧条件期间监测 16 天。与 rhEPO 微剂量给药后的基线值相比，ALAS2 mRNAs 增加了 300%。此外，ALAS2 mRNAs 在缺氧条件下没有显着增加。相比之下，rhEPO 微剂量和缺氧后CA1 mRNA 增加，而SLC4A1 mRNA 在任何一种情况下都没有显着增加。此外，此处描述的分析是使用干血斑 (DBS) 进行的，这在样本收集、运输和存储物流方面具有优势。本研究表明，ALAS2 mRNA 水平是使用 ABP 的血液学模块检测 rhEPO 微剂量的敏感和特异性转录组生物标志物，并且该方法与使用 DBS 进行反兴奋剂分析兼容。