Laboratoire de contrôle du dopage, INRS-Institut Armand-Frappier, Laval, Québec, Canada.
Drug Test Anal. 2013 Nov-Dec;5(11-12):870-6. doi: 10.1002/dta.1494. Epub 2013 May 29.
Recombinant erythropoietin (rhEPO) has been misused for over two decades by athletes, mainly but not only in endurance sports. A direct rhEPO detection method in urine by isoelectric focusing (IEF) was introduced in 2000, but the emergence of third-generation erythropoiesis-stimulating agents and so-called biosimilar rhEPOs, together with the sensitivity of human endogenous EPO (huEPO) pattern to enzymatic activities and its modification following short strenuous exercise, prompted the development of a complementary test based on SDS-PAGE analysis. While Mircera and NESP are easily detected with the existing IEF and SDS-PAGE methods, some samples containing both epoetin-α/β and huEPO present profiles that are still difficult to interpret. As doping practices have moved to micro-dosing, these mixed patterns are more frequently observed. We investigated the impact of enzymatic desialylation on the urinary and serum EPO profiles obtained by SDS-PAGE with the aim of improving the separation of the bands in these mixed EPO populations. We observed that the removal with neuraminidase of the sialic acid moieties from the different EPOs studied reduced their apparent molecular weight (MW) and increased the migration distance between huEPO and rhEPO centroids, therefore eliminating the size overlaps between them and improving the detection of rhEPO.
重组促红细胞生成素（rhEPO）被运动员滥用已有二十多年，主要是在耐力运动中，但也不仅限于此。2000 年，人们引入了一种通过等电聚焦（IEF）直接检测尿液中 rhEPO 的方法，但第三代促红细胞生成刺激剂和所谓的生物类似 rhEPO 的出现，以及人类内源性 EPO（huEPO）模式对酶活性的敏感性及其在剧烈短时间运动后的修饰，促使人们开发了一种基于 SDS-PAGE 分析的补充检测方法。虽然 Mircera 和 NESP 可以通过现有的 IEF 和 SDS-PAGE 方法轻松检测到，但一些含有 epoetin-α/β 和 huEPO 的样本的图谱仍然难以解释。随着兴奋剂实践转向微剂量，这些混合图谱更频繁地被观察到。我们研究了酶去唾液酸化对 SDS-PAGE 获得的尿液和血清 EPO 图谱的影响，目的是改善这些混合 EPO 群体中条带的分离。我们观察到，用神经氨酸酶去除研究中不同 EPO 上的唾液酸部分会降低它们的表观分子量（MW），并增加 huEPO 和 rhEPO 质心之间的迁移距离，从而消除它们之间的大小重叠，并提高 rhEPO 的检测。
