The interference of heparin on IEF‐PAGE of erythropoietins

Abstract

Because of the risk of suffering a stroke or heart attack, some athletes and their medical supervisors admitted having used anticoagulants (e.g. acetylsalicylic acid) in combination with doping with recombinant erythropoietins (rhEPO). Heparin is one of the oldest and cheapest anticoagulants. The anticoagulative effect of heparin is a result of the binding of heparin to the plasma protein antithrombin III and the subsequent inactivation of blood clotting factors (e.g. factor IIa, IXa, Xa, XIa, XIIa). Heparin-a polyanion-is known to interact with carrier ampholytes used in IEF-PAGE. Two different types of heparin pharmaceuticals are used for medical purposes: unfractionated heparins (UFH) and low molecular weight heparins (LMWH). Their influence on IEF- and SDS-PAGE was investigated. Only UFH had a profound impact on IEF-PAGE, leading to excessive smearing or complete abolishment of the EPO IEF-profile and shifting of acidic EPO-isoforms in the endogenous region of the gel. No such effect was observable for SDS-PAGE. Remedies include immunoaffinity purification of EPO before IEF-PAGE or the treatment of the urinary retentate with solid urea. A combined usage of IEF- and SDS-PAGE is recommended for confirming the presence of rhEPO in urine and for further analysis of smearing (and therefore suspicious) samples. This two-method approach is already in accordance with the technical document on EPO-analysis (TD2009EPO) of the World Anti-Doping Agency (WADA).