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).
Subject(s)
Anticoagulants/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Erythropoietin/metabolism , Erythropoietin/urine , Heparin/metabolism , Substance Abuse Detection/methods , Erythropoietin/analysis , Erythropoietin/isolation & purification , Heparin, Low-Molecular-Weight/metabolism , Heparitin Sulfate/metabolism , Humans , Isoelectric Focusing/methods , Recombinant Proteins , Sensitivity and SpecificityABSTRACT
The detection of doping with MIRCERA (the brand name for Continuous Erythropoietin Receptor Activator, or CERA) is hampered by the limited excretion of the rather large molecule (approximately 60 kDa) in urine. Blood (serum, plasma) in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) appears to be the ideal matrix for detecting all forms of doping with erythropoiesis-stimulating agents (ESAs) because the apparent molecular masses of ESAs are different from the mass of human serum erythropoietin (shEPO). While SDS-PAGE has proven the most sensitive method for the detection of doping with Dynepo, the sensitivity of SDS-PAGE for MIRCERA is drastically decreased. By exchanging the SDS for SARCOSYL (SAR) in the sample and running buffers the sensitivity problem was solved. SARCOSYL, a methyl glycine-based anionic surfactant, is only binding to the protein-part of MIRCERA but not to its polyethylene glycol (PEG)-chain, while SDS binds to both parts. In consequence, the monoclonal anti-EPO antibody (clone AE7A5) no longer interacts with the fully SDS-solubilized MIRCERA molecules. Only those molecules that contain SDS bound to the protein-chain are detected. Due to the inability of SARCOSYL to solubilize PEG-molecules, MIRCERA can be detected on SARCOSYL-PAGE with the same sensitivity as non-PEGylated epoetins. In a typical SAR-PAGE experiment, 200 microL of serum are used, which allows the direct detection of MIRCERA, recombinant epoetins (such as NeoRecormon, Dynepo, NESP), and shEPO in a single experiment and with high (i.e. femtogram) sensitivity.
