Detection of Recombinant Epoetin and Darbepoetin Alpha after Subcutaneous Administration in the Horse

Lasne, Françoise; Popot, Marie-Agnès; Varlet‐Marie, Emmanuelle; Martin, Laurent; Martin, Jean-Antoine; Bonnaire, Yves; Audran, Michel; Céaurriz, J. de

doi:10.1093/jat/29.8.835

Cited by 31 publications

(37 citation statements)

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“…administration, single or several doses), which make comparisons difficult. The accredited EPO doping method for human athletes based on isoelectric focusing IEF, showed that s.c. administration of 36 IU Eprex/kg bodyweight was not detectable in equine urine from the three horses after 48 h from last injection [26]. By LC-MS/MS (limit of identification at 200 ng/L), the single i.v.…”

Section: Detecting Administration Of Rhuepomentioning

confidence: 88%

“…Other studies using the IEF method showed that a single s.c. administration of 0.37 μg/kg of Aranesp to one horse was detectable in equine urine up to 5 days postadministration [26]. In a study using LC-MS/MS, an identification limit of 100 ng/L was obtained for Aranesp and an i.v.…”

Section: Detecting Administration Of Aranespmentioning

confidence: 97%

“…For EPO doping control in horses, a rapid screening method for rHuEPO using a human EPO immunoassays with low reactivity for eEPO compared with HuEPO has been used [26] to select samples to be confirmed with the more resource demanding methods. Exogenous rHuEPOs and analogues present in equine samples have been detected by isoelectric focusing (IEF) with double-immunoblotting [26], due to their differences in oligosaccharide composition.…”

Section: Introductionmentioning

confidence: 99%

“…Exogenous rHuEPOs and analogues present in equine samples have been detected by isoelectric focusing (IEF) with double-immunoblotting [26], due to their differences in oligosaccharide composition. Equine and human EPO have 84 % identity in the amino acid structure [27] which has been utilised for LC-MS/MS analyses of peptide sequences specific for HuEPO appearing after trypsin digestion of EPO [28][29][30][31][32].…”

Section: Introductionmentioning

confidence: 99%

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Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test

Lönnberg

Bondesson

Cormant³

et al. 2012

Anal Bioanal Chem

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Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of erythropoiesis-stimulating agents, like the rHuEPO glycoprotein varieties Eprex and Aranesp, to horses. After administration of 40 IU Eprex kg(-1) day(-1) to seven horses during 6 days, the presence of Eprex in horse plasma was detected up to 2-5 days after last injection. In urine samples collected from two horses, Eprex was detected up to 3 days. A single injection of Aranesp (0.39 μg/kg) was detected up to 9 days in plasma and up to 8 days, the last day of testing, in the urine sample. The LC-FAIMS-MS/MS system, with 1 day reporting time, confirmed the presence of Eprex up to 1 day after last injection for six out of seven horses and the presence of Aranesp up to 5 days after last injection in plasma samples. The MAIIA system showed to be a promising tool with high sensitivity and extremely short reporting time (1 h).

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Section: Detecting Administration Of Rhuepomentioning

confidence: 88%

Section: Detecting Administration Of Aranespmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test

Lönnberg

Bondesson

Cormant³

et al. 2012

Anal Bioanal Chem

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“…Such protein modifications do not necessarily result in a loss of biological activity [1][2][3][4][5]. For example, in vivo human erythropoietin (Epo) is more active as a dimer [6] and inhomogeneous glycolysation patterns [2] can be used for doping control [7,8]. In most cases, however, the band with the expected molecular size of the monomer is selected for quantification of Western blots.…”

Section: Introductionmentioning

confidence: 99%

Quantifying Western blots: Pitfalls of densitometry

et al. 2009

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Although Western blots are frequently quantified, densitometry is not documented and appears to be based merely on traditions and guesswork. Confirming previous experience, none of 100 randomly selected and systematically scanned most recent papers provided sufficient information on how Western blot results were translated into statistical values. The importance of such information, however, becomes evident from our correlations of plasma erythropoietin values of various mammals determined using RIA and Western blot densitometry. Different common densitometry procedures applied to the identical Western blot revealed p-values of these correlations ranging from 0.000013 to 0.76 reflecting the necessity of a scientifically sound basis for densitometry of Western blots. At present, the current lack of any definitions in densitometry opens the door to uncontrollable acquisition of any desired p-value. Here we provide data that define what should be considered, what avoided and what documented when quantifying Western blots.

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Blood cells RNA biomarkers as a first long‐term detection strategy for EPO abuse in horseracing

Bailly-Chouriberry¹,

Noguier²,

Manchon³

et al. 2010

Drug Testing and Analysis

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Recombinant human erythropoietins (rHuEPOs) are glycoproteins drugs, produced by the pharmaceutical industry to restore production of red blood cells by stimulating human bone marrow for which this pathology has been diagnosed. It is suspected that these molecules are diverted as doping agents in horseracing to enhance oxygen transport and aerobic power in racehorses. Although indirect double-blotting or direct liquid chromatography-mass spectrometry (LC-MS) methods have been developed to confirm the presence of rHuEPO in a sample, the short detection time (48 h) is still a problem for doping control. In this context, gene profiling investigation through Serial Analysis of Gene Expression (SAGE) has been conducted on seven thoroughbreds treated with Eprex. This functional genomic method has been performed from total blood cells collected from each animal to assess the mRNA expression consecutive to rHuEPO injections. Sample pooling was chosen as a powerful, cost-effective, and rapid means of identifying the most common and specific changes in terms of gene expression profile and to eliminate individual variation. Consequently, three SAGE libraries were constructed, before, during, and after Eprex treatment. More than 71 440 mRNA signatures were observed and subjected to statistical analysis; 49 differentially expressed genes were identified and analyzed by qPCR. From the selected gene list, were defined as potential biomarkers in terms of their low inter-individual variation and capacity as strong markers of rHuEPO administration up to 60 days after the beginning of the doping period. In this paper, a new strategy is proposed to the horseracing industry to prevent rHuEPO abuse.

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Detection of Recombinant Epoetin and Darbepoetin Alpha after Subcutaneous Administration in the Horse

Cited by 31 publications

References 1 publication

Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test

Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test

Quantifying Western blots: Pitfalls of densitometry

Blood cells RNA biomarkers as a first long‐term detection strategy for EPO abuse in horseracing

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