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StopDoping Foundation Current projects Finalized projects Athlete's biological passport Detection of homologous blood transfusion Nutritional supplements contamination Anabolic steroids and recovery Synthetic glucocorticosteroids GHB origin by GC/C/IRMS Effects of exercise on the isoelectric patterns of erythropoietin Conjugated steroids analysis by LC-MS CERA project Testosterone metabolism and ethnicity Testosterone metabolism by GC/C/IRMS Publications

Effects of exercise on the isoelectric patterns of erythropoietin


Objectives
This study aimed at improving our understanding of the physiology and the metabolism specific to effort urines. These urines will be characterized in a formal manner in the context of EPO anti-doping testing and a potential marker for effort urines will be proposed.

Introduction
Erythropoietin (EPO) is a naturally secreted hormone which causes an increase in the number of erythrocytes. Because EPO enhances performance in endurance sports, synthetic EPO use is prohibited by the World Anti-Doping Code. Natural and synthetic forms of the EPO protein differ in the number of surface charges. These differences lie at the basis of the current anti-doping tests that reveal the presence of synthetic EPO in the urine. A sample is declared positive for EPO if the isoelectric pattern obtained after the test fulfills three positivity criteria established by WADA. These criteria define the distribution, positions and intensities of the bands for each endogenous or synthetic EPO isoform. In practice, certain negative samples (B in the illustration) fail to display the normal negative isoelectric pattern (A in the illustration). Indeed, in some cases, and under the influence of different factors, a negative sample yields a distribution which resembles that of synthetic EPO (R in the illustration). While the exact cause of this phenomenon is still unknown, it appears to take place preferentially when urines are collected immediately after an intense physical effort. This observation explains the use of the term effort urines to describe the phenomenon.

Result of an EPO screening electrophoresis gel

Methodology
In the initial clinical phase of this study, 7 elite cyclists carried out characteristic, vigorous efforts in order to generate the phenomenon of effort urines in a controlled fashion. Blood and urine samples were collected throughout the protocol. Urine and blood samples were analyzed for the presence of EPO in a standard manner. Immunological dosages for proteins were also performed. Notably, a protein marker for disturbed urinary excretion was tested, in order to propose a complementary and alternative measure for effort urines.

Results
As expected, the clinical part of this study allowed to obtain samples presenting all the characteristics of effort urines. Notably, all the urines collected either during or after the effort demonstrated a shift of endogenous EPO isoforms towards the basic area the gel. This shift was characterized by an increase of the percentage of basic isoforms and was correlated with changes in several other parameters such as total protein concentration, retinol-binding protein (RBP) concentration and -2 microglobulin concentration. RBP seems to constitute the most appropriated marker to discriminate an effort urine from another urine. Inversely, analyses of EPO in blood demonstrated that seric EPOs did not show any change following effort.

Conclusion
On the basis of these results, a metabolic hypothesis was proposed to explain the occurrence of effort urines. For a long time, strenuous efforts have been known to disturb the renal function, and post-effort urinary proteinuria constitutes a well-described phenomenon. Therefore, we suggest that a partial inhibition of the tubular reabsorbtion process, due to the effort, allows some seric EPO to avoid reabsorbtion and consequently, to be excreted in urine. This mix of seric and urinary EPO may explain the occurrence of a shift towards the basic area of the gel. This hypothesis is supported by the striking increase of RBP, which is a well-known marker of a disturbed distal tubular reabsorbtion, in effort urines. RBP concentration is close to zero in a normal urine, while it was present in huge concentrations in all observed effort urines. This protein is therefore proposed as a discriminative marker allowing to distinguish between a normal and an effort urine.

Publications
1. Lamon S, Martin L, Robinson N, Saugy M, Lasne F - Effects of exercise on the isoelectric patterns of erythropoietin - Submitted July 2008 in the Clinical Journal of Sports Medicine

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