LAUSSANNE-RBP

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patterns of erythropoietin

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Effec ts o f exerc ise on t he isoe lect r ic pa t te rn s o fe r y th r opo ie t i n

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This study aimed at improving our understanding of the physiology andthe metabolism specific to effort urines. These urines will be characterized

in a formal manner in the context of EPO anti-doping testing and apotential marker for effort urines will be proposed.

I n t r o d u c t i o n

Erythropoietin (EPO) is a naturally secreted hormone which causes an

increase in the number of erythrocytes. Because EPO enhancesperformance in endurance sports, synthetic EPO use is prohibited by theWorld Anti-Doping Code. Natural and synthetic forms of the EPO proteindiffer in the number of surface charges. These differences lie at the basisof the current anti-doping tests that reveal the presence of synthetic EPOin the urine.

A sample is declared positive for EPO if the isoelectric pattern obtained

after the test fulfills three positivity criteria established by WADA. Thesecriteria define the distribution, positions and intensities of the bands foreach endogenous or synthetic EPO isoform. In practice, certain negative

samples (B in the illustration) fail to display the normal negativeisoelectric pattern (A in the illustration). Indeed, in some cases, andunder the influence of different factors, a negative sample yields adistribution which resembles that of synthetic EPO (R in the illustration).While the exact cause of this phenomenon is still unknown, it appears totake place preferentially when urines are collected immediately after anintense physical effort. This observation explains the use of the term«effort urines» to describe the phenomenon.

Methodo logy

In the initial clinical phase of this study, 7 elite cyclists carried outcharacteristic, vigorous efforts in order to generate the phenomenon of

effort urines in a controlled fashion. Blood and urine samples werecollected throughout the protocol. Urine and blood samples were analyzedfor the presence of EPO in a standard manner. Immunological dosages forproteins were also performed. Notably, a protein marker for disturbedurinary excretion was tested, in order to propose a complementary andalternative measure for effort urines.

Resul ts

As expected, the clinical part of this study allowed to obtain samplespresenting all the characteristics of effort urines. Notably, all the urinescollected either during or after the effort demonstrated a shift of endogenous EPO isoforms towards the basic area the gel. This shift wascharacterized by an increase of the percentage of basic isoforms and wascorrelated with changes in several other parameters such as total proteinconcentration, 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 notshow any change following effort.

Conclus ion

On the basis of these results, a metabolic hypothesis was proposed toexplain the occurrence of effort urines. For a long time, strenuous effortshave been known to disturb the renal function, and post-effort urinaryproteinuria constitutes a well-described phenomenon. Therefore, wesuggest that a partial inhibition of the tubular reabsorbtion process, dueto the effort, allows some seric EPO to avoid reabsorbtion andconsequently, to be excreted in urine. This mix of seric and urinary EPOmay 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 efforturines. RBP concentration is close to zero in a normal urine, while it was

present in huge concentrations in all observed effort urines. This protein istherefore proposed as a discriminative marker allowing to distinguishbetween a normal and an effort urine.

Pub l i ca t i ons

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

Result of an EPO screening

electrophoresis gel

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