Correspondence letterDrug Testing

and Analysis

Received: 16 July 2014 Accepted: 19 July 2014 Published online in Wiley Online Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1704

Iron concentration increases after moderateendurance exercise: implications for screeningof blood transfusion in sports

* Correspondence to: Giuseppe Lippi, U.O. Diagnostica Ematochimica, AziendaOspedaliero-Universitaria di Parma, Via Gramsci, 14, 43126 - Parma, Italy.E-mail: glippi@ao.pr.it; ulippi@tin.it

Figure 1. Variation of iron in serum before, immediately thereafter and24h after the end of a half-marathon run in 37 trained athletes. The close cir-cles (●) represent the median values.

To the Editor

In a recent and interesting study, Leuenberger et al.[1] concluded thatiron assessment in ethylenediamine tetraacetic acid (EDTA) plasmamay be regarded as a simple and reliable approach for the screeningof blood transfusion misuse in sports. Interestingly, a fixed thresholdof 45μg/dL iron in plasma EDTA was associated with 1.00 specificityand 0.93 sensitivity. It is noteworthy that little information is availableso far about the effect of endurance sports on iron concentration inblood. Therefore, we investigated whether circulating iron valuesmay be altered by moderate endurance exercise.

The study population consisted of 37 trained athletes (13 femaleand 24male; mean age 45years, range 28–64 years), who underwenta 21.1 km, half-marathon run. The distance was run on a hilly and de-manding route (197m vertical gain, with slopes averaging 1.8% andpeaks up to 7%), in a daywith 42min of sunshine during the exercise,temperatures between 14 and 17 ° C, wind force between 3 and5km/h, and humidity between 60 and 80%. Blood samples werecollected in serum tubes containing no additives before the run,immediately thereafter, and 24h after the end of the run. They wereimmediately transported to the clinical chemistry laboratory of theAcademic Hospital of Verona under controlled conditions of temper-ature (18–22 ° C) and humidity (<50%). Blood tubes were centri-fuged according to local practice (i.e., 1500xg for 10min at roomtemperature). Serum iron concentration was then measured on aCobas C501 (Roche Diagnostics GmbH, Mannheim, Germany), witha method similar to that used by Leuenberger et al.,[1] and basedon sample incubation in phosphate buffer containing ascorbicacid and FerroZine (i.e., 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid) (Roche Diagnostics GmbH, Mannheim, Germany).Results were expressed as median and interquartile range (IQR)and difference were analyzed with Wilcoxon-Mann-Whitney test,using Analyse-it (Analyse-it Software Ltd, Leeds, UK).

The results of this study are shown in Figure 1. The concentration ofserum iron increased significantly from the baseline (18μg/L; IQR,15–21μg/L) to the period immediately after the run (24μg/L; IQR,20–26μg/L; p< 0.001). The concentration of serum iron returned tovalues non-significantly different from the baseline (19μg/L; IQR,13–23; p=0.209) 24h after the run. The plasma volume change was+0.6% (IQR, �0.6 to 1.2%) in the period immediately after the runand 0.4% (�0.2 to 1.1%) 24h after the run (both p> 0.05), respectively.

In a previous study, Schumacher et al. reported that serum ironwas increased by approximately 26% in seven trainedmale athletesperforming a prolonged cycling exercise.[2] According to our data,the concentration of iron in serum increased significantly byapproximately 29% (IQR, 15–38%) after a moderate enduranceexercise in trained athletes, a variation that slightly exceeded thewithin-subject biologic variation of this parameter (±26%).[3] It ishence noteworthy that this minor but significant increase of iron

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in serum (and thereby in blood) may be sufficient to overcome thechelator effect of EDTA reported in the study of Leuenberger et al.,[1]

thus paving the way for additional investigation to assess the varia-tion of iron in EDTA plasma and the relative thresholds for the screen-ing of blood transfusion misuse in sports.[1,4] The plasma volumechange could not completely explain the increase of serum iron atthe end of the run. It seems reasonable then to attribute this changeto the modest but still significant foot-strike haemolysis that is fre-quently observed after marathon and half-marathon running, andtriggers the release of intracellular iron from injured erythrocytes.[5]

Provided that our data can be confirmed in EDTA plasma, thesefindings also confirm the appropriateness of the current practice ofanti-doping testing, which excludes samples takenwhen strenuousexercise has been performed 2h prior to the test. Accordingly, thenon-significant variation from baseline that could be observed 24 hafter exercise strengthens the applicability of this test despite exer-cise, provided that the current sampling standards are respected.

Yours,

GIAN LUCA SALVAGNO,a GIUSEPPE LIPPI,b* CANTOR TARPERI,c

GIAN CESARE GUIDIa and FEDERICO SCHENAcaLaboratory of Clinical Biochemistry, Department of Life andReproduction Sciences, University of Verona, Verona, Italyb Laboratory of Clinical Chemistry and Hematology, AcademicHospital of Parma, Parma, Italyc Department of Neurological, Neuropsychological, Morphologicaland Movement Sciences, University of Verona

Copyright © 2014 John Wiley & Sons, Ltd.

G. L. Salvagno et al.

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References[1] N. Leuenberger, C. Ansermet, T. Pottgiesser, N. Baume, N. Robinson,

M. Saugy, Y.O. Schumacher. A fast automated screening method forthe detection of blood transfusion in sports. Drug Test. Anal. 2014.DOI: 10.1002/dta.1676

[2] Y.O. Schumacher, A. Schmid, D. König, A. Berg. Effects of exercise onsoluble transferrin receptor and other variables of the iron status. Br. J.Sports Med. 2002, 36, 195.

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[3] C. Ricós, V. Alvarez, F. Cava, J.V. García-Lario, A. Hernández, C.V. Jiménez,J. Minchinela, C. Perich, M. Simón. Current databases on biologicalvariation: pros, cons and progress. Scand. J. Clin. Lab. Invest. 1999, 59, 491.

[4] G. Lippi, R. Aloe, P. Avanzini, G. Banfi. Measurement of iron in serum andEDTA plasma for screening of blood transfusion in sports. Drug Test.Anal. 2014 Jul 15. DOI: 10.1002/dta.1696. [Epub ahead of print].

[5] G. Lippi, F. Schena, G.L. Salvagno, R. Aloe, G. Banfi, G.C. Guidi. Foot-strike haemolysis after a 60-km ultramarathon. Blood Transfus.2012, 10, 377.

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