Download PDF
Int J Sports Med 2008; 29(1): 1-6
DOI: 10.1055/s-2007-989369
Physiology & Biochemistry

© Georg Thieme Verlag KG Stuttgart · New York

Discrimination of Recombinant and Endogenous Urinary Erythropoietin by Calculating Relative Mobility Values from SDS Gels

M. Kohler1 , C. Ayotte2 , P. Desharnais2 , U. Flenker1 , S. Lüdke1 , M. Thevis1 , E. Völker-Schänzer1 , W. Schänzer1
  • 1Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Cologne, Germany
  • 2Laboratoire Controle du Dopage, INRS-Institut Armand, Montreal, Canada
Further Information

Publication History

accepted after revision November 16, 2007

Publication Date:
30 November 2007 (online)

Also available at
    Permissions and Reprints

Abstract

Erythropoietin (EPO) promotes the production of red blood cells, the key factor in the regulation of the oxygen transport, and has been abused by athletes for performance enhancement in endurance sports. Current methods to detect EPO misuse are based on isoelectric focussing (IEF), double blotting, and chemiluminescence detection. A new approach utilizing SDS-PAGE mobilities of target analytes is presented. Employing two internal standards (novel erythropoiesis stimulating protein and recombinant rat EPO), the assay provides a tool which allows the calculation of relative mobility values for endogenous urinary EPO and recombinant epoetins (e.g., Dynepo™) and, thus, the distinction of these analytes in doping control samples. A reference group of 53 healthy volunteers and samples originating from a Dynepo™ (epoetin delta) excretion study conducted with a single person were analyzed and led to a significant discrimination of endogenous urinary and recombinant EPO. A clear differentiation was accomplished over a period of four days post-administration of a single injection of 50 IU/kg body weight. Hence, the method may be useful as a screening procedure in doping control or as complementary confirmation tool to the established IEF assay.

Key words

EPO - Dynepo™ - electrophoretic mobility - sport - doping - MALDI‐TOF

References

  • 1 R-Foundation .R: A language and environment for statistical computing. WU Wien: http://www.r-project.org. 02..10..2007
    Google Scholar
  • 2 Balaguer E, Demelbauer U, Pelzing M, Sanz-Nebot V, Barbosa J, Neususs C. Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis - electrospray - time-of-flight mass spectrometry.  Electrophoresis. 2006;  27 2638-2650
    CrossrefPubMedGoogle Scholar
  • 3 Bates D M, Chambers J M. Nonlinear models. Chambers JM, Hastie TJ Statistical Models. Pacific Grove, California; Wadsworth & Brooks/Cole 1992
    Google Scholar
  • 4 Bates D M, Watts D G. Nonlinear Regression Analysis and its Application. Weinheim; Wiley-VCH 1988
    Google Scholar
  • 5 Becker R A, Chambers J M, Wilks A R. The New's Language. Pacific Grove, California; Wadworth & Brooks/Cole 1988
    Google Scholar
  • 6 Deicher R, Horl W H. Differentiating factors between erythropoiesis-stimulating agents: a guide to selection for anaemia of chronic kidney disease.  Drugs. 2004;  64 499-509
    CrossrefPubMedGoogle Scholar
  • 7 Desharnais P, Ayotte C. Towards the screening of urinary EPO by SDS-PAGE. Schänzer W, Geyer H, Gotzmann A, Mareck U Proceedings of the 25th Cologne Workshop on Dope Analysis. Cologne; Sportverlag Strauß 2007 in press
    Google Scholar
  • 8 Egrie J C, Browne J K. Development and characterization of novel erythropoiesis stimulating protein (NESP).  Nephrol Dial Transplant. 2001;  16 (Suppl 3) 3-13
    PubMedGoogle Scholar
  • 9 Fukuda M N, Sasaki H, Lopez L, Fukuda M. Survival of recombinant erythropoietin in the circulation: the role of carbohydrates.  Blood. 1989;  73 84-89
    PubMedGoogle Scholar
  • 10 Gimenez E, Benavente F, Barbosa J, Sanz-Nebot V. Towards a reliable molecular mass determination of intact glycoproteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.  Rapid Commun Mass Spectrom. 2007;  21 2555-2563
    CrossrefPubMedGoogle Scholar
  • 11 Higuchi M, Oh-eda M, Kuboniwa H, Tomonoh K, Shimonaka Y, Ochi N. Role of sugar chains in the expression of the biological activity of human erythropoietin.  J Biol Chem. 1992;  267 7703-7709
    PubMedGoogle Scholar
  • 12 Jelkmann W. Erythropoietin.  J Endocrinol Invest. 2003;  26 832-837
    PubMedGoogle Scholar
  • 13 Kwan J T, Pratt R D. Epoetin delta, erythropoietin produced in a human cell line, in the management of anaemia in predialysis chronic kidney disease patients.  Curr Med Res Opin. 2007;  23 307-311
    CrossrefPubMedGoogle Scholar
  • 14 Lai P H, Everett R, Wang F F, Arakawa T, Goldwasser E. Structural characterization of human erythropoietin.  J Biol Chem. 1986;  261 3116-3121
    PubMedGoogle Scholar
  • 15 Lasne F. Double-blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures.  J Immunol Methods. 2003;  276 223-226
    CrossrefPubMedGoogle Scholar
  • 16 Lasne F, Martin L, Crepin N, de Ceaurriz J. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones.  Anal Biochem. 2002;  311 119-126
    CrossrefPubMedGoogle Scholar
  • 17 Long D L, Doherty D H, Eisenberg S P, Smith D J, Rosendahl M S, Christensen K R, Edwards D P, Chlipala E A, Cox G N. Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity.  Exp Hematol. 2006;  34 697-704
    CrossrefPubMedGoogle Scholar
  • 18 Lottspeich F, Zorbas H. Bioanalytik. Heidelberg; Spektrum 1998: 233-234
    Google Scholar
  • 19 Martin K J. The first human cell line-derived erythropoietin, epoetin-delta (Dynepo), in the management of anemia in patients with chronic kidney disease.  Clin Nephrol. 2007;  68 26-31
    PubMedGoogle Scholar
  • 20 Rahbek-Nielsen H, Roepstorff P, Reischl H, Wozny M, Koll H, Haselbeck A. Glycopeptide profiling of human urinary erythropoietin by matrix-assisted laser desorption/ionization mass spectrometry.  J Mass Spectrom. 1997;  32 948-958
    CrossrefPubMedGoogle Scholar
  • 21 Reichel C. Identification of Zinc-alpha-2-glycoprotein binding to clone AE7A5 anti-human EPO antibody by means of nano-HPLC and high-resolution high-mass accuracy ESI‐MS/MS. Schänzer W, Geyer H, Gotzmann A, Mareck U Proceedings of the 25th Cologne Workshop on Dope Analysis. Cologne; Sportverlag Strauß 2007 in press
    Google Scholar
  • 22 Sanz-Nebot V, Benavente F, Gimenez E, Barbosa J. Capillary electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry for analysis of the novel erythropoiesis-stimulating protein (NESP).  Electrophoresis. 2005;  26 1451-1456
    CrossrefPubMedGoogle Scholar
  • 23 Sasaki H, Bothner B, Dell A, Fukuda M. Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA.  J Biol Chem. 1987;  262 12059-12076
    PubMedGoogle Scholar
  • 24 Singh R N, Seavey B K, Lewis U J. Heterogeneity of human growth hormone.  Endocr Res Commun. 1974;  1 449-464
    PubMedGoogle Scholar
  • 25 Skibeli V, Nissen-Lie G, Torjesen P. Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin.  Blood. 2001;  98 3626-3634
    CrossrefPubMedGoogle Scholar
  • 26 Smith W B, Dowell J A, Pratt R D. Pharmacokinetics and pharmacodynamics of epoetin delta in two studies in healthy volunteers and two studies in patients with chronic kidney disease.  Clin Ther. 2007;  29 1368-1380
    CrossrefPubMedGoogle Scholar
  • 27 Solberg H E, Grasbeck R. Reference values.  Adv Clin Chem. 1989;  27 1-79
    PubMedGoogle Scholar
  • 28 Stübiger G, Marchetti M, Nagano M, Grimm R, Gmeiner G, Reichel C, Allmaier G. Characterization of N- and O-glycopeptides of recombinant human erythropoietins as potential biomarkers for doping analysis by means of microscale sample purification combined with MALDI‐TOF and quadrupole IT/RTOF mass spectrometry.  J Sep Sci. 2005;  28 1764-1778
    CrossrefPubMedGoogle Scholar
  • 29 Stübiger G, Marchetti M, Nagano M, Reichel C, Gmeiner G, Allmaier G. Characterization of intact recombinant human erythropoietins applied in doping by means of planar gel electrophoretic techniques and matrix-assisted laser desorption/ionisation linear time-of-flight mass spectrometry.  Rapid Commun Mass Spectrom. 2005;  19 728-742
    CrossrefPubMedGoogle Scholar
  • 30 Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kochibe N, Kobata A. Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells.  J Biol Chem. 1988;  263 3657-3663
    PubMedGoogle Scholar
  • 31 Thevis M, Schänzer W. Identification and characterization of peptides and proteins in doping control analysis.  Curr Proteomics. 2005;  2 191-208
    CrossrefPubMedGoogle Scholar
  • 32 Tomiya N, Narang S, Lee Y C, Betenbaugh M J. Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines.  Glycoconj J. 2004;  21 343-360
    CrossrefPubMedGoogle Scholar
  • 33 Wen D, Boissel J P, Tracy T E, Gruninger R H, Mulcahy L S, Czelusniak J, Goodman M, Bunn H F. Erythropoietin structure-function relationships: high degree of sequence homology among mammals.  Blood. 1993;  82 1507-1516
    PubMedGoogle Scholar
  • 34 Wognum A W, Lansdorp P M, Eaves A C, Krystal G. An enzyme-linked immunosorbent assay for erythropoietin using monoclonal antibodies, tetrameric immune complexes, and substrate amplification.  Blood. 1989;  74 622-628
    PubMedGoogle Scholar
  • 35 Wognum A W, Lansdorp P M, Eaves C J, Krystal G. Use of a sensitive bioimmunoabsorbent assay to isolate and characterize monoclonal antibodies to biologically active human erythropoietin.  Blood. 1988;  71 1731-1737
    PubMedGoogle Scholar

Prof. PhD Wilhelm Schänzer

German Sport University Cologne
Center for Preventive Doping Research Institute of Biochemistry

Carl-Diem-Weg 6

50933 Cologne

Germany

Phone: + 49 2 21 49 82 49 20

Fax: + 49 22 14 97 32 36

Email: wilhelm@biochem.dshs-koeln.de

      >