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

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.

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
• 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
• 3 Bates D M, Chambers J M. Nonlinear models. Chambers JM, Hastie TJ Statistical Models. Pacific Grove, California; Wadsworth & Brooks/Cole 1992
• 4 Bates D M, Watts D G. Nonlinear Regression Analysis and its Application. Weinheim; Wiley-VCH 1988
• 5 Becker R A, Chambers J M, Wilks A R. The New's Language. Pacific Grove, California; Wadworth & Brooks/Cole 1988
• 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
• 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
• 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
• 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
• 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
• 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
• 12 Jelkmann W. Erythropoietin.  J Endocrinol Invest. 2003;  26 832-837
• 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
• 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
• 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
• 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
• 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
• 18 Lottspeich F, Zorbas H. Bioanalytik. Heidelberg; Spektrum 1998: 233-234
• 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
• 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
• 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
• 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
• 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
• 24 Singh R N, Seavey B K, Lewis U J. Heterogeneity of human growth hormone.  Endocr Res Commun. 1974;  1 449-464
• 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
• 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
• 27 Solberg H E, Grasbeck R. Reference values.  Adv Clin Chem. 1989;  27 1-79
• 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
• 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
• 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
• 31 Thevis M, Schänzer W. Identification and characterization of peptides and proteins in doping control analysis.  Curr Proteomics. 2005;  2 191-208
• 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
• 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
• 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
• 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