Procedures for monitoring recombinant erythropoietin and analogues in doping control

The present report summarizes the main analytical strategies developed to identify the presence of recombinant erythropoietin (EPO) administered as a doping agent. Indirect evidence is based on the analysis of blood parameters (haemoglobin, haematocrit, reticulocytes, macrocytes, etc.) and serum markers (concentration of EPO and serum transferrin receptors, etc.). The problem of intertechnique comparison for reliable results evaluation is emphasized, especially for serum markers. Charge differences between isoforms of recombinant EPO and native urinary EPO are the grounds for the isoelectric focusing–double blotting–chemiluminescence detection method presently approved for doping control. Works addressing its advantages and limitations are presented and commented on. The chemical bases of the differential detection are highlighted and some future approaches for detection are also presented. The appearance and detectability of EPO analogues and mimetics susceptible for abuse are also addressed.

[1]  Ken Sharpe,et al.  Standardization of reticulocyte values in an antidoping context. , 2004, American journal of clinical pathology.

[2]  S. Elliott,et al.  Comparison of the isoelectric focusing patterns of darbepoetin alfa, recombinant human erythropoietin, and endogenous erythropoietin from human urine. , 2002, Clinical chemistry.

[3]  I. Macdougall,et al.  Meeting the challenges of a new millennium: optimizing the use of recombinant human erythropoietin. , 1998, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[4]  P. Lundberg,et al.  Are current methods of measurement of erythropoietin (EPO) in human plasma or serum adequate for the diagnosis of polycythaemia vera and the assessment of EPO deficiency? , 1998, Scandinavian journal of clinical and laboratory investigation.

[5]  David T. Martin,et al.  Reticulocyte parameters as potential discriminators of recombinant human erythropoietin abuse in elite athletes. , 2000, International journal of sports medicine.

[6]  B. Ekblom,et al.  Detection in blood and urine of recombinant erythropoietin administered to healthy men. , 1995, Medicine and science in sports and exercise.

[7]  F. Bressolle,et al.  Effects of erythropoietin administration in training athletes and possible indirect detection in doping control. , 1999, Medicine and science in sports and exercise.

[8]  M. Bollen,et al.  False-positive detection of recombinant human erythropoietin in urine following strenuous physical exercise. , 2006, Blood.

[9]  J. Segura,et al.  Recombinant Erythropoietin and Analogues: A Challenge for Doping Control , 2004, Therapeutic drug monitoring.

[10]  Christian Thomas,et al.  Biochemical markers and hematologic indices in the diagnosis of functional iron deficiency. , 2002, Clinical chemistry.

[11]  Edmond J. Breen,et al.  New urinary EPO drug testing method using two-dimensional gel electrophoresis. , 2005, Clinica chimica acta; international journal of clinical chemistry.

[12]  A. Collins,et al.  New epoetin molecules and novel therapeutic approaches. , 1999, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[13]  G. Allmaier,et al.  Detection of isoforms of recombinant human erythropoietin by various plant lectins after isoelectric focusing , 2005, Electrophoresis.

[14]  W. Schmidt,et al.  How Valid is the Determination of Hematocrit Values to Detect Blood Manipulations? , 2000, International journal of sports medicine.

[15]  R. Konrad,et al.  New automated chemiluminescent assay for erythropoietin , 2000, Journal of clinical laboratory analysis.

[16]  G Lippi,et al.  Laboratory Screening for Erythropoietin Abuse in Sport: an Emerging Challenge , 2000, Clinical chemistry and laboratory medicine.

[17]  F. Wians,et al.  Soluble transferrin receptor (sTfR) concentration quantified using two sTfR kits: analytical and clinical performance characteristics. , 2001, Clinica chimica acta; international journal of clinical chemistry.

[18]  J. Kamerling,et al.  Sialylated carbohydrate chains of recombinant human glycoproteins expressed in Chinese hamster ovary cells contain traces of N‐glycolylneuraminic acid , 1990, FEBS letters.

[19]  R. Gutiérrez Gallego,et al.  Assessing the instability of the isoelectric focusing patterns of erythropoietin in urine , 2006, Electrophoresis.

[20]  Ken Sharpe,et al.  A third generation approach to detect erythropoietin abuse in athletes. , 2006, Haematologica.

[21]  H. Schellekens.  How similar do 'biosimilars' need to be? , 2004, Nature Biotechnology.

[22]  V. Wiwanitkit,et al.  Methodology evaluation of a new immunoturbidimetric method for measuring serum soluble transferrin receptor. , 2001, Asian Pacific journal of allergy and immunology.

[23]  Huwei Liu,et al.  Ionene-dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry. , 2005, Journal of separation science.

[24]  M. Saugy,et al.  New urinary EPO drug testing method using two-dimensional gel electrophoresis. , 2006, Clinica chimica acta; international journal of clinical chemistry.

[25]  D. Catlin,et al.  False-positive Epo test concerns unfounded. , 2006, Blood.

[26]  C. Breymann.  Erythropoietin test methods. , 2000, Bailliere's best practice & research. Clinical endocrinology & metabolism.

[27]  J. Boeynaems,et al.  Measurement of soluble transferrin receptor by immunoturbidimetry and immunonephelometry. , 2000, Clinical biochemistry.

[28]  C J Gore,et al.  A novel method utilising markers of altered erythropoiesis for the detection of recombinant human erythropoietin abuse in athletes. , 2000, Haematologica.

[29]  Y Miura,et al.  Blood cells. , 1984, Cell structure and function.

[30]  C. Neusüss,et al.  Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry. , 2006, Analytical chemistry.

[31]  D Macmillan,et al.  Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin. , 2001, Chemistry and Biology.

[32]  V. Wray,et al.  Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells. , 1993, European journal of biochemistry.

[33]  H. Miyazaki,et al.  Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. , 1988, The Journal of biological chemistry.

[34]  R. Sasaki,et al.  Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins. , 1988, Biochemistry.

[35]  J. Adamson,et al.  Design and Chemical Synthesis of a Homogeneous Polymer-Modified Erythropoiesis Protein , 2003, Science.

[36]  J. Marsden,et al.  Unreliability in immunoassays of erythropoietin: anomalous estimates with an assay kit , 1993, British journal of haematology.

[37]  M. Vahter,et al.  Evaluation of kits for measurement of the soluble transferrin receptor. , 1999, Scandinavian journal of clinical and laboratory investigation.

[38]  Kerry R Emslie,et al.  Second-generation blood tests to detect erythropoietin abuse by athletes. , 2003, Haematologica.

[39]  L. Buck,et al.  Enhancement of therapeutic protein in vivo activities through glycoengineering , 2003, Nature Biotechnology.

[40]  W. Jelkmann.  Erythropoietin after a century of research: younger than ever , 2007, European journal of haematology.

[41]  F. Lasne.  No doubt about the validity of the urine test for detection of recombinant human erythropoietin. , 2006, Blood.

[42]  J. Egrie,et al.  Development and characterization of novel erythropoiesis stimulating protein (NESP). , 2001, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[43]  T. Lappin,et al.  Erythropoietin--the first 90 years. , 1996, Clinical and Laboratory Haematology.

[44]  D. Boger,et al.  Erythropoietin mimetics derived from solution phase combinatorial libraries. , 2002, Journal of the American Chemical Society.

[45]  K. Punnonen,et al.  Evaluation of new immunoenzymometric assay for measuring soluble transferrin receptor to detect iron deficiency in anemic patients. , 1997, Clinical chemistry.

[46]  S. Zlotkin,et al.  Disparity of serum transferrin receptor measurements among different assay methods , 1998, European Journal of Clinical Nutrition.

[47]  J Keul,et al.  Haemoglobin, Haematocrit and Red Blood Cell Indices in Elite Cyclists. Are the Control Values for Blood Testing Valid? , 2000, International journal of sports medicine.

[48]  V. Skibeli,et al.  Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin. , 2001, Blood.

[49]  K. Punnonen,et al.  Automated immunoturbidimetric method for measuring serum transferrin receptor. , 1999, Clinical chemistry.

[50]  K. Maiese,et al.  New avenues of exploration for erythropoietin. , 2005, JAMA.

[51]  T. Hayakawa,et al.  Structural analysis of sulfated N-linked oligosaccharides in erythropoietin. , 2001, Glycobiology.

[52]  F. Lasne,et al.  Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures. , 2001, Journal of immunological methods.

[53]  Jeffrey F. Gorzek,et al.  Detection of recombinant human erythropoietin in urine by isoelectric focusing. , 2003, Clinical chemistry.

[54]  J. Kamerling,et al.  Sulfated di-, tri- and tetraantennary N-glycans in human Tamm-Horsfall glycoprotein. , 1998, European journal of biochemistry.

[55]  Evaluation of immunoassays for the measurement of erythropoietin (EPO) as an indirect biomarker of recombinant human EPO misuse in sport. , 2004, Journal of pharmaceutical and biomedical analysis.

[56]  F. Lasne,et al.  Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones. , 2002, Analytical biochemistry.

[57]  C. Gore,et al.  Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis. , 2001, Haematologica.

[58]  I. Macdougall.  CERA (Continuous Erythropoietin Receptor Activator): a new erythropoiesis-stimulating agent for the treatment of anemia. , 2005, Current hematology reports.

[59]  W. Roberts,et al.  Performance characteristics of the IMMUNLITE 2000 erythropoietin assay. , 2004, Clinica chimica acta; international journal of clinical chemistry.

[60]  J. Marx,et al.  Haematocrit in Elite Athletes , 1999, International journal of sports medicine.

[61]  B. Berglund,et al.  Effect of short‐term and intermittent normobaric hypoxia on endogenous erythropoietin isoforms , 2003, Scandinavian journal of medicine & science in sports.

[62]  H. Schellekens,et al.  When biotech proteins go off-patent. , 2004, Trends in biotechnology.

[63]  F. Lasne,et al.  Recombinant erythropoietin in urine , 2000, Nature.

[64]  Huub Schellekens,et al.  'Biogenerics': the off-patent biotech products. , 2002, Trends in pharmacological sciences.

[65]  M. Pelzing,et al.  Glycoform characterization of intact erythropoietin by capillary electrophoresis‐electrospray‐time of flight‐mass spectrometry , 2005, Electrophoresis.

[66]  F. Pigozzi,et al.  Identification of blood erythroid markers useful in revealing erythropoietin abuse in athletes. , 2001, Blood cells, molecules & diseases.

[67]  Evaluation of immunoassays for the measurement of soluble transferrin receptor as an indirect biomarker of recombinant human erythropoietin misuse in sport. , 2004, Journal of immunological methods.

[68]  M. Bednarek,et al.  Mimicry of erythropoietin by a nonpeptide molecule. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[69]  P. Mayeux,et al.  Biology of erythropoietin. , 1998, Haematologica.

[70]  R. Sherwood,et al.  Evaluation of Six Erythropoietin Kits , 1999, Annals of clinical biochemistry.