Erythropoietin: induction of synthesis to signal transduction

Roberts, Delia; Smith, DM

doi:10.1677/jme.0.0120131

Cited by 16 publications

(4 citation statements)

References 60 publications

(113 reference statements)

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“…HuEPO is the main factor responsible for the proliferation of erythrocytes in the human body. Approximately 90% of HuEPO production takes place in the kidneys whenever a tissue oxygen sensor detects oxygen depletion (1,2 ). For patients with kidney failure, the loss of the main HuEPO production site leads to severe anemia, which was treated by blood transfusion until 1988 when rHuEPO became available.…”

confidence: 99%

Detection of Recombinant Human Erythropoietin in Urine by Isoelectric Focusing

Andreas¹,

Catlin

Green³

et al. 2003

Clinical Chemistry

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Background: Doping with erythropoietic proteins such as recombinant human erythropoietin (rHuEPO) and darbepoetin alfa is a serious issue in sport. There is little information on the time course of detection of rHuEPO in urine and on methods to evaluate electrophoresisbased data. Methods: We used a recently described isoelectric focusing method for detecting rHuEPO and endogenous EPO in urine obtained from individuals treated with placebo or epoetin alfa. The latter was administered subcutaneously at 50 IU/kg on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. Blood and urine samples were collected during the morning of study days ؊3, 0, 2, 4, 7, 9, 11, 14, 16, and 18 and on days 2, 3, 4, and 7 postadministration. We developed visual and numerical (two-band ratio) techniques to evaluate the electropherograms for the presence of rHuEPO. Results: Compared with the placebo group, the epoetin alfa-treated group responded with increases in hematocrit, reticulocytes, macrocytes, serum EPO, and serum soluble transferrin receptor. The electropherograms showed that the pattern of bands arising from urinary rHuEPO is different from that of endogenous urinary EPO. Both the two-band ratio and the visual technique detected rHuEPO in all 14 epoetin alfa-treated individuals 3 days after the last dose. On the 7th day after the last dose, both techniques detected rHuEPO in approximately one-half of the participants. rHuEPO was not detected in the placebo-treated individuals. Conclusions: The isoelectric focusing method detects rHuEPO in most urine samples collected 3 days after nine doses of epoetin alfa. The numerical two-band

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confidence: 99%

Detection of Recombinant Human Erythropoietin in Urine by Isoelectric Focusing

Andreas¹,

Catlin

Green³

et al. 2003

Clinical Chemistry

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“…A massa molecular da estrutura glicosilada, que contém cerca de 40% de carboidratos, é de 30-34kDa, e somente da cadeia peptídica é de 18kDa (1)(2)(3)(4)(5). A atividade especí-fica é de não menos que 100.000UI/mg de proteína (6).…”

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Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Schmidt

Ramos

Silva

et al. 2003

Arq Bras Endocrinol Metab

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RESUMORealizou-se a identificação de eritropoietina humana recombinante em produtos farmacêuticos comerciais por eletroforese em gel de poliacrilamida e detecção com anticorpos específicos, demonstrando-se típi-ca banda larga, semelhante ao padrão de rhEPO da Farmacopéia Européia. Igualmente por focalização isoelétrica e imunodetecção, observaram-se 5-6 isoformas características de acordo com o laboratório produtor. A avaliação de potência efetuada através de ensaio biológico em camundongos normocitêmicos forneceu valores entre 67,6% e 119,4% em relação à declarada. A precisão dos ensaios combinados, calculada pela ponderação, variou de 200 a 389. Os testes de endotoxinas bacterianas, toxicidade e pH apresentaram valores variáveis conforme o lote. Concluiu-se destacando a importância dos testes e ensaios de controle para assegurar a qualidade lote a lote e garantir a eficácia terapêutica dos produtos farmacêuticos. The identification of rhEPO in pharmaceutical products was carried out by polyacrylamide gel electrophoresis and detection with specific antibodies that revealed a typical single broad band similar to that obtained with the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The isoelectric focusing with immunodetection revealed extensive heterogeneity with 5-6 isoforms, characteristic according to the manufacturer. The potency was assessed by the normocythaemic mouse assay with values within 67.6% and 119.4% calculated against the stated potency. The precision evaluated by the weight was within 200 and 389, for the combined assays. The toxicity, bacterial endotoxins, and pH gave variable results according to the batch. In conclusion, we emphasize the importance of the batch-to-batch tests and assays that would guarantee the quality and therapeutic efficacy of the pharmaceutical products.

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“…This sialoglycoprotein hormone consists of 165 amino acids that form a single polypeptide chain containing two intra-chain disulfide bonds (Cys and Cys [29][30][31][32][33] ) and four potential glycosylation sites, three of which are N-linked (Asn 24 , Asn 38 and Asn 83 ) and one O-linked (Ser 126 ) (1,2). The molecular mass of EPO is 30-34 kDa, but the molecular mass of the peptide chain is only about 18 kDa.…”

Section: Introductionmentioning

confidence: 99%

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

Ramos

Schmidt

Andrade

et al. 2003

Braz J Med Biol Res

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The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the endpoint using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4% for the brilliant cresyl blue method, 14.1-30.8% for the selective hemolysis method and 8.5-19.7% for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products. Correspondence

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Erythropoietin: induction of synthesis to signal transduction

Cited by 16 publications

References 60 publications

Detection of Recombinant Human Erythropoietin in Urine by Isoelectric Focusing

Detection of Recombinant Human Erythropoietin in Urine by Isoelectric Focusing

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

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