Molecular and electrical heterogeneity of circulating human erythropoietin measured by sensitive enzyme immunoassay

Sohmiya, Motoi

doi:10.1046/j.1365-2362.2000.00638.x

Cited by 5 publications

(5 citation statements)

References 15 publications

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“…15,26,52,55,73,74 Tam et al 55 analyzed serum from anemic patients by IEF demonstrating charge patterns similar to those in the current study, whereas Sohmiya and Kato 74 recently showed that normal human serum after IEF contained 4 more basic forms of EPO. Although these discrepancies could be attributed to the pathologic conditions of the patients, they may also reflect variations within or between healthy persons.…”

Section: Discussionsupporting

confidence: 75%

Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin

Skibeli

Nissen-Lie

Torjesen

2001

Blood

202

160

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Erythropoietin (EPO) from sera obtained from anemic patients was successfully isolated using magnetic beads coated with a human EPO (hEPO)-specific antibody. Human serum EPO emerged as a broad band after sodium dodecyl sulfatepolyacrylamide gel electrophoresis, with an apparent molecular weight slightly smaller than that of recombinant hEPO (rhEPO). The bandwidth corresponded with microheterogeneity because of extensive glycosylation. Two-dimensional gel electrophoresis revealing several different glycoforms confirmed the heterogeneity of circulating hEPO. The immobilized anti-hEPO antibody was capable of binding a representative selection of rhEPO glycoforms. This was shown by comparing normal-phase high-performance liquid chromatography profiles of oligosaccharides released from rhEPO with oligosaccharides released from rhEPO after isolation with hEPO-specific magnetic beads. Charge analysis demonstrated that human serum EPO contained only mono-, di-, and tri-acidic oligosaccharides and lacked the tetra-acidic structures present in the glycans from rhEPO. Determination of charge state after treatment of human serum EPO with Arthrobacter ureafaciens sialidase showed that the acidity of the oligosaccharide structures was caused by sialic acids. The sugar profiles of human serum EPO, describing both neutral and charged sugar, appeared significantly different from the profiles of rhEPO. The detection of glycan structural discrepancies between human serum EPO and rhEPO by sugar profiling may be significant for diagnosing pathologic conditions, maintaining pharmaceutical quality control, and establishing a direct method to detect the misuse of rhEPO in sports. IntroductionHuman erythropoietin (EPO) is a glycoprotein that is synthesized mainly in the kidney and that stimulates erythropoiesis through actions on erythroid progenitor cells. [1][2][3] Human EPO (hEPO) was the first hematopoietic growth factor to be cloned. 4,5 Recombinant hEPO (rhEPO) has been available as a drug since 1988 and is used in the clinical treatment of anemia, especially anemia caused by renal failure. In sports the misuse of rhEPO has been suspected among athletes for several years. 6 Active hEPO consists of a single 165-amino acid polypeptide chain with three N-glycosylation sites at Asn24, Asn38, and Asn83, respectively, and one O-glycosylation site at Ser126. The average carbohydrate content is approximately 40%. 7-9 Glycosylation is important for the biologic activity of EPO. Removal or modification of the glycan chains results in altered in vivo and in vitro activity. [9][10][11][12][13][14] Furthermore, the number of sialic acid residues and the branching pattern of the N-linked oligosaccharides modify the pharmacodynamics, speed of catabolism, and biologic activity of hEPO. 11,15,16 Oligosaccharide units, or glycans, of glycoproteins serve a variety of functions, including folding of nascent polypeptide chains in the endoplasmic reticulum, protection of the protein moieties from the action of proteases, and modulation of biologic act...

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Section: Discussionsupporting

confidence: 75%

Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin

Skibeli

Nissen-Lie

Torjesen

2001

Blood

202

160

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“…We established a sensitive EIA for EPO, and could evaluate precisely plasma EPO levels with a minimal detectable level of 0·75 IU/l using a 20‐µl plasma sample (Sohmiya & Kato, 2000). We have reported previously that short‐term administration of rhGH increased both plasma EPO levels and reticulocyte counts in patients with chronic renal failure (Sohmiya et al ., 1998a).…”

Section: Discussionmentioning

confidence: 99%

“…Normal ranges of plasma IGF‐I levels in our laboratory were 202·8 ± 70·6 µg/l and 198·6 ± 91·8 µg/l in males and females, respectively. Plasma EPO levels were measured by sensitive EIA, as described previously (Sohmiya & Kato, 2000). Sensitivity was 0·75 IU/l using 20 µl of plasma sample.…”

Section: Methodsmentioning

confidence: 99%

Effect of long‐term administration of recombinant human growth hormone (rhGH) on plasma erythropoietin (EPO) and haemoglobin levels in anaemic patients with adult GH deficiency

Sohmiya

Kato

2001

Clinical Endocrinology

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We conclude that EPO secretion was stimulated in the initial 2 weeks after the start of CSI of rhGH in anaemic patients with adult GH deficiency. Increased Hb concentrations after long-term administration of rhGH might be explained by direct stimulatory effects of rhGH and IGF-I on erythroid cells, which was accompanied by suppressed EPO secretion, in combination with a more generalized indirect impact of rhGH on physical activety. These findings suggest a beneficial effect of rhGH replacement in anaemic patients with adult GH deficiency.

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“…at high altitude residence) or when the O2 affinity of the blood increases. Like other plasma glycoproteins EPO circulates as a pool of isoforms that differ in glycosylation, molecular mass, biological activity and immunoreactivity (51,52). There is a diurnal fluctuation of the concentration of circulating EPO, with values being about 40% higher at midnight than in the morning (53).…”

Section: Sites and Mechanisms Of Epo Production And Degradationmentioning

confidence: 99%

Molecular Biology of Erythropoietin

2004

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The glycoprotein hormone erythropoietin (EPO) is an essential viability and growth factor for the erythrocytic progenitors. EPO is mainly produced in the kidneys. EPO gene expression is induced by hypoxia-inducible transcription factors (HIF). The principal representative of the HIF-family (HIF-1, -2 and -3) is HIF-1, which is composed of an O2-labile alpha-subunit and a constant nuclear beta-subunit. In normoxia, the alpha-subunit of HIF is inactivated following prolyl- and asparaginyl-hydroxylation by means of alpha-oxoglutarate and Fe(2+)-dependent HIF specific dioxygenases. While HIF-1 and HIF-2 activate the EPO gene, HIF-3, GATA-2 and NFkappaB are likely inhibitors of EPO gene transcription. EPO signalling involves tyrosine phosphorylation of the homodimeric EPO receptor and subsequent activation of intracellular antiapoptotic proteins, kinases and transcription factors. Lack of EPO leads to anemia. Treatment with recombinant human EPO (rHuEPO) is efficient and safe in improving the management of the anemia associated with chronic renal failure. RHuEPO analogues with prolonged survival in circulation have been developed. Whether the recent demonstration of EPO receptors in various non-hemopoietic tissues, including tumor cells, is welcome or ominous still needs to be clarified. Evidence suggests that rHuEPO may be a useful neuroprotective agent.

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Molecular and electrical heterogeneity of circulating human erythropoietin measured by sensitive enzyme immunoassay

Abstract: These findings indicate the first evidence for molecular and electrical heterogeneity of circulating EPO in normal human plasma without any concentrating procedure.

Cited by 5 publications

References 15 publications

Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin

Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin

Effect of long‐term administration of recombinant human growth hormone (rhGH) on plasma erythropoietin (EPO) and haemoglobin levels in anaemic patients with adult GH deficiency

Molecular Biology of Erythropoietin

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