Molecular charge heterogeneity of human serum erythropoietin

The charge heterogeneity of human serum erythropoietin (S-Epo) was studied in 89 serum specimens from 78 subjects by zone electrophoresis in 0.17% agarose suspension at pH 8.6. The electrophoretic elution profiles of S-Epo were determined with a radioimmunoassay for Epo. The number of Epo peaks indicated that at least 20-30 different forms of Epo were present in a single serum specimen. The median charge of Epo, estimated as its median electrophoretic mobility, was determined for each serum specimen. This median charge was measured in 10 healthy adults, 46 patients with anaemia, six patients with secondary polycythaemia, one patient with polycythaemia vera treated by phlebotomy, and six healthy newborn infants (cord sera). Forty-four of the patients with anaemia had a median charge of S-Epo within the reference range for healthy adults, while all the patients with polycythaemia and the newborn infants had less negatively charged forms of S-Epo. In nine patients in whom the S-Epo level had a circadian rhythm, the forms of S-Epo in the evening were less negative than those in the morning. The median charge of recombinant preparations was much less negative than that of S-Epo in healthy individuals, while that of the 2nd International Reference Preparation was more negative than in any of the 78 subjects analysed. A significant change to less negatively charged S-Epo forms was observed 24 h after a subcutaneous injection of recombinant Epo in one patient, who before the injection had a normal median charge and concentration of S-Epo. In conclusion, Epo exhibits a considerable charge heterogeneity in individual serum specimens, the forms of S-Epo in the morning may differ from those in the evening, those in adults differ from those in newborn infants, and those in patients with anaemia differ from those in polycythaemia. The results also suggest that the methods used in this study may be useful for detecting the presence of injected recombinant Epo in the blood in persons with a normal endogenous Epo production.