W.A. Neal

The apparent heterogeneity of erythropoietin

Abstract Concentrates of urinary erythropoietin, from two patients with paroxysmal nocturnal hemoglobinuria, were electrofractionated exhaustively. Erythropoietin (ESF1) was collected (biweekly for 8 weeks) from the anode section between Amicon membranes with molecular weight (MW) cutoffs at 20,000 to 30,000. An erythropoietin generating factor(s) (EGF)s was collected from the cathode section between Amicon membranes with MW cutoffs at 30,000 to 50,000. A relatively large erythropoiesis stimulating fraction (ESF2) remained in the center section for several more weeks. The electrofractionation process was continued, however, and after 12 weeks all activity moved to the anode (ESF1) and cathode sections (EGF). A previous report indicated the EGF fraction to contain proerythropoietin and an enzyme that converts proerythropoietin to erythropoietin, a reaction that is potentiated by a normal serum cofactor (9). Preparative acrylamide gel electrophoresis of urinary erythropoiesis regulatory factors was used to indicate the need of a component at about 32,000 MW (possibly also the normal serum cofactor) for the conversion, which is prevented by dithiothreitol and/or N-ethylmaleimide by apparently acting on the conversion factor. Erythropoietin appeared to be a monomer near to 23,000 MW, and proerythropoietin a dimer near to 45,000 MW. A complex of the monomer and dimer was above 50,000 MW. The apparent heterogeneity of erythropoietin can be explained by the erythropoietin monomer, the proerythropoietin dimer, and complexes of both (ESF2).

A comparison of four preparations of erythropoiesis regulatory factors

Abstract A comparison was made of two erythropoietin preparations distributed by the National Heart and Lung Institute with two preparations from our laboratory. One NIH preparation contained a mixture of at least two erythropoiesis regulatory factors, an erythropoietin-generating factor and erythropoietin. A second NIH preparation contained erythropoietin but had no detectable generating factor.

The production of prostaglandins with an erythropoietin generating factor(s)

Abstract Thin-layer chromatography of ERFs extracts indicates components in ERFs, after treatment by a bioconversion technique which converts linoleic and/or arachidonic acids to prostaglandins, with the same mobility as prostaglandins E2 and A2. Linoleic acid can be detected in ERFs prior to bioconversion. PGE1 and PGE2 potentiate erythropoiesis to about the same extent as PGF2α (found in erythropoiesis inhibitory fractions, EIF) inhibits erythropoiesis. The inhibition produced by PGF2α can be prevented with a neutralizing antiserum to EIF. Our data suggest that fatty acids and prostaglandins are part of the pathway for the production of erythropoietin by an enzyme and reduced glutathione.

Immobilization and kinetic studies of an erythropoietin-generating factor☆

Abstract An erythropoietin-generating factor was bound to glass beads and incubated with normal human plasma. About the same amount of erythropoietin was produced with the bound EGF as was produced with EGF in solution when incubated with normal serum. The bound EGF lost all potentiating ability after producing a limited amount of erythropoietin activity. A previous postulate for a proerythropoietin and an activating enzyme in the EGF fraction, that was potentiated by a serum cofactor, appeared appropriate. Kinetic studies suggested a similarity between EGF and the renal erythropoietic factor.

Some solubility studies of erythropoiesis regulatory factors (ERF)s

Abstract Erythropoiesis regulatory factors, isolated from the urines of a male patient with paroxysmal nocturnal hemoglobinuria (PNH) and a female patient with an anemia secondary to multiple myeloma (MM), were treated with an ethanol: acetone solution for chemical characterizations. The described treatment appeared to inactivate reversibly an erythropoiesis generating factor (EGF, molecular weight 30,000) and irreversibly an erythropoiesis stimulating factor (ESF 2 mol wt >50,000) from the PNH patient. The inactivated EGF appeared to be reactivated in the presence of several adrenocorticosteroids or batyl alcohol. The treatment with ethanol: acetone did not inactivate EGF or ESF 1 (mol wt 20,000) from the MM patient nor an inhibitor of erythropoiesis (mol wt 10,000).

The association of prostaglandins E2 and F2α with erythropoiesis regulatory factors

Abstract Prostaglandins E2 and F2α have regulatory roles, respectively, in the potentiation and inhibition of erythropoietin. Antisera to PGF2α and EIF, an erythropoiesis inhibitory factor, can be used to resolve the reason for improvement in erythropoiesis following renal dialysis. Concurrent loss of PGE2 with Ep during treatment with neuraminidase suggests that PGE2 is bound to Ep by way of sialic acid.

Erythropoiesis inhibitory factor(s) from human plasma and urine: The production and application of a neutralizing anti-EIF serum☆

Abstract The erythropoiesis inhibitory fraction (EIF) isolated from different plasmas is equally effective on a weight basis. However, the yield of the plasma EIF fraction varies relative to the source. In general, the amounts of the inhibitor fraction seem to increase with, but not parallel to, increased erythropoiesis. A young girl with recessive erythrocytosis, and a renal transplant patient, however, have a relative deficiency of the EIF fraction which could explain the erythrocytosis.

Partial characterization of an erythropoiesis inhibitory factor

An inhibitory factor of erythropoiesis, obtained from normal human urine, is indicated to be a complex of a fragment of alpha 1-acid glycoprotein and prostaglandin F2 alpha. Immunoelectrophoresis reveals two protein components in the EIF complex which separate during acrylamide gel electrophoresis. A gamma-globulin (MW 185,000) is a carrier of the complex. A fragment of alpha 1-acid glycoprotein (MW 9300) retains the inhibitory factor, PGF2 alpha. Noncovalent forces bind the PGF2 alpha to the protein, and PGF2 alpha can be extracted with benzene.