Jasper P. Lewis

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 Influence of Antiserum to Human Erythropoietin on the Production of Hemoglobin C in Goats

Summary The production of Hb-C was studied, with and without the presence of antibodies to ESF(s), in goats made anemic by injections of phenylhydrazine. Although the ESF(s) activity was possibly slightly decreased by the relatively weak anti-ESF serum, the production of Hb-C was not altered appreciably. Twin goats were injected with ESF(s) dissolved in rabbit serum without and with potent neutralizing antibodies to ESF. There was a marked increase in Hb-C production in the goat receiving the ESF in normal serum, but the Hb-C in the goat receiving antiserum with the ESF activity neutralized was minimal.

Potentiation of Purified Erythropoietin with Serum Proteins

1 Non‐neutralizing antiserum, normal rabbit, human and mouse sera potentiate the erythropoietic activity of anaemic human urinary Fraction II + III. This substantiates the concepts introduced by Kuratowska, Lewartowski and Lipinski. 2 Orosomucoid (alpha‐1 acidic‐glycoprotein) similarly potentiates the erythropoietic activity of anaemic human urinary Fraction II + III. 3 The erythropoietic activity of Standard B was not enhanced by the addition of serum proteins. 4 The erythropoietic activity of human urinary concentrates was enhanced by the addition of normal serum proteins and suggestive enhancement of anaemic human serum and plasma was noted.

The Isolation of Erythropoiesis Regulatory Factors by an Electrofractionation Technique Combined with Selective Membrane Permeability

Summary A urine concentrate from a patient with paroxysmal nocturnal hemoglobinuria contained four regulators of erythro-poiesis that could be isolated by an electro-fractionation technique combined with selective membrane permeability. An erythropoie-sis-stimulating factor (ESF) passed toward the anode through a membrane with a cutoff at a molecular weight of 30,000, but was retained by a membrane with a cutoff at 20,000, while another ESF was retained by a membrane with a cutoff at 50,000. An ESF-generating factor passed toward the cathode through a membrane with a cutoff at 50,000 but was retained by a membrane with a cutoff at 30,000. An inhibitor of erythropoiesis (EIF) passed toward the cathode through a membrane with a cutoff at a mol wt of 30,000 but was retained by a membrane with a cutoff at 10,000. The EIF brought about maximal depression of erythropoiesis 6 days after the first of four daily injections, but erythropoiesis gradually returned to normal 3 days later, though the EIF injections were continued. The EIF appeared to be mostly lipid and about 7.5% protein. The authors express their appreciation to members of the engineering departments at the Veterans Administration Hospitals in Augusta, GA and St. Louis, MO for their assistance in the construction of the electrofractionation apparatus used in this work.

Potentiation of purified erythropoietin with serum proteins. II. Serial dose response relationships.

Summary Erythropoietin (urinary fraction II + III) was diluted with either saline or normal serum and injected into assay animals as a single dose or as 2 or 4 fractional doses either intraperitoneally or subcutaneously. Erythropoietic activity increased as the dose was fractionated; however, the erythropoietin plus serum showed greater activity than erythropoietin plus saline in all dosage schedules. This suggests that normal serum actually enhances the activity of erythropoietin rather than merely acting as an adjuvant causing delayed absorption of the material. Grateful acknowledgement is made to Miss Bettie Williams and Mrs. Carol Cope for fine technical assistance, to Dr. Charles B. Bragassa, Mrs. Rebecca Bedingfield and Mr. Bob Ellis of the Computer Center, Medical College of Georgia, for statistical analyses and to Mrs. Margaret Hiers and Mrs. Shirl Melton for secretarial assistance.

Erythropoietin and Haemoglobin Studies on AKR Mice

Summary. DEAE‐Sephadex column chromatography has been used to study the haemoglobins of AKR untreated mice, polycythaemic mice injected with saline and polycythaemic mice treated with an extract of human erythropoietin. All elution patterns of the haemolysates showed four haemoglobins. The haemolysates of each group had an elution pattern different from the other groups. Chromatographic studies of an ageing haemolysate indicated the formation of a minor haemoglobin from a major haemoglobin to be linear for a period of about 18 days.

The Selective Inactivation of an ESF-Generating Factor (EGF) in the Presence of Erythropoietin (ESF)

Summary An ESF-generating factor (EGF), erythropoietin (ESF) and a mixture of both were incubated with dithiothreitol (DTT, a protective reagent for sulfhydryl groups). Ten μM DTT completely prevented the production of 0.26 ± 0.01 IU of ESF by EGF but did not inactivate ESF. DTT was used to indicate the relative amounts of EGF and ESF activity in a mixture of both.

ERYTHROPOIETIC ACTIVITY IN HUMAN URINE CONCENTRATE.

Summary Increased erythropoietic specific activities remained in urine which had been concentrated by flash evaporation, adjusted to high ionic strength, and precipitated at high ethanol concentration. The apparent stability of erythropoietin was attributed to a protective carrier relatively stable under the conditions described. Erythropoietic activity from urine concentrate was detected in the dialyzate from a membrane which retained thyrotropic hormone and allowed glucagon to escape. The dialysis data added credence to the postulate of a free and bound erythropoietin. The authors are indebted to Miss Dorothy A. Alford for helpful assistance.

The Selective Membrane Filtration of an ESF-generating Factor (EGF) in the Presence of Erythropoietin (ESF)

Summary A mixture of an erythropoiesis stimulating factor (ESF) and an ESF-generating factor (EGF), isolated from the urine of a patient with paroxysmal nocturnal hemoglobinuria (PNH), was separated by selective membrane filtration. The optimum pH for activity of the EGF fraction from the urine of the PNH patient was about 7.4, the same as that previously found for the EGF from the urine of a patient with an anemia secondary to multiple myeloma (MM). The urine from the PNH patient contained an ESF that appeared larger than EGF, whereas the urine from the MM patient did not; the urine from both patients, however, contained an ESF that appeared smaller than EGF as previously noted.