Robert D. Lange

OBSERVATIONS ON ERYTHROPOIETIC-STIMULATING FACTOR (ESF) IN THE PLASMA OF UREMIC AND NONUREMIC ANEMIC PATIENTS

Excerpt During the last decade the clinical and experimental observations of numerous investigators have established the concept that erythropoiesis is regulated in part by humoral factors. Recent ...

Response to Erythropoietin.

Alpen and Cranmore (1) and Erslev (2) have concluded that erythropoietin increases red cell output by accelerating the rate of stem cell differentiation into erythroid precursors. Observations on erythropoiesis in polycythemic mice by Gurney, et al.(3) have supported this conclusion. However, observations by several investigators (4-7) seem to indicate that the action of the erythropoietic humoral factor (s) is not limited to regulation of stem cell differentiation. Experiments reported here tested the effect of erythropoietin on later phases of erythroid formation and the results suggest an action here as well as in the early stem cell phase. Methods. Female Sprague-Dawley rats, weighing 180 to 200 g, were made polycythemic by one or several transfusions of packed homologous red blood cells. Each transfusion equaled 50% of the animals original red cell mass. At various intervals following the initial transfusion the polycythemic rats were given a single injection of human urine erythropoietin (8) or purified sheep erythropoietin (9).‡ Subsequent changes in erythropoiesis were assessed by reticulocyte counts (10) and measurement of the 18-hour erythrocyte radio-iron incorporation(8). Erythroid elements per 1000 nucleated cells were enumerated in smears of marrow from control polycythemic animals. In 3 experiments, polycythemic rats were given single intraperitoneal injections of 2 ml of urine concentrate on either the 5th, 15th or 31st day after their first transfusion. Control polycythemic rats were injected with saline. At the end of each successive 24-hour period following erythropoietin or saline injection, representative animals from each group were killed and the reticulocyte counts, hematocrits and erythrocyte radio-iron incorporation were determined. The radio-iron was injected 18 hours before the rats were killed. All groups given urine concentrate included at least 5 rats.

Immunological Studies of the Renal Erythropoietic Factor (Erythrogenin)

An immunological study of the renal erythropoietic factor (REF or erythrogenin) has been described. The experiments indicate that the erythropoietic activity of REF, as assayed in the polycythaemic mouse, is neutralized in vitro by addition of serum obtained from a rabbit previously immunized with a REF preparation. These anti‐REF sera had no effect on the biological activity of ESF; however, a depression in erythropoiesis was observed after injection of anti‐REF into normal mice. It is concluded that the injection of an antibody developed against the REF into normal mice interrupts normal erythropoiesis by reducing REF levels; the reduced REF levels lead to decreased amounts of ESF.

ANGIOTENSIN II AND ERYTHROPOIESIS.

Summary1. Polycythemic mice and hypophysectomized rats were used as assay animals to measure the erythropoietic stimulating activity of synthetic angiotensin II (A-S) and of angiotensin II made from a crude extract of the hog kidney (A-CE). 2. No increase in erythropoietic activity, as measured by the red blood cell incorporation of Fe59, was found following injection of these substances. The assay animals were responsive to a concentrated sheep plasma erythropoietin. 3. These results suggest that the reninangiotensin system does not encompass erythropoietin but do not rule out the possibility that a second material is released by the juxtaglomerular cells.Summary 1. Polycythemic mice and hypophysectomized rats were used as assay animals to measure the erythropoietic stimulating activity of synthetic angiotensin II (A-S) and of angiotensin II made from a crude extract of the hog kidney (A-CE). 2. No increase in erythropoietic activity, as measured by the red blood cell incorporation of Fe59, was found following injection of these substances. The assay animals were responsive to a concentrated sheep plasma erythropoietin. 3. These results suggest that the reninangiotensin system does not encompass erythropoietin but do not rule out the possibility that a second material is released by the juxtaglomerular cells.

Studies of the renal erythropoietic factor using a hemagglutination-inhibition system.

Summary A hemagglutination-inhibition system utilizing gamma-globulins extracted from anti-ESF serum for the determination of erythropoietin (ESF) is reported. Use of this technique showed that the renal erythropoietic factor (REF) is different immunochemically from ESF. Small but detectable levels of ESF were found in normal serum and the incubation of REF with serum resulted in the generation of ESF. The small increases in red cell 59Fe incorporation noted in assay mice treated with the REF alone is probably due to the endogenous production of the ESF.

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.

Variant von Willebrandʼs Disease and Pregnancy

The clinical course and coagulation profile of a pregnant patient with variant von Willebrands disease were followed from the second trimester through puerperium. The clinical course was characterized by a normal delivery and absence of abnormal bleeding or need for replacement therapy. The coagulation profile demonstrated an increase in factor VIII procoagulant activity, factor-VIII-related antigen, and platelet aggregation activity in response to ristocetin prior to delivery. Postpartum, these factors decreased to prepregnancy values with distinctly different patterns. Factor VIII procoagulant activity continued to rise for 5 days after delivery and then decreased with a half-life of approximately 6 days. Factor-VIII-related antigen began to decrease just prior to delivery, displaying a half-life or approximately 6 days. Ristocetin cofactor activity, however, dropped immediately postpartum and displayed a half-life of approximately 6 hr. The ristocetin cofactor activity was associated with factor-VIII-related antigen, which displayed a significantly smaller molecular weight than does normal factor-VIII-related antigen. Larger aggregates of factor-VIII-related antigen. Larger aggregates of factor-VIII-related antigen did not appear during the pregnancy, and ristocetin cofactor activity could not be demonstrated in fragments of less than 0,8 x 10(6).