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Erythropoietin activity in serum was measured using 59Fe incorporation into erythrocytes in protein-starved, hypoxic mice. The activity in serum from 20 patients with untreated myelomatosis was not significantly different from that in 31 saline controls. Only three patients had detectable erythropoietin levels in serum: 0.24 IU/ml, 0.27 IU/ml and 0.50 IU/ml (standard B), respectively. The venous haematocrit was correlated positively with the glomerular filtration rate as measured by 51Cr EDTA-clearance. No correlation could be established between venous haematocrit and serum albumin or serum transferrin. The results are in agreement with the assumption of a defective erythropoietin activity due to renal failure in myelomatosis.

Sjaastad, Ø.V., T. Framstad and A.K. Blom: Effect of iron on erythropoietin pro-duction in anaemic piglets. Acta vet. med. 1996, 37,133-138. - In the present study the response of plasma erythropoietin to iron injection in anaemic piglets was examined. At the age of 16 days, 4 piglets from the same litter were given 180 mg iron subcutane-ously. After iron injection, blood samples for estimation of erythropoietin activity in plasma, haemoglobin concentration, and reticulocyte counts were taken every 6 or 12 h for 3 Vi days. Plasma erythropoietin activity was estimated by a monoclonal enzyme-im-munoassay (ELISA), developed for human erythropoietin. On the day of iron injection, haemoglobin concentration ranged between 41 and 48 g/1, reticulocyte counts from 9 to 17 percent, and plasma erythropoietin between 22 and 144 mU/ml. In 3 of the 4 piglets, iron injection resulted in a 2-6 fold increase in erythropoietin activity. Maximal eryth-ropoietin activities were observed 24-42 h after injection, and after 66 h, the activities were close to the pretreatment values. It is concluded that in our experiment, iron, per se, has stimulated erythropoietin production.

We have simultaneously determined the plasma levels of erythropoietin (EPO) in 40 chronic dialysis patients by an in vitro bioassay and a radioimmunoassay (RIA) and have made a comparative study of the results. For comparison with regard to the normal EPO level, 20 samples for the bioassay and 131 samples for the RIA study from healthy persons were also examined.In the in vitro bioassay, the EPO plasma titers were determined by the method of Sakata et al., using fetal mouse liver cells obtained from 12-day-old mice. The mean EPO level of the dialysis patients was 74.4±29.1mU/ml by the bioassay and 15.7±1.3mU/ml by the RIA. In the bioassay, in 27 (67.5%) of the 40 dialysis patients, the EPO titer levels were lower than the normal lower limit (90mU/ml). Conversely, in the RIA, the EPO levels were higher than the normal lower limit in all except one patient.The plasma EPO value in the bioassay showed a positive correlation with the hemoglobin (Hb) concentration and the corrected reticulocyte count, but no significant correlation between the two methods was found. On the other hand, in the comparative study limited to 21 individuals who did not show such low EPO levels as less than 70mU/ml in the bioassay, there was a significant relationship between the results of the two methods.Therefore, it is strongly suggested that the plasma EPO level obtained by RIA does not exclusively reflect the real erythropoietic activity in dialysis patients with chronic glomerulonephritis and the presence of some inhibitors in uremic plasma as well.

Summary. In a series of experiments with polycythaemic mice the influence of liver enzyme inhibitors on erythropoietin activity was studied. SKF 525-A was found to increase the half disappearance time of the activity of administered sheep erythropoietin. Both the administration of SKF-525-A and Disulfiram were found to increase the half disappearance time of endogenous erythropoietin activity. It is concluded that these compounds block the inactivation of erythropoietin in the liver. A model in which the action of such inhibitors on the disappearance of endogenous erythropoietin activity can be tested, is proposed.

The in vitro study of erythropoiesis is often hampered by impurities associated with commerical preparations of erythropoietin. This report describes the activities associated with commercial erythropoietin, as well as a rapid and convenient method, preparative isoelectric focusing, to enhance the purity of low specific activity erythropoietin preparations.

Wir fanden im Blut von Patienten mit aplastischer Anamie hohere Erythropoetinaktivitat als im Blut von Patienten mit Blutungsanamie gleichen Schweregrades.

The effects of synthetic Angiotensin II on erythropoiesis were investigated. 1. Two daily intravenous injections of Angiotensin II, 100 µg./Kg. of body weight, revealed no acceleration of Fe59 incorporation into erythrocytes of either normal rabbits or hypophysectomized rats. 2. When given by intravenous drip, 100 µg./Kg. of Angiotensin II significantly accelerated the radioiron incorporation. 3. The renal blood flow was markedly reduced throughout the period of the dropwise injection; the same effect was transitory after single intravenous injection. The elevation of plasma erythropoietin activity was observed in hypertransfused polycythemic rabbits following the dropwise injection of Angiotensin II. From these results, it is concluded that erythropoietic activity of Angiotensin II results from an increased erythropoietin production. The increased production may have been induced by a renal ischemia through administration of Angiotensin II.

SummaryStudies in three hypophysectomized monkeys revealed markedly reduced red cell volume, plasma volume, percentage of nucleated erythron in the bone marrow, and plasma erythropoietin activity. Corticosteroids and thyroxine, given alone or in combination, had no significant effect on these abnormalities; the combined growth hormone and thyroxine therapy, however, restored all the abnormalities to normal.

The properties of erythropoietin from sheep plasma and human urine were compared by gel filtration in columns of Sephadex G-150 calibrated with proteins of known molecular weight. Erythropoietin activity eluted as a single component in good yield at an elution volume consistent with a molecular weight of 60 000 in each case. No activity was detected in other fractions of the eluate, indicating the homogeneity of erythropoietin from both sources.

Erythropoietin activity was elevated in some patients with iron-deficiency anaemia but in none with idiopathic thrombocytopenic purpura. Thrombopoietin was elevated only in patients with idiopathic thrombocytopenic purpura. No correlation existed between erythropoietin and thrombopoietin activities measured on the same serum sample. Although erythropoietin activity bore some relationship to the degree of anaemia, thrombopoietin could not be related. The finding suggests that erythropoietic and thrombopoietic activities are different factors.

Erythrocytosis has been observed to occur infrequently in association with uterine fibromyomas. Study of this case revealed erythrocytosis prior to surgery and a remission was noted after myomectomy. Elevated erythropoietin activity in this patient's serum was demonstrated. The erythropoietin activity in the tumor tissue extract was 10 times higher than in the control tissue extract. We postulate that the tumor itself was responsible for the erythrocytosis observed. The mechanisms proposed to explain polycythemia in such a tumor were revised and our conclusion is that the tumor produced the erythropoietin autonomously and is not subjected to feedback control mechanism.

The erythropoietin activity and the erythropoiesis inhibiting factor(s) (EIF) were studied in the serum of 10 children and adolescents suffering from terminal renal insufficiency. The influence of haemodialysis on these factors was examined as well. As a test model for the estimation of erythropoietin activity and EIF we used hypertransfused polycythaemic mice. No erythropoietin activity was detectable in the serum of children and adolescents. A 40%-inhibitory effect on erythropoiesis existed in 8 out of 10 uraemic sera prior to dialysis. After haemodialysis the inhibition is eliminated in 4 of the 9 sera examined, and reduced to half in the 5 remaining sera. The findings suggest that the inhibitory effect is possibly caused by one or several EIF of the middle molecule group.

In mice administered with estradiol benzoate (EB) at 0.2 or 5 μg dose levels, the number of erythroid colony-forming units (CFU-E) in marrow declines progressively starting from 12 through 48 hr. On the other hand hypoxic erythropoietin (Ep) production, although potentiated in animals primed with relatively large dosages of EB (5–25 μg), is significantly diminished after treatment with lower amounts of estrogen (0.2 μg). In mice primed with large amounts of EB, the enhancement of Ep activity apparently compensates the depleting influence on marrow CFU-E, thus leading to a nearnormal erythroid response to hypoxia. On the other hand, a sharp drop of this parameter is observed in animals primed with the lower dosage (0.2 μg), i.e. the depleting influence on the CFU-E pool is not compensated here by potentiation of the Ep response.

Erythropoietin activity in lymph and plasma of normal and anemic dogs and sheep was tested. No erythropoietin activity in lymph from thoracic duct or renal lymph vessel in the normal animals was determined. In normovolemic anemic dogs the erythropoietin activity in plasma or lymph from thoracic duct was increased 4 and 8 h after bleeding. The rise of erythropoietin activity in the lymph did not differ from that of plasma.

Erythropoietin (ESF) activity was determined during acute and chronic uremic states with and without evidence of excretory renal function in humans. These observations demonstrated that detectable endogenous levels of ESF were absent in humans during an acute and chronic uremic state. Unlike the humans, the experimental animals produced ESF at a diminished rate. Also, in experimental animals, factors appear by 48–60 hr which make the bone marrow and spleen less responsive to exogenous ESF. Therefore, the human and nonhuman data show that the anemia of the uremic state can be explained by two factors: (1) an initial inhibition of end-organ ESF responsiveness, and (2) a failure in ESF production. The temporal difference in the effect of these two aspects of erythropoietin activity is perhaps the basis for the variability in measured ESF activity in acute and chronic uremic states.

Crude erythropoietin was subjected to isoelectric focusing over the pH ranges 3–10 and 3–5. All the biological activity was located between pH 3.5 and 4.0. The erythropoietin activity displayed heterogeneity, being divided into three or four peaks within this pH range. Asialoerythropoietin, prepared by acid or enzymic hydrolysis, focused in the pH gradient between pH 5.0 and 6.5 and displayed a similar degree of heterogeneity. Partially desialated erythropoietin focused over a range of intermediate pH values. The large shift in pI on desialation and the demonstration of intermediate partially desialated species suggest a high content of sialic acid in erythropoietin. All desialated species of erythropoietin having isoelectric points greater than 4.0 were without in vivo biological activity. Oxidation of asialoerythropoietin with galactose oxidase completely restored in vivo biological activity.

• A child with homozygous sickle cell disease and transposition of the great vessels had erythrocytosis associated with markedly increased plasma erythropoietin activity. Her clinical course was complicated by neurologic manifestations but not by recurrent sickle cell vaso-occlusive episodes. The fetal hemoglobin level which had been greater than 25% during the first two years of life gradually decreased to less than 10%. She died at 3 years of age of congestive heart failure and severe anemia. The only sickle cell painful crisis occurred during her terminal illness. It is likely that the high levels of fetal hemoglobin decreased sickling and thus allowed erythrocytosis to develop. Fetal hemoglobin may also have prevented frequent vaso-occlusive events despite the high hematocrit level. ( Am J Dis Child 132:149-151, 1978)