Anemic Plasma Research Articles

Measurement of mouse plasma erythropoietin by an improved ELISA

We have examined whether mouse plasma erythropoietin (EPO) can be measured by an improved enzyme-linked immunosorbent assay (ELISA) using milk proteins (Block Ace) both as a blocking reagent and as a diluent for standard recombinant human EPO (rHuEPO) and for plasma samples. Block Ace brought about high slope sensitivity of the standard curve, with a low background. The dose-response curves of normal or anaemic mouse plasma and of rHuEPO were linear and parallel to each other. The anaemic plasma had an additive effect with rHuEPO by increasing the absorbance at 405 nm. The coefficients of variation in the intra- and interassays ranged from 4.2% to 15.3%. The plasma EPO levels in 22 normal mice were 18.3±10.3 mU/ml. An inverse relationship between the logarithm of plasma EPO concentrations and blood haemoglobin concentrations, red blood cell counts or packed cell volumes was found in normal mice and in mice with iron deficiency anaemia (IDA). These results show the validity for the use of the new improved ELISA method for measuring circulating murine EPO.

Response to anaemia in experimental Trypanosoma vivax infection of sheep

Trypanosoma vivax produced a progressive macrocytic normochromic anaemia in sheep during the acute phase of infection. Reticulocytes were absent from the blood of healthy sheep and of sheep with T. vivax-induced anaemia. However, anaemia induced artificially (AHA) in sheep by in vitro heat treatment of red cells, which was comparable in classification and degree to the anaemia of T. vivax infection, produced a reticulocytosis of 1·5 ± 1·0 per cent. When plasma from the anaemic blood of sheep infected with T. vivax for two and four weeks was inoculated subcutaneously into mice, the reticulocyte response was similar to that of mice that received no sheep plasma and inferior to that elicited by normal sheep plasma. The anaemic plasma from sheep infected with T. vivax for three weeks induced a moderate reticulocyte response in mice which was, however, less intense than that induced by plasma from sheep with artificially induced anaemia of comparable intensity. These results indicate that, although the macrocytosis suggests that T. vivax-induced anaemia in sheep is slightly responsive, this response is suboptimal since reticulocytes were lacking in the blood of the sheep and their plasma was weakly erythrogenic in mice. This contrasts with the mild reticulocytosis in sheep with AHA of the same intensity and classification, whose plasma also stimulated considerable erythropoiesis in mice. The poor stimulation by plasma from T. vivax-infected sheep at two and four weeks post-infection suggests subnormal erythropoietin at these periods of infection.

The Chemistry and Physiology of Erythropoietin

This chapter discusses the chemistry and physiology of erythropoietin. Erythropoietin is the primary regulator of erythropoiesis in mammals. It is a glycoprotein hormone produced primarily in the kidney and secreted into the circulation, acting on the bone marrow to induce the differentiation of hematopoietic stem cells along erythroid lines. Mammalian erythropoietins show relatively little species specificity with regard to biologic activity. Anemic plasma and serum can stimulate erythropoiesis in polycythemic hosts of either the same or unrelated mammalian species, thereby allowing the use of a standardized assay animal—the polycythemic mouse or the fasted rat. Likewise, tissue and circulating erythropoietins from several mammalian species react with an antibody to the human urinary hormone and inhibit binding of the pure human urinary erythropoietin tracer in the radioimmunoassay. Species differences have been observed, both in biologic and immunologic activity. For example, in a series of studies, preparations of sheep erythropoietin did not give dose-response curves parallel to those for human material assayed in the polycythemic mouse system.

Induction of hemosome formation and hemoglobin biosynthesis in HeLa cells.

Tissue cultures of HeLa cells in Hank's medium suspension, previously mixed with a solution containing reticulocyte polysomes and/or free globin, hemoglobin and blood plasma from bled rabbits, were homogenized, and an electrophoretic pattern of hemoglobin band was obtained from the supernatant; hemoglobin denatures under the tissue culture conditions. HeLa cells show typical hemosomes, identidical to those found in immature erythrocytes of embryos, and adult mammals with hemolytic anemias. When HeLa cell suspensions are mixed with solutions void of anemic blood plasma, or containing normal rabbit blood plasma, anomalous organelles are formed in the cells, and no hemoglobin biosynthesis occurs.

Activity of inhibitors on plasmatic erythropoietin and on the renal and splenic erythropoietic factors in the rabbit.

The author has studied the action of Cleland's reagent and Koshland's reagent on the erythropoietic factors present in the kidneys and the spleen of normal rabbits, in the plasma of bled rabbits and in the mixtures obtained through incubation of normal plasma with kidney and spleen homogenates. The results show that Cleland's reagent inhibits the activity of the renal and splenic erythro-stimulant factors, whereas it has no effect upon the erythropoietin in anaemic plasma and in plasma-kidney and plasma-spleen incubation mixtures. The activity of erythropoietin, on the other hand, is inhibited by Koshland's reagent, which has no influence on the renal and splenic erythro-stimulant factors.

Observations on the Relationship between Haemolysates and Erythropoiesis in Mice and Rabbits

Studies were made on the reticulocyte responses of groups of five polycythaemic F 1 mice to injections of anaemic and normal mouse and rabbit plasmas, and anaemic and normal lysed mouse and rabbit red cells. Only anaemic mouse and rabbit plasmas produced a marked reticulocytosis. Two groups of six mice were given twenty-one daily injections of anaemic mouse plasma or normal lysed mouse red cells. Anaemic plasma produced a marked increase in the rate of erythropoiesis, whereas the lysed red cells produced no such increase. It was concluded that if lysed red cells are erythropoietic then they do not act in the same way as erythropoietin (ESF). On a effectué des études sur les réactions des réticulocytes de groupes de cinq souris F 1 polycythémiques aux injections de plasma anémique et normal de souris et de lapins, ainsi que de globules rouges, lysés, anémiques et normaux, de souris et de lapins. Seul le plasma anémique des souris et des lapins produisit une réticulocytose. Deux groupes de six souris F 1 reçurent vingt-et-une injections journalières de plasma anémique de souris ou de globules rouges, lysés et normaux, de souris. Le plasma anémique produisit un accroissement prononcé du taux d’érythropoièse, tandis que les globules rouges lysés n’eurent pas de tel effet. On conclut de là que si les globules rouges lysés sont érythropoiétiques, alors, ils n’agissent pas de la même façon que l’érythropoiétine (ESF). Es wurden Untersuchungen über das Rektikulozyten-Ansprechen von jeweils fünf polycuthaemischen F 1 Mäusen auf Injektionen mit anaesmischem und normalem Mäuse- und Kaninchenplasma, ebenso wie anaemischen und normalen lytischen roten Zellen von Mäusen und Kaninchen angestellt. Nur anaesmisches Mäuse- und Kaninchenplasma produzierte eine deutliche Rektikulozytosis. Zwei Gruppen, bestehend aus sechs F 1 Mäusen, erhielten täglich einundzwanzig Injektionen mit anaesmischem Mäuseplasma oder normalen lytischen Mäusezellen. Anaemisches Plasma führte zu einer deutlichen Erhöhung der Erythropoese, während lytische rote Zellen keine derartige Steigerung aufwiesen. Daraus wird geschlossen, daß, wenn lytische rote Zellen erythropoitisch sind, sie sichnicht wie Erythropoetin (ESF) verhalten. Se realizaron estudios sobre las respuestas de los reticulocitos en grupos de cinco ratones policitémicos F 1 a las inyecciones de plasmas de ratones y conejos anémicos y normales, y células rojas de ratones y conejos anémicos y normales lisados. Sólo los plasmas de los ratones y conejos anémicos produjeron reticulocitosis marcada. A dos grupos de seis ratones F 1 se les dieron veintiuna inyecciones diarias de plasma de ratón anémico o de células rojas de ratón normal lisado. El plasma anémico produjo un marcado aumento en el volumen de la eritropoyesis, mientras que las células rojas lisadas no produjeron tal aumento. Se llegó a la conclusión de que, si las células rojas Usadas son eritropoyéticas, no obran de la misma manera que la eritropoyetina (ESF).

Effect of post-treatment with erythropoietin(s) on survival and erythropoietic recovery in irradiated mice.

DEATHS from acute radiation injury with doses of 500–1,000 r. are assumed to be a consequence of a typical bone-marrow syndrome characterized by septicaemia, haemorrhagic diathesis and anaemia. Attempts have been made to alleviate these conditions. Antibiotics have been used successfully to prevent septicaemia1,2, but the excellent results from the injection of fresh haemopoietic tissue3,4 are complicated by the fact that the irradiated animals recover their ability to react against foreign proteins5. This may result in a homograft reaction in the recipient6,7. Agents have therefore been sought to induce recovery in the bone-marrow of irradiated animals8–10. Stimulation of erythropoiesis in sub-lethally irradiated rats by anaemic plasma was presumed to be caused by its erythropoeitin content11, but no systematic investigation has been made to test the value of erythropoietin(s) in radiation injury. This investigation was undertaken to examine the effect of post-treatment with erythropoietin(s) on the survival and erythropoietic recovery of lethally irradiated mice.

Reversion by erythropoietin of the suppression of erythroid clones caused by transfusion-induced polycythemia.

The in vivo intrasplonic cloning method for bone marrow cells was applied to studios on the regulation of erythropoiesis. Experiments were made to test whether erythropoietin would reverse the specific suppression of erythroid clones, caused by polycythemia. It was found that the injection of anemic plasma (erythropoietin) elicited the re-formation of erythroid clones. The kinetics of the reversal is discussed in relation to the target cell for erythropoietin and its possible morphogenetic action.

IN VITRO STUDIES ON THE EFFECT OF ERYTHROPOIETIN ON GLUCOSAMINE-1-14C INCORPORATION INTO RAT BONE MARROW CELLS.

It has been shown that glucosamine-l-14C is incorporated into the stroma (acid-insoluble fraction) of rat bone marrow cells in culture. Erythropoietin preparations at stages of purification ranging from untreated anemic plasma to one highly purified material (approximately 900 U/mg protein) caused increased glucosamine uptake into these bone marrow cell cultures. It is therefore assumed that erythropoietin is the agent responsible for this effect, which first becomes measurable after about 3 hr of incubation. A fixed amount of incorporating capacity appears after erythropoietin is added to the culture. The response to erythropoietin is a logarithmic function of the dose over a considerable part of the range tested. The radioactivity incorporated into the stroma has been identified in 3 classes of compounds: a) material not solubilized by 0.05N H2SO4 at 80 C (44% of total radioactivity in the original sample); b) material so far not identified which stayed at the origin of the paper-chromatographic system ...

Die humorale Steuerung der Erythropoiese

The field of research relating to erythropoiesis has recently so expanded that the yearly appearance of a new volume recapitulating and updating our information has ceased to cause surprise. The task is here very ably performed. The present volume, after an introduction by one of the pioneers in the field, Professor Ferdinand Hoff, discusses nervous and humoral regulation of erythropoiesis, specific humoral control of erythropoiesis by erythropoietin, and nonspecific humoral influences on erythropoiesis. There is a very extensive bibliography. It is necessary to distinguish between two aspects of the work, the author's own contributions (some of them published here for the first time) and his review of the literature. The author's own contributions range over a wide field and include studies on such subjects as the nature of cells responding to injections of erythropoietin; the effect of anemic plasma filtrates on oxygen consumption in the chick erythrocyte; the effects of

The Use of Mice for the Assay of Erythropoietin in Sheep Plasmas

In a series of preliminary observations in which sheep and rabbit recipients were used, it was found impossible to demonstrate conclusively the presence of Erythropoietin (.ESF) in the plasma of sheep rendered anaemic by bleeding or by injection of phenylhydrazine. Both anaemic and normal plasma produced a fall in the recipients’ red cell count, and this could have produced the reticulocytosis which invariably followed. However, mice were used successfully to demonstrate ESF in anaemic sheep plasma, and the results obtained following the use of 2 simple assay techniques on a variety of plasmas and plasma extracts are described. A polycythaemic mouse assay gave encouraging results, but was a little cumbersome. An assay based on the production of a reticulocytosis in intact mice was simpler but the interpretation of results depended to a large extent on the control of variance in the colonies of mice used. Extracts producing a good measure of response to the above tests were injected into mice daily for 21 days to investigate their erythrocyte-stimulating ability. A concentrated extract of anaemic-by-phenylhydrazine sheep plasma produced a polycythaemia in mice without altering the mean corpuscular volume or mean corpuscular haemoglobin concentration.

Immunological neutralization of various erythropoietins.

SummaryIncreased serum erythropoietin levels were produced in mice, rats, rabbits, and a sheep in response to hypoxia or phenylhydrazine anemia. In all cases, including that of the rabbit, the serum erythropoietin was neutralized on addition of serum from rabbits previously immunized with an erythropoietin extract of human urinary origin. Also, an erythropoietin extract prepared from phenylhydrazine anemic sheep plasma was neutralized by such serum. These data suggest that erythropoietins of the various animal species studied are similar.

Factors controlling hemopoiesis: erythropoietic effects of "anemic" plasma.

Abstract Normal rats given multiple intraperitoneal injections of the filtrate of phenylhydrazine-induced anemic rabbit plasma that had been boiled for one minute manifested a parallel increase in all peripheral erythroid values together with myeloid erythrocytic hyperplasia. The newly formed erythrocytes were normal in size. After boiling for 30 minutes, filtrates of the same anemic plasma induced erythrocytosis because of the production of microcytes, reticulocytosis, and increased marrow nucleated red cell counts without augmentation of the recipients' hemoglobin or hematocrit levels. Anemic plasma that had been processed by prolonged boiling evoked identical responses by parenteral and oral routes. When given by gastric tube, the anemic plasma that had been boiled for one minute did not enhance the recipients' circulating hemoglobin levels. Instead, these animals developed an erythromicrocytic response similar to that attributed to the thermostable plasma fraction. These data provide further support for the existence of two humoral erythropoietic factors. It is suggested that a relatively thermolabile factor, which is inactive orally, governs hemoglobin production by affecting the rate of erythrocytic differentiation from the primitive pluripotential marrow cells. The thermostable factor, which is active orally, apparently determines the number of mitoses which erythrocytic precursors undergo during maturation. Together, these two agents appear to regulate the size and hemoglobin content of each cell in addition to the amount of hemoglobin and number of erythrocytes formed.

Studies on Erythropoiesis. XI. Reticulocyte Response of Transfusion-Induced Polycythemic Mice to Anemic Plasma from Nephrectomized Mice and to Plasma from Nephrectomized Rats Exposed to Low Oxygen

Abstract Mice with transfusion-induced polycythemia have been used to assay erythropoietic activity in plasma derived from mice and rats subjected to various stimuli and experimental procedures. 1. Anemic, anoxic, or cobalt mouse plasma increased erythropoiesis from the zero baseline to about the normal range after four 0.5 cc. injections. No significant effect is observed from the same number of injections of normal rat or mouse plasma or normal saline. Similarly, plasma from rats starved for 4 days had no erythropoietic activity, but erythropoietic activity was present in the plasma of pregnant mice during the last trimester of pregnancy. 2. Plasma from mice made anemic by phenylhydrazine and then subjected to bilateral nephrectomy contained slight erythropoietic activity (less than 0.2 per cent reticulocytes). The plasma of sham-operated anemic animals had considerably more erythropoietic activity (2.0 per cent). The plasma was collected for assay 10 hours after the operative procedures. 3. Plasma from nephrectomized rats exposed to hypoxic anoxia for 8, 16, or 24 hours, had slight erythropoietic activity when measured by the peripheral reticulocyte response of the recipients (0.18, 0.11, and 0.00 per cent reticulocutes, respectively). The same plasma had no erythropoietic activity when judged by the Fe59 red cell incorporation response in starved rats. Plasma from rats subjected to bilateral ureter ligation and similarly exposed had an erythropoietic activity (0.7, 1.38, and 1.26 per cent reticulocytes, respectively), comparable with that of the plasma from normal rats exposed to hypoxic anoxia (0.9, 1.71, and 1.78 per cent, respectively). The low and inconstant erythropoietic activity in the plasma of nephrectomized mice and rats subjected to anemic or hypoxic anoxia is more or less comparable with the response occasionally produced by normal plasma. We are, therefore, disinclined to consider the data as evidence against the renal origin of erythropoietins, but the alternative possibilities are discussed.

Effect of Erythropoiesis on P32 Uptake by Deoxyribonucleic Acid of Spleen in the Rat.

Summary1. A method for evaluating erythropoietic stimulation has been described. 2. Following a standard hemorrhage there is a 2- to 5-fold increase in P32 uptake by splenic DNA. 3. Exposure to anoxic anoxia increases P32 uptake by the DNA of spleen. 4. Injection of anemic rabbit or hypoxic rabbit plasma increased P32 uptake by splenic DNA in normal receptor rats.

Question of purity of erythropoietic factor concentrates.

SummaryThe bulk of erythropoietic activity of anemic plasma can be concentrated in a fraction which represents less than 0.5% of plasma proteins. Yet from the similarity in yield and electrophoretic behavior of the corresponding inactive fraction from normal plasma, it appears likely that the erythropoietic factor constitutes only a small portion of the present concentrates.

Isolation of an erythropoietic fraction from the plasma of rabbits made severely anaemic with phenylhydrazine.

THE plasma of rabbits made severely anaemic with phenylhydrazine, with less than 3.7 gm. haemoglobin per 100 ml. of blood, contains erythropoietic material. The erythropoietic activity has been demonstrated in heterologous species, for example, rats1, mice2 and dogs3, in the filtrate obtained after boiling the anaemic rabbit plasma at pH 5.5 for a few minutes.

Erythropoietic Stimulating Activity of Urine from Anemic Human Subjects.

SummaryOf 17 unconcentrated urine samples taken from 7 anemic humans, approximately 25% displayed erythropoietic stimulating properties. Blood volume and bone marrow studies confirmed the erythropoietic nature of the responses obtained. Urine from 4 children with normal hemoglobin levels showed no ability to stimulate erythropoiesis. When compared to anemic human plasma, anemic human urine is a more dilute and less constant source of erythropoietically active material.

Effect of plasma from bled and phenylhydrazine treated animals on plasm iron turnover; a test for hemopoietine.

Conclusions1) Plasma obtained from phenylhydrazine treated rabbits markedly increases plasma iron turnover. The supernatant obtained after boiling this plasma retains its effect on iron turnover. 2) Bled rabbits' plasma does not affect iron turnover, but has a clear cut lowering effect on plasma iron. Dialyzed plasma of bled rabbits, however, increases plasma iron turnover, while acetone extracts of bled rabbits' plasma, significantly depress plasma iron and plasma iron turnover. 3) The fraction of Fe59 appearing in the red cells at 24 and 48 hr is increased by normal plasma, bled rabbits' plasma, phenylhydrazine treated rabbits' plasma and is not a good index of the state of erythropoiesis. 4) Analysis of the effect of anemic rabbits' plasma on iron metabolism as studied with tracer doses of Fe59 gives information on the factors controlling iron metabolism and presents a practical method for bioassay of the plasma erythropoietic factor “Hemopoietine.”

Fe59 assessment of erythropoiesis in mice following injections of anemic rabbit plasma extracts.

Fe59 assessment of erythropoiesis in mice following injections of anemic rabbit plasma extracts.