High Erythropoietic Activity Research Articles

Excessive Erythrocytosis Is Not Associated With Altered Iron Homeostasis in Men From the World's Highest City.

Excessive Erythrocytosis Is Not Associated With Altered Iron Homeostasis in Men From the World's Highest City.

The Role of Erythropoiesis on Hepcidin Level in Polytransfused b-Thalassemia Major Patients

AbstractBackground: Hepcidin peptide hormone is a main con-troller of iron homeostasis, itbecomes elevated in case of iron overload, however despite iron overload in b-Thalassemia major (b-TM) patients a contradictory decrease in Hepcidin occurs. The incompatible Hepcidin level is the main responsible factor causing the iron overload status in iron-loading anemias such as (b-TM) which contributes to organ dysfunctionand to iron toxicity.Aim of Study: To evaluate the conflicting effect of increased erythropoiesis in contrast to the effect of iron overloading on Hepcidin serum level and its correlation with iron status in (b-TM) patients.Subjects and Methods: For all patients and controls Complete Blood Count (CBC), serum assaysof Hepcidin, iron, ferritin, transferrin, HCVAbs, HBs Ag, CRP and serum level were performed.Results: Hepcidin level was significantly lowered in (b-TM) patients compared to controls with high significant difference (p=0.00). There was no correlation between serum Hepcidin and ferritin level, neither was between Hepcidin and serum iron, transferrin, Hb level and reticulocyte count in the study group. Hepcidin/Ferritin ratio showed high significance difference (p<0.000) with mean value in the study group 0.056 vs. 4.75 in controls pointing to an incom-patible levels of Hepcidin to iron overload status. Hepcidin level wasnegative correlatedwith age (p<0.05).Conclusion: Hepcidin was markedly decreased in the study group with no significant correlation between serum Hepcidin and ferritin level as a marker of iron overload in thalassemia major. Hepcidin deficiency is the main contributing factor of iron overload in b thalassemia which results from a strong suppressive effect of the high erythropoietic activity on Hepcidin expression. Hepcidin-ferritin ratio was markedly depressed <1 in all b thalassemia major patients which indicating suppression of Hepcidin out of proportion with the degree of iron loading.

Multisystem, Induced Pluripotent Stem Cell (iPSC) Modeling Reveals a Role for Growth Differentiation Factors (GDFs) in the Etiology of β Thalassemia and Ineffective Erythropoiesis

β Thalassemia is one of the most common monogenic diseases in man encompassing a heterogeneous group of naturally occurring, inherited mutations characterized by abnormal globin gene expression. Iron overload is the principle cause of morbidity and mortality in β thalassemia. The hepatic hormone hepcidin regulates iron homeostasis modulating iron concentration in the plasma and its distribution in tissues throughout the body. Dysregulation of hepcidin production underlies many iron disorders with emerging evidence suggesting that deficiency of the hormone may result from the strong suppressive effect of high erythropoietic activity on hepcidin expression. Current treatment modalities for iron overload include phlebotomy and iron chelation. In β thalassemia, phlebotomy is not feasible and regular chelation is the principal treatment for iron overload. Iron chelators have side effects ranging from mild to very serious, and compliance is often suboptimal. Hepcidin diagnostics and the development of novel therapeutic options are clearly desirable and may help in the management of patients with β thalassemia.Hepcidin dysregulation, along with the ineffective erythropoiesis and anemia noted in β thalassemia highlight the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Using induced pluripotent stem cell (iPSC) technology, cell lines can be established that are genetically identical to the individual from whom they are derived, allowing for disease modeling and the development of novel therapeutics in the exact genetic context of the patient. We have generated disease-specific iPSC lines from patients with β thalassemia major. Harnessing the pluripotency of iPSCs, we demonstrate the modeling of this multisystem disease through the directed differentiation of patient-specific iPSCs into hepatocytes that produce hepcidin as well as erythroblasts produced via a platform that allows for exponentially greater production of blood cells in comparison to existing methodologies (Smith et. al, Blood, 2013). We demonstrate that β thalassemia iPSC-derived erythroblasts secrete greater amounts of GDFs 11 and 15, and that exposure of the patient’s own iPSC-derived hepatocytes to disease-specific erythroblast supernatants results in a marked decrease in hepcidin expression recapitulating essential aspects of the disease in vitro. Furthermore, exposure of developing iPSC-derived erythroblasts to recombinant GDFs results in the production of immature cells that fail to reach maturity, providing a potential novel mechanism contributing to the development of ineffective erythropoiesis. Taken together, these results validate this iPSC-based, patient-specific in vitro system as a platform for testing new diagnostic approaches as well as novel therapeutic strategies targeting the correction of hepcidin dysregulation. DisclosuresNo relevant conflicts of interest to declare.

β-Thalassemia and Polycythemia vera: Targeting chronic stress erythropoiesis

β-Thalassemia and Polycythemia vera are genetic disorders which affect the synthesis of red blood cells, also referred to as erythropoiesis. Although essentially different in clinical presentation – patients with β-thalassemia have an impairment in β-globin synthesis leading to defective erythrocytes and anemia, while patients with Polycythemia vera present with high hemoglobin levels because of excessive red blood cell synthesis – both pathologies may characterized by lasting high erythropoietic activity, i.e. chronic stress erythropoiesis. In both diseases, therapeutic strategies targeting chronic stress erythropoiesis may improve the address phenotype and prevent secondary pathology, such as iron overload. The current review will address the basic concepts of these strategies to reduce chronic stress erythropoiesis, which may have significant clinical implications in the near future.

Iron and hepcidin: a story of recycling and balance

To avoid iron deficiency and overload, iron availability is tightly regulated at both the cellular and systemic levels. The liver peptide hepcidin controls iron flux to plasma from enterocytes and macrophages through degradation of the cellular iron exporter ferroportin. The hepcidin-ferroportin axis is essential to maintaining iron homeostasis. Genetic inactivation of proteins of the hepcidin-activating pathway causes iron overload of varying severity in human and mice. Hepcidin insufficiency and increased iron absorption are also characteristic of anemia due to ineffective erythropoiesis in which, despite high total body iron, hepcidin is suppressed by the high erythropoietic activity, worsening both iron overload and anemia in a vicious cycle. Hepcidin excess resulting from genetic inactivation of a hepcidin inhibitor, the transmembrane protease serine 6 (TMPRSS6) leads to a form of iron deficiency refractory to oral iron. Increased hepcidin explains the iron sequestration and iron-restricted erythropoiesis of anemia associated with chronic inflammatory diseases. In mice, deletion of TMPRSS6 in vivo has profound effects on the iron phenotype of hemochromatosis and beta-thalassemia. Hepcidin manipulation to restrict iron is a successful strategy to improve erythropoiesis in thalassemia, as shown clearly in preclinical studies targeting TMPRSS6; attempts to control anemia of chronic diseases by antagonizing the hepcidin effect are ongoing. Finally, the metabolic pathways identified from iron disorders are now being explored in other human pathologic conditions, including cancer.

Hepcidin in β‐thalassemia

Iron overload is the principal cause of morbidity and mortality in β‐thalassemia with or without transfusion dependence. Iron homeostasis is regulated by the hepatic peptide hormone hepcidin. Hepcidin controls dietary iron absorption, plasma iron concentrations, and tissue iron distribution. A deficiency in this hormone is the main or contributing factor of iron overload in iron‐loading anemias such as β‐thalassemia. Hepcidin deficiency results from a strong suppressive effect of the high erythropoietic activity on hepcidin expression. Although in thalassemia major patients iron absorption contributes less to the total iron load than transfusions, in non‐transfused thalassemia, low hepcidin, and the consequent hyperabsorption of dietary iron is the major cause of systemic iron overload. Hepcidin diagnostics and future therapeutic agonists may help in management of patients with β‐thalassemia.

T lymphocyte subsets of the umbilical cord blood of dogs

The hematological parameters red blood cells (RBC) and total white blood cells (WBC) counts, hematocrit, hemoglobin concentration, and RBC indexes (median corpuscular volume and median corpuscular hemoglobin concentration) were determined and T CD5+ lymphocytes and CD4+ and CD8+ subpopulations of the umbilical cord blood (UCB) of dogs were quantified by the cytofluorimetric technique. Nine adult Beagles, from two do five-year old, were used as control. The umbilical cord blood (UCB) was collected from 20 neonate dogs. The method for the UCB collection was adequate to obtain sufficient quantity of blood for the accomplishment of the hematological analyses and lymphocyte quantification. Cytoscopic preparations of the UCB suggested high erythropoietic activity. There was no difference for the global leukocyte and lymphocyte counts between the groups. UCB T lymphocyte counts were lower than those obtained for adult dogs. The proportion of CD4:CD8 showed a great dominance of T CD4+ cells over T CD8+ lymphocytes in UCB.

Hypocholesterolemia in chronic anemias with increased erythropoietic activity

Hypocholesterolemia of unknown etiology has been previously described in various chronic anemias. Few small studies also suggested that those patients have a lower incidence of atherosclerotic events. The aim of our study was to determine the extent of hypocholesterolemia in various types of anemias. We studied 59 patients with chronic anemias associated with high-erythropoietic activity (thalassemia intermedia, congenital dyserythropoietic anemia type I, congenital spherocytosis), 8 patients with low-erythropoietic activity anemias (acquired aplastic anemia, Fanconi anemia, and Diamond Blackfan anemia), and 20 healthy controls. Mean serum cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, hemoglobin, serum ferritin, soluble transferrin receptor (STR), and serum erythropoietin levels were determined in each patient. All patients with chronic anemia and increased erythropoietic activity had hypocholesterolemia, whereas none of those with low erythropoietic activity was hypocholesterolemic. Mean serum cholesterol, HDL cholesterol, and LDL cholesterol levels were found to be significantly lower in the high-erythropoietic activity group (80+/-19 mg/dl; 31+/-10 mg/dl; 35+/-14 mg/dl, respectively) compared with the control group (P<0.001; 0.001; 0.001, respectively) and the low-erythropoietic activity group (P<0.001; 0.001; 0.01, respectively). Significant inverse correlation (R2=0.507) was observed between serum cholesterol and STR levels, which in the absence of iron deficiency reflect bone marrow activity. Taken together, our results imply that hypocholesterolemia accompanies anemias with high-erythropoietic activity. We suggest that the high-erythropoitic activity-associated hypocholesterolemia is due to increased cholesterol requirements by the proliferating erythoid cells. Further studies are needed to elucidate the exact mechanism and the possible clinical consequences of this phenomenon.

Characterization and quantification of blood cells from the umbilical cord of dogs

Models for the study of hematopoietic stem cells in dogs provide important information for bone marrow transplantation in humans. Recent studies have reported the importance of human umbilical cord blood (UCB) as an alternative to allogenic bone marrow for hematopoietic reconstitution. However, there are no studies on the UCB cells of dogs. The aim of this experiment was to characterize and quantify the blood cells of the umbilical cord of dogs. The blood of the umbilical cord of 20 neonatal dogs, delivered at term, with a median gestation time of 58 days, was collected with a 5-mL syringe containing EDTA. Total RBC, WBC, and platelet counts, HCT, hemoglobin (Hgb) concentration, and RBC indices were determined using an automatic cell counter. The differential leukocyte count was determined manually in blood smears stained with May-Grünwald-Giemsa. Reticulocyte percentages were determined on blood smears stained with brilliant cresyl blue and counterstained with May-Grünwald Giemsa. The MCHC and numbers of RBCs, WBCs, neutrophils, and eosinophils in UCB were lower as compared with reference values for the peripheral blood of healthy neonatal and adult dogs; whereas, the MCV and reticulocyte percentages were higher. Erythrocyte macrocytosis and hypochromasia in UCB were consistent with marked reticulocytosis and indicative of high erythropoietic activity. The results of this study are an important first step in the characterization of UCB from neonatal dogs.

COMPARATIVE RESPONSE OF EPO AND SOLUBLE TRANSFERRIN RECEPTOR AT HIGH ALTITUDE

Erythropoietin (EPO) and soluble transferrin receptors (sTFR) are both sensitive indicators for erythropoietic activity. EPO is produced in the kidney by cells located between the tubuli in the interstitial space of the renal cortex. It is regulated at the level of its gene chiefly by tissue oxygenation. EPO triggers erythropoiesis via stimulating the proliferation, differentiation, and maturation of the erythroid precursors in bone marrow; that is, the burst-forming and colony-forming unit erythroid. About 120 million erythrocytes are destroyed every minute in the adult human body. To avoid anemia, an incredible fine balanced equilibrium between this destruction and the production of new red blood cells has to be ensured. About 500–1000 EPO receptors are present mainly on the surface of purified human erythroid precursor cells (4). Since hypoxia obviously plays a key role in stimulating EPO production and release, the biochemical mechanisms of oxygen sensing, in general, have been the target of intensive research during the last decades. It was found that a transcriptional factor, called the hypoxia-inducible factor (HIF), is essential in promoting cellular adaptation to changes in oxygen availability and regulating the hypoxic gene expression. Further studies revealed that in oxygenated and iron-repleted cells the HIF-alpha subunits are usually rapidly destroyed. This destruction involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pHVL) E3 ligase complex. By hypoxia and iron chelation, in contrast, this process is suppressed and a transcriptional activation is induced. Most recently it could be shown that the interaction between pVHL and a specific domain of the HIF-1alpha subunit is regulated via hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme termed HIF-alpha prolyl-hydroxylase (HIF-PH). This HIF-PH seems to function directly as a cellular oxygen sensor (2,3). TFR is a transmembrane glycoprotein that controls the uptake of transferrin-iron into premature red blood cells in a density of several hundred thousands per cell (1). Most important is that i) the soluble TFR reflects the total mass of tissue receptors and ii) the concentration of soluble TFR correlates directly with the erythropoietic activity and inversely with the amount of iron available for erythropoiesis (functional iron status). Therefore, if EPO increases, erythropoiesis increases and with an increased erythropoiesis the TFR receptor concentrations start to rise due to a concomitant increasing transfer of iron in the red blood precursor cells. From a physiological and pathophysiogical point of view, especially for exercise physiology and clinical medicine, it is very important to understand the time course of this process under various conditions (5). Therefore in this issue of Medicine & Science in Sports & Exercise®, Robach et al. (6) investigated the serum EPO and serum TFR concentrations in healthy humans during i) a progressive (31 d) decompression in a hypobaric chamber up to a simulated altitude of Mount Everest (8848 m) and ii) during and after a 1-wk stay at 4350 m. The first set-up was chosen to analyze the effect of step-by-step increasing hypoxia on both parameters, whereas the latter design focused on the acute effects during a shorter stay at a given altitude. The authors found that serum TFR mirrors the EPO response for a given level of hypoxia but clearly differs from EPO response during transitory phases. Therefore, Robach et al. conclude that the analysis of serum TFR may account for altitude-related erythropoiesis, at a time when EPO is blunted. That is why the measurement of TFR concentrations may be used i) as an index of altitude-related erythropoiesis, when EPO response is already blunted and ii) is helpful to detect a recent high erythropoietic activity due to high altitude exposure and/or exogenous administration (EPO doping) at a time when other parameters (EPO concentrations, hematocrit (Hct), hemoglobin (Hb), red cell volume) are not different from baseline values.

On the origin of the increased tissue iron content in graded magnesium deficiency states in the rat

To investigate the mechanism of tissue Fe accumulation in graded Mg deficiency rats were fed on diets of different Mg contents (70, 110, 208, 330, and 850 mg Mg/kg) for 10, 20, and 30 d during rapid growth. There was no significant impact of Mg deficiency or high luminal Mg concentrations on intestinal59Fe transferin vitroorin vivo. Plasma Mg concentrations and body weight started to decrease after 10 d. Significant haemolytic anaemia was observed after 20 d with siderosis in liver and spleen developing in parallel. Anaemia showed no features of Fe deficiency or infiammation. Comparison between the 70 mg Mg/kg group and animals that received the same quantity of a Mg-adequate diet (850 mg Mg/kg) permitted estimation of quantities of Fe liberated by haemolysis and the increased Fe content in liver and spleen. Both variables showed a high degree of correlation, indicating that the excess of liberated haemoglobin Fe was stored in the tissue. The erythropoietic activity was high during rapid growth, i.e. at days 10 and 20 and decreased significantly after 30 d in all except the most Mg-deficient groups. However, haemolytic anaemia developed because even the high erythropoietic activity in the 70 and 110 mg Mg/kg groups was not sutlicient to recycle all haemoglobin Fe liberated by haemolysis. After 30 d of Mg-deficient feeding the erythrocyte Mg content had decreased to 40% of control values. According to the literature Mg-deficient erythrocytes have a decreased survival time which is likely to be the cause of the observed haemolysis.

術前貯血式自己血輸血基準の考案 外科的顎矯正術への適応

Standards for stored autologous blood transfusion were established recently based on the clinical experience and research data for 140 autologous blood transfusions, which have been carried out in orthognathic surgery patients between 1985 and 1992 at the First Department of Oral and Maxillofacial Surgery, Kagoshima University Dental Hospital, Japan. Regarding standards designed to avoid anemia after phlebotomy, one time collection volume was decided using the following formula: predicted Hb level after collection=(circulating blood volume-one time collection volume) ÷circulating blood volume×Hb level before collection ≥10 g/dl. Fifteen female patients with mandibular prognathism were studied hematologically following a 400 ml autologous blood collection and a transfusion to evaluate the efficiency of this new standard.Two units of whole blood were collected 15 days before each patient's operation. All units of collected blood were transfused without homologous blood transfusion. RBCs, Hb and Hct, which were reduced after the phlebotomy, normalized the day before operation. The rate of reticulocytes was tended to increase during the period of blood storage and for one week after operation. The iron level in the serum was minimum on the day following operation, suggesting the need for postoperative iron supplementation. Increased serum erythropoietin levels, similar to reticulocytes, showed high erythropoietic activity from the first day following the phlebotomy to the postoperative period. There were no differences between the actual minimum Hb level and the predicted Hb level after blood collection of 400 ml. The Hb level on the first day after operation was significantly higher than the calculated Hb level on the same day without blood transfusion (p<0.01).These results suggested the usefulness of our standards.

84 IMMUNOREACTIVE ERYTHROPOIETIN AND ERYTHROPOIESIS STIMULATING FACTOR(S) IN PLASMA FROM HYPERTRANSFUSED NEONATAL AND ADULT MICE

The objective was to study whether the high erythropoietic stimulatory activity in plasma from neonatal mice is erythropoietin (Ep) alone or Ep in combination with other factors. Plasma from hypertransfused (hy.tr.) neonatal (20d) and adult (13-20w) WLO-mice were compared by a RIA and a cell culture assay for Ep. The bioassay reflects erythropoiesis stimulating factor(s) (ESF), defined as the net activity of Ep and other stimulatory and possible inhibitory factors. The RIA determines immunoreactive Ep (iEp). There was no difference between the mean iEp levels of hy. tr. neonatal and adult animals (P>0.3). ESF was not detectable in hy.tr. adult mice, while significant levels were found in neonatal animals. Thus, the mean ESF level of hy.tr. neonatal mice was significantly above that of adult animals (P<0.001). The data show that plasma from hy.tr. neonatal mice contain one or more erythropoietic stimulatory factors not detected by the RIA for Ep. It is concluded that part of the high erythropoietic stimulatory activity in plasma from neonatal mice is due to non-Ep factors.

Quantitative evaluation of erythropoietic activity in dysmyelopoietic syndromes.

Based on the morphological appearances of the bone marrow and peripheral blood, 43 patients with dysmyelopoietic syndromes were categorized into four types: refractory anaemia with excess of blasts, chronic myelomonocytic leukaemia, primary acquired sideroblastic anaemia and refractory anaemia with cellular marrow, without excess of blasts and/or ring sideroblasts. Ferrokinetics allowed three distinct groups of patients to be defined. All cases of refractory anaemia with excess of blasts and chronic myelomonocytic leukaemia were classified in the same group. They were characterized by relative marrow failure and had a high likelihood of developing acute leukaemia. At the other end of the spectrum, individuals with primary acquired sideroblastic anaemia had high erythropoietic activity which was largely ineffective. They had a benign clinical course without evidence of leukaemic transformation. In the middle group, in terms of erythropoietic activity, lay patients with refractory anaemia with cellular marrow and a few individuals with primary acquired sideroblastic anaemia. Their clinical course and risk of developing acute leukaemia were intermediate between the other two groups. These findings indicate that separate entities may exist within the spectrum of dysmyelopoietic syndromes. In clinical practice, they may be recognized by morphological studies and other simple laboratory means.

A simple test for erythropoietin.

In Gelfiltration on Sephadex G-100 crude protein prepared from the urine of anemic patients incubated with 59Fe+++ -ions was separated and a distinct fraction isolated having high erythropoietic activity and a high 59Fe+++ -absorption. Increased amount of erythropoietin resulted in a higher incorporation of 59Fe+++ -ions in this fraction. From this we propose to replace the time consuming and very expensive bioassay for erythropoietin in poly-cythemic mice by gelfiltration of the material under investigation preincubated with 59Fe+++ -ions to test the Fe+++ uptake in the forementioned fraction.

Regulation of fetal liver erythropoiesis

The liver is the main erythropoietic tissue of the human fetus at midterm. The regulatory mechanisms involved in red cell formation should explain three main aspects of fetal erythropoiesis. First, the initiation of erythropoiesis during the formation of the embryonic liver from the hepatic endoderm. Second, the conservation of a high erythropoietic activity in the fetal liver for several weeks until achievement of a fully developed bone marrow. And third, the switch mechanism from fetal hemoglobin to adult hemoglobin. There is experimental evidence for the induction of red cell formation in cells of the hepatic mesenchyme due to the interaction of these cells with the liver parenchymal cells. The nature of the factors involved in this process remain to be investigated but it has been found that at least one of them may be erythropoietin, a protein actively synthesized in cultures of fetal hepatocytes. Erythropoietin has been found in the fetal serum of mammals but seems to play a role late in the erythropoietic development of the liver. Liver erythroid cells isolated from young fetuses (8–12 weeks of gestation) show a poor response towards erythropoietin when compared with cells isolated from older fetuses. This observation suggests the involvement of other factors different from erythropoietin at early gestation. One of these factors is testosterone which stimulates heme and hemoglobin synthesis in liver cells isolated from fetuses of 10–13 weeks of gestation. Another set of factors are the hormones which interact with cellular β-andrenergic receptors. These factors act to regulate red cell formation in cells of fetuses obtained at 8 and 11 weeks of gestation. The switch mechanism from fetal to adult hemoglobin remains one of the main unresolved problems of fetal erythropoiesis. Erythropoietin does not change the ratios of fetal to adult hemoglobin in cell cultures of human fetal liver. Testosterone and estradiol added to these cells in vitro can change the ratio of both hemoglobins but since the change is small these steroid hormones may play only a secondary role in the switch mechanism of globin chains. The fetal lamb seems to have a switch mechanism different from the human because experimental bleeding (to increase erythropoietin concentration) elevates the adult hemoglobin levels. It has been suggested that in the sheep the regulation of adult hemoglobin synthesis (α 2β 2) may be indirectly controlled by an imbalance of γ to α chain mRNA's, which may change from 2 to 1 at early gestation to a ratio of 1:1 after 100 days of gestation. If a similar mechanism is operative in man remains to be investigated.

The Production of Hemoglobin C in Sheep Carrying the Gene for Hemoglobin A: Hematologic Aspects

Abstract The first part of this study describes the hematologic and physiologic changes observed in sheep homozygous for the hemoglobin A during severe blood loss anemia. It was found possible thus experimentally to replace Hb A entirely with a new hemoglobin type, Hb C. The following additional observations were made: (1) Hb C could not be distinguished from Hb A by submitting the appropriate red blood cells to an "acid elution" technic. These two hemoglobin types were found to be more resistant to this treatment than a second adult hemoglobin type, Hb B, while the fetal hemoglobin of the new-born lamb was found to be highly resistant. In sheep heterozygous for Hb A and Hb B, both hemoglobin types were equally distributed among all red blood cells. (2) During stages of severe blood loss relatively small quantities of acid resistant red blood cells of larger size were demonstrable in homozygous Hb A sheep; these cells were considered to be reticulocytes. Additional observations regarding variations in red cell parameters are also presented. (3) The oxygen affinities and the Bohr effects of blood samples and red cell hemolysates containing over 90 per cent Hb C are presented and compared with those of samples containing only Hb A, Hb B or the hemoglobin of the newborn lamb. Attempts to produce Hb C in sheep homozygous for Hb A by means other than phlebotomy are described in the second part of this report. Small amounts of Hb C were demonstrable in a sheep homozygous for Hb A after repeated injections of a urinary extract of human origin with high erythropoietic activity. Administration of cobalt and of thyroxin did not result in the formation of significant amounts of Hb C.