Plasma Erythropoietin Levels Research Articles

Lack of angiotensin II-facilitated erythropoiesis causes anemia in angiotensin-converting enzyme-deficient mice.

While nephrologists often observe reduced hematocrit associated with inhibitors of angiotensin-converting enzyme (ACE), the basis for this effect is not well understood. We now report that two strains of ACE knockout mice have a normocytic anemia associated with elevated plasma erythropoietin levels. (51)Cr labeling of red cells showed that the knockout mice have a normal total blood volume but a reduced red cell mass. ACE knockout mice, which lack tissue ACE, are anemic despite having normal renal function. These mice have increased plasma levels of the peptide acetyl-SDKP, a possible stem cell suppressor. However, they also show low plasma levels of angiotensin II. Infusion of angiotensin II for 2 weeks increased hematocrit to near normal levels. These data suggest that angiotensin II facilitates erythropoiesis, a conclusion with implications for the management of chronically ill patients on inhibitors of the renin-angiotensin system.

Mechanisms of local regulation of erythropoiesis within bone marrow erythroblastic islands in rats

The purpose of the study was to examine the mechanism of macrophage involvement in erythropoietic process within bone marrow erythroblastic islands (EI). Changes of the tissue oxygen supply following removal of blood cells (2% BW) or transfusion of 2.5ml/100g BW of a 70% suspension of washed isogenous red cells/2days were modeled in rats. Plasma erythropoietin level (by RIA), the total number of EI/femur and specific indices of erythropoiesis, based on EI of different classes of maturity were estimated during a 10-day period. In bled animals the initial decrease in hematocrit was accompanied by an increased number of absolute EI/femur—628.3±102×103/femur (day 3) vs. 406.7±169×103/femur (P<0.01) and proliferating EI appeared elevated on days 3–7. The rate of CFU-E differentiation into erythroblasts in EI and the rate of repeated participation of macrophages in new EI reconstruction were increased during the first days after bleeding (P<0.05). Polyglobulia in hypertransfused rats was accompanied by a significant attenuation of EI formation. The number of islands was a 135.6±25×103/femur (day 5) vs. 347.6±65×103/femur (P<0.001) in controls. A persistent reduction of young and reconstructive islands, and of the related indices of erythropoiesis was observed. The role of macrophages to organize islands of different classes of maturity was estimated. Our results support the assumption about the specific regulatory role of the centrally-situated macrophages. Macrophage-erythroblast interactions were influenced by the oxygen supply to the tissues after stimulation of reduction of erythropoiesis.

Hypoxia-stimulated erythropoietin production in mice with hyperoxia-depressed erythropoiesis

A great deal of evidence supports the concept that plasma erythropoietin levels (pEPO) in mammals are related to the O2 supply to tissues relative to their O2 needs. Current evidence also suggests that a modulatory action on O2-dependent EPO secretion is exerted by the erythroid progenitor/precursor cell populations in the erythropoietic organs through a negative feedback system. In this sense, enhanced EPO response to acute hypoxemia has been observed in our laboratory in mice with either pharmacological or post-hypoxic depression of erythropoiesis. The aim of the present investigation was to estimate hypoxia-stimulated EPO production in mice with hyperoxia-depressed red cell production. Female CF#1 mice aged 70d were divided in control (C) and experimental (E) groups. The former was maintained in plastic cages in a normal environments, while the latter was placed in an environment of 60%O2-40%N2. in an 85 dm3-atmosphere chamber with an air flow of 1 L/min. Carbon dioxide was removed from the chamber by washout and absorption with calcium hydroxide. Erythropoiesis was evaluated in 10 C and E mice by iron kinetics performed 3 h after i.v. injection of a tracer dose of 59Fe. The fraction of iron going to erythroid tissue (Hogdson test) was 65.7 % (p<0.0001) lower in E than in C mice, showing the erythropoiesis-lowering effect of hyperoxia after a 72-hour exposure. Hematocrit values were similar in both groups. O2-dependent EPO production was derived from pEPO (ELISA, Medac Diagnostika, Germany) changes after a 4-hour exposure to 506 mb in a decompression chamber. Plasma EPO was 173.2 % higher (p<0.05) in E than in C mice. Data support the concept that the rate of erythropoiesis modulates O2-dependent EPO secretion.

Fetal erythropoietin levels in pregnancies complicated by meconium passage: Does meconium suggest fetal hypoxia?

<h2>Abstract</h2> <b>Objective:</b> We sought to determine whether umbilical cord plasma erythropoietin levels were different in deliveries complicated by meconium passage and to determine whether this response is influenced by gestational age. <b>Study Design:</b> Fetal erythropoietin levels were measured in 203 appropriately grown neonates at 37 to 43 weeks of gestation; among those, 70 had passed meconium. <b>Results:</b> Meconium passage in the entire population was associated with elevated fetal erythropoietin levels (68 vs 31 mIU/mL; <i>P</i> <.001). Cord blood gases, pH, base deficit, and PO₂, as well as the 1- and 5-minute Apgar scores, were not different between the meconium and no-meconium groups. Gestational age and birth weights were significantly higher in the meconium group. Stepwise multiple regression analysis with meconium and gestational age used as the independent variables showed both meconium and gestational age to be independently associated with fetal erythropoietin levels (<i>r</i> = 0.356, F=14.5; meconium, <i>P</i> <.001; gestational age, <i>P</i> <.01). <b>Conclusions:</b> These results suggest that meconium passage can be associated with chronic fetal hypoxia as demonstrated by elevated fetal erythropoietin levels, independent of gestational age. (Am J Obstet Gynecol 2000;183:188-90.)

Fetal erythropoietin levels in pregnancies complicated by meconium passage: Does meconium suggest fetal hypoxia?

Objective: We sought to determine whether umbilical cord plasma erythropoietin levels were different in deliveries complicated by meconium passage and to determine whether this response is influenced by gestational age. Study Design: Fetal erythropoietin levels were measured in 203 appropriately grown neonates at 37 to 43 weeks of gestation; among those, 70 had passed meconium. Results: Meconium passage in the entire population was associated with elevated fetal erythropoietin levels (68 vs 31 mIU/mL; P <.001). Cord blood gases, pH, base deficit, and PO2, as well as the 1- and 5-minute Apgar scores, were not different between the meconium and no-meconium groups. Gestational age and birth weights were significantly higher in the meconium group. Stepwise multiple regression analysis with meconium and gestational age used as the independent variables showed both meconium and gestational age to be independently associated with fetal erythropoietin levels (r = 0.356, F = 14.5; meconium, P <.001; gestational age, P <.01). Conclusions: These results suggest that meconium passage can be associated with chronic fetal hypoxia as demonstrated by elevated fetal erythropoietin levels, independent of gestational age. (Am J Obstet Gynecol 2000;183:188-90.)

Effect of long-term treatment with recombinant human growth hormone on erythropoietin secretion in an anemic patient with panhypopituitarism

We studied the effect of treatment with recombinant human GH in an anemic patient with panhypopituitarism in which hemoglobin (Hb) concentration remained as low as 11.0 g/dl in spite of appropriate replacement with thyroid and adrenocortical hormones. Recombinant human GH was subcutaneously and constantly infused for 12 months using a portable syringe pump at a rate of 0.25 U/kg/week. After the treatment with human GH plasma erythropoietin (EPO) levels increased from 12.2 to 25.1 mIU/ml, with a concomitant increase of Hb concentration to 13.6 g/dl. When the administration of human GH was interrupted, both plasma EPO levels and Hb concentrations decreased. There was a close correlation between plasma GH and EPO levels before and during the human GH administration (y=2.444x+1 3.423, r=0.641, p<0.05). Plasma GH levels were well correlated with Hb concentrations before and during human GH administration (y=0.529x+11.313, r=0.690, p<0.01). Plasma IGF4 levels were also correlated with Hb concentrations (y=0.007x+10.874, r=0.832, p<0.001), but not with plasma EPO levels. These findings suggest that GH treatment may be useful in anemic patients with panhypopituitarism.

Effects of phenylhydrazine or phlebotomy on peripheral blood, bone marrow and erythropoietin in Wistar rats.

This study was conducted to characterize better the response of rats to blood loss and hemolysis and to incorporate automated methods into the routine evaluations of those responses. Serial phlebotomies of 1.5-2.0 ml of blood per day for 5 days, or intraperitoneal injection of 50 mg kg(-1) phenylhydrazine (PHZ) for 3 days, were used to cause anemia associated with blood loss or hemolysis, respectively. Maximum decreases in red blood counts were observed on Day 3 in PHZ-treated animals (68%) and Day 4 in blood-loss animals (35%). In the routine complete blood count (CBC), hemoglobin, hematocrit/hemoglobin ratio and erythrocyte indices could be used to discriminate between the two treatments. Free plasma hemoglobin in PHZ-treated animals resulted in marked elevations of mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) with a 2:1 hematocrit/hemoglobin ratio rather than the anticipated 3:1 ratio. Although both groups of animals had elevated white blood cell counts, PHZ-treated animals also had monocytosis and basophilia. Reticulocyte counts were more sensitive than erythropoietin (EPO) concentrations in predicting erythroid changes. Maximum mean reticulocyte values were ca. 24% in serially phlebotomized animals and >99% in PHZ-treated rats. Plasma EPO levels were 4-10-fold higher than EPO levels in urine, kidney or liver. Flow cytometric differentials of rat bone marrow using 2, 7-dichlorofluorescin successfully predicted erythroid hyperplasia in both experimental groups. Erythrocyte indices returned to normal within 14 days and the remaining CBC parameters were normal within 28 days for both treatment groups. Reticulocyte counts remained slightly elevated on Day 28, but were normal when assessed at Day 56 in blood-loss and PHZ-treated animals.

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.

Modulation of erythropoiesis in rat bone marrow erythroblastic islands by cyclooxygenase inhibition

We designed our study to explore how the inhibition of prostaglandins (PGs) could affect erythropoiesis in bone marrow erythroblastic islands (EIs). To this end, we used hypoxic-stimulated rats—hypobaric hypoxia (42.55 kPa/6 h)—pretreated or not with indomethacin (4 mg/kg/3 days). Blood sampling was done at 0 h, 24 h, and 72 h after hypoxia. The study included estimations of the plasma erythropoietin (EPO) level (by radioimmunoassay), peripheral blood, number of EI from classes I to V per femur, rate of immature cell’s differentiation into erythroblasts, and rate of repeated participation of macrophages in new El reconstruction. Plasma EPO rose significantly (p < 0.01) in all hypoxic rats: 40.5 ± 10.15 mU/ml and 46.75 ± 16.28 mU/ml and at 0 h versus 13.83 ± 6.82 mU/ml in controls. An increased rate of cell differentiation into erythroblasts in EIs (p < 0.01), an enhanced reconstruction in involuted EIs, and a reduced number of maturing EIs (p < 0.01) were observed in all hypoxic animals. However, in indomethacin-pretreated rats, the stimulation of bone marrow erythropoiesis was better expressed. Our results favor the concept that PG inhibition does not attenuate the erythropoietic response to hypoxia and support the hypothesis about the important role of EI macrophages as a local regulator of bone marrow erythropoiesis.

Decreasing Reticulocyte Counts Associated with Declining Post-Dose Erythropoietin Plasma Levels in Anaemia of Prematurity

A prospective sequential, multicentre trial was conducted to determine the association between erythropoietin (EPO) plasma levels and the erythropoietic response to recombinant human erythropoietin (r-HuEPO) during long-term treatment of premature infants. Twenty-nine infants, gestational ages 26–34 weeks and postnatal ages more than 14 days, received 600 IU r-HuEPO per kg per week divided into three doses subcutaneously for haemoglobin levels less than 120 g/l or haematocrit less than 36% over a period of 4 weeks. Eight additional patients were studied for a total of 10 weeks. EPO plasma concentrations and haematologic parameters were measured prior to the onset of treatment and at 2-weekly intervals thereafter. Treatment with r-HuEPO resulted in a median increase in corrected reticulocyte counts of 2.5% (range 0.2–4.6%) above patient’s baseline, thereafter a decrease was observed. In the 8 patients followed for 10 weeks reticulocyte counts declined significantly during weeks 6–10 when compared with the first 4 weeks (p < 0.005). Median 72-hour post-dose EPO plasma levels increased significantly (p < 0.0001) to 57.3 mU/ml (range 5.0–160) above patient’s baseline after the first injection, but declined progressively thereafter until they approached baseline values at week 10. Conclusion: R-HuEPO treatment after the first month was associated with a decrease in post-injection plasma levels and a decrease in erythropoietic response. This decrease in erythropoietin’s efficacy and the decline observed in post-dose EPO plasma levels may be causally related.

Erythropoietin mRNA in hepatocellular carcinomas and kidney in male B6C3F1 mice with secondary polycythemia.

The purpose of this study was to investigate the cause of polycythemia occurring in hepatocellular carcinoma-bearing control male B6C3F1 mice from 2-yr carcinogenicity studies. Erythrocyte counts and plasma levels of erythropoietin in mice with hepatocellular carcinomas were significantly increased compared with the values in non-tumor-bearing mice. Erythropoietin mRNA in 4 of 5 non-tumor-bearing mice was detected in the kidney, but no visible signals for hepatic erythropoietin mRNA in 5 of 5 non-tumor-bearing mice were detected by the reverse transcriptase competitive polymerase chain reaction method. Erythropoietin mRNA was expressed in neoplastic hepatocytes from 8 of 9 hepatocellular carcinoma-bearing mice, and this expression was accompanied by decreased expression of erythropoietin mRNA in the kidneys from these mice. The present findings show that polycythemia in hepatocellular carcinoma-bearing mice occurs secondary to excess synthesis of erythropoietin mRNA by neoplastic hepatocytes.

Vitamin B 12 levels in pregnancy influence erythropoietin response to anemia

Objective: to discover whether vitamin B 12 levels influence erythropoietin (EPO) response during pregnancy. Study design: 117 pregnant women after the 27th week were divided into three groups according to log vitamin B 12 concentrations. EPO (by enzyme-linked immunosorbent assay), Hemoglobin (Hb) and medium corpuscular Hb concentration (MCHC) were measured in these patients. The tests used were: calculation of simple statistic, regression coefficient and t-independent test with level of significance. An exclusive partitioned cluster method (K-means procedure) was used. Results: For the lowest vitamin B 12 levels there is an unexpected lack of difference in plasma EPO levels between anemic and nonanemic patients. In fact EPO levels were high even in nonanemic women. The only parameter of the blood count that seems to change in relation to vitamin B 12 concentration is the MCHC. Conclusions: These results suggest that low vitamin B 12 levels inhibit the suppression of EPO response in nonanemic pregnant women probably through MCHC modifications.

Proinflammatory cytokines lowering erythropoietin production.

The plasma level of erythropoietin (Epo) in anemic patients suffering from inflammation is often low in relation to the blood hemoglobin concentration. Various proinflammatory cytokines have been tested for their action on the synthesis of Epo. Interleukin 1 (IL-1) and tumor necrosis factor-alpha(TNF-alpha) suppress Epo gene expression in isolated perfused rat kidneys and in human hepatoma cell cultures. IL-6 inhibits in the kidney, and conflicting results have been reported for its effect on Epo synthesis in hepatic cells. Several other cytokines tested were without effect. Thus, mainly IL-1 and TNF-alpha seem to be responsible for the defect in Epo production in severe systemic and renal inflammatory diseases.

Possible mechanism for the anemia induced by candesartan cilexetil (TCV-116), an angiotensin II receptor antagonist, in rats

Candesartan cilexetil (TCV-116), an angiotensin II receptor antagonist, was administered orally to male F344/Jcl and Crj:CD (SD) rats at 1000 mg kg −1 day −1 for 1–28 days, and the possible mechanism for the anemia induced by TCV-116 was investigated. In the TCV-116 group, the erythrocyte count, hematocrit value and hemoglobin concentration were decreased by 7–8% as compared with the values in the control group after dosing for 28 days. The plasma and renal erythropoietin levels, the reticulocyte count in the peripheral blood and the erythroid cell count upon bone marrow examination were decreased on day 7, but there were no accompanying histopathological renal lesions. Renal blood flow was increased, and mean blood pressure was decreased after TCV-116. These results suggest that the primary cause of the anemia induced by TCV-116 treatment is the increase in renal blood flow followed by a decrease in erythropoietin production.

Elevated Umbilical Cord Plasma Erythropoietin Levels in Prolonged Pregnancies

Objective: To determine if umbilical cord plasma erythropoietin levels are elevated in pregnancies that continue beyond their expected date for delivery. Methods: Erythropoietin was measured using an enzyme-linked immunosorbent assay in 124 appropriately grown newborns delivered from 37–43 weeks’ gestation. Each woman had an uncomplicated labor and delivery course without evidence of fetal stress or meconium. The comparison was made between pregnancies ending at 37–40 weeks’ gestation and those at 41–43 weeks’ gestation. Results: There was no difference between the two groups in cord blood gases or Apgar scores at 1 and 5 minutes. Cord plasma erythropoietin levels were significantly higher in pregnancies delivered after 41 completed weeks’ gestation (41 or more weeks: 48.0 ± 7.1 mIU/mL, n = 45 versus 37–40 weeks: 26.1 ± 4.2 mIU/mL, n = 79, P < .001). When compared with pregnancies delivered between 37 and 40 weeks, erythropoietin levels were significantly elevated in pregnancies delivered at either 41, 42, or 43 weeks’ gestation. Conclusion: In pregnancies reaching 41 weeks and beyond, cord plasma erythropoietin levels are significantly increased, indicating altered fetal oxygenation in some of these pregnancies. These results support the current practice of close fetal surveillance of prolonged pregnancies.

Changes in the plasma erythropoietin level in rats following fasting, ageing, and anaemia

Changes in the plasma erythropoietin (Epo) level in fasted and aged rats, and in male F344 rats following phenylhydrazine-and captopril-induced anaemia were investigated using an enzyme immunoassay. In male F344 rats, the plasma Epo level was decreased upon fasting, and was higher at night than in the day. In both sexes, the level was decreased from 5 to 10 weeks of age and remained almost constant from 10 to 19 weeks of age. The level in male Sprague-Dawley rats was higher than that in male Wistar and F344 rats at 6 and 10 weeks of age. After phenylhydrazine was administered to male F344 rats, anaemia was observed, and the plasma Epo level increased rapidly, followed by a delayed increase in the reticulocyte count. The plasma Epo level then decreased gradually to the initial level 2 weeks after dosing. Upon repeated administration of captopril, a decreased plasma Epo level on day 7 and anaemia on day 28 were observed in male F344 rats. These results suggest that monitoring the plasma Epo level would be useful in investigating the mechanism by which a chemical induces anaemia. It is also necessary to consider the feeding conditions, sampling time, age, sex, and strain of the rats when interpreting the Epo level.

Effects of aging on erythropoietin secretion in female rats

The secretion of erythropoietin (EPO) in old (22–25 months), middle (mid)-aged (15–17 months), adult (6–7 months), and young (3 months) female rats was studied. Rats of different ages were bled by cardiac puncture for measuring plasma EPO, estradiol, and progesterone levels. The metabolic clearance rate of EPO was determined by a single-injection method. The effects of ovariectomy and replacement of estradiol (E) and/or progesterone (P) on plasma EPO concentration were also investigated. Analysis of direct effects of E and/or P on EPO from kidney tissues was carried out in an in vitro study. Plasma EPO levels were higher in both mid-aged and old rats than in young rats ( P<0.01). The metabolic clearance rate of EPO was also greater in adult, mid-aged and old rats than in young rats ( P<0.01). Ovariectomy increased rat plasma EPO concentration which might be inhibited by E ( P<0.01), P ( P<0.01), or E and P ( P<0.05) replacement. Neither E (10 −10 or 10 −9 M) nor P (10 −8 or 10 −7 M) alone altered EPO release from kidney tissue in vitro. Combination of E (10 −10 M) and P (10 −8 or 10 −7 M) stimulated the EPO release from kidney tissue ( P<0.01). These data suggest that the mechanism of the secretion of EPO by kidneys in female rats during aging is ovarian steroid hormones independent.

Uterine environment and breed effects on erythropoiesis and liver protein secretion in late embryonic and early fetal swine.

In this study we investigated erythropoiesis and fetal liver protein secretion during late embryonic (Day 24 and Day 30) and early fetal (Day 40) development in pigs from domestic white crossbred (WC) gilts with a normal (intact; INT) or crowded (unilateral hysterectomized/ovariectomized; UHO) uterine environment, or from prolific Chinese Meishan (MS) gilts. Increased fetal weight, fetal liver weight, placental weight, total red blood cells, hematocrit, blood hemoglobin content, and maternal plasma erythropoietin (EPO) levels were observed as gestation advanced. Cultured fetal liver secretion of transferrin and a protein of Mr 12500 and pI 7.5 also increased as gestation advanced. Fetal plasma EPO declined between Day 30 and Day 40. Differential counts of circulating erythroid precursors revealed a decline in basophilic erythroblasts and polychromatic erythroblasts between Day 24 and Day 40, an increase in orthochromatic erythroblasts on Day 30 followed by a drop on Day 40, and an increase in the percentage of reticulocytes/ erythrocytes from < 1.0% to approximately 90% of circulating red blood cells between Day 24 and Day 40. Differences among the treatment groups included a lower fetal survival percentage in UHO (vs. INT or MS) on Day 40, and higher maternal hematocrits, fetal weights, fetal hematocrits, fetal EPO levels, and liver transferrin secretion in WC vs. MS pigs. MS pigs had a lower percentage of polychromatic erythroblasts overall and a higher percentage of orthochromatic erythroblasts on Day 24 followed by a higher percentage of erythrocytes on Day 40 than WC pigs, suggesting a more mature erythron (circulating red blood cells plus erythropoietic tissue) in the MS pigs. Covariate analysis indicated that MS had larger placentae per unit of body weight than did WC. Conclusions were that 1) Days 24-40 of gestation is a critical time for fetal erythropoiesis in pigs as well as survival in a crowded uterine environment, 2) the MS breed may differ in the development of the fetal erythropoietic system because of altered fetal or uterine physiology, and 3) the UHO procedure did not significantly affect erythropoiesis in the fetuses studied but did alter fetal survival and the relationship between fetal weight and both hematocrit and hemoglobin on Day 40.

Umbilical cord plasma erythropoietin levels in pregnancies complicated by maternal smoking

Objective: Our goal was to determine whether maternal smoking was associated with elevated umbilical cord erythropoietin, a marker for chronic hypoxia. Study Design: Plasma erythropoietin levels were measured in umbilical cord plasma of 222 newborns. There were 48 mothers who smoked and 174 nonsmokers. Results: When all pregnancies were included, mean cord plasma erythropoietin levels were significantly higher in the smokers (78.0 ± 15.3 mIU/ml) compared with the nonsmoking group (35.2 ± 4.0 mIU/ml; p < 0.005). Regression analysis showed a significant positive correlation between the number of cigarettes smoked per day and cord plasma erythropoietin levels ( r = 0.26, p < 0.0001). Smoking was associated with a significantly elevated risk (relative risk = 2.6, 95% confidence interval 1.7 to 10.9, p < 0.005) of fetal growth restriction. When pregnancies with fetal growth restriction were excluded from the analysis, the difference between the two groups remained significant (smokers 81.3 ± 18.6, n = 38; nonsmokers 24.3 ± 1.4, n = 164; p < 0.03). Conclusions: These results illustrate that smoking during pregnancy is associated with fetal growth restriction and significantly elevated umbilical cord erythropoietin levels. (Am J Obstet Gynecol 1998;178: 433-5.)

Erythroid accelerating activity of rat serum in early stage of drug induced hemolysis.

An increase in the number of erythroblasts can be seen to some extent in the bone marrow of rats in the early stage of experimentally induced hemolytic anemia prior to any elevation in the plasma erythropoietin (Epo) level. This observation suggests that there is another erythroid stimulating factor present other than Epo. We studied the enhancing effect of serum, taken sequentially during experimentally induced hemolysis in rats, on erythroid proliferation, differentiation and maturation in vitro. Single intraperitoneal injection of 60 mg/kg of acetylphenylhydrazine (APH) induced self-limited hemolytic anemia in rats, in which the hematocrit dropped rapidly with a nadir at day 4 after APH injection, followed by a gradual increase with return to normal level by day 8. Serum obtained consecutively every day after APH injection from day 1 to day 7 was applied to an in vitro culturing system of erythroid progenitors. Addition of day 1 serum, in which an elevation of Epo level had not occurred, to a conventional methyl-cellulose culture of rat bone marrow mononuclear cells (BM-MNCs) resulted in a significant increase in the number of colonies derived from colony forming unit erythroid, but not in burst forming unit erythroid. This erythropoietic activity of the serum was particularly evident in the presence of Epo. In the liquid culture of BM-MNCs, day 1 serum also showed some enhancing effect on erythroblast formation. We were able to see significant differences in these erythroid enhancing activities induced by serum drawn on day 1 in comparison to the serum drawn on subsequent days. These results suggest that an unknown erythroid enhancing factor besides Epo stimulates erythropoiesis in the early stage of hemolytic anemia or sudden hypoxia before there is a measurable rise in the serum Epo level. We propose that this factor be termed erythroid accelerating factor (EAF).