Erythropoietin & erythroid progenitors in rats exposed to chronic hypoxia.

In order to better understand the mechanisms affecting erythropoietin (Epo) synthesis and red cell mass increase under chronic hypoxia, we examined Epo production and erythroid progenitors (CFU-E) in rats exposed to normobaric hypoxia for four weeks. Hypoxia induced the rise of hematocrit (Htc), hemoglobin (Hb) concentration and the red blood cell (RBC) number with a plateau in hematocrit values after two weeks. After 24 h of hypoxia, Epo levels were increased 20 fold, followed by a significant decrease. After the first week of hypoxia, the values were still higher than in the controls, but after two weeks Epo levels did not differ significantly from the normal values. The fall of Epo levels coincided with the plateau values of hematocrit. The changes in the CFU-E number followed the changes in Epo concentration: a two fold increase after 24 h of hypoxia; a further increase during the next two weeks reaching a peak on day 14, and then a progressive decrease at the time when Epo concentration was at a normal level. Although decreased, but still higher than normal, the CFU-E number during the last two weeks of hypoxia could be necessary for the maintenance of an achieved steady state under persistent hypoxic conditions with normal Epo concentration sufficient to maintain the existing rate of erythropoiesis.

Elevated erythropoietin levels in cord blood of newborns with Down's syndrome.

Plasma erythropoietin (Ep) was determined in umbilical cord blood in 18 infants with Down's syndrome. The 16 infants with Down's syndrome who were delivered after labor had significantly elevated plasma Ep levels compared to 36 control infants born after labor (p < 0.001). Six of the ten infants with Down's syndrome who had their packed cell volume (PCV) measured in the first 24 h of life were polycythemic based on a PCV of > or = 0.65. The presence of congenital heart disease in 9 of the 18 infants with Down's syndrome was not associated with a higher plasma Ep or PCV levels. Plasma Ep was correlated with neonatal PCV in the combined group of control and Down's syndrome infants (p = 0.003). Increased plasma Ep levels observed in infants with Down's syndrome suggested chronic fetal hypoxemia as a likely explanation for the high incidence of neonatal polycythemia observed in this group.

Mild hypoxaemia does not alter red blood cell production in fetal sheep.

1. Fetal sheep at 120 days gestation were fitted with upper and lower body arterial and venous catheters in addition to a flow sensor and occluder placed around the aorta below the renal arteries. 2. After 7 days of recovery, the occluder was partially inflated to reduce aortic blood flow to 70% of control. Blood flow reduction was maintained at this level for the remainder of the experiment. 3. Blood samples were taken after 60 min of blood flow reduction and again after 3 or more days of blood flow reduction. 4. There was no change in upper body arterial or venous blood pressure. Lower body arterial blood pressure decreased, as expected. Arterial PO2 decreased while packed cell volume and haemoglobin concentration increased. There was no change in plasma erythropoietin concentrations or plasma renin activity. 5. While both red cell mass and haemoglobin mass increased during the period of the study, the rate of increase was no different from the rate of blood volume increase.

Erythropoietin in human fetuses with immune hemolytic anemia and hydrops fetalis.

OBJECTIVE: To determine whether plasma erythropoietin is increased in fetuses with anemia due to Rh isoimmunization. METHODS: Hemoglobin and erythropoietin were measured in samples obtained by funipuncture from 15 fetuses with Rh isoimmunization (gestational age 26.2 +/- 5.0 weeks, mean +/- standard deviation) and from 13 control fetuses (23.1 +/- 6.7 weeks). Hemoglobin and erythropoietin also were determined in umbilical cord blood collected at birth from 20 term fetuses delivered by elective cesarean. RESULTS: Fetuses with Rh isoimmunization had lower hemoglobin and higher plasma erythropoietin measurements than mid-gestation controls (6.1 +/- 3.9 versus 10.7 +/- 1.5 g/dL and 105.5 +/- 168.1 versus 12.5 +/- 3.1 mU/mL, P < .05, respectively). Hemoglobin and plasma erythropoietin increased with gestational age in control fetuses. There was an inverse association between hemoglobin and plasma erythropoietin in control and Rh-isoimmunized fetuses (r = -0.56, P < .005). Using multiple linear regression, hemoglobin and gestational age were associated independently with plasma erythropoietin (overall F2,25 = 12.3, multiple r2 = 0.49, P < .001). Despite marked decreases in hemoglobin, fetuses below 24 weeks' gestation had minimal increases in plasma erythropoietin compared to fetuses above that gestational age. Mildly anemic Rh-isoimmunized fetuses (hemoglobin 11.6 +/- 2.0 g/dL) delivered vaginally had significantly higher erythropoietin levels in umbilical cord plasma than Rh-isoimmunized fetuses with comparable hemoglobin (10.9 +/- 3.5 g/dL) delivered by elective cesarean without labor (1246 +/- 856 versus 106 +/- 66 mU/mL, respectively, P < .05). CONCLUSION: Fetuses with anemia at mid to late gestation respond with increases in plasma erythropoietin, but these changes are substantially attenuated before 24 weeks' gestation.

Quantitative assessment of erythropoiesis and functional classification of anemia based on measurements of serum transferrin receptor and erythropoietin.

We evaluated the quantitative value of a simple model of erythropoiesis, based on the basic assumptions that the red blood cell (RBC) mass determines erythropoietin (Epo) production, which in turn stimulates erythropoietic activity. The RBC mass was quantitated by direct isotopic measurement (RCM), Epo production by serum Epo levels, and erythropoiesis by the ferrokinetic measurement of the erythron transferrin uptake (ETU), the serum transferrin receptor (TfR) level, and the reticulocyte (retic) index, and was completed by an evaluation of overall marrow erythron cellularity. We studied a total of 195 subjects, including 31 normal individuals, 38 patients with polycythemia, and 126 patients with various forms of anemia. Instead of only quantitating Epo and erythropoiesis in absolute terms, we also evaluated them in relation to the degree of anemia or polycythemia, and expressed the results as a ratio of observed values to values predicted from the regression equations between hematocrit (Hct) on the one hand, and Epo, TfR, and ETU on the other, obtained in a carefully selected subpopulation. The slope of the regression of TfR (as well as ETU) versus Hct was very similar to the slope of the regression of Epo versus Hct. Average EPO and TfR (as well as ETU) values predicted from the regression equations were quite comparable to observed values in most groups of subjects, with exceptions predictable from knowledge of the pathophysiology of these hematologic disorders. We identified four major patterns of erythropoiesis, ie, normal, hyperdestruction (with variants of hemolysis or ineffective erythropoiesis), intrinsic marrow hypoproliferation, and defective Epo production. Dissecting out groups of patients showed much greater heterogeneity than when patients were analyzed by group. This was particularly true in the case of a hypoproliferative component being combined with hyperdestruction, giving what we called a "mixed disorder of erythropoiesis." We conclude that the pathophysiology of anemia can be assessed by a simple measurement of Hct, retic index, Epo, and TfR levels, with Epo and TfR being more informative when expressed in relation to the degree of anemia. The model is particularly useful for detecting the presence of multiple mechanisms of anemia in the same patient. However, it has limitations inherent to the relative invalidity of TfR in iron deficiency, the imprecision of a retic count, and the difficulty in distinguishing hemolysis from ineffective erythropoiesis in some patients and in recognizing a component of hyperdestruction in hypoproliferative anemia.

Hematopoietic growth factors in anemia of Belgrade laboratory (b/b) rats.

In this study, the extent to which growth factor production and microenvironment might be responsible for defective erythropoiesis and granulopoiesis in anemic b/b rats is investigated. Radioimmunoassay-determined serum erythropoietin (Epo) levels are high in b/b rats and closely related to degree of anemia. The low number of erythroid progenitors in b/b rats despite a high Epo level suggested that the defective erythropoiesis could be due to a low level of burst-promoting activity (BPA). A pokeweed mitogen-stimulated medium (PWM-SCM) was prepared with b/b rat spleen cells and used in normal and anemic rat bone marrow and spleen cultures to determine BPA and other growth factor levels. No erythroid burst-forming unit-derived colonies were found but granulocyte-macrophage colony-forming units were counted in significant number, suggesting that the production of growth factors that supports the growth of granulopoietic progenitors is not significantly disturbed. Because BPA is produced mainly by T-lymphocytes, the low BPA level in b/b rat PWM-SCM raised the question of the functional capacity of T-lymphocytes. Investigations showed a decrease in the proliferative activity of b/b rat spleen mitogen-activated T-lymphocytes to about 20% of controls as well as a decrease in interleukin-2 activity in b/b rat spleen cell supernatants. These results point to defective T-lymphocytes. A study of bone marrow fibroblastoid cell colonies (CFU-F) revealed significantly lower CFU-F counts in the b/b rats. This finding is indicative of a disturbed microenvironment, which could also to some extent be responsible for decreased growth factor production and depressed hematopoiesis in the b/b rat.

Gross composition and plasma and extracellular water volumes of tissues of a reference mouse.

The laboratory mouse is a primary animal model for experimental radiation biology and pharmacology. The usefulness of the mouse for those purposes is enhanced if detailed data are available to define a Reference Mouse [weight and composition of soft tissues and bones and their in-life content of plasma and extracellular water (ECW)]. Only fragmentary data are available for wet weights and plasma volumes of soft tissues and bones of mice; there are no reports of total volume or distribution of ECW in mouse tissues. To remedy those defects, wet weight and composition of all major organs and soft tissues were measured, and measurements were made or estimates obtained for wet weights and composition of all bones of the young adult (12 to 13 wk old) female Swiss-Webster mouse. 125I-transferrin was used as a tracer for plasma, and 22Na was used as a tracer for ECW. Tissue weight and tracer measurements were conducted using the metabolic balance approach and a freezing technique that avoids blood loss during dissection. Results compare favorably with published weights and plasma volumes of tissues of mature mice of both genders and other strains. Total plasma volume (48.9 +/- 4.4 microL g-1) and Na-space (232 +/- 15 microL g-1), and the specific plasma and ECW volumes of vascular mouse tissues, exceed those of rat tissues. Applications of the data are presented: (1) interpretation of plutonium uptake kinetics in the mouse; (2) estimation of masses of mineralized bone tissue (1.92 g), bone marrow (1.2 g), and endosteal (BS) cells (0.2 g) of the mouse.

Serum erythropoietin levels in hemodialysed patients after administration of recombinant human erythropoietin.

In anemic patients on regular hemodialysis (HD), correction of anemia with recombinant human erythropoietin (rHuEpo) administered intravenously (iv) or subcutaneously (sc) was followed over a 2-month period. Monitoring serum Epo post-dose concentrations after the first iv rHuEpo injection and following another regular injection after 2 months of therapy with rHuEpo iv in 9 patients showed that the Epo elimination half-life was reduced from 7.48 h to 4.68 h. In the same patients the initially low percentage of erythroblasts and mature erythroid progenitors increased during 2 months of rHuEpo therapy. Because Epo molecules bound to Epo receptors are internalized in target cells we suggest that the expansion of the Epo responsive cell pool could explain the shorted Epo elimination time after 2 months of rHuEpo treatment. By monitoring serum Epo concentration following sc rHuEpo injection in 7 HD patients it was found that the modest increase in serum Epo levels (30-60 mU/ml) was sufficient to correct anemia.

Anemia in Balkan endemic nephropathy.

The severity of anemia in patients at different stages of the evolution of two tubulointerstitial nephropathies, Balkan endemic nephropathy and chronic pyelonephritis, was compared to clarify the previous observations that anemia appears earlier and is more severe in Balkan endemic nephropathy than in other renal diseases. The role of erythropoietin insufficiency as the cause of anemia in endemic nephropathy was studied as well. The severity of anemia increased with the impairment of renal function in endemic nephropathy and was similar to anemia in chronic pyelonephritis. However, in patients with endemic nephropathy at the initial stage of renal insufficiency significantly lower red cell concentrations were found compared with control subjects from the endemic region. In contrast, patients with pyelonephritis did not have decreased red cell concentrations at the early phase of their renal failure, suggesting that earlier appearance of anemia is characteristic for endemic nephropathy. To confirm this finding a study involving larger number of patients would be necessary. The serum erythropoietin levels, inappropriately low for the degree of anemia in patients with renal failure, were unrelated to the type of tubulointerstitial nephropathy.

Circulating erythropoietin levels after bone marrow transplantation: inappropriate response to anemia in allogeneic transplants.

We studied 24 recipients of autologous bone marrow transplantation (ABMT) or allogeneic BMT (BMT) to determine whether impaired erythropoietin (Epo) response to anemia could delay full erythropoietic recovery. Observed Epo levels were compared with predicted levels based on the relationship between Epo and hematocrit in 125 control subjects. Circulating Epo levels were normal during conditioning and the early posttransplant period. Between days 21 and 180, Epo levels remained normal in ABMT patients but were inappropriately low for the degree of anemia in BMT patients. Median time to full erythropoietic engraftment was longer in BMT than in ABMT recipients. Circulating Epo returned to appropriate levels after day 180, except in patients with active cytomegalovirus infection. We conclude that impaired Epo response to anemia can contribute to delayed erythropoietic recovery after allogenic BMT. Renal toxicity of ciclosporin, interaction between host and donor marrow, and cytomegalovirus infection might play a role. This study could support the use of recombinant human Epo to accelerate erythropoietic engraftment after BMT.

Erythropoietin and improvement of anemia in long-term hemodialysis patients.

Two groups, with 4 patients each were selected for study out of 155 patients on regular hemodialysis (HD): Group I, with hematocrit (PCV) less than 20% and group II, with PCV greater than 30%. The patients in both groups had been anemic at the start of HD treatment, but a significant improvement in their anemia had occurred only among the patients in the Group II. The main difference between the two patient groups, other than the degree of anemia, was found to be in serum erythropoietin (Ep) levels. No significant differences were observed between the two groups in serum urea, creatinine, parathyroid hormone or CFU-E growth inhibition. Acquired cystic disease of the kidney was found in five patients from group I, and in 11 patients from group II. The correlation between the number of cysts in the kidneys and the patient's PCV and serum Ep levels proved significantly positive. The results presented could be regarded as another proof that diseased kidney is capable of functioning as an Ep producing organ despite the loss of excretory function.

Maternal alcohol abuse is associated with elevated fetal erythropoietin levels.

The erythropoietin levels in mixed cord serum of 40 infants born to drinking women were compared with those of 24 infants born to abstinent women. Twenty infants born to drinkers had signs of fetal alcohol effects. Thirty-five percent of the erythropoietin levels in mixed cord serum of infants of drinking mothers were above the normal range. Further, the elevation in fetal erythropoietin level correlated with maternal alcohol intake; infants of mothers consuming at least 300 g of ethanol weekly (28) had significantly higher (P less than .025) umbilical erythropoietin levels (median 66 mU/mL, range 10-2500) compared with infants of mothers consuming 150-300 g of ethanol weekly (median 37 mU/mL, range 23-215) or infants of control women (median 32 mU/mL, range 11-73). The subgroup analysis between infants with and without fetal alcohol effects showed no differences in umbilical erythropoietin levels. Maternal alcohol ingestion during pregnancy is associated with elevated umbilical erythropoietin levels, but whether this is a direct effect of ethanol or is induced by chronic fetal hypoxemia remains unclear.

Direct relationship of antepartum glucose control and fetal erythropoietin in human type 1 (insulin-dependent) diabetic pregnancy.

In the present study the antepartum relationship between maternal diabetic glucose control and fetal hypoxaemia was examined in 44 Type 1 (insulin-dependent) diabetic and 23 non-diabetic control pregnancies. Maternal HbA1C was used to assess maternal integrated blood glucose control while fetal metabolic control was evaluated by antepartum glucose, insulin, and C-peptide determinations in amniotic fluid at elective caesarean delivery. Fetal hypoxaemia was assessed indirectly by fetal umbilical vein plasma erythropoietin level at delivery. A prospectively developed statistical pathway model was used to examine the relationship of these variables. In applying forced stepwise multiple regression with this model, we observed in the diabetic subjects that mean maternal HbA1C during the last month of pregnancy correlated significantly with fetal umbilical venous erythropoietin at delivery (r = 0.57, p less than 0.001). Additional significant contributions to umbilical venous erythropoietin were found for amniotic fluid glucose and amniotic fluid insulin when these two independent variables were added in stepwise fashion (p less than 0.01). We conclude that in diabetic pregnancy, antepartum control of maternal hyperglycaemia is a significant factor associated with fetal hypoxaemia. We speculate that this effect is mediated through perturbations which accelerate fetal metabolism and which is expressed by amniotic fluid levels of glucose and insulin.

Erythropoietin and anemia in the progression of Balkan endemic nephropathy and other renal diseases.

We have investigated anemia in patients at different stages of the evolution of three chronic renal diseases: Balkan endemic nephropathy (BEN), chronic pyelonephritis (PN) and chronic glomerulonephritis (GN). A total of 88 patients with creatinine clearances from 9 to 118 ml/min and hemoglobin concentrations from 70 to 160 g/l were studied with regard to the relationship, if any, between erythropoietin production and the type and stage of nephropathy. Anemia in BEN was a particular focus of interest since it had been stated that in BEN, anemia precedes renal failure. Our data neither prove nor disprove this statement. A significant positive correlation between creatinine clearance and hemoglobin concentration was found in all three nephropathies, indicating that in the patients studied the severity of anemia increased with the impairment of renal function regardless of the underlying disease. Serum levels of immunoreactive erythropoietin were in the normal range in 54 patients, moderately increased in 20 and slightly decreased in 14. The erythropoietin level appears to be unrelated to the stage of renal failure or the type of nephropathy. The only exception was the subgroup where the patients with glomerulonephritis and normal renal function had increased serum erythropoietin levels and significantly higher parameters of red blood cell concentration than the patients from the same subgroup with tubulointerstitial nephropathies. In patients with severe renal failure and anemia, serum erythropoietin levels were inappropriately low for the degree of anemia, indicating that erythropoietin plays a role in the pathogenesis of the anemia.

Correlation of plasma erythropoiesis stimulating factor(s) and immunoreactive erythropoietin levels during rapid growth in the mouse.

During the early neonatal period of rapid growth in the mouse, increased plasma levels of erythropoiesis stimulating factor(s) (ESF) have been found when measured by an in-vitro bioassay technique. It is unclear whether these increased ESF levels represent increased levels of circulating erythropoietin (Ep) alone or Ep in combination with other less-defined erythropoietic stimulatory factors. To examine this issue, plasma from neonatal mice of varying post-natal ages and from normoxic and hypoxic adult mice was studied. We found that plasma Ep levels measured by radioimmunoassay (RIA) correlated significantly with in-vitro bioassayed ESF levels (r = 0.84, P less than 0.0001, n = 21). Although an in-vivo bioassay for plasma Ep proved too insensitive for rigorous correlation with data from the RIA and in-vitro bioassay, the in-vivo data were in qualitative agreement with the other two, more sensitive, assays. In all three assays the highest plasma levels were observed in the 20-day-old mice and in adult mice which had been subjected to hypobaric hypoxia for 8 h. Based on the strong agreement of the results obtained with the RIA and the in-vitro bioassay in both neonatal and adult mouse plasma, we conclude that the high plasma ESF levels of 20-day-old mice measured with the in-vitro bioassay are largely immunochemically identifiable Ep. However, the data also suggest the presence of non-Ep factors in neonatal plasma which stimulate the in-vitro bioassay.

Fetal responses to long-term hypoxemia in sheep.

The effect of acute or short-term hypoxia on fetal cardiovascular hemodynamics has been well known; however, little is known about the effect of long-term hypoxemia. To determine the fetal hemodynamic responses to this stress we studied two groups of animals: 1) pregnant ewes (n = 20) at 110-115 days of gestation subjected to hypoxia for up to 28 days and 2) pregnant ewes (n = 4) that served as normoxic controls. We chronically catheterized the fetal brachiocephalic artery and vein. Five to 6 days after surgery, control measurements were made of mean arterial blood pressure, heart rate, arterial PO2, O2 saturation, hemoglobin, hematocrit, blood volume, and the concentrations of erythropoietin, cortisol, epinephrine, and norepinephrine. The next day the ewes were placed in a chamber with an inspired O2 fraction of 12-13%. Within a few minutes fetal arterial PO2 decreased from control value of 29.7 +/- 2.1 to 19.1 +/- 2.1 Torr, where it remained. Hemoglobin increased from 10.0 +/- 1.0 to 12.9 +/- 1.9 g/dl by day 7, where it remained. This was associated with an increase of erythropoietin from 22.8 +/- 2.2 to 144 +/- 37 mU/ml within 24 h, but by day 7 it had returned to levels slightly above normal. Epinephrine also increased moderately and remained elevated throughout the study. However, values of mean arterial pressure and heart rate did not differ from controls. Perhaps surprisingly, these fetuses were able to compensate so that at term their body weights were normal, 3.77 +/- 0.2 kg.

Amniotic fluid erythropoietin predicts fetal distress in Rh-immunized pregnancies.

Repeated amniotic fluid erythropoietin measurements in 23 Rh-immunized pregnancies were done to evaluate erythropoietin levels of amniotic fluid as an indicator of fetal distress (umbilical artery, pH 7.14 or less, or 1-minute Apgar score of 4 or less). Amniotic fluid erythropoietin levels did not vary significantly between 168 and 273 gestational days in the pregnancies without fetal distress. Increasing levels of amniotic fluid erythropoietin predicted highly reliably severe fetal distress at birth. Whether erythropoietin levels of amniotic fluid can also predict fetal distress in other pathologic pregnancies needs further study.