In vitro studies of erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes.

Erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes (MDS) was evaluated by measuring the in vitro response of primitive (BFU-E) and relatively mature (CFU-E) erythroid progenitors from 12 patients and from eight healthy donors to recombinant human erythropoietin (rhEPO), and by quantifying relationships between circulating EPO levels and progenitor cell frequencies in MDS marrow. Half-maximal growth of MDS CFU-E and BFU-E was detected at a 4-fold higher rhEPO concentration than required by control erythroid progenitors. Nine of the patients evaluated exhibited maximal growth of erythroid colonies at 5- to 20-fold higher than control saturating rhEPO concentrations. Circulating EPO levels in MDS patients were elevated, with a mean value approximately 35-fold higher than that of controls. The frequency of MDS marrow CFU-E and BFU-E was 57 +/- 42% and 18 +/- 9% of the mean control values, respectively. Correlation analysis of the relationships between MDS EPO levels and erythroid progenitors indicated that the anemia in MDS is not attributable to an abnormality in the capacity of EPO to induce the generation of CFU-E, but may be influenced by the BFU-E population, whose severe deficiency results in insufficient influx of EPO-responsive cells. Our findings therefore suggest that treatment of MDS patients with rhEPO may be of limited benefit, since the generation of BFU-E from more primitive ancestors and the initial growth requirements of these cells are not under the regulatory influence of this hormone.

In vivo effects of recombinant human erythropoietin on circulating human hemopoietic progenitor cells.

Recombinant human erythropoietin (rhEpo), now available, has become increasingly more important for clinical use, e.g., in the treatment of anemia of chronic renal failure, and has been shown to reverse anemia in these patients. When patients with anemia of chronic renal failure were treated with rhEpo at dosages between 40 and 120 U/kg three times per week, the numbers of circulating erythroid burst-forming units (BFU-E) and granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming units (CFU-GEMM) significantly increased during the first week of therapy. In contrast, the incidence of circulating granulocyte-monocyte CFU (CFU-GM) was not significantly altered.

Correction of amino acid metabolism by recombinant human erythropoietin therapy in hemodialysis patients.

We assessed the effect of correction of anemia with recombinant human erythropoietin (rhEPO) on plasma amino acid levels in maintenance hemodialysis patients. The plasma amino acid pattern was estimated by high performance liquid chromatography in 18 healthy persons and 14 hemodialysis patients before and up to 12 weeks after rhEPO therapy. There was a correction of the plasma serine values (67 +/- 16 to 87 +/- 22; P less than 0.05) and a corresponding decrease in the serine precursors, glycine (317 +/- 113 to 228 +/- 56; P less than 0.05) and hydroxyproline (26 +/- 21 to 15 +/- 13; P less than 0.10). The low plasma branched-chain amino acids, valine (137 +/- 33 to 154 +/- 50; P less than 0.10) and leucine (72 +/- 22 to 80 +/- 27; P less than 0.20), also were corrected. The elevated values of ornithine (78 +/- 16 to 62 +/- 19; P less than 0.1) and arginine (94 +/- 14 to 72 +/- 14; P less than 0.1) fell. The diminished glutamine values (470 +/- 125 to 563 +/- 115; P less than 0.1) and the decreased tyrosine/phenylalanine ratio (0.78 +/- 0.17 to 0.98 +/- 0.21; P less than 0.05) rose. Thus, the administration of rhEPO not only affects erythropoiesis, but also corrects the plasma amino acid pattern towards normal.

The use of epoetin beta in anemic predialysis patients with chronic renal failure.

UNLABELLED: Two clinical studies were conducted to investigate the efficacy and safety of epoetin beta in 266 [corrected] anemic predialysis patients. Epoetin beta was administered subcutaneously either daily or thrice weekly. Mean duration of treatment was 211 days (interquartile range: 105 to 350 days). RESULTS: Renal anemia could be corrected and the regular transfusion need could be eliminated in all patients. There was no difference in the dose requirement per week between daily and thrice weekly administration of epoetin beta. Regarding the entire study population, there was no acceleration of the progression of renal failure during epoetin beta treatment nor were there any notable changes in laboratory values other than retention values. Epoetin beta was safe and well tolerated; the most important adverse event was the development or aggravation of hypertension.

Biochemical markers as surrogates in clinical trials in patients with metastatic bone disease and osteoporosis.

Biochemical markers of bone turnover have been employed in phase II dose-finding trials to determine the therapeutic window of the highly potent bisphosphonate ibandronate. For intravenous dose finding in metastatic bone disease 158 patients with breast cancer were treated by single bolus injection of up to 3.0 mg and by single infusion of 4 and 6 mg of ibandronate. In the oral double-blind dose-finding trial, 108 patients received up to 50 mg of ibandronate or placebo once daily for 28 days. For dose-finding in osteoporosis 126 postmenopausal women with osteoporosis were treated in a double-blind trial by four injections of up to 2.0 mg of ibandronate or placebo every 3 months. In the phase II dose-finding trial, 180 postmenopausal women with osteoporosis received up to 5 mg of ibandronate or placebo daily for 12 months. During these trials, the bone resorption markers urinary Ca, pyridinoline (Pyd), deoxypyridinoline (Dpd), type I collagen cross-linked N-telopeptide and type I collagen cross-linked C-telopeptide revealed clear dose-dependent responses in accordance with the appropriate dose of ibandronate. The bone formation markers serum osteocalcin, carboxy-terminal propeptide of type I collagen and bone-specific alkaline phosphatase demonstrated also dose-dependent changes in long-term treatment. Thus, for dose finding and monitoring of ibandronate treatment in osteoporosis and in patients with malignant metastatic bone disease urinary type I collagen cross-linked N-telopeptide and C-telopeptide are the most responsive biochemical markers of bone resorption. For bone formation monitoring serum osteocalcin and serum bone-specific alkaline phosphatase should be employed.

Changes in glucose 6-phosphate dehydrogenase and phosphofructokinase activity during maturation and ageing of red blood cells in children with chronic renal insufficiency.

Red blood cells (RBC) of children with terminal renal insufficiency show a reduction of the apparent 51Cr RBC half-life (16.5 +/- 5.5 days). The enzymatic activities of phosphofructokinase (PFK) and glucose 6-phosphate dehydrogenase (G6PD) in the RBC of these patients do not differ from those in health persons. The RBC of uraemic and healthy children were separated by density gradient centrifugation in dextran medium according to maturity and age. RET count, mean cellular Hb concentration (MCHC), PFK and G6PD activities were determined in the separated cell populations. MCHC increases with increasing cell density while the number of RET decreases. There is a distinct decrease in the G6PD activity of low-density cells compared to high-density cells. The extent of the decrease in enzymatic activity is about the same for healthy persons as for uraemic patients. A more rapid decrease in G6PD activity per unit of time can be assumed when the enzymatic activity is plotted against the distinctly shortened life span of the RBC of the uraemic children. There is a possible relationship between G6PD activity in old RBC and the premature destruction of these cells. PFK activity apparently does not decrease with increasing cell age.

Adverse events of subcutaneous recombinant human erythropoietin therapy: results of a controlled multicenter European study.

In a controlled European multicenter study, clinical tolerance of subcutaneously administered recombinant human erythropoietin (rh-EPO) therapy and its influence on the course of illness in 362 hemodialyzed patients (162 males, 200 females) from 16 European dialysis centers was studied. Of these, 181 patients served as a control group in the first year and received rh-EPO therapy in the second year. Of the 837 adverse events that occurred, 277 were classified as serious and 560 as nonserious. Thirty-two deaths have been reported for the study population: 18 in the control group and 14 in the therapy group. The individual analysis of the serious adverse events including death demonstrates a protective effect of rh-EPO on the high-risk cardiovascular situation of dialysis patients. Hypertension was no problem, and under rh-EPO therapy an increase in resistance to infection was observed. Subcutaneous rh-EPO treatment might have an even better safety profile than intravenous application.

[Operations with a heart-lung machine in adult members of Jehovah's Witnesses]. / Operationen mit der Herz-Lungen-Maschine bei erwachsenen Patienten der Glaubensgemeinschaft "Zeugen Jehovahs".

Members of Jehovah's Witnesses refuse blood transfusions and blood products under any circumstances. Because of an improvement in blood salvage techniques in our centre, they are not excluded from open-heart surgery. In recent years recombinant human erythropoietin (rhEPO) has been applied to correct perioperative anemia in these patients. METHODS. Seventeen members of Jehovah's Witnesses who were more than 18 years of age were operated on using various blood salvage technique, e.g., haemoseparation and a high dose of Aprotinin. We present the first three patients treated with 4 x 500 U of i.v. rhEPO/kg body wt. given within 11 days preoperatively. Thirteen of the patients operated on had elevated preoperative risk factors, for instance poor left ventricle, severe aortic valve stenosis, metabolic syndrome, age older than 70 years, etc. In other centres that perform cardiac operations on members of Jehovah's Witnesses, these risk factors represent contraindications for open-heart surgery in these patients. RESULTS. Patients with rhEPO treatment showed a preoperative hematocrit increase of 7 Vol.% within 10 days and no postoperative complications. At the 6th postoperative hour the hematocrit returned to the starting values; in patients without rhEPO, however, the hematocrit generally had not increased to preoperative values even by the 8th day after operation. In 9 patients with preoperative elevated risk factors and a postoperative relative decrease in hematocrit below 33% we observed an uncomplicated postoperative period. Four patients with these risk factors, a pronounced decrease in hematocrit and blood loss postoperatively had various severe complications. CONCLUSIONS. Preoperative treatment with a high dose of rhEPO to enhance the hematocrit and maturity by precursor red blood cells in patients with a hematocrit below 45 Vol.% is a possibility to compensate for the blood loss perioperatively and to avoid complications from a decrease in oxygen transport capacity. The anaemia and high blood loss postoperatively are the main causes for a slightly elevated operation risk in members of Jehovah's Witnesses in all heart centres that perform cardiac operations on these patients. Nevertheless, Jehovah's Witnesses should be not excluded from cardiac operations, since open-heart surgery without use of homologous blood is becoming a routine procedure.

Red blood cell density distribution in uremic patients on acetate and bicarbonate hemodialysis.

Renal anemia is the result of reduced erythropoietin (EPO) biosynthesis in the diseased kidney and also in part the result of a reduced life span of red blood cells (RBCs). An increase in density and a decrease in enzyme equipment (aspartate aminotransferase; GOT) of RBCs reflect cell age. In the following study, the density distribution (median density D50; determined by Percoll density gradients) and GOT activities of RBCs were measured in patients on acetate (HDA; n = 15) and bicarbonate (HDB; n = 51) hemodialysis. Hemoglobin (Hb) concentrations were: in the HDB group, 9.1 +/- 3.4 g/dl; in the HDA group, 6.2 +/- 1.2 g/dl, and, in a control (C) group of healthy persons, 14.0 +/- 1.5 g/dl. 14 HDB patients with severe anemia received EPO therapy during 1 year. D50 were found as follows: group C, 1.0674 +/- 0.0016 g/ml; HDB, 1.0674 +/- 0.0015 g/ml, and HDA, 1.0660 +/- 0.0012 g/ml (HDA vs. group C: p less than 0.05; HDA vs. HDB: p less than 0.05. D50 were elevated in the subgroups of HDA and HDB patients with severe anemia (Hb less than 8 g/dl). During activated erythropoiesis by EPO therapy, D50 decreased from 1.06739 +/- 0.0015 to 1.0656 +/- 0.0014 g/ml. The GOT activities in RBCs demonstrated a rejuvenation of the RBC population in the HDB group (6.4 +/- 2.5 U/g Hb) and HDA group (5.9 +/- 3.1 U/g Hb) compared to group C (3.9 +/- 1.3 U/g Hb).(ABSTRACT TRUNCATED AT 250 WORDS)

[Megacalycosis in childhood]. / Die Megakaliose im Kindesalter.

Congenital megacalycosis is a renal dysplasia characterised by hypoplasia of the medullary pyramids and associated non-obstructive dilatation of the calyces. Calyceal dilatation promotes via disturbed urodynamics the manifestation of chronic interstitial nephritis and calculus formation. Therapy is primarily not necessary, but in the presence of stone development and chronic infections surgical procedures may become indispensable. Pathogenesis, diagnosis, differential diagnosis and therapy of megacalycosis in two children are discussed.

Effect of human recombinant erythropoietin on human hemopoietic progenitor cells in vitro.

Recombinant human erythropoietin (rhEpo), now available, might become increasingly more important for clinical use, e.g., in the treatment of anemia of chronic renal failure. As a prerequisite of clinical trials, we analyzed the stimulatory and suppressive effects of rhEpo on human hemopoiesis by adding rhEpo to in vitro cultures of nonadherent low-density bone marrow cells obtained from normal persons and from patients undergoing hemodialysis for chronic renal failure. rhEpo was shown to be an effective stimulus for erythroid and multilineage colony formation. The dose-response curve was similar for erythroid progenitors BFU-E from normal controls and patients with chronic renal failure. rhEpo had no effect on megakaryocytic colony formation nor on the megakaryocytic differentiation of multilineage stem cells. Because of a good stimulatory activity on erythroid and multilineage stem cells and lack of toxic effects, rhEpo might be useful in the treatment of certain kinds of anemia.

[Erythropoietin and erythropoiesis inhibition factor in the newborn and infants with cyanotic heart abnormalities]. / Erythropoietin und Erythropoesehemmfaktor bei Neugeborenen und Süglingen mit zyanotischen Herzmissbildungen.

In the present study, hematocrit, pO2 in the capillary blood, reticulocyte count, erythropoietin activity in plasma and activity of the erythropoiesis inhibition factor in 24-h collective urine were determined in newborns and infants with cyanotic malformations of the heart (transpositions of the big arteries). The plasma of the healthy newborns shows immediately after birth an erythropoietin activity below 1%, as measured by the 59Fe incorporation. The erythropoietin activity of the plasma of infants with cyanotic malformations of the heart is enhanced up to the 14th day of life. In the period of day 14--30 it corresponds to the values of healthy newborns despite the persistence of hypoxia. After the 30th day of life there is again demonstrable an increased erythropoietin activity in the plasma of infants with cyanotic malformations of the heart. Obviously, the low values of 59Fe incorporation from day 14--30 are due to the occurrence of the erythropoiesis inhibition factor. In the urine of infants with cyanotic malformations of the heart there could be demonstrated distinct inhibitions of the stimulated erythropoiesis of the polycythemic mouse. The present studies suggest that the erythropoiesis inhibition factor occurs in postnatal development irrespectively of the degrees of O2-supply of the tissue, and is possibly dependent on the gestational age.

On the inhibitory effect of the serum of uraemic children on erythropoiesis.

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

The estimation of efficacy of oral iron supplementation during treatment with epoetin beta (recombinant human erythropoietin) in patients undergoing cardiac surgery.

We estimated the efficacy of oral iron therapy during treatment with rhEPO in patients undergoing cardiac surgery who were contraindicated for autologous blood donation. Seventy-six patients were enrolled in this double-blind, placebo-controlled trial and assigned to the 2 treatment groups (5x500 U/kg body weight rhEPO or placebo intravenously over 14 d before surgery). During the treatment period all patients received 300 mg Fe2+ (iron glycine sulfate) orally per day. rhEPO therapy produced significant increases in hemoglobin concentration (Hb), reticulocyte count, hematocrit (Hct) and the hypochromic red blood cells (HRBC), and a decrease in transferrin saturation (41%) compared to the placebo group before surgery. However, the preoperative increase in HRBC was independent of the baseline ferritin and even correlated positively with the preoperative increase in Hct (r=0.47, p<0.01). In rhEPO patients there were inverse correlations between baseline serum iron and the preoperative increases in Hb (r=-0.39, p<0.05), Hct (r=-0.50, p<0.01) and HRBC (r=-0.53, p<0.001). With this treatment regimen the HRBC appear to reflect the degree of erythropoietic stimulation rather than functional iron deficiency. The preoperative increases in reticulocytes, HRBC and Hb/Hct in patients with ferritin <100 mg/l or transferrin saturation <16% showed no significant difference compared to their complementary groups. The preoperative decrease in storage iron and the inverse correlation between the baseline ferritin and the preoperative change in ferritin (r=-0.94, p<0.0001) in the rhEPO group indicate that the iron requirement for hemoglobin synthesis is probably covered by the breakdown of stored iron and an increase in the rate of absorption of orally administered Fe2+. Intravenous rhEPO treatment with 5x500 U/kg body weight in combination with 300 mg oral Fe2+/d given over 14 d before surgery is a suitable regimen to increase Hb by about 1.61 g/dl and Hct by 0.06.

Avoidance of allogeneic blood transfusions by treatment with epoetin beta (recombinant human erythropoietin) in patients undergoing open-heart surgery.

In a double-blind, randomized, placebo-controlled trial, we evaluated the ability of epoetin beta (recombinant human erythropoietin) to avoid allogeneic blood transfusions (ABT) and the associated risks in patients undergoing primary elective open-heart surgery and in whom autologous blood donation (ABD) was contraindicated. Seventy-six patients overall were enrolled onto the trial and were randomly assigned to the two treatment groups, 5 x 500 U/kg body weight (BW) epoetin beta or placebo intravenously over 14 days preoperatively. All patients received 300 mg Fe2+ orally per day during the treatment period. Preoperatively, the mean hemoglobin increase was 1.50 g/dL greater in epoetin beta patients than in placebo patients (95% confidence interval, 1.10 to 1.90 g/dL), allowing a rapid return to the baseline value by the seventh postoperative day in most epoetin beta patients. The mean volume of blood collected by intraoperative isovolemic hemodilution was 562 mL (red blood cell mass, 274 mL) in the epoetin beta group and 218 mL (red blood cell mass, 94 mL) in the placebo group, respectively. Only four patients (11%) in the epoetin beta group received an ABT, compared with 19 (53%) in the placebo group (P = .0003). Epoetin beta was most useful in patients with a perioperative blood loss greater than 750 mL, in those with a baseline hematocrit value less than 0.42, and in those aged > or = 60 years. The iron supplementation proved adequate despite the fact that a significant decrease in ferritin (median, 48.1%) and transferrin saturation (median, 40.5%) was observed in epoetin beta patients preoperatively. No influence of epoetin beta therapy on blood pressure, laboratory safety variables, or the frequency of specific adverse events was observed. Intravenous epoetin beta treatment of 5 x 500 U/kg BW in combination with 300 mg Fe2+ orally per day administered over 14 days preoperatively is an adequate therapy for increasing mean hemoglobin levels by approximately 1.50 g/dL and reducing the allogeneic blood requirement in patients undergoing elective open-heart surgery and in whom ABD is contraindicated.

[Effect of uremic plasma fractions of different molecular sizes on the filterability of red blood cells]. / Der Einfluss urämischer Plasmafraktionen unteschiedlicher Molekülgrösse auf die Filtrabilität roter Blutzellen (rBZ).

Behaviour of the filtration of red blood cells (rbc) passed through a filter paper was investigated. The rbc were incubated in fractions of blood plasma with different molecular mass (MM). By means of an Amicon membrane XM-50 the separation of plasma from healthy and uremic patients (children) was performed into two parts of different molecular sizes one of these was the concentrate (MM more than 50 000 = K) and the other was the ultrafiltrate (MM below 50 000 = UF). By a second step the UF was divided (Amicon membrane UM-50 into an UF1 (MM below 500) and into an UF2 (MM 500 to 50 000). In addition a hemofiltrate (MM below 5 000) was used for the incubation too. This hemofiltrate was made form pooled plasma of healthy and uremic patients, respectively. Rbc was incubated in both different fractions of plasma and hemofiltrate at a temperature of 37 degrees C and for a duration of 60 min. The pH-level and the osmolality were physiological. Under the influence of uremic UF the flexibility of rbc was clearly decreased. The UF1 had the most effect of the decrease of flexibility. Therefore the small substances which are easy dialysable play also an important role in the damage of rbc in the uremia.

One year's experience with recombinant erythropoietin in children undergoing continuous ambulatory or cycling peritoneal dialysis.

Fourteen patients (aged 5.9-22.1 years) undergoing continuous ambulatory or cycling peritoneal dialysis were treated with recombinant human erythropoietin (rhEPO), which was given intravenously once a week at a dosage of 300 units/kg. The mean haematocrit level increased from 18.5% to 27.5% and the reticulocyte count from 19% to 62% within 1 month. After an average time of 3.1 months rhEPO dosage could be adjusted to 100 units/kg per week to keep the haematocrit level at 30%. Only 1 patient had an exacerbation of hypertension, which required a dosage reduction; other side-effects were not noted.

Erythropoiesis and erythrocyte age distribution in hemodialysis patients undergoing erythropoietin therapy.

Renal anemia is caused in part by a reduced life span of red blood cells (RBCs) and by reduced erythropoietin biosynthesis in the damaged kidney. The RBC age can be determined by density gradient centrifugation and estimation of cell-age-dependent enzyme activities, as aspartate aminotransferase. The RBC age distribution influences the median density (D50) of RBCs and the blood rheology in coherence with the hematocrit. In our study, the median density was determined by Percoll density gradient centrifugation in 18 healthy subjects (D50 = 1.0674 +/- 0.0016 g/ml) and in 14 hemodialysis patients (D50 = 1.0674 +/- 0.0016 g/ml in the course of recombinant human erythropoietin (rhEPO) therapy. During the first 4 weeks of therapy, a strong rejuvenation of RBCs was observed whereby the D50 reached a minimum after 2 weeks (D50 = 1.0655 +/- 0.0022 g/ml; p less than 0.05 vs. value before therapy) and a steady state after 4 weeks (D50 = 1.0658 +/- 0.0013 g/ml; p less than 0.1 vs. value before therapy). In 5 of the patients with elevated plasma parathyroid hormone (i-PTH) concentrations greater than 10 pmol/l, a significantly (p less than 0.05) reduced amount of younger RBCs (D50 = 1.0675 +/- 0.0016 g/ml) was observed in the first 2 weeks of rhEPO therapy as compared to patients with i-PTH less than 10 pmol/l (D50 = 1.0677 +/- 0.0019 g/ml). Thus, erythropoiesis in the early phase of rhEPO therapy is strongly influenced by elevated plasma i-PTH concentrations. Therefore, a gradual increase in rhEPO doses is preferable before therapy at elevated doses with an uncontrolled increase in RBC amount.