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Serum erythropoietin (EPO) and its relationship to hemoglobin (Hb), iron status markers and iron supplementation during normal pregnancy was assessed in a longitudinal, placebo-controlled study on 118 women, 61 took daily tablets containing 66 mg ferrous iron from the second trimester until delivery and 57 took placebo. Blood samples were obtained at 4-week intervals until delivery as well as post-partum. In the placebo-treated women, median serum EPO rose from 22.5 U/l at inclusion to 35.0 U/l at delivery (P = 0.0001). In the iron-treated women, median serum EPO rose from 23.9 to 29.9 U/l (P = 0.0001). Serum EPO showed a steeper increase in the placebo-treated women than in the iron-treated women (P < 0.05). After delivery, serum EPO became normal in both groups (P = 0.0001). Median Hb was lower in placebo-treated (iron depleted) than in iron-treated (iron repleted) women (P < 0.05). In the placebo-treated women there was a negative correlation and in the iron-treated women a positive correlation between serum EPO and Hb. In the placebo-treated women, inverse correlations existed between serum EPO and serum transferrin saturation and serum ferritin, reflecting the consequences of iron deficiency, whereas the iron-treated women displayed no correlation. A physiological, nonhypoxia-induced increase in EPO production accounts for the basic expansion of the red cell mass during pregnancy. In placebo-treated women, iron deficient erythropoiesis constitutes an additional hypoxic stimulus, which induces a further increase in serum EPO.

In this study, we examined the cord serum erythropoietin (EPO) level in newborn infants in relation to venous cord blood gases and iron status markers (cord serum ferritin, cord serum transferrin saturation). The subjects were 90 healthy newborn term infants with a normal birth and 90 healthy women with an uncomplicated pregnancy and delivery. Within 14-18 weeks of gestation, 47 prospective mothers, allocated at random, received tablets containing 66 mg ferrous iron daily, and 43 received a placebo. Serum EPO was measured in women prior to delivery. Serum EPO, serum ferritin and serum transferrin saturation were analyzed in cord blood from the newborn. Blood gases (PO2, PCO2 and standard HCO3, PH) were measured in venous cord blood and Apgar scores were recorded. The cord serum EPO level in the newborn was not significantly affected by the iron supplementation to the mothers. In the entire series, the geometric mean cord serum EPO was 36 U/I, (median 32 U/I, 5-95% 16-150, range 12-380). There was no correlation between cord serum EPO and APgar score, cord blood PO2, PCO2, standard HCO3 and pH. In the newborn of placebo-treated mothers, log cord serum EPO was inversely correlated with log cord serum ferritin (r = -0.54, P < 0.0002) and cord serum transferrin saturation (r = -0.39, P < 0.011), suggesting that iron deficiency may occur in this newborn group.

This study investigated the human erythropoietin (EPO) response to short-term hypocapnic hypoxia, its relationship to a normoxic or hypoxic increase of the haemoglobin oxygen affinity, and its suppression by the addition of CO2 to the hypoxic gas. On separate days, eight healthy male subjects were exposed to 2 h each of hypocapnic hypoxia, normocapnic hypoxia, hypocapnic normoxia, and normal breathing of room air (control experiment). During the control experiment, serum-EPO showed significant variations (ANOVA P = 0.047) with a 15% increase in mean values. The serum-EPO measured in the other experiments were corrected for these spontaneous variations in each individual. At 2 h after ending hypocapnic hypoxia (10% O2 in nitrogen), mean serum-EPO increased by 28% [baseline 8.00 (SEM 0.84) U.l-1, post-hypoxia 10.24 (SEM 0.95) U.l-1, P = 0.005]. Normocapnic hypoxia was produced by the addition of CO2 (10% Co2 with 10% O2) to the hypoxic gas mixture. This elicited an increased ventilation, unaltered arterial pH and haemoglobin oxygen affinity, a lower degree of hypoxia than during hypocapnic hypoxia, and no significant changes in serum-EPO (ANOVA P > 0.05). Hypocapnic normoxia, produced by hyperventilation of room air, elicited a normoxic increase in the haemoglobin oxygen affinity without changing serum-EPO. Among the measured blood gas and acid-base parameters, only the partial pressures of oxygen in arterial blood during hypocapnic hypoxia were related to the peak values of serum-EPO (r = -0.81, P = 0.01). The present human EPO responses to hypoxia were lower than those which have previously been reported in rodents and humans. In contrast with the earlier rodent studies, it was found that human EPO production could not be triggered by short-term increases in pH and haemoglobin oxygen affinity per se, and the human EPO response to hypoxia could be suppressed by concomitant normocapnia without acidosis.

This study tested the hypothesis that the diurnal variations of serum-erythropoietin concentration (serum-EPO) observed in normoxia also exist in hypoxia. The study also attempted to investigate the regulation of EPO production during sustained hypoxia. Nine subjects were investigated at sea level and during 4 days at an altitude of 4350 m. Median sea level serum-EPO concentration was 6 (range 6-13) U.l-1. Serum-EPO concentration increased after 18 and 42 h at altitude, [58 (range 39-240) and 54 (range 36-340) U.l-1, respectively], and then decreased after 64 and 88 h at altitude [34 (range 18-290) and 31 (range 17-104) U.l-1, respectively]. These changes of serum-EPO concentration were correlated to the changes in arterial blood oxygen saturation (r = -0.60, P = 0.0009), pH (r = 0.67, P = 0.003), and in-vivo venous blood oxygen half saturation tension (r = -0.68, P = 0.004) but not to the changes in 2, 3 diphosphoglycerate. After 64 h at altitude, six of the nine subjects had down-regulated their serum-EPO concentrations so that median values were three times above those at sea level. These six subjects had significant diurnal variations of serum-EPO concentration at sea level; the nadir occurred between 0800-1600 hours [6 (range 4-13) U.l-1], and peak concentrations occurred at 0400 hours [9 (range 8-14) U.l-1, P = 0.02]. After 64 h at altitude, the subjects had significant diurnal variations of serum-EPO concentration; the nadir occurred at 1600 hours [20 (range 16-26) U.l-1], and peak concentrations occurred at 0400 hours [31 (range 20-38) U.l-1, P = 0.02]. This study demonstrated diurnal variations of serum-EPO concentration in normoxia and hypoxia, with comparable time courses of median values. The results also suggested that EPO production at altitude is influenced by changes in pH and haemoglobin oxygen affinity.

In a randomized, double-blind, placebo controlled study of the effect of iron supplementation during pregnancy, iron status (hemoglobin (Hb), serum (S-)transferrin saturation, S-ferritin) and S-erythropoietin (EPO) were assessed in 120 healthy pregnant women at 14-16 weeks of gestation, and just before delivery; 63 women were treated with 66 mg iron daily, and 57 with placebo. There were no differences in baseline values in the two groups. At term, the iron treated group had significantly higher Hb, transferrin saturation, S-ferritin (median 22 micrograms/l vs. 14 micrograms/l, (p < 0.0001) and lower S-EPO compared to the placebo treated group. In the iron group, 30.2% had exhausted iron stores (i.e. S-ferritin < 20 micrograms/l), 6.3% latent iron deficiency (S-ferritin < 20 micrograms/l and transferrin saturation < 15%), and no patients had iron deficiency anemia (S-ferritin < 20 micrograms/l and transferrin saturation < 15% and HB < 110 g/l). In the placebo group, 93.0% had exhausted iron stores, 54.4% latent iron deficiency, and 17.5% iron deficiency anemia; S-EPO was inversely correlated to iron status markers: Hb, rs = -0.51, p < 0.001; transferrin saturation, rs = -0.65, p < 0.0001; S-ferritin, rs = -0.31, p < 0.01, suggesting that the elevation in S-EPO was secondary to iron deficient erythropoiesis. Newborns to iron treated mothers had higher cord S-ferritin, median 155 micrograms/l, than newborns to placebo treated mothers, median 118 micrograms/l (p < 0.02); there were no differences in birth weight, transferrin saturation, or S-EPO.(ABSTRACT TRUNCATED AT 250 WORDS)

Objectives: To explore the possible pathogenetic role of erythropoietin (EPO) in the anaemia associated with cyclosporin (Cs) in newly diagnosed patients with insulin-dependent diabetes mellitus (IDDM)., Design: A multicentre randomized placebo controlled prospective trial of Cs immunosuppression for 12 months in IDDM patients., Setting: Patients were recruited from the out-patient clinics of diabetes centres in Europe and Canada., Subjects: Patients 9-35 years old with a clinical diagnosis of ketonuric IDDM entering less than 6 weeks after diagnosis. 188 patients were originally recruited; 105 patients completed the investigation, 52 patients being treated with Cs, and 53 patients receiving placebo., Interventions: Random allocation to receive either Cs or placebo. The initial dose of Cs was 10 mg kg-1 BW day-1. Therapy was maintained for 12 months., Main Outcome Measures: B-Haemoglobin, s-creatinine, and s-EPO concentrations were monitored before, during and after therapy with either Cs or placebo., Results: Blood-haemoglobin (Hgb) fell from 8.5 +/- 0.8 to a nadir of 7.8 +/- 0.9 mmol l-1 at 6 months (P < 0.0001) in IDDM patients treated with Cs but not in the placebo patients (8.5 +/- 0.8 to 8.8 +/- 0.9 mmol l-1, NS). The mean serum EPO levels remained unaltered throughout the 6-month period of Cs and placebo therapy. No significant differences in serum EPO levels between Cs and placebo-treated diabetic patients were found after 6 months of treatment., Conclusions: The light anaemia associated with Cs therapy in IDDM patients is not related to an insufficient production of EPO, but is caused by other, as yet unknown mechanisms, unrelated to the nephrotoxic action of this drug.

A consistent elevated level of erythropoietin was found in eight patients with R.A., who continuously for 10 years had a low hemoglobin (< 8 mmol/l) compared with nine patients with R.A., who had a normal hemoglobin (> or = 8 mmol/l) (p = 0.017). A significant inverse correlation between erythropoietin and hemoglobin (r = -0.61) was found in the low-hemoglobin group, but not in the normal-hemoglobin group. Erythropoietin was not found to be correlated to clinical variables.

The relationship between the renin-angiotensin system and erythropoietin was studied in twenty patients with renal artery stenosis and hypertension. Ten of the patients had a unilaterally activated renin-angiotensin system (group 1), while ten patients had not (group 2). Plasma erythropoietin was simultaneously measured in a brachial artery and both renal veins before and 5 and 30 min after an intravenous injection of 1.25 mg enalaprilat. The mean (+/- SD) arterial erythropoietin concentration was 27.3 +/- 16.8 mU/ml in group 1 and 14.1 +/- 11.3 mU/ml in group 2 patients (P less than 0.05). There was no significant change after enalaprilat i.v. in either group. The venous erythropoietin concentration in plasma from the stenotic kidney did not differ from that of the contralateral kidney. The higher erythropoietin concentration in group 1 patients may be explained by a systemic stimulatory effect of the renin-angiotensin system on erythropoietin production. As no side-differences were found, renal vein as well as peripheral erythropoietin measurements cannot be used as a tool in the diagnosis of the functional significance of a renal artery stenosis.

The effect of iron supplementation, 66 mg elemental iron daily, from the 16th week of gestation to delivery, on iron status markers during uncomplicated pregnancies was assessed in a randomised, double-blind, placebo controlled study of 207 healthy women (100 iron treated, 107 placebo treated) and their newborn babies. Haemoglobin (Hb) and serum (S-) human placental lactogen (HPL) were measured in all 207 females, while transferrin saturation, S-ferritin and S-erythropoietin (EPO) were measured in 120 females at monthly intervals. Hb: from 27th week of gestation to eight weeks post partum, the placebo treated group had significantly lower Hb levels than the iron treated group (p less than 0.001). Iron status markers: in the placebo group (n = 57), 92% developed exhausted iron stores (i.e. S-ferritin less than or equal to 20 micrograms/l), 65% latent iron deficiency (i.e. S-ferritin less than or equal to 20 micrograms/l and transferrin saturation less than 15%), and 18% iron deficiency anaemia (i.e. S-ferritin less than or equal to 20 micrograms/l, transferrin saturation less than 15% and Hb less than 110 g/l). In the iron treated group (n = 63), 54% developed exhausted iron stores, 6% latent iron deficiency, and none iron deficiency anaemia. S-EPO: the placebo group had significantly higher values than the iron treated group from the 27th week of gestation to one week post partum (p less than 0.01). S-HPL: levels were identical in placebo and iron treated females. Babies of iron treated mothers had higher S-ferritin than babies of placebo treated mothers (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Six patients with acute renal failure, in five cases due to acute crescentic glomerulonephritis and in one case due to total bilateral renal cortical necrosis, were studied. All had serum erythropoietin (EPO) concentrations in the normal range, despite a relatively severe anaemia. Half-life and plasma clearance of intravenously injected recombinant human erythropoietin (rhEPO) were determined. The results indicate that the lack of compensatory increase in serum EPO to the anaemic stimulus is not due to increased catabolism, but to decreased synthesis of the renal hormone. Two patients were treated with rhEPO (Eprex). In marked contrast to untreated controls, both patients responded with vigorous reticulocytosis and normalization of haemoglobin levels while they were still in severe renal failure. These results are similar to our previous findings in patients with acute renal failure due to tubular necrosis. Under all three conditions the defective EPO synthesis is probably the dominant pathogenetic factor for the largely aregeneratory anaemia of prolonged cases, and for the sluggish restoration of red cell mass during recovery of renal function. It is concluded that defective synthesis of EPO is not only a permanent and irreversible feature of severe chronic renal failure, but that it is also present, usually in a transient and reversible form, in different types of acute renal failure.

The relations between serum-erythropoietin (se-EPO), blood hemoglobin and lung function were investigated in patients with chronic obstructive pulmonary disease (COPD). In a randomized, double-blind, placebo-controlled design, the hypothesis was tested that se-EPO might increase after treatment with a calcium antagonist. In 18 patients with COPD, median se-EPO (19.2 mU ml-1) was in the low normal range. The correlations between se-EPO and blood hemoglobin (p = -0.63, p = 0.024) and between se-EPO and lung function indices were negative (n.s.) and the correlation between blood hemoglobin and lung function indices (n.s.) were positive. The hypothesis is proposed that the normal hemoglobin found in most patients with COPD is a result of a balance between a trend towards a decreased red cell mass, as found in chronic diseases, and a trend towards an increased red cell mass due to the erythropoietic effect of EPO. Se-EPO was not changed by the calcium antagonist, isradipine.

Indirect evidence points to extrarenal organs, presumably the liver, as the site of degradation of erythropoietin (EPO). The metabolism of both fully glycosylated and desialated intrinsically labelled 35S-Cysteine recombinant human erythropoietin (rhEPO) was therefore studied in isolated Wistar rat livers perfused in a recirculating mode for 180 min with a hemoglobin-free medium containing rhEPO. Perfusate and bile levels of rhEPO were measured by RIA. Total 35S-radioactivity in liver, bile and perfusate as well as non-acid precipitable radioactivity in perfusate were determined. In addition, detection of 35S-radioactivity was performed after subcellular fractionation of rat livers perfused with desialo-35S-Cysteine rhEPO. While concentrations of fully glycosylated 35S-Cysteine rhEPO did not exhibit any detectable decrease during perfusion, desialo-35S-Cysteine rhEPO was rapidly cleared from the perfusate. After 60 min of perfusion, only 32% of the initial levels of both immunoreactive rhEPO and total radioactivity remained in the perfusate. Quantitative hepatic accumulation of desialated tracer was demonstrated. Subcellular fractionation showed extensive hepatic degradation of the desialated tracer. Furthermore, during perfusion progressively larger amounts of small molecular weight degradation products of the tracer were found in the perfusate. Bile excretion of both fully glycosylated and desialated tracer was negligible. The significance of hepatic metabolism of desialo-35S-Cysteine rhEPO was supported by reduced removal of desialo-35S-Cysteine rhEPO from plasma in hepatectomized rats. It is hypothesized that continuous in vivo desialation is a crucial rate-limiting step in the degradation of circulating EPO.

Immunoreactive levels of serum erythropoietin (EPO) have been measured in 95 patients with multiple myeloma (MM) and 12 patients with Waldenström's macroglobulinaemia (MW). Of the MM patients 23% were uraemic (mostly light and moderate renal failure) and 61.7% were anaemic. In the anaemic nonuraemic MM patients the mean serum EPO titre was 106.8 +/- 30.4 mU/ml which, when related to the extent of anaemia, was found to be appropriately elevated for the degree of anaemia (the mean haemoglobin (B-HGB) concentration was 6.66 +/- 1.31 mmol/l). The mean serum EPO concentration in uraemic and anaemic MM patients was 39.2 +/- 9.2 mU/ml, which was markedly lower than serum EPO levels in the non-uraemic MM patients, but still higher than in nonanaemic control subjects (22.5 +/- 8.5 mU/ml). The mean B-HGB concentration in uraemic MM patients was 6.04 mmol/l. In the anaemic MM patients with severe renal failure (S-creatinine levels greater than 400 mumol/l) the compensatory secretion of EPO was inadequate in relation to the degree of anaemia. The data indicate that unless the hypoproliferative anaemia of MM is accompanied by considerable renal failure the anaemia does not appear to be associated with a deficient biogenesis of EPO. Likewise the anaemia found in patients with MW also seems, generally, to elicit an appropriate increase in EPO secretion. Reasonably clinically stable MM patients with anaemia and uraemia may be candidates for replacement therapy with recombinant human erythropoietin (rhEPO).

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.

It has been suggested that angiotensin-converting enzyme (ACE) inhibitors halt the progression of chronic renal failure. During the first months of a controlled trial of this hypothesis a fall in haemoglobin (Hb) was observed in patients treated with the ACE inhibitor enalapril. It was investigated whether this was related to changes in serum erythropoietin (EPO). Data were analysed in 59 consecutive patients during an observation period of 90 days. In enalapril-treated patients (n = 27) Hb fell gradually from a median value of 7.6 to 6.7 mmol/l at 90 days of treatment. In the control group of patients on conventional antihypertensive treatment (n = 32) median Hb was unchanged (7.6 mmol/l) throughout the observation period (p less than 0.001 enalapril vs control). In the enalapril-treated group median EPO concentration fell from 32 to 24 U/l at 90 days of treatment, whereas in conventionally treated patients median EPO was 34 U/l and 35 U/l, respectively (p less than 0.05 enalapril vs control). Neither glomerular filtration rate nor arterial blood pressure differed significantly in the two groups. Furthermore, there were no signs of bone marrow suppression, increased haemolysis or change in plasma volume. In conclusion, a decrease in Hb was found after start of treatment with enalapril in patients with progressive chronic renal failure, possibly caused by a suppression of EPO production.

Immunoreactive serum erythropoietin concentrations were measured in 35 patients with anaemia associated with active rheumatoid arthritis. Based on an evaluation of stainable iron in the bone marrow (marrow iron grade 0-4) and serum ferritin concentrations (concentrations less than or equal to 60 micrograms/l compatible with iron deficiency) the anaemia was found to be complicated by iron deficiency in 19/35 (54%) of the patients. The mean serum erythropoietin level (57.6 (SD) 27.3) U/l) was sufficiently raised for the degree of anaemia irrespective of the size of the marrow iron stores. Thus the data do not support the contention that suppressed secretion of erythropoietin is involved in the pathogenesis of anaemia of chronic disorders. There was a significant inverse correlation between the haemoglobin concentration and log serum erythropoietin in the patients with rheumatoid arthritis. In the patients with adequate iron stores, but not in the iron depleted patients, there was a tendency for serum erythropoietin concentrations to correlate positively both with C reactive protein and erythrocyte sedimentation rate. Red cell distribution width (mean (SD) 16.3 (1.8)%) was above normal (11.5-14.5%) both in the iron replete and the iron depleted patients, and the mean red cell distribution width values did not differ significantly among the two subpopulations. The plasma lactoferrin concentration (mean (SD) 137.6 (109.9) micrograms/l) was normal and did not differ significantly between the iron deficient patients and those with adequate iron.

Serum erythropoietin (EPO) concentrations were markedly depressed relative to the degree of anaemia in 10 patients with acute tubular necrosis, and remained low long after restoration of excretory renal function as estimated by glomerular filtration rate. Evidence is presented that the low serum EPO level is due to defective synthesis and not to increased catabolism. It is suggested that the predominantly are generatory anaemia found in prolonged cases of acute tubular necrosis, and the slow restoration of red cell mass during recovery, are due to the deficient synthesis of EPO. A positive erythropoietic response in a therapeutic trial with recombinant human erythropoietin (rhEPO) appears to support this hypothesis.

Single dose intravenous and subcutaneous pharmacokinetics of recombinant human erythropoietin (rhEPO) has been determined in 6 chronic haemodialysis patients. Four patients, all on maintenance therapy with rhEPO, were consecutively treated both intravenously and subcutaneously with injections of rhEPO in a dose of 50 U/kg. Two previously untreated patients received 150 U/kg of rhEPO intravenously and subcutaneously. After intravenous injection of 50 U/kg of rhEPO a mean serum half-life value of 5.4 +/- 0.9 hr was found. The corresponding half-life after injection of 150 U/kg was 7.6 hr. The peak concentration of serum EPO after subcutaneous injection was 12.5-20 times lower than the corresponding intravenous Cmax. After administration of 150 U/kg subcutaneously the absorption of EPO was monitored to completion at 120 hr. The complete bioavailability of subcutaneous rhEPO after injection of 150 U/kg was 31.7%. Whether this low and protracted subcutaneous absorption of rhEPO is accounted for by either impeded absorption or partial skin degradation of rhEPO is not known.

A sensitive competitive radioimmunoassay for the determination of circulating human erythropoietin (Epo) is presented. This RIA of human Epo is founded on the extensive application of a recombinant protein approach. Thus, highly purified recombinant human Epo is employed both as a tracer, immunogen and occasionally as standard in the RIA. The lower limit of detection is approximately 8 mU/ml and well below the mean value of Epo in normal individuals (25.6 +/- 6.6 mU/ml). The intra- and interassay variation are 6.3 and 9.2%, respectively. The documentation of consistent alterations in Epo plasma levels of selected categories of anemic patients establishes the clinical relevance of the immunoassay. The radioimmunoassay of human Epo fulfills reasonable requirements of a clinical routine analysis in terms of both specificity, sensitivity and practicability.

A human erythropoietin (Epo) cDNA fragment encoding the complete erythropoietin peptide sequence was fused to the 3'-end of the lacZ gene in the polylinker region of the high expression vector, pUR 278. Escherichia coli bacteria were transformed with the recombinant plasmid harboring the hybrid Epo-beta-D-galactosidase gene. After induction with isopropyl-thiogalactoside large amounts of the fusion protein, Epo-beta-D-galactosidase were synthesized in the transformed bacteria. The fusion protein was partially purified and shown to exhibit intact galactosidase enzymatic activity. Although no biological activity of the Epo counterpart of the fusion protein was detected both in an in vivo and in an in vitro bioassay, the fusion protein served as an effective antigen for the production of anti-erythropoietin antibodies. Antifusion protein antibodies raised in rabbits were shown to react with the intact human Epo molecule from erythropoietin producing culture supernatants. The affinity of these anti-fusion protein antibodies was sufficiently high to permit the development of a sensitive radioimmunoassay for human Epo. This fusion protein approach is a relatively straightforward and rapid method of generating antibodies with specificity for any protein encoded by a cloned eukaryotic gene.

A markedly anaemic, haemodialysis patient with a mixed deficiency of erythropoietin (EPO) and iron developed a life-threatening worsening of anaemia during an episode of severe rectal bleeding. Despite frequently recurring episodes of considerable ischaemic cardiac pain, the patient's religious conviction did not permit blood transfusion. Consequently, combination therapy with both recombinant human EPO (rhEPO) and oral iron was instituted. In this haemodialysis patient, the vitally significant replacement therapy of rhEPO and iron was followed by a steady and uncomplicated restoration of subnormal hematocrit and complete disappearance of cardiac ischaemia.

Production of immuno and biologically active erythropoietin was documented to occur in the human hepatoblastoma cell line HepG-2. The expression of the erythropoietin gene was further verified by Northern blot analysis using a single stranded RNA probe. In vitro studies showed that erythropoietin production by these cells was not stimulated by hypoxia or cobalt chloride, but was related to the proliferative activity of the cells in culture. In addition it was found that the secretion of erythropoietin was almost completely abrogated by tunicamycin, an inhibitor of N-linked glycosylation. This effect of tunicamycin was also observed in a permanently transfected cell line that secretes erythropoietin in large quantities.

An open non-randomized clinical trial with recombinant human erythropoietin (rhEpo) was conducted in 10 chronic haemodialysis patients with considerable iron overload. Pretreatment serum erythropoietin levels were significantly decreased for the degree of anaemia (22.0 +/- 7.6 mUnits (U) ml-1). Initially all patients received 120 U kg-1 of rhEpo intravenously three times a week and after a period of 45 days the haematocrit rose from 25 +/- 3 to 36 +/- 5. A reduced dose of 30 U kg-1 was then instituted which stabilized the haematocrit at this level. The development of anti-erythropoietin antibodies to rhEpo could not be demonstrated. Endogenous serum erythropoietin levels remained unchanged during therapy with rhEpo. The serum ferritin concentration, which was initially 3118 +/- 1556 micrograms l-1, was significantly reduced to 2203 +/- 1299 micrograms l-1 after an 80-d treatment period. The side-effects in three patients included the occasional sensation of increased body heat without fever. In one previously hypertensive patient the antihypertensive therapy had to be temporarily increased, but otherwise both the mean diastolic and systolic blood pressure remained constant during the observation period. No clotting of arteriovenous fistulas occurred.

This study provides clear documentation of in vivo biogenesis of erythropoietin (Epo) by a human renal carcinoma. A middle-aged woman with a clear cell renal carcinoma of the left kidney developed severe polycythemia. This polycythemia was accompanied by markedly elevated levels of immunoreactive erythropoietin both in the peripheral venous blood, and in blood derived from the left renal vein during nephrectomy. Exstirpation of the non-invasive renal carcinoma was followed by complete restoration of both hematocrit and erythropoietin plasma concentration to normal levels. The fall in plasma erythropietin concentration immediately after nephrectomy (T/2 less than or equal to 3 hours) was probably a valid representation of plasma erythropoietin metabolism in this patient. Direct evidence of erythropoietin production in individual renal carcinoma cells was provided by immunoperoxidase studies demonstrating focal cytoplasmatic accumulation of immunoreactive erythropoietin in the tumor cells.