Erythropoietin.

Maintenance of the red cell volume is a fundamental aspect of ensuring oxygen supply to the tissues. The balance between the very dynamic processes of erythropoiesis and erythrocyte loss is precarious and yet normal individuals experience a remarkably constant haematocrit. This is achieved by a very elegant and sensitive homeostatic mechanism which links tissue oxygen delivery to red cell production. The glycoprotein hormone erythropoietin (EPO) is the principle controller of this process. It is now clear that even minor underproduction of EPO will result in anaemia. The most widespread example of this is the anaemia of end-stage renal failure. The pharmacological use of recombinant human EPO (rHuEPO) in this setting is now well established and has had a dramatic impact on the quality of life of patients with renal disease. With the more widespread use of EPO in other clinical conditions and the advent of novel therapeutic approaches, this is an opportune moment to review the physiology and patho-physiology of this fascinating and essential hormone.

Patients with pulmonary and cardiac disease show an elevated proportion of immature reticulocytes.

The value of reticulocyte analysis has been largely confined to classification of anaemia. In the present study, we have investigated the value of undertaking automated fluorescent reticulocyte analysis (Abbott Cell-Dyn 4000) in patients with pulmonary or cardiac diseases. A control group of nonanaemic (n = 367) and anaemic patients (n = 57) was established and thereafter compared with a group of patients with pulmonary disease (172 without anaemia, 92 with anaemia) and another group with cardiac disease (520 without anaemia, 254 with anaemia). The Hb level and reticulocyte RNA content (as measured by the immature reticulocyte fraction, IRF) of the study subjects were inversely correlated (r = -0.41). The mean IRF of nonanaemic patients with pulmonary (mean = 0.331, SD=0.124) and cardiac disease (mean = 0.266, SD = 0.079) were both significantly higher (P < 0.05) than in control subjects (mean = 0.220, SD = 0.062). Nonanaemic and anaemic patients with cardiac disease both showed significantly higher (P < 0.05) reticulocyte percentage counts than the equivalent anaemic and nonanaemic controls. Each of these phenomena can all be attributed to hypoxia mediated EPO production. Our observation that 35% of patients with pulmonary disease have elevated levels of IRF, may prove useful in screening for tissue hypoxia in nonanaemic patients. Furthermore, serial monitoring of the IRF may prove valuable in observing the efficacy of therapy in these patients.

Red cell ferritin, a marker of iron deficiency in hemodialysis patients.

Estimation of red cell ferritin (RCFer) may give a good indication of iron supply to the erythron and it may therefore be clinically useful for the detection of functional iron deficiency. In a cross-sectional study of hemodialysis patients on erythropoietin (EPO) therapy and regular oral iron we have compared the RCFer levels with conventional indicators of iron status. The patients studied, 19 female, 48 male, mean age 62 +/- 3.6 years (range 20-83 years) were characterized by the following mean parameters: aluminum 1.24 +/- 0.12 mumol/L, PTH 115.7 +/- 39 pg/mL, vitamin B12 626 +/- 71.2 ng/L, serum folate 18.8 +/- 2.2 micrograms/L, and hemoglobin 9.8 +/- 0.3 g/dL (range 7.3-12.4). The median serum ferritin (SF), RCFer, total iron binding capacity (TIBC), transferrin saturation (TS), and serum iron were 68 micrograms/L, 14.1 ag ferritin/red cell, 57 mumol/L, 20% and 11.5 mumol/L, respectively. Eleven patients had a reduced RCFer (< 7 ag ferritin/red cell), 5 had a SF of < 15 micrograms/L and 22 a TS of < 16%. The occurrence of functional iron deficiency was suggested by the presence of 10 subjects with reduced RCFer despite normal SF levels (15-240 micrograms/L). Four patients with reduced SF showed acceptable levels of RCFer, suggesting that some patients may maintain an adequate iron supply despite diminished iron stores. Despite oral iron therapy, a significant number of patients (63%) on regular hemodialysis remain relatively iron deficient with a serum ferritin of less than 100 micrograms/L. It has previously been proposed that oral iron provides adequate supplementation during increased demand caused by EPO stimulation. The present study has demonstrated overt iron deficiency in five subjects and suggests functional iron deficiency in a further seven (22% of total patients). We therefore conclude that oral iron therapy cannot maximize the response to EPO.

Re-establishment of erythropoietin responsiveness in end-stage renal failure following renal transplantation.

Re-establishment of erythropoietin (EPO) secretion following renal transplantation is poorly understood. The development of sensitive EPO radioimmunoassay has enabled further study of this phenomenon. Forty-one adult patients were studied during the first 16 weeks following renal transplantation. Twenty six received cyclosporin monotherapy and 15 also received prednisolone and azathioprine. Serum creatinine, haemoglobin (Hb), ferritin and EPO were assayed pre-operatively, daily for 1 week, weekly for 1 month, and fortnightly to 16 weeks. An expected EPO value, for any Hb level, was derived by linear regression analysis in 144 patients with iron deficiency anemia. An observed to expected ratio (O/E) was calculated, a value of 1.0 implying appropriate responsiveness. Hb increased from 8.6 +/- 2.0 (SD) to 12.3 +/- 2.1 g/dl (p < 0.001) over 16 weeks, an increase unaffected by ferritin status. Mean EPO concentration increased during the first week with a peak at day 4 (22.1 +/- 13.3 to 44.6 +/- 40.0 mu/ml, p < 0.05), a change apparent only in patients with immediate graft function (24 cases). There was no correlation between EPO and Hb pre-operatively; however a significant inverse relationship was established by week 16 (r = -0.404, p < 0.02). The median O/E ratio (0.22) at baseline increased progressively to 1.0 at 16 weeks (p < 0.001); ratios were significantly greater in the immediate versus delayed function group throughout (p < 0.05). In the former group an O/E ratio of 1.0 was reached at 10 weeks when mean serum creatinine was 142 +/- 48 mumol/l. Patients with poor ongoing renal function (9 cases, serum creatinine > 250 mumol/l at 16 weeks) had impaired Hb recovery (10.1 +/- 1.6 vs 12.7 +/- 2.0 g/dl at 16 weeks, p < 0.05). EPO values were not different in those patients but median O/E ratios were significantly depressed (p < 0.05) throughout, the maximum O/E ratio being 0.75. Recovery of renal function is accompanied by a beneficial Hb response driven by EPO synthesis in the transplant. The O/E ratio provides a useful index to assess EPO responsiveness. Appropriate secretion was achieved during the first 4 months and optimized by immediate and satisfactory graft function.

Quantitative reticulocyte analysis may be of benefit in monitoring erythropoietin treatment in dialysis patients.

Reticulocyte responses to low-dose erythropoietin (EPO) were monitored using automated flow cytometric analysis. Sixteen adult dialysis patients were treated with 1,000 U of recombinant human EPO (rHuEPO), subcutaneously, thrice weekly (mean dose 15.7, SD 3.7 U/kg). The reticulocyte count (baseline 31.1, SD 19.1 x 10(9)/L) increased in 14 patients in the first week, with a peak response occurring at Week 2 (mean 57.3, SD 26.5 x 10(9)/L, p < 0.01). There was a wide spectrum of response, the maximal absolute increment ranging from 6.8-69.7 x 10(9)/L (maximal percentage increase 19-863%). Overall there was no relationship between the early increment in reticulocyte count and hemoglobin (Hb) response over the ensuing 4 months. Nine patients became transfusion independent (mean Hb increasing from 6.9, SD 0.8-9.2, SD 1.2 g/dl). Two patients had poor reticulocyte increments and no significant change in Hb. The remaining 5 patients responded partially with a brisk reticulocyte response and a marked reduction in transfusion dependency, but without a sustained increase in Hb. On investigation, all had gastrointestinal bleeding (melena in 1, commencing after initiation of treatment, positive fecal occult bloods in 4), whereas none of the other patients showed evidence of blood loss. It is notable that the erythron was sensitive to this modest dose of rHuEPO in the majority of patients as evidenced by the reticulocyte response. The results provide useful information in the management of patients on rHuEPO. A small or inapparent reticulocyte response suggests a confounding factor; a poor Hb response in the presence of active reticulocyte synthesis points to occult blood loss or hemolysis.

The effect of cytokines on the in-vitro erythropoietin bioassay.

The effects of various concentrations of granulocyte macrophage colony stimulating factor (GM-CSF), gamma interferon (gamma IFN) and interleukins 1 alpha (IL-1 alpha), 1 beta (IL-1 beta) and 3 (IL-3) on the anaemic mouse spleen cell bioassay for erythropoietin (EPO) were investigated. Addition of IL-3 and GM-CSF at various concentrations had no effect on EPO stimulated 3H thymidine incorporation. However the addition of IL-1 alpha, IL1-beta and gamma IFN (3.3 x 10(-8) gl-1) caused a significant (P < 0.01) inhibition of EPO stimulated thymidine incorporation. This suggests that the EPO bioassay may be influenced by variable levels of some inflammatory cytokines in serum. Previous studies have shown that the bioassay is influenced by serum transferrin levels and thus serum immunoassays remain the technique of choice for specific estimates of EPO. Since EPO bioassays are not specific, they should be reserved for situations in which an estimate of the total erythropoietic activity of serum is required.

Reduced erythropoietin response to anaemia in elderly patients with normocytic anaemia.

We studied the effect of age on the relationship between haemoglobin and serum erythropoietin (EPO) levels in anaemic patients. 568 patients over 70 years of age were compared with 137 patients under 70 and a reference group of 144 patients of all ages with proven iron deficiency. EPO was measured using a radioimmunoassay. We found that elderly patients with a normocytic anaemia (N = 375) had a statistically lower EPO response than younger patients with normocytic anaemia (N = 61) (p < 0.05) or patients of all ages with iron-deficiency anaemia (p < 0.05). There was no difference between the sexes. Elderly patients with microcytic or macrocytic anaemia had a normal EPO response as compared to the "gold standard" of iron deficiency. These findings suggest that a proportion of elderly patients with normocytic anaemia has an impaired EPO response.

Anaemia in children following cardiac transplantation: treatment with low dose human recombinant erythropoietin.

Anaemia is common in children following cardiac transplantation. In a series of 5 children with anaemia beyond the immediate post-operative period one had a hypochromic, microcytic anaemia which corrected with oral iron. The other four had normochromic, normocytic anaemias unresponsive to iron or folate supplementation and associated with inappropriately low levels of erythropoietin. Subcutaneous administration of low dose human recombinant erythropoietin to these four patients resulted in correction of their anaemia. Our findings suggest that erythropoietin deficiency is an important cause of anaemia in transplant recipients and should be sought in cases of anaemia refractory to conventional haematinic therapy. In cases of proven erythropoietin deficiency, treatment with erythropoietin is effective, acceptable to patients and preferable to repeated blood transfusion.

Renal haemodynamic, hormonal and excretory effects of low-dose atrial natriuretic factor infusion in renal transplant recipients.

1. In experimental studies, activation of renal sympathetic nerves attenuates the natriuretic response to atrial natriuretic factor. We therefore investigated the response to low-dose infusion of atrial natriuretic factor in renal transplant recipients. 2. Eight male cyclosporin-treated renal transplant recipients received human-alpha atrial natriuretic factor (1-28) at a dose of 1.2 pmol min-1 kg-1 or placebo for 2 h in a placebo-controlled, randomized, cross-over study. The plasma atrial natriuretic factor concentration rose from 18.5 to 49.2 pmol/l in association with an immediate natriuresis (a rise of 49.1 mumol/min in the first 30 min, P less than 0.05; peaking at a 61% increase from baseline, P less than 0.01), diuresis (from 3.37 to 7.46 ml/min) and a threefold rise in urinary cyclic GMP excretion. 3. In response to infusion of atrial natriuretic factor, the packed cell volume rose by 4.2% (P less than 0.001) and the filtration fraction by 5% (from 22 to 27%, P less than 0.05), but there was no significant change in renal plasma flow, glomerular filtration rate or mean arterial blood pressure. Likewise, the plasma catecholamine concentrations, plasma renin activity and serum erythropoietin concentration remained unchanged. 4. The sensitivity of the renal allograft to plasma atrial natriuretic factor concentrations in the high physiological range suggests a role for endogenous atrial natriuretic factor in the modulation of graft function. Furthermore, the immediate natriuretic response to atrial natriuretic factor in the effectively denervated transplant kidney, in contrast to the delayed response seen in normal subjects, may imply that sympathetic nerves have an inhibitory effect on the renal response to atrial natriuretic factor in normal man.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in serum erythropoietin levels during allogeneic bone marrow transplantation.

Serial serum erythropoietin levels were measured in 10 consecutive patients undergoing allogeneic bone marrow transplantation. Observed erythropoietin levels are compared with those predicted from a large control population of anaemic patients not receiving chemotherapy. There was an initial acute rise in serum erythropoietin, peaking between days 1 and 4 after marrow transfusion, which was unrelated to changes in haemoglobin concentration. Patients maintained serum erythropoietin concentrations at around twice the predicted level for the first 2 weeks following transplantation, with a gradual fall into the expected range by wk 3. Erythropoietin levels did not change with episodes of bacterial infection or acute graft-versus-host disease. A patient with severe aplastic anaemia had initial successful engraftment with normalisation of erythropoietin levels, but showed a marked and amplified rise in erythropoietin 2 wk before falling peripheral blood counts indicated failure of the bone marrow graft.

Storage and preparation of samples for erythropoietin radioimmunoassay.

The evaluation of a radioimmunoassay for erythropoietin developed using recombinant material as immunogen and radiotracer is presented. A series of serum samples prepared and stored under varying conditions showed that immunoreactive erythropoietin levels were stable at room temperature for at least 10 days and at -20 degrees C for 5 months. The optimum time for separating sera from samples was between 6 and 24 h after venepuncture. Serum EPO values were significantly higher than those measured in heparin or potassium EDTA plasma.

Erythropoietin deficiency in acute renal failure.

Erythropoietin (Epo) was sequentially measured by radioimmunoassay in 11 patients with acute renal failure (ARF) of varied aetiology. Epo rapidly decreased to a level inappropriately low for the haemoglobin, the reduced Epo value persisting throughout the oliguric phase and for up to 2 weeks after the restoration of apparently normal renal function. Epo values found in ARF were: at referral 18.2 +/- 9.5, mid-oliguria 14.4 +/- 6.8, diuresis 15.6 +/- 5.8, and recovery 25.1 +/- 15.8 mU/ml. Results are compared with 34 patients with end-stage chronic renal failure, 42 with non-renal anaemia, and 96 normal subjects. Epo deficiency alone is an inadequate explanation of the rapid reduction in haemoglobin at the onset of ARF, but would appear to be an important factor in the maintenance of anaemia in prolonged ARF and accounts for the slow increase in haemoglobin following recovery.