Acute effects of recombinant human erythropoietin on plasma levels of proendothelin-1 and endothelin-1 in haemodialysis patients

Background: The pathogenesis of rHuEpo-induced hypertension in haemodialysis (HD) patients still remains uncertain. Endothelin-1 (ET-1) is produced from proendothelin-1 (proET-1) by an endothelin-converting enzyme. Since proET-1 is known to have approximately 1/100 the potency of ET-1 for contracting an isolated blood vessel, the change in the activity of endothelin-converting enzyme (ECE) has been proposed as an important factor in the pathophysiology of various hypertensive diseases. However there is no report on whether a change in the rate of conversion of proET-1 to ET-1 may be involved in the pathogenesis of rHuEpo-induced hypertension. The purpose of this study was to ascertain the potential role of ECE in the development of rHuEpo-induced hypertension. Methods: The levels of plasma erythropoietin, proET-1, ET-1, and mean arterial blood pressure (MAP) were measured following a single dose of rHuEpo (100 U/kg) in HD patients with 24-h ambulatory blood pressure monitoring. Different routes of administration (19 intravenous group, 10 subcutaneous group) were compared to a placebo-injected control group (10 HD patients). Results: Plasma erythropoietin levels reached maximal value 5 min after i.v injection of rHuEpo (13.1±2.4 vs 2780.9±290.1 mU/ml, P<0.01), whereas it was 6h in the s.c. group (14.7±3.8 vs 38.8±17.7 mU/ml, P<0.05). A significant increase in MAP was noted 30 min after rHuEpo injection, which lasted for 3 h in the i.v. group. However, no significant changes in MAP were noted in patients given rHuEpo subcutaneously. Both the plasma concentrations of proET-1 and ET-1 started to increase from 10 min after i.v. rHuEpo administration, with the pro-ET-1 reaching a peak level at 30 min (13.5±7.4 vs 21.6±3.8 pg/ml, P<0.05) and the ET-1 at 1 h (4.2±2.6 vs 9.9±4.8 pg/ml, P<0.05). In patients with significant interdialysis hypertension following a single i.v. injection of rHuEpo, the molar ratio of ET-1 over proET-1 (ET-1/proET-1) was significantly higher than in patients without hypertension. In addition, the increase in ET-1 levels was significantly greater in patients with interdialysis hypertension, while changes in proET-1 level were similar in both hypertensive and non-hypertensive groups. Changes in interdialysis MAP (Dgr;IDMAP) was significantly correlated with &Dgr;ET-1 during the interdialysis period, but not with &Dgr;proET-1. Conclusion: Differences in ET-1/proET-1 ratio in relation to changes in MAP after a single intravenous administration of rHuEpo suggest a potential role for ECE in the pathogenesis of rHuEpo-induced hypertension. Key words: endothelin-1; endothelin-converting enzyme; haemodialysis; proendothelin-1; rHuEpo-induced hypertension      

Desferrioxamine improves burst-forming unit-erythroid (BFU-E) proliferation in haemodialysis patients

Background: In chronic renal failure, desferrioxamine (DFO) may improve erythropoiesis independent from its aluminium (Al) chelating effect. The mechanism of this action is still unknown. Methods: To verify whether DFO influences proliferation of erythropoietic precursors, we studied 10 patients on chronic haemodialysis, free from malignancies or other haematological diseases, iron deficiency, bone marrow fibrosis, and Al toxicity. Al accumulation was excluded by the DFO test. Peripheral blood samples were drawn for basal burst-forming unit-erythroid (BFU-E) assay. Mononuclear cells were isolated by density gradient centrifugation with Ficoll-Hypaque, and incubated for 15 days with three different experimental conditions: (a) low-dose recombinant human erythropoietin (rHuEpo) (3 U/ml); (b) high dose rHuEpo, (30 U/ml); (c) both DFO (167 &mgr;g/ml) and rHuEpo (3 U/ml). We determined TIBC, transferrin, ferritin, reticulocytes, hypochromic erythrocytes, soluble transferrin receptor (sTR), haemoglobin (Hb), and haematocrit (Hct) at baseline and then every 14 days. Patients received 5 mg/kg DFO infused during the last hour of each dialysis session for 6 weeks; six patients remained in the study for an additional 6 more weeks. BFU-E assays were set up after 6 and 12 weeks of DFO therapy. Results: At baseline DFO had small effect on BFU-E proliferation (33.9±25 vs 30.4±25.9) and high-dose rHuEpo had a significant effect (45.15±27 vs 30.4±25.9, P<0.01). After 6 weeks of DFO therapy a significant increase in BFU-E proliferation was observed in all culture conditions (78.25±32 vs 30.45±25.9 standard culture, P<0.01; 110.9±30 vs45.15±27 high dose rHuEpo, P<0.01; 98.75±32 vs 45.15±27 DFO culture, P<0.01). Moreover, the increase in BFU-E proliferation was significant greater with DFO culture than standard culture (P<0.01). The same trend was found at the third BFU-E assay, performed in only six patients, when all culture conditions showed a further increase of erythroid precursor proliferation. However, the DFO culture was not significantly greater than the standard culture, while the high-dose rHuEpo was significantly greater than the DFO culture. Patients in group 1 (n=10), had a significant increase in reticulocytes (1.5±0.6 vs 1.72±0.3, P<0.01) and of hypochromic erythrocytes (HE) (5.6±5.1 vs 14.4±12.7, P<0.01), while sTR, Epo, Hb, and Hct were only minimally increased. Ferritin decreased significantly (448±224 vs 196±215, P<0.01) and TIBC and transferrin were unchanged. Conclusions: Thus DFO increases erythroid activity by BFU-E proliferation and increases reticulocytes in haemodialysis patients. Such an effect may be related to increased iron utilization. DFO may be a useful tool for anaemic patients with good iron stores and without Al overload. Key words: desferrioxamine; erythroid progenitors; erythropoiesis; haemodialysis      

High dose enalapril impairs the response to erythropoietin treatment in haemodialysis patients

Background: The resistence to recombinant human erythropoietin (rHuEpo) therapy in haemodialysis (HD) patients has multifactorial aetiologies; erythropoietin insufficiency, dialysis insufficiency, iron deficiency, and secondary hyperparathyroidism. Angiotensin-converting enzyme (ACE) inhibitors induce anaemia in patients with essential hypertension, congestive heart failure, chronic renal insufficiency, and renal transplants. Data exist suggesting that ACE inhibitors impair erythropoiesis in HD patients. Therefore the aim of this study was to investigate the impact of enalapril on rHuEpo requirement. Methods: In the present prospective non-randomized study of 12 months, we compared the effects of enalapril and nifedipine on rHuEpo requirement in 40 hypertensive patients receiving rHuEpo for more than 6 months on maintenance haemodialysis. Twenty normotensive rHuEpo-dependent patients served as a control group. All patients with severe hyperparathyroidism or iron deficiency were excluded. The mean (±SD) haemoglobin concentration was >10 g/dl in all groups. The mean weekly rHuEpo dose increased in the enalapril group (P<0.0001 vs before) and remained constant in the nifedipine and control groups (P=NS vs before). Statistically, there was no differences with regard to iPTH levels, dialysis parameters, iron status, and underlying renal diseases among all groups. Conclusion: High-dose enalapril increases rHuEpo requirement and should be reserved for dialysis patients with hypertension uncontrollable with other antihypertensive medications or dialysis patients with cardiac failure.     

The impact of early normalization of haematocrit by erythropoietin on renal damage in the remnant kidney model

Background: Correction of anaemia in moderate to advanced renal failure is still a matter of debate because of postulated detrimental effects of erythropoietin on the progression of renal damage. Methods: The renal effects of early normalization of haematocrit (Htc) by erythropoietin (rHuEpo) were investigated from the time of 5/6 nephrectomy up to 8 weeks post-intervention in three groups of remnant kidney model rats: untreated controls (CON), rats receiving 100 UI/kg body-wt of rHuEpo i.p. twice a week (EPO), and rats receiving rHuEpo in which periodic phlebotomies maintained Htc similar to the value of the control group (PHL). The latter group was included to evaluate the direct effects of rHuEpo on renal damage, i.e. independent from Htc correction. Results: Two weeks after renal ablation (basal), Htc decreased in CON and PHL (from 49.3±1.4% to 43.2±1.1, P<0.05 and from 49.6±1.1 to 43.3±1.5% P<0.05 respectively), while it remained consistently normal in EPO rats (78.9±1.2% to 48.8±1.5%, P<0.05 vs other groups). Thereafter Htc did not change throughout the remaining period in all groups. At the end of the study, with respect to basal, resting blood pressure increased significantly by the same extent in CON (+13±2%) and EPO rats (+15±5%), while it remained constant in PHL rats. Notably, creatinine clearance significantly decreased in CON (-53±8% vs basal) and EPO (-38±8% vs basal), while it did not change in PHL rats. Likewise the degree of proteinuria as well as renal morphologic alterations and glomerular hypertrophy/sclerosis was similar in CON and EPO rats, and was significantly more severe than in the phlebotomized group. The only difference detected between CON and EPO group was the greater mesangial hypercellularity in rHuEpo-treated rats. Conclusion: In uraemic rats, chronic treatment with rHuEpo aimed at normalization of Htc beginning the early stage of renal failure does not inevitably account for a rise in systemic blood pressure. In addition, neither erythropoietin per se nor the correction of haematocrit accelerates the progression of renal damage when blood pressure remains constant.     

The required dose of erythropoietin during renal anaemia treatment is related to the degree of impairment in erythrocyte deformability

Background: Renal anaemia is rapidly corrected by recombinant human erythropoietin (rHuEpo) therapy, but the dose required varies greatly. Since impaired erythrocyte deformability may be one factor contributing to the development of renal anaemia, the interrelationship between that variable and the rHuEpo requirement was examined. Methods: Twenty-five patients treated with hemodialysis and rHuEpo for at least 6 months were included in the study. The Hb value had been stable and the rHuEpo dose unchanged the last two months. Using a rotational viscometer, the fluidity of erythrocytes, separated from plasma and re-suspended in isotonic buffered saline to a standardized haematocrit, was taken as a measure of erythrocyte deformability. Results: The average weekly dose of s.c. epoetin alpha was 186±93 U/kg body weight (range 56-370). The dose was correlated to the reticulocyte fraction (R-0.69, P=0.0001). When the rHuEpo dose was used as dependent variable and blood haemoglobin concentration, serum (S) albumin, S ferritin, S aluminium, S PTH, S urea, Kt/V/week, erythrocyte fluidity, and plasma viscosity were used as independent variables in a stepwise multiple regression analysis, only erythrocyte fluidity remained significantly negatively correlated to the rHuEpo dose (R=0.5, P=0.01). Despite a tendency towards higher doses of rHuEpo in patients with a C-reactive protein concentration exceeding 20 mg/l, the Hb was lower in these patients. Conclusions: We conclude that the interindividual differences in bone marrow response to rHuEpo were small in these patients. Impaired erythrocyte deformability and inflammation seem to be factors associated with increased rHuEpo requirement.     

Does long-term treatment of renal anaemia with recombinant erythropoietin influence oxidative stress in haemodialysed patients?

Background. Patients with end-stage renal failure undergoing haemodialysis (HD) are exposed to oxidative stress. Increased levels of malondialdehyde (MDA) were demonstrated in plasma of uraemic patients, indicating accelerated lipid peroxidation (LPO) as a consequence of multiple pathogenetic factors. The aim of our investigation was to examine the role of renal anaemia in oxidative stress in HD patients. Methods. MDA and 4-hydroxynonenal (HNE) were measured in three groups of patients undergoing HD: group I comprised eight patients with a blood haemoglobin (Hb) <10 g/dl (mean Hb=8.1±1.3 g/dl), and group II were eight patients with a Hb <10 g/dl (mean Hb=12.4±1.9 g/dl); none of these 16 patients had been treated with human recombinant erythropoietin (rHuEpo). Group III comprised 27 patients with a mean Hb of 10.5±1.6 g/dl after long-term rHuEpo treatment. Results. Mean plasma concentrations of both MDA and HNE were significantly higher (P<0.0001) in all 43 HD patients than in 20 healthy controls (MDA 2.85±0.25 vs 0.37± &mgr;M, HNE 0.32± vs 0.10±0.01 &mgr;M). Comprising the three groups, it was shown that HD patients with a Hb <10 g/dl had significantly higher plasma levels of LPO products (MDA 3.81±0.86 &mgr;M, HNE 0.45±0.07 &mgr;M) than HD patients with a Hb > 10 g/dl (MDA 2.77±0.58 &mgr;M, HNE 0.25±0.05 &mgr;M), and than HD patients treated with rHuEpo (MDA 2.50±0.12 &mgr;M, HNE 0.29±0.03 &mgr;M). Furthermore, an inverse correlation between plasma concentration of LPO products and haemoglobin levels was seen (r=0.62, P<0.0001). Conclusion. Radical generation in HD patients might be caused in part by renal anemia itself. Treatment with rHuEpo may decrease radical generation effectively in HD patients due to the increase in the number of red blood cells and blood haemoglobin concentration. Keywords: erythropoietin; haemodialysis; HNE; lipid peroxidation; MDA; renal anaemia      

Changes of endogenous erythropoietin level and iron status during a 30-month hemodialysis treatment of a group of patients

In our earlier paper [1] we found that among 50 hemodialysis patients(HD pts) 48% (24 pts) control anemia with hemoglobin (Hb)concentration >9.5 g/dl and hematocrit (Hct) >30%without recombinant human erythropoietin (rHuEpo) therapy. These HD ptshad significantly higher mean endogenous erythropoietin (eEpo) level andlower iron reserves (IR) than HD pts who need rHuEpo therapy. The aim ofthis study was to judge whether the possibility to control anemia in ptsnot requiring rHuEpo therapy changes during a 30-month HD treatment.Serum eEpo and ferritin were measured every 6 months. After 30 months ofHD treatment 18 pts remained in this group – 5 pts died, Iunderwent transplantation. During the study period 4/18 ptspermanently had a very low level of eEpo (under detection limit),7/18 had the level of eEpo within normal range for healthy control,7/18 pts had a high level of eEpo (up to 3 times higher than themean for healthy control). Pts who had the highest level of eEpo had thelowest IR. After 30 months IR were significantly lower than at thebeginning of observation (292 ± 87 vs 143 ± 127 mg).Important negative correlation between eEpo and IR was observedthroughout the whole period of study: r = –0.4820,p < 0.02 at the start of the study, and r =–0.6126, p < 0.007 after 30 months of treatment. Thestudy shows that the possibility to control anemia in pts not treatedwith rHuEpo did not change significantly during 30 months of HDtreatment. Endogenous Epo level in HD pts not treated with rHuEpo variedbetween different pts: it was permanently low in some pts, permanentlyhigh in others and stayed normal in remaining pts.     

Iron absorption in erythropoietin-treated haemodialysis patients; effects of iron availability, inflammation and aluminium

Background: The response to recombinant human erythropoietin (rHuEpo) is determined primarily by the availability of iron. In contrast to i.v. iron, oral iron supplementation is often insufficient for an optimal response. Method: We studied iron absorption and the effects of iron status, aluminium status and inflammation in 19 chronic haemodialysis patients on maintenance rHuEpo therapy. Iron mucosal uptake after 24 h, iron retention after 2 weeks and mucosal transfer of iron were determined with a whole-body counter using an oral dose ⁵⁹Fe. Iron absorption was measured once without, and once after the ingestion of 2 g aluminium hydroxide. Results: On the basis of transferring saturation, two groups of dialysis patients were distinguished: a group with a functional iron deficiency (n=9), and an iron-deficient dialysis patients group, mucosal uptake, mucosal transfer, and iron retention were 49.9%±29.4, 0.73±0.29, and 41.6%±32.2, being significantly lower than in a non-uraemic iron deficient population (P <0.01, P <0.05, P <0.01 respectively). In the iron-replete dialysis patients group, mucosal uptake, mucosal transfer, and iron retention were 20.0±12.3, 0.59±0.18, and 11.1±6.7, mucosal uptake and iron retention being lower than in a normal iron-replete population (P <0.0005 and P <0.003 respectively). Dialysis patients with high C-reactive protein (CRP) values showed lower iron absorption. Iron absorption data correlated significantly with transferrin saturation and CRP in the iron-deficient group, and with serum ferritin in the iron-replete group. Iron absorption decreased after an aluminium hydroxide challenge in the iron-deficient patients to the lower levels of the iron-replete subjects. Body aluminium stores, estimated by the desferrioxamine test, did not correlate with parameters of iron absorption. Conclusion: The absorption of iron in dialysis patients is decreased in haemodialysis patients, which may, at least in part, be due to inflammation. Aluminium ingestion further reduces absorption in functional iron-deficient patients. Key words: anaemia; erythropoietin; iron absorption; haemodialysis      

Importance of iron supply for erythropoietin therapy

BACKGROUND.: rHuEpo and iron therapy corrects renal anaemia. However, dosage,route of administration, and monitoring of iron and rHuEpo therapyin uraemic patients remains controversial. METHODS.: Therefore a 22-month i.v. iron substitution trial, subdividedinto four study periods, was initiated in 64 iron-depleted chronichaemodialysis (HD) patients receiving i.v. rHuEpo therapy. Withinthe first period (6 months) patients were treated with high-doseiron (100mg at the end of HD treatment, mean cumulative i.v.iron saccharate dosage was 2538±810 mg per patient) inorder to replete the iron stores. During the 2nd period (6 months)the available iron pool was maintained with low-dose iron byadministration of 10, 20, or 40 mg iron at each HD, dependingon haemoglobin, serum ferritin and transferrin saturation levels.During the 3rd period (4 months), the iron-replete patientswere randomized to i.v. or s.c. route of rHuEpo administration.During the 4th period (3 months) iron substitution was omittedto exclude severe iron overload. RESULTS.: In the first study period, high-dose iron therapy dramaticallyreduced the weekly rHuEpo requirement by 70% of the initialdose (from 217±179 to 62.6±70.2 U/kg/week). Inthe 2nd period iron storage pools were easily maintained. Serumferritin and transferrin saturation levels remained stable duringthis study period. Randomization for thrice-weekly i.v. or s.c.administration of rHuEpo in the 3rd study period revealed comparableefficacy for both administration routes in iron-replete patients.In well-nourished patients (serum albumin >40 g/1) withouthyperparathyroidism (parathyroid hormone levels < 100 pg/ml),50–60 U/kg/week rHuEpo were required in contrast to >100 U/kg/week in patients with hyperparathyroidism. In the 4thstudy period, withdrawal of iron administration led to a rapiddecrease of serum ferritin and transferrin saturation levels,indicating the absence of severe iron overload. CONCLUSIONS.: Long-term thrice-weekly i.v. low-dose iron therapy (10–20mg per HD treatment) proved to be a very effective, economicaland safe treatment schedule for iron-replete HD patients. Intravenousand s.c. rHuEpo therapy was equally efficacious in iron-replete,well-nourished patients. HD patients with increased parathyroidhormone levels require significantly more rHuEpo than HD patientswith parathyroid hormone levels values <100 pg/ml).     

Single-dose Pharmacokinetics of Recombinant Human Erythropoietin in Patients with Various Degrees of Renal Failure

The pharmacokinetic profile of recombinant human erythropoietin(rHuEpo) was studied after a single intravenous dose of 150U/kg in ten patients with various degrees of renal function:group I, creatinine clearance >80 ml/min, n=2; group II,creatinine clearance 10–50 ml/min, n=6; group III, creatinineclearance <3 ml/min (patients undergoing haemodialysis) n=2.Erythropoietin concentrations in serum and urine samples obtainedover 48 h were measured by RIA. rHuEpo was cleared from circulationin an exponential fashion, the half-life ranged from 6.5 to12.7 h (mean 9.03 h) and was not different between the groups.The apparent volume of distribution varied from 0.041 to 0.0991/kg(mean 0.0701/kg) this corresponds to 1.5 times the plasma volumeand was unrelated to kidney function. Renal clearance (groupsI, II) accounted for less than 3% of total body clearance, bothparameters were unaffected by decreasing renal function. These results indicate that, in accordance with animal data,the elimination of rHuEpo occurs mainly through non-renal mechanisms.     

Influence of long-term recombinant human erythropoietin (rHuEpo) therapy on plasma leptin and neuropeptide Y concentration in haemodialysed uraemic patients

Background: In patients with chronic renal failure, rHuEpo therapy ameliorates anaemia and improves wellbeing, exercise tolerance, and appetite. Both leptin and neuropeptide Y play an important role in regulation of appetite and energy balance in humans. Methods: The present study aimed to assess the influence of 12 months rHuEpo therapy on plasma leptin and neuropeptide Y concentrations in 15 haemodialysed patients (HDP) (6F, 9M; mean age 4.8±2.9 years; mean BMI 23.6±1.1 kg/m²; mean duration of HD 3.3±0.6 months) (Epo group). A second group (no-Epo group) consisted of 17 HDP (9F, 8M; mean age 44±3.2 years; mean BMI 24.3±1.0 kg/m²; mean duration of HD 2.5±0.4 months not treated with rHuEpo for 12 months. Basal plasma leptin and neuropeptide Y concentrations were estimated by RIA at the beginning and after 3, 6, 9 and 12 months of rHuEpo therapy (Epo group) or clinical observation (No-Epo group). The control group consisted of 30 healthy subjects (15 females, 15 males, mean age-38.2±1.7 years, mean BMI 24.7±0.7 kg/m²). Results: Baseline plasma leptin concentrations in HDP were higher, although statistically not significant than leptinaemia in healthy subjects. After 3, 6, and 12 months of rHuEpo therapy plasma leptin concentrations were significantly lower than at the beginning of the study. Baseline plasma neuropeptide Y concentrations in HDP did not differ significantly from controls. After 3 and 6 months of the study period plasma neuropeptide Y concentrations increased significantly in patients of both the Epo and No-Epo group. This increase was, however significantly higher in rHuEpo-treated than in untreated patients. Conclusions: (1) rHuEpo treatment in haemodialysed patients with chronic renal failure is followed by a significant decline of leptinaemia and disappearance of the physiological positive BMI/leptinaemia relationship. (2) Suppression of leptinaemia induced by rHuEpo may be of clinical relevance in haemodialysed patients with chronic renal failure.     

High blood soluble receptor p80 for tumour necrosis factor-alpha is associated with erythropoietin resistance in haemodialysis patients.

BACKGROUND: Inflammation is one of the major causes of resistance to erythropoietin (rHuEpo) treatment. Tumour necrosis factor-alpha (TNF-alpha), one of the most potent proinflammatory cytokines, is known to inhibit human erythropoiesis directly in vitro. Although blood levels of soluble receptors for TNF-alpha (sTNFRs) are elevated in haemodialysis (HD) patients, the role of sTNFR for rHuEpo responsiveness in HD patients remains to be clarified. METHODS: We measured serum sTNFR (p55 and p80) levels in 83 stable outpatients undergoing regular HD (age 62+/-1, HD duration 15+/-1 years). After dividing the patients into three groups according to rHuEpo dose: (low (L) <60, n=31; moderate (M) > or =60 to <120, n=31; high (H) > or =120 U/kg/week rHuEpo, n=21), we examined the relationship between serum sTNFR levels and the degree of renal anaemia and rHuEpo dosage. RESULTS: Haemoglobin was significantly higher in patients receiving low rHuEpo dosage (L, 10.5+/-0.2; M, 9.7+/-0.1; H, 9.5+/-0.2 g/dl, P<0.01 vs M and H groups). There were no differences in blood TNF-alpha, sTNFR p55, C-reactive protein, albumin, ferritin, or intact parathyroid hormone levels among the three groups. Body mass index and creatinine generation rate, a marker of whole-body muscle volume, were significantly reduced in group H (P<0.01). Serum sTNFR p80 levels were significantly higher in group H (4.88+/-0.45 ng/ml) than in L (3.73+/-0.14 ng/ml) and M (3.67+/-0.21 ng/ml) groups (P<0.05). The blood interleukin (IL)-6 level was also increased in patients requiring high rHuEpo doses (L, 5.5+/-0.5; M, 6.4+/-0.5; H, 10.2+/-2.0 pg/ml, P<0.05 vs L and H groups). A stepwise regression analysis revealed that gender and sTNFR p80 were significant predictors of rHuEpo dosage. A significant direct relationship was found between rHuEpo dose and sTNFR p80 (r=0.499) and IL-6 (r=0.439) values in women (P<0.01) but not in men. CONCLUSIONS: These findings suggest that high blood sTNFR p80 may contribute to the development of rHuEpo resistance in female patients undergoing long-term HD.     

Erythropoietin and oxidative stress in haemodialysis: beneficial effects of vitamin E supplementation

Oxidative stress can produce profound alterations to cellular membrane lipids, impairing cell metabolism and viability. This phenomenon, previously observed in haemodialysis patients, had been proposed as a significant factor in regard to haemodialysis-related shortened red blood cells (RBC) survival. In the present study, several parameters associated with oxidative stress were evaluated in a group of haemodialysis patients either receiving erythropoietin therapy (n=12, mean erythropoietin dose 88±24 U/kg/week) or not receiving such therapy (n=20), and in 38 controls. Malonyldialdehyde (MDA, nmol/ml), an end-product of lipid peroxidation, and RBC anti-oxidant systems were measured, including RBC &agr;-tocopherol (RBC vitamin E, mg/l), RBC glutathione (GSH, nmol/mgHb), and RBC superoxide dismutase activity (SOD, U/mgHb). Plasma vitamin E concentrations were also evaluated. Finally, oral vitamin E supplementation (500 mg daily), an exogenous antioxidant, was administered for 6 months to seven patients from the dialysis group receiving erythropoietin while oxidative parameters were repeatedly evaluated and erythropoietin requirements monitored, in order to appreciate the therapeutic relevance of an antioxidant supplementation. An elevation of serum MDA was observed in all haemodialysis patients and a significant decrease in RBC vitamin E, despite normal serum vitamin E levels. Furthermore, the reduction in RBC vitamin E was more important in patients treated with erythropoietin. Vitamin E supplementation resulted in a significant increase in RBC vitamin E (from 0.3±0.1 to 1.2±0.2 mg/l of pellet) and a reduction in erythropoietin dose (from 93±24 to 74±26 U/kg/week) while maintaining stable haemoglobin concentrations. These results suggest that the oxidative stress could be one of the resistance factors to erythropoietin response in haemodialysis and that vitamin E supplementation could have a sparing effect on erythropoietin dosage requirement. Key words: antioxidant; erythropoietin; haemodiafiltration; lipid peroxidation; oxidative stress; vitamin E      

Crossover comparison of intravenous and subcutaneous erythropoietin in haemodialysis patients.

To examine the suggestion that s.c. administration of recombinant human erythropoietin (rHuEpo) may be more effective than i.v. administration, we changed the route of administration in 11 patients, previously established on a stable dose of rHuEpo given twice or thrice weekly, from i.v. to s.c. administration without altering the dose. All patients were iron replete (serum ferritin greater than 100 micrograms/l). In one patient the haemoglobin concentration declined at the time of conversion due to poor compliance, and another patient died shortly after conversion. In the remainder there was a significant increase in haemoglobin concentration from 9.30 (SD 0.78) at the time of conversion to 9.84 (0.59) at 1 month, 10.35 (1.22) at 2 months, and 10.39 (1.42) at 3 months. The increase in haemoglobin concentration was greater than 1 g/dl at 3 months in only five of the patients. Serum ferritin prior to conversion was similar in 'responders' and 'non-responders', but all responders had a transferrin saturation of greater than 16%, whereas three of four non-responders had transferrin saturation of less than or equal to 16%. Subcutaneous administration of rHuEpo is more effective, dose for dose, than i.v. administration, but poor iron mobilization may limit the response.     

Pharmacokinetics of recombinant human erythropoietin in children with renal failure.

The single-dose pharmacokinetics of recombinant human erythropoietin (rHuEpo; 40 units/kg1) were investigated in children (9-16 years) with end-stage renal failure. After an intravenous (i.v.) dose, serum rHuEpo concentrations declined in a monoexponential manner with a mean half-life (t1/2) of 5.6 +/- 3 h (+/- SEM; n = 9). Serum clearance and the apparent volume of distribution were estimated to be 10.1 +/- 0.9 ml h-1 kg-1 and 79.5 +/- 5.0 ml kg-1 (n = 9) respectively. Subcutaneous (s.c.) delivery resulted in serum values that peaked at 10 h, and thereafter concentrations declined slowly with a t1/2 of 21.1 +/- 4.5 h (n = 9). Serum rHuEpo concentrations were maximal at 14 h after i.p. administration and the t1/2 was 9.5 +/- 1.0 h (n = 3). The mean fraction absorbed of SC rHuEpo was 0.40 whereas after i.p. administration this fraction was only 0.17. These results show that after both s.c. and i.p. delivery, disposition of the hormone is rate-limited by absorption, and bioavailability for these extravascular routes is poor. In addition, comparison of the results with those available for adults indicates that rHuEpo is better absorbed but more rapidly cleared in children.     

Influence of diclofenac (group of nonsteroidal anti-inflammatory drugs) on fracture healing

Introduction Nonsteroidal antirheumatics (NSAR; NSAID) are often used in patients with fractured bones for analgetic reasons. This animal experiment was performed to determine the influence of NSAR on the process of fracture healing. As an alternative, tramadol, the centrally acting analgetic without peripheral effects, was included in this experiment.Materials and methods Wistar rats were operated on by a transverse osteotomy of the proximal tibia of the left leg. The fracture was stabilized by intramedullary nailing (healing period 21 days). All drugs were applied orally twice a day. The animals were divided into four groups with 10 rats each: Group 1 was treated with placebo (P), group 2 with tramadol (T; 20 mg/kg body weight/day), group 3 with diclofenac sodium (DS; 5 mg/kg bw/day) for 7 days followed by 14 days of placebo, group 4 with diclofenac sodium (DL; 5 mg/kgbw/day) over 21 days. On day 21 the rats were killed, and each leg was examined by X-ray, then the tibia was examined by CT scan, three-point bending, and histology.Results The results of CT and three-point bending showed that rats treated by diclofenac presented with delayed fracture healing compared with those treated by placebo or tramadol. Bone density in CT was highest in group 1 (mean 611.4±50.1 mg/ml), followed by group 2 (mean 542.5±29.5 mg/ml). Groups 3 (mean 411±34.0 mg/ml; p=0.006) and 4 (mean 395.2±15.4 mg/ml; p=0.009) were significantly lower. The stability of the bones, as measured by the breaking force (F_max), was highest in group 1 (mean 45.8±19.0 N), followed by group 2 (mean 39.0±7.9 N; NS); group 3 (mean 20.6±7.8 N; p=0.01) was significantly lower than the placebo animals, followed by group 4 (mean 26.5±8.3 N; p=0.03). Similar results were shown for bending stiffness: group 1 (mean 1404.6±611.4 Nmm/mm), group 2 (mean 1033.2±232.1 Nmm/mm; NS), group 3 (mean 564.2±457 Nmm/mm; p=0.045), and group 4 (mean 494.8±340.2 Nmm/mm; p=0.028). There were no significant differences between groups 1 and 2 and between groups 3 and 4, respectively. Diclofenac serum levels on day 21 in rats with long-term diclofenac application (mean 301.4±83.3 ng/ml) were comparable to those in humans.Conclusion Oral application of diclofenac significantly delayed fracture healing in rats. This effect might be comparable to other NSAR and fracture healing in humans.     

Thrombogenicity of an elastomer-coated aortofemoral Dacron prosthetic graft in humans

To determine the potential benefit of Dacron prostheses impregnated with silicon polymer, an elastomer-coated prosthetic graft (Intervascular [I]) was implanted in 30 patients with aortic occlusive arterial disease or infrarenal abdominal aortic aneurysm. These patients were compared with two other groups who underwent vascular reconstruction with either a knitted double-velour prosthesis (Meadox [M]) or a knitted prosthesis (Bard-USCI [U]). The different prostheses were randomly allocated just prior to their insertion. Average blood loss was 1063.33 ± 1065.69 ml for the I group, 888.33 ± 575.85 ml for the M group, and 908.33 ± 471.80 ml for the U group (NS). The duration of the operation was 160 ± 56 minutes for the I group, 142 ±37 minutes for the M group, and 153 ± 65 minutes for the U group (NS). The average follow-up was 12 months. As calculated by the actuarial method, primary patency at 36 months was 71.5% for the I group, 100% for the M group, and 98.2% for the U group (p < 0.001). Secondary patency was 73.7% for the I group and 100% for the M and U groups, respectively (p <0.001). This study shows that the thrombogenicity of the elastomer-coated aortofemoral vascular prosthetic graft was significantly higher than that of the two other prostheses. Increased intraoperative blood loss and longer duration of operation were related to the aortic abnormality being treated (aneurysm or occlusion) rather than to the type of prosthetic graft being used.Presented at the Annual Meeting of the Société de Chirurgie Vasculaire de Langue Française, Reims, France, June 19– 20, 1992.     

Effect of recombinant human erythropoietin on adrenergic activity in normotensive hemodialysis patients

Ten normotensive hemodialysis patients with severe anemia participated in the study. Human recombinant erythropoietin (rHuEpo) was administered i.v. 3 times a week in doses of 50 U/kg of body weight. During 12 weeks of observation, the mean hematocrit value increased from 19%, before start of therapy, to 32%. Simultaneous monitoring of serum plasma noradrenaline (NA) concentration showed an elevation from 202 to 281 pg/ml. An increase of NA concentration after a cold pressure stimulating test (CP) was not statistically significant after as compared to before treatment, but became statistically significant after 12 weeks of rHuEpo therapy (281 pg/ml before to 441 pg/ml after CP test, p < 0.01). The mean arterial blood pressure increased from 92 - 109 mmHg after 12 weeks of rHuEpo therapy (p < 0.001). We have demonstrated significantly increased NA blood concentrations after 12 weeks of rHuEPO therapy in normotensive patients, which correlated with increased MAP. This may suggest that the observed increase of noradrenaline concentration as a vasoactive substance after the CP test may contribute to hypertension during rHuEPO therapy.     

Enhanced platelet reactivity with erythropoietin but not following transfusion in dialysis patients

Although the haemostatic defects that occur in uraemia are complex,a major factor is the anaemia of renal failure. This may nowbe corrected by recombinant human erythropoietin (rHuEpo) therapyrather than by repeated blood transfusion. Platelet reactivityto shear stress and collagen was measured using non-anticoagulatedblood to study the effect of erythropoietin or blood transfusionon platelet function. Twenty dialysis patients were commencedon 25–50 U/kg rHuEpo twice weekly. The dose was adjustedafter 3 months to maintain target Hb 10–10.5 g/dl. A further15 patients were studied before and 10–12 days after receivingblood transfusion. Baseline platelet reactivity was subnormalin both groups versus control (P<0.0001). In the rHuEpo group,a significant increase in platelet reactivity was observed at2 months (P<0.005) which disappeared at 3 months. This wasnot related to the increase in Hb (7.3±0.3 to 10.2±0.3g/dl, P<0.0001). There was no change in platelet reactivityafter transfusion, despite an increase in Hb (6.2±0.2to 8.8±0.2 g/dl, P<0.0001) similar to that occurringin the rHuEpo group. We conclude that after rHuEpo therapy butnot after transfusion a transient increase in platelet reactivityoccurs which is dissociated from changes in platelet and redcell numbers.