Intravenous iron-gluconate during haemodialysis modifies plasma ß2-microglobulin properties and levels
Regina Michelis1,
Shifra Sela1,3 and
Batya Kristal2,3
1 Eliachar Research Laboratory and 2 Nephrology Department, Western Galilee Hospital, Nahariya and 3 Bruce Rappaport School of Medicine, Technion, Haifa, Israel
Correspondence and offprint requests to: Professor Batya Kristal, Head of Nephrology and Hypertension Department, Western Galilee Hospital, Nahariya, 22100, Israel. Email: Batya.Kristal{at}naharia.health.gov.il
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Abstract
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Background. Intravenous iron replacement therapy is routinely used for correction of anaemia in patients with end-stage renal failure. Free or labile iron, present both in parenteral iron formulations and in blood of haemodialysis (HD) patients, has the potential to induce severe oxidative processes. This study evaluated the acute in vivo effect of intravenous iron administration on the oxidation of plasma ß2-microglobulin (ß2m) and on its plasma levels after HD.
Methods. Iron-gluconate was administered intravenously to 14 patients receiving HD with low-flux cellulose-triacetate membranes during the first hour of the 4 h HD treatment. Each patient underwent three different dialysis treatments, during which an infusion of 62.5, 125 or 0 mg (control) of iron-gluconate was administered in random order. Plasma ß2m levels and iron parameters were monitored immediately before and after each HD treatment. The molecular isoforms of ß2m were studied by two-dimensional gel electrophoresis and western analysis. Levels of oxidized ß2m were evaluated by reaction with 2,4-dinitrophenylhydrazine and western analysis.
Results. Both doses of iron-gluconate caused remarkable changes in the molecular properties of ß2m, including shift in isoelectric point, molecular mass and degree of oxidation. Iron administration also limited the decline in plasma ß2m levels to <7.5%, compared with 27.9±2.7% during HD without iron.
Conclusions. Intravenous iron-gluconate led to a characteristic increase in molecular weight and in negative charge of ß2m, both of which can be assumed to be consistent with reduced membrane sieving coefficients and membrane adsorption, and thus with reduced clearance of ß2m.
Keywords: advanced glycation end-products; carbonyl; iron-gluconate; low flux; protein oxidation
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Introduction
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Intravenous (i.v.) iron replacement therapy is routinely used for correction of anaemia in patients with end-stage renal failure [1,2]. Free or labile iron, present both in parenteral iron formulations [3] and in blood of haemodialysis (HD) patients [4], has the potential to induce severe oxidative processes. Such oxidative damage is accelerated due to the pre-existing oxidative stress caused by the dialysis process per se, demonstrated by lipid peroxidation products [5] and protein oxidation products [6,7]. Iron deposits were found in joints of HD patients, in addition to ß2-microglobulin (ß2m) amyloids [8,9].
ß2m (11.8 kDa) accumulates in plasma of uraemic patients due to impaired renal excretion, oxidative stress and inflammation, and can reach up to 92 mg/l in HD patients, while normal levels are <2 mg/l [10]. Aggregation of ß2m into amyloid fibrils and their deposition in musculoskeletal tissues causes dialysis-related amyloidosis [11]. The molecular basis for ß2m aggregation has been attributed to various modifications, including formation of advanced glycation end-products (AGEs) [12].
The present HD methods (such as low-flux HD, high-flux HD and haemodiafiltration) fail to normalize circulating ß2m levels, although its plasma level can be lowered during each treatment [1315]. The removal of ß2m during a dialysis treatment depends on the permeability properties (molecular weight) and adsorption capacity of the dialyser membrane [14,16]. The adsorption of a given protein to the dialyser membrane, in turn, depends on the molecular properties of the protein, such as its isoelectric point (pI) [17], and on intrinsic properties of the membrane [18].
The present study was designed to evaluate the in vivo effect of i.v. iron therapy on ß2m oxidation during a HD treatment and to elucidate the molecular mechanisms underlying the interactions between iron and plasma ß2m.
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Subjects and methods
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Subjects
We studied 14 patients (eight males/six females, six diabetics) on chronic HD treatment for a mean of 33±6 months, aged 72±3 years (mean±SE). All patients underwent HD treatment thrice weekly; each dialysis treatment lasted 4 h and was carried out on cellulose-triacetate hollow fibre dialysers (Nipro produced by Nissho, Osaka, Japan) using bicarbonate dialysate. All patients received maintenance i.v. iron-gluconate therapy (Ferrlecit®; Rhone-Poulenc Rorer, Cologne, Germany) and recombinant human erythropoietin treatment (400012 000 U/week, Recormon®; Roche Diagnostics Gmbh, Mannheim, Germany). None of the patients developed hypotension or other symptoms related to the iron administration. The mean haemoglobin value was 11.2±0.4 g/dl and the corresponding transferrin saturation was 28.1±2.1%. The water for dialysis was compatible with the standards of the Association for the Advancement of Medical Instrumentation. Patients with evidence of infection, malignancy or severe hyperparathyroidism were excluded. Informed consent was obtained from all patients, and the institutional Helsinki committee approved the protocol.
Study protocol
Each patient included in the study underwent three different dialysis treatments, during which an i.v. infusion of 62.5, 125 [1,2] or 0 mg (control) of iron-gluconate (Ferrlecit®) was administered in random order. Each iron treatment was repeated twice with at least a 1 week interval between the protocols [1]. Iron-gluconate was administered during the first hour of the dialysis treatment. No other medications were administered during the treatments. For each patient, the blood flow rate and amount of ultrafiltration were the same for all studied treatments, with a mean single pool Kt/V of 1.2±0.2. Blood samples were drawn from the arterial line immediately (within 10 min) before and after dialysis and assayed for iron parameters and ß2m levels. Sample collection from all the patients was completed within 2 months.
Gel filtration chromatography of plasma proteins
All chemicals were obtained from Sigma (St Louis, MO), unless specified otherwise. Plasma samples were subjected to gel filtration so as to obtain ß2m-enriched fractions: a 5 ml column of Sephacryl S-200-HR was prepared and the column was washed with 5 vols of column buffer (50 mM Tris, 100 mM NaCl, pH 7.0). A 100 µl aliquot of plasma was diluted with Tris buffer to a final concentration of 50 mM Tris at pH 7.0, centrifuged for 5 min at 12 000 g and the supernatant loaded onto the column. Proteins were eluted in column buffer in 0.5 ml fractions. The ß2m-containing fractions were acetone precipitated and used for further analyses.
Detection and quantification of carbonylated ß2m
Detection of carbonylated ß2m could not be performed as previously described [6] due to the insolubility of ß2m during the procedure. For the modified procedure, acetone-precipitated proteins from ß2m-enriched fractions after gel filtration were resuspended in reducing Laemmli buffer. After SDSPAGE and identification of ß2m bands by western analysis, the membrane area containing the ß2m bands was excised and washed briefly in a solution containing 25 mM Tris, 140 mM NaCl, 0.05% Tween-20, pH 7.4 (TBST). The membrane strip was submerged in dinitrophenylhydrazine solution (20 mM dinitrophenylhydrazine in 20% trifluoroacetic acid) for 15 min at room temperature and then neutralized in 2 M Tris solution. The membrane strip was then briefly washed in TBST and used for a second western analysis with anti-dinitrophenylhydrazine serum [6]. Untreated and in vitro oxidized commercial ß2m were similarly analysed, as controls. Carbonyl signal was detected on X-ray films as described above. The intensities of carbonyl and ß2m signals were quantified by densitometry using the BioCapt and Bio-Profil (Bio-1D) softwares. The ratios of carbonyl to protein signals were determined in all the samples. For each experiment, the ratio of carbonyl to protein of the before dialysis sample was set as 100% and the ratios in all other samples were calculated relative to this sample. In the in vitro oxidation experiments, the untreated sample was considered as 100% and the oxidized sample was expressed as a percentage of this control.
Preparation of oxidized ß2m
Commercial ß2m (from human urine) was oxidized in vitro using a metal-catalysed oxidation system of iron/ascorbate [5 mM ascorbate and 100 µM FeCl3 in phosphate-buffered saline, 5 h at 37°C] to yield a highly oxidized (carbonylated) protein. The reaction was stopped at 4°C by addition of EDTA (pH 8.0) to a final concentration of 1 mM. The oxidizing reagents were removed by acetone precipitation and the proteins were analysed by western blotting as described below.
Gel electrophoresis and western analysis
Plasma samples were separated by SDSPAGE and then transferred to nitrocellulose filters. To evaluate the molecular properties and changes in ß2m isoforms, proteins from five HD patients were first subjected to gel filtration chromatography, in which ß2m-enriched fractions were obtained, and then separated by two-dimensional PAGE at the Smoler Proteomics Center (Faculty of Biology, Technion, Israeli Institute of Technology, Israel). The first dimensional separation was by isoelectric focusing in a pH gradient between 3 and 10 and the second dimension was molecular weight-based separation by SDSPAGE. The proteins were then transferred to nitrocellulose filters for detection of ß2m by western blot analysis with rabbit anti-human ß2m (purified immunoglobulin fraction, ICN Pharmaceuticals, Aurora, OH) and goat anti-rabbithorseradish peroxidase conjugate. The chemiluminescence signal was detected on X-ray films.
ß2m and iron parameters
Transferrin, ferritin, total iron and plasma ß2m levels were determined on chemical analysers. Haemoglobin and haematocrit determinations were performed using a Beckmann Coulter analyser (LH 750). Transferrin saturation was calculated as 70 x total iron/measured transferrin.
Plasma variable calculations
The results of ß2m before dialysis are given as measured. In order to correct for the haemoconcentration caused by water loss during HD, the values of ß2m measured after dialysis were corrected using the correction factor f: f = (1 HctA)/(1 HctB) x (HctB/HctA) where HctA and HctB are the haematocrit after and before dialysis, respectively.
Statistics
All the results are given as the mean±SE. Paired sample t-test was used for analysing the changes in ß2m levels and transferrin saturation during HD. Wilcoxon signed ranks test was used for analysing the effect of the iron dose on ß2m fall, patients age and prevailing plasma ferritin levels. Pearson correlation test was used for analysing the correlation between dialytic age and ß2m levels and between ferritin and ß2m levels.
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Results
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Level of carbonyl groups on plasma ß2m
Iron-induced oxidation of plasma ß2m during HD was evaluated by following carbonyl groups as a marker of oxidation. For this analysis, plasma samples from six patients, before and after HD, were separated and analysed for carbonyl groups on ß2m as described above. Of the six patients examined, four patients received 125 mg and two received 62.5 mg of iron. The findings and representative western results are shown in Figure 1. After administration of 125 and 62.5 mg iron-gluconate, ß2m was composed of 51 and 22% more carbonyl groups, respectively, than ß2m obtained before HD. A significant increase of 41% in the mean carbonyl intensity was observed relative to ß2m obtained before HD and a 54% increase was observed relative to ß2m obtained after HD without iron (n = 6). In vitro oxidation of commercial ß2m (by metal-catalysed oxidation) similarly resulted in an increased carbonyl signal (Figure 1).

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Fig. 1. ß2m and carbonyl formation. Plasma ß2m from six patients was separated by gel filtration. Proteins obtained before (1), after dialysis without iron (2) or with iron administration (3) were precipitated, separated by SDSPAGE and used for western analysis with anti-ß2m serum (ß2m signal). After detection, the membrane strip containing ß2m was excised, reacted with 2,4-dinitrophenylhydrazine and used for a second western analysis, with anti-DNP serum (carbonyl signal). As a control, untreated (U) and in vitro oxidized (Ox) commercial ß2m samples were separated by SDSPAGE and analysed similarly. The intensities of carbonyl and ß2m signals were quantified by densitometry, and the ratio of carbonyl to protein was calculated as a percentage of the control. Western analysis signal from a representative patient before (lane 1), after dialysis without iron (lane 2) or with administration of 125 mg of iron (lane 3) is shown.
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ß2m isoform profile
ß2m from plasma samples and the commercial sample were studied using SDSPAGE and western analysis with anti-ß2m serum. In addition to the major 12 kDa form of ß2m, high molecular mass bands of >100 kDa were detected in plasma and bands of
24 kDa appeared in the commercial sample (Figure 2A). The major form of ß2m in plasma (
12 kDa) was further analysed and characterized.

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Fig. 2. Molecular changes in ß2m during HD. ß2m was identified in patients plasma (P) and in a commercial preparation (C) by western analysis. Arrows indicate the major form of ß2m (12 kDa) and additional ß2m bands. Molecular size markers are indicated on the left (A). ß2m-enriched fractions were obtained by gel filtration chromatography and the major ß2m isoforms were studied by 2D-PAGE and western analysis (B). Arrows indicate various ß2m isoforms, and molecular size markers are indicated on the right.
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Figure 2B shows representative results of three samples from the same patient before dialysis, after dialysis without iron and after dialysis with iron administration (125 mg), separated by 2D-PAGE and studied by western analysis. Before dialysis, ß2m showed a pattern of several distinct isoforms, which differed in their pI values (Figure 2B). One minor isoform (no. 5) showed increased molecular mass. The pattern of ß2m isoforms was conserved after HD without iron. However, this pattern changed dramatically after iron administration: the most abundant ß2m spots disappeared (nos 14), the protein spot with a higher molecular mass became more acidic (no. 5) and a new ß2m spot appeared (no. 6), with a higher molecular mass than normal ß2m and with more acidic pI than the other isoforms. This form of ß2m was observed uniquely after HD with iron administration. A similar ß2m pattern was observed in all patients studied.
ß2m isoforms profile after in vitro oxidation
The effect of in vitro oxidation on the pattern of ß2m isoforms was studied in a commercial ß2m preparation. The highly carbonylated protein was studied by 2D-PAGE, and ß2m isoforms identified in western analysis are illustrated in Figure 3. The pattern of untreated commercial ß2m differed slightly from that observed in patient plasma, evident by the presence of additional protein isoforms with very acidic pI values or increased molecular weight (Figure 3 Untreated, arrows 35). In vitro oxidation caused dramatic changes in the pattern of ß2m isoforms, resulting in the disappearance of several isoforms (Figure 3, arrows 1, 2 and 5) and formation of several new isoforms (arrows 614) with more acidic (nos 611) or somewhat more basic (nos 1214) pI values than the major isoform of ß2m. Similar changes in ß2m pattern were observed after oxidation in vitro of plasma ß2m (data not shown).

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Fig. 3. Molecular changes in ß2m after oxidation in vitro. The molecular changes in commercial ß2m due to metal-catalysed oxidation in vitro were studied using 2D-PAGE and western analysis. Arrows indicate the various ß2m isoforms, and the 12 kDa molecular size marker is indicated on the right.
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The effect of iron administration on plasma ß2m levels
The mean level of plasma ß2m before dialysis was 26.9±1.4 mg/l. The baseline ß2m values did not differ among different HD treatments (P>0.5). Figure 4A shows the levels of ß2m before and after each dialysis treatment. The mean levels of ß2m decreased significantly (P<0.001) from 26.2±1.6 to 19.0±1.4 mg/l during HD without iron. This decrease corresponded to 27.9±2.7% of the initial ß2m level (Figure 4B). However, no significant reduction in ß2m levels was observed when iron-gluconate was administered (Figure 4A). The reduction in ß2m level was blunted, such that the decrease was only <7.5% below baseline: from 25.5±1.9 to 23.4±2.0 mg/l (before and after dialysis, respectively) when 62.5 mg of iron-gluconate were administered and from 28.5±1.8 to 25.8±2.2 mg/l for the 125 mg iron dose (Figure 4A). The blunted decrease of ß2m levels after iron therapy was significantly less than the decrease during the HD without iron, irrespective of iron dosage (P<0.005, Figure 4B). There was no significant difference between iron doses regarding their effect on plasma ß2m levels.

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Fig. 4. Plasma ß2m levels before and after HD treatments. The levels of ß2m (A) were determined for each patient before (b) and after (a) dialysis. ß2m levels after HD were corrected for haemoconcentration. The heavy lines indicate mean values. Significant and non-significant (NS) P-values are provided. The mean percentage reduction in plasma levels of ß2m during HD without (open squares) or with iron (filled squares) is presented in (B).
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Iron profile
The mean transferrin saturation value before dialysis was 28.1±2.1% (Figure 5). Following iron-gluconate administration, the values after the HD treatment showed a significant, dose-dependent increase to 67.1±4.1 and 83.8±5.0% for the 62.5 and 125 mg iron dose regimens, respectively. No change was observed after dialysis without iron (28.1±2.9%). Determination of transferrin saturation provided quality control for the proper execution of the study protocol. Only in two treatments with 125 mg iron was transferrin oversaturation observed.

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Fig. 5. Transferrin saturation before and after the HD treatments. Transferrin saturation was determined before (b) and after (a) dialysis. #Significant changes (P<0.0001) during HD.
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The effect of dialytic age on ß2m levels
The correlations between ß2m levels and either dialytic age or patient age were examined. There was a correlation between dialytic age and the levels of pre-dialysis ß2m (Figure 6). In contrast, no correlation could be demonstrated between the age of the patients and the baseline levels of ß2m.

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Fig. 6. Relationship of dialytic age to baseline ß2m levels. The correlation between baseline plasma levels of ß2m and dialytic age (in months) is presented.
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The effect of ferritin levels on ß2m decrease
The mean ferritin level before dialysis was 435±82 µg/l. The effect of serum ferritin on reduction of ß2m levels during HD was examined post hoc. Patients with ferritin levels of >300 µg/l showed significantly (P<0.03) greater reduction of ß2m levels during control HD (without iron) than patients with ferritin levels of <300 µg/l (Figure 7A). However, reduction of ß2m levels was blunted after iron administration in both groups of patients, i.e. regardless of ferritin levels (Figure 7A).

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Fig. 7. Relationship of plasma ferritin levels to plasma ß2m levels during dialysis. The percentage of ß2m reduction during HD without iron (open squares) or with iron (filled squares) is shown in patients with plasma ferritin levels >300 mg/l vs patients with <300 mg/l (A). The correlation between the baseline levels of ß2m and ferritin is presented in (B).
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A negative, significant correlation was found between the mean levels of ferritin and ß2m before dialysis, as shown in Figure 7B (r = 0.64, P = 0.007). Other iron parameters such as transferrin levels, saturation or total plasma iron did not correlate with either baseline ß2m levels or reduction of ß2m during HD.
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Discussion
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We have examined the effects of two clinically recommended (DOQI) iron-gluconate doses administrated during a dialysis treatment on the oxidation and levels of plasma ß2m. Iron administration during HD resulted in molecular modifications of ß2m, reflected by alteration of the pI value, increased carbonyls level and changes in molecular mass, which can be assumed to be consistent with reduced membrane sieving coefficients and membrane adsorption, and thus with reduced clearance. Our study confirms earlier reports that cellulose triacetate low-flux HD is relatively inefficient, reducing circulating ß2m levels by only 30% [1315]. Nevertheless, even this modest reduction in plasma ß2m concentration was blunted by the administration of iron-gluconate during HD. As a result of i.v. iron, ß2m levels before and after the HD treatment were virtually indistinguishable. The inhibitory effect of iron therapy on ß2m reduction during dialysis was similar for 62.5 and 125 mg doses.
Based on the iron-induced changes in pI and the increase in carbonyl levels following i.v. iron, we assume that iron administration aggravated oxidation reactions pre-existing in these patients, including the oxidation of ß2m. A limitation of the presented findings lies in the potential inaccuracy of quantification of protein carbonyls by densitometry. Nevertheless, to the best of our knowledge, this is the first study demonstrating the effect of i.v. iron on the oxidation of plasma ß2m. This is consistent with earlier observations in vivo that i.v. iron increases the level of carbonyls on proteins such as fibrinogen [6] and albumin [7]. Our finding is in agreement with an earlier report that in vitro oxidation of ß2m isoforms resulted in generation of AGE products that are manifested by an acidic shift in ß2m pI [12]. The disappearance of some ß2m isoforms may well be due to oxidation resulting in formation of new isoforms. Although we have not demonstrated the formation of AGEß2m, we showed the increase in an intermediate product for AGEs, namely carbonyl-modified ß2m.
The iron-induced modifications of ß2m were associated with a marked blunting of plasma ß2m fall during a HD treatment. We imply that the iron-induced protein oxidation could account for this observation, since the passage of oxidized (AGE-modified) ß2m through various dialysis membranes was previously shown to be reduced [19]. Another possible cause of reduced plasma ß2m fall may be due to diminished adsorption of oxidized ß2m to HD membranes, as suggested by in vitro studies [19]. The alteration of ß2m pI may also impair the adsorption of ß2m to the dialysis membrane [17], accounting for the reduced removal of the protein from the circulation during a dialysis with iron therapy.
To rule out the possibility that ß2m synthesis or shedding during dialysis masks the fall in ß2m levels, we administered iron-gluconate to chronic kidney disease patients (not on renal replacement therapy). In these experiments, iron did not cause an increase in the levels of plasma ß2m measured 3 h later (data not shown). Thus, an iron-dependent increase in ß2m synthesis or shading is not likely to account for our observations with HD patients.
Our findings may have some relevance to dialysis-related amyloidosis. Iron-gluconate can directly modify ß2m in vivo, resulting in formation of oxidized ß2m that, according to Miyata et al. [12], favours the deposition of amyloid. Secondly, as iron apparently inhibits ß2m removal during HD, it may induce a slow accumulation in the levels of circulating ß2m, that is strongly supported by the correlation to the overall dialytic age (Figure 6) and not to the patients age [10,20]. High concentrations of intact ß2m, which have been shown to form amyloids in vitro [21], and the oxidation of ß2m can lead to amyloid deposition.
Patients with high ferritin levels showed greater reduction of ß2m during HD. We think that high ferritin levels minimize the concentrations of catalytically active iron in plasma, probably by its chelation, although transferrin is the accepted candidate for iron binding in the circulation. Our assumption is supported by several facts: (i) ferritin binds the toxic ferrous (Fe2+) ion, which is not bound by transferrin [22]; and (ii) in HD patients, ferritin levels are often increased and transferrin levels are decreased [6,23], thus shifting the normal ratio between these two iron-binding proteins in favour of ferritin.
Based on this study, the long-term deleterious effects of iron therapy on ß2m should be explored further, particularly its effects on the onset of amyloidosis. Iron cumulative dose may be important in this regard, given that both iron-gluconate doses inhibited the decrease in ß2m. Additional protocols can be addressed so as to minimize oxidation of ß2m: rate and timing of iron administration and the option to use antioxidants (either free or membrane-bound) during dialysis.
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Acknowledgments
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We thank Dr Natalie Grinberg and the dialysis unit staff for their help in adapting the study protocol, Mrs Orly Yakir for the statistical analysis, Mrs Hadia Abu-hala and Mrs Meital Mazor for their help in the analyses, and Professor B. D. Myers for reviewing and proofreading the manuscript. Part of this study was presented at the European Iron Club meeting in Vienna, 2003 and at the 12th International Congress on Nutrition and Metabolism in Renal Disease, Padua/Venice, Italy, 2004.
Conflict of interest statement. None declared.
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Received for publication: 14. 9.04
Accepted in revised form: 20. 4.05