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J Am Soc Nephrol 15:1648-1655, 2004
© 2004 American Society of Nephrology

CLINICAL SCIENCE

Non–Transferrin-Bound Iron in the Serum of Hemodialysis Patients Who Receive Ferric Saccharate: No Correlation to Peroxide Generation

Barbara Scheiber-Mojdehkar*, Barbara Lutzky*, Roland Schaufler{dagger}, Brigitte Sturm* and Hans Goldenberg*

*Department of Medical Chemistry, Medical University of Vienna, Austria; and {dagger}Department of Nephrology and Dialysis, Wilheminenspital, Vienna, Austria

Correspondence to Prof. Dr. Hans Goldenberg, Department of Medical Chemistry, Medical University of Vienna, Waehringerstrasse 10, A-1090 Vienna, Austria. Phone: +43-1-4277/60802; Fax: +43-1-4277/60881; E-mail: Hans.Goldenberg{at}univie.ac.at


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Intravenous iron (iv.Fe) is used to optimize response to recombinant human erythropoietin (r-HuEPO) in ESRD, but no consensus exists with respect to the best regimen to avoid transferrin "oversaturation," oxidative stress, and the occurrence of non–transferrin-bound iron (NTBI). Iv.Fe was stopped for 1 wk in 35 hemodialysis (HD) patients who were routinely receiving iv.Fe and r-HuEPO. The iv.Fe group received 100 mg of ferric saccharate (Venofer) at the end of the first HD session, whereas the time-control group was treated under the same conditions but received no iv.Fe. Serum samples were taken before the first HD session, immediately and 60 min after iv.Fe administration, and before the next HD session. Sera were analyzed for NTBI and peroxides; transferrin saturation was analyzed by urea-PAGE and Western blot. In an in vitro model system with HepG2 cells, the effects of ESRD serum on the labile iron pool (LIP) were assayed using the fluorescence calcein assay. NTBI significantly increased after iv.Fe-administration and returned to baseline values before the next HD-session. There was a shift from apo- to monoferric transferrin, but no "oversaturation" of transferrin after iv.Fe-treatment. Peroxides increased in both groups after HD. Hemodialysis decreased bioavailable iron for the LIP in HepG2-cells, whereas serum of iv.Fe-treated HD patients highly increased the LIP in these cells. A total of 100 mg of iv.Fe led to NTBI generation but not to an oversaturation of transferrin. Peroxide concentrations significantly increased during HD but were not correlated to iv.Fe administration and seemed to result from other sources of oxidative stress related to HD. NTBI can enter liver cells and increase the potentially harmful LIP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESRD typically results in anemia, primarily as a result of deficient renal production of erythropoietin (EPO). Most patients who undergo hemodialysis (HD) therefore are treated with recombinant human erythropoietin (r-HuEPO) (1,2). The most important confounding factor limiting the effectiveness of rHuEPO is absolute or functional iron deficiency. Iron is usually substituted by intravenous iron (iv.Fe), because oral supplementation is inefficient (3–6). Despite the acknowledged safety of iv.Fe preparations such as ferric saccharate (7–9), the occurrence of non–transferrin-bound iron (NTBI) in the plasma of patients has been reported, either directly (10–14) or indirectly, by detection of enhanced symptoms of oxidative stress (15–18), of neutrophil damage (19), of support of bacterial growth (11), or increased incidence of infectious diseases (20,21).

There is wide agreement that patients who undergo regular HD treatment experience increased oxidative stress (22). NTBI in the circulation could in principle participate in redox reactions that give rise to reactive oxygen species. NTBI therefore is implicated to exacerbate oxidative stress in ESRD patients who receive iv.Fe therapy (23,24).

The chemical nature of NTBI is not really known. Binding to citrate or albumin was suggested by some authors (25–27). Although the binding affinity of most of them, such as albumin, is relatively low compared with transferrin, they are present at high concentrations, which makes them effective competitors with transferrin (28,29).

The preparations themselves do not yield iron to plasma transferrin in vitro (30,31); thus, processing by reticuloendothelial cells or by the liver presumably precedes iron release into the labile plasma iron pool. Recently, we showed the ability of parenteral iron preparations to deliver iron to nonreticuloendothelial cells (32) and their effect on the labile iron pool (LIP) of the human hepatoma cells HepG2 (33). Because elevations of the LIP are implicated in the generation of oxidative cell injury (34), these findings may have important implications on the possible toxicity of parenteral iron preparations. This is particularly true for liver hepatocytes, because the liver is also the main sink for excess iron either from transferrin or from non-transferrin sources.

ESRD patients usually have low plasma transferrin concentrations (35). Nevertheless, the transferrin saturation (TFS) is below the normal range. Reports on apparent oversaturation of transferrin in iv.Fe therapy (36–38) seem to rest on analytical errors in the determination of plasma iron (30,31).

There are two possibilities to measure NTBI: Indirect measurement involves the chemical reactivity of NTBI, for instance, the bleomycin method (39–41), whereas direct measurement typically involves the use of scavenging molecules to mobilize loose, nonspecifically bound iron (42,43). The results of such tests largely depend on the design of the assay. The recently described NTBI assays based on quenching the fluorescence of metal-binding dyes (12,13) open the possibility to obtain reliable results in statistically significantly great numbers, which does not require HPLC (44).

This study was designed to relate the levels of NTBI to TFS and the generation of peroxides in ESRD patients who undergo iv.Fe therapy under controlled conditions. In an in vitro model system with HepG2 cells using ESRD sera, the effect of dialysis and in vivo iv.Fe administration on the LIP was assayed using the fluorescence calcein assay.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Design
All chemicals were purchased from Sigma (St. Louis, MO) or Merck if not indicated otherwise. During this study, all patients underwent dialysis with an Asahi hollow fiber dialyzer APS 650 with biocompatible polysulfone membrane ({gamma}-sterilized) and were heparinized with a high molecular weight heparin (Heparin Immuno; Baxter) by continuous intravenous infusion.

Parenteral iron administration was stopped 1 wk before the start of the study (washout phase). Thirty-five HD patients who routinely receive iv.Fe in combination with r-HuEPO in the maintenance phase were included in this study after having given their informed consent. Patients were divided into two groups: The iv.Fe-group (27 patients) received an infusion of 100 mg of ferric saccharate (Venofer; Vifor Int., St. Gallen) during 10 min at the end of the HD session. This dose corresponds to the highest recommended single dose for this product (by the manufacturer). The time-control group (eight patients) was treated under the same conditions but received no iv.Fe. Four blood samples were collected (Figure 1): (1) before the first HD session; (2) directly after iv.Fe infusion (t = 0 min); (3) after 60 min (t = 60 min); and (4) before the next HD session, which was approximately 2 d after the first session. From the time-control group, serum samples were collected at the same times. Whole blood samples were drawn in Vacuette with Z Serum Separator clot activator (Greiner bio-one; Graz, Austria) and after 10 min were centrifuged at 2000 x g for 10 min. Serum samples were frozen immediately after centrifugation and kept at –20°C until analysis. All sera were analyzed for NTBI; the relative amount of apo-, monoferric-, and diferric transferrin; and the presence of peroxides. Sera from five ESRD patients from the iv.Fe group were also assayed for bioavailable iron by the fluorescence calcein assay (see below).



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  		Figure 1. Study design and blood sample collection. The scheme depicts the basic steps of the study: Step 1: No intravenous iron (iv.Fe) administration was performed for 1 wk (washout phase). Step 2: Patients were divided into two groups: the iv.Fe group and the time-control group. Step 3: Collection of four blood samples from each patient: before hemodialysis (A), after hemodialysis (HD; time control group) or directly after iv.Fe infusion at the end of the HD session (iv.Fe group; B), 1 h after collection of sample B (C), and before the next HD (after ~2 d; D).

 
Laboratory Assessments
Clinical routine methods were used for the determination of serum iron (by the Guanidin-FerroZine-method, INTEDRA), serum ferritin (by enzyme immunoassay, COBASCORE), and serum transferrin concentration (by immunoturbidimetry, INTEDRA) in serum samples taken before the first hemodialysis session and analyzed in the clinical laboratory at the hospital (Table 1).


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  		Table 1. Baseline iron indices and clinical characteristics of HD patientsa
 
Determination of Peroxides
To avoid a loss of total peroxide activity during prolonged storage, serum samples were stored for no more than 1 wk at –20°C after collection. For determination of peroxide concentration, the "Peroxide-activity" assay (POX ACT; Tatzber KEG, Klosterneuburg, Austria) was used, which is based on the reaction of horseradish peroxidase (HRP) with peroxides, using tetramethylbenzidine as the chromogen substrate. Ten microliters of the serum was incubated with a mixture of HRP, tetramethylbenzidine, and phosphate buffer for 20 min, and absorbances were determined in a multiwell plate reader (Victor from Wallac) at 450 nm. Using a hydrogen peroxide (H2O2) standard curve, total peroxide concentrations were expressed as micromoles of H2O2 equivalents per liter of serum.

Measurement of NTBI
NTBI was measured according to the method published by Breuer et al. (12), which is based on the binding of NTBI from the sera to desferrioxamine (DFO)-coated wells in the presence of a mobilizing agent (oxalate). The binding of NTBI to DFO in the wells is detected with a nonfluorescent iron-calcein complex, which can donate iron to DFO not occupied by NTBI from the serum sample. Because calcein has a lower affinity to iron than DFO, calcein is rendered iron-free and the fluorescence of free calcein can be measured.

In brief, serum samples were analyzed by two variants of the test, using different concentrations of oxalate as mobilizing reagents. Serum samples (20 µl) were incubated either with 230 µl of 100 mmol/L sodium oxalate, 20 mmol/L Hepes (pH 7.4), and 2.5 mmol/L MnCl2 (method A) or with 200 mmol/L oxalate, 20 mmol/L MnCl2, 1 µmol/L FeCl3, and 20 mmol/L Hepes (pH 7.4; method B) in DFO-coated 96-well plates (black with clear bottom; Greiner, Vienna, Austria) for 2 h at 37°C. After two washings with distilled water and incubation with 5 mmol/L EDTA (pH 8.0) and two more washings with distilled water, 0.1 ml of freshly prepared calcein-iron complex, consisting of 600 nmol/L calcein (Molecular Probes, Eugene, OR), and 540 nmol/L ferrous ammoniumsulfate in 20 mmol/L Hepes, 150 mmol/L NaCl (pH 7.3) was added and incubated for 2 h at 37°C in the dark. Then the fluorescence in the wells was read in a fluorescence multiwell plate reader (Victor from Wallac) with excitation/emission filters of 485/530 nm. The DFO-coated 96-well plates were prepared in advance by incubation of the plates with 0.1 ml of 75 mg of hydroxyethylstarch-coupled-DFO/ml (Biomedical Frontiers, Minneapolis, MN) in 10 mmol/L sodium phosphate (pH 8.6) for 72 h at 4°C, followed by two washings with distilled water. Calibration of the assay was performed with a freshly prepared 2 mmol/L Fe-NTA complex and a serial 1:1 dilution in water. Contaminating iron was removed from all buffers by treatment with 5% (vol/wt) Chelex 100 Resin (BioRad Laboratories, Vienna, Austria) for 20 min. A serial dilution of ferric saccharate in HEPES-buffered saline (HBS) (pH 7.3) up to 600 µmol/L iron was assessed for DFO-chelatable iron with methods A and B.

Quantification of Apo-, Monoferric, and Diferric Transferrin
The routinely used method to calculate TFS from serum iron and transferrin content can give false values in serum samples that contain iv.Fe. In this study, TFS therefore was analyzed by urea-PAGE and Western blot. In this system, differently iron-loaded transferrin isoforms (apo- and holo-transferrin, plus the C- and N-terminal partially saturated monoferric transferrins) display a different electrophoretic mobility and then can be detected by immunoblot using antitransferrin antibodies. Urea-PAGE was performed with slight modifications according to the method described by Makey and Seal (45). We modified this method by using a Tris-borate-electrophoresis buffer without EDTA to avoid removal of iron from transferrin by this chelator (46,47). Serum samples were separated on 6% polyacrylamide gels that contained 6 M urea with Tris-borate electrophoresis buffer (100 mmol/L Tris, 10 mmol/L boric acid [pH 8.4]) at 150 V (const.) for 2 h at 4°C in a Mini Protean II electrophoresis chamber (BioRad Laboratories). Blotting on nitrocellulose membrane (0.45 µm; BioRad) was performed according to the method of Plekhanov (48). Detection of transferrin on the Western blot was performed with a rabbit anti-human transferrin antibody (DAKO) as first antibody (1:500 dilution) and goat anti-rabbit HRP conjugated antibody (DAKO) as second antibody (1:7500 dilution). Then the blots were incubated with SuperSignal (Pierce), and the chemiluminescence signal was detected in a FluoroS MultiImager (BioRad). The relative density of the bands was analyzed with the MultiAnalyst software (BioRad). The total density of the bands recognized by the anti-transferrin antibody representing apo-, holo- and monoferric transferrin was set as 100%.

Fluorescence Calcein Assay for ESRD Serum Samples
The influence of ESRD serum on the LIP was assayed by the fluorescence calcein assay (49). Human hepatoma HepG2 cells were cultured in DMEM containing 10% FCS, 2 mmol/L L-glutamine, and 50 µg/ml gentamicin on 48-well tissue culture plates at a density of 1 x 106 cells/ml. After 2 d, the cells were in the log phase and were used for the measurement of the LIP.

Cells were incubated with ESRD sera for 2.5 h at 37°C. Then the cells were washed with DMEM containing 50 µmol/L DTPA and two more washings with DMEM alone to remove surface-bound iron. The cells were subsequently loaded with 0.25 µmol/L calcein-AM for 15 min at 37°C in DMEM buffered with 20 mmol/L Hepes. The cell monolayer was washed free of excess calcein-AM and reincubated with DMEM containing 20 mmol/L Hepes and a fluorescence-quenching anti-calcein antibody (10 µl/ml medium) to eliminate all extracellular fluorescence (33). Calcein fluorescence was measured in a fluorescence plate reader (Victor II; Perkin Elmer; excitation 485 nm, emission 535 nm) at 37°C. After stabilization of the signal, the amount of intracellular iron bound to calcein (Ca-Fe) was assessed by addition of 100 µmol/L of the fast permeating chelator isonicotinoyl salicylaldehyde hydrazone (a gift from Dr. Prem Ponka, McGill-University, Montreal, Canada).

Statistical Analyses
Statistical analysis was performed with GraphPad Prism software. Data are presented as mean ± SEM unless stated otherwise. P < 0.05 was considered significant. A paired t test was applied for analyzing differences to the baseline during the treatment in one group. Significant differences to the baseline are marked in the figures with * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). An unpaired t test was used for analyzing differences between iv.Fe-treated and the time-control group. Significant differences are marked with + (P < 0.05), ++ (P < 0.01), and +++ (P < 0.001) in the figures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Dialysis and Iv.Fe Administration on Oxidative Stress
Total peroxide concentration was significantly increased in ESRD samples collected directly and at 60 min after HD compared with predialysis samples. Before the next HD session, total peroxide concentrations returned to the baseline value. Administration of 100 mg of ferric saccharate at the end of the HD session did not further increase total peroxide concentration in the iv.Fe group (Figure 2). Peroxide generation therefore is not correlated to iv.Fe administration; the observed increase seems to result from other sources of oxidative stress related to HD.



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  		Figure 2. Effect of HD and iv.Fe on total peroxide concentration. The concentration of total peroxides was analyzed by the POX-ACT test as described in Materials and Methods. The concentration of total peroxides is significantly increased in both groups after HD (samples B and C), whether they were treated with iv.Fe or not.

 
Effect of Iv.Fe Administration on TFS
To obtain reliable information about TFS in the presence of iv.Fe, we used urea-PAGE to determine TFS (Figure 3). TFS significantly increased directly after iv.Fe administration and to a lesser extent in the samples collected after 1 h and returned to the baseline level before the next HD session. There was always a large amount of unsaturated transferrin present after iv.Fe treatment, whereby ~75% of total transferrin was in the only partly saturated monoferric isoform (Figure 4, A through C).



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  		Figure 3. Determination of transferrin saturation (TFS) by urea-PAGE and Western blot. After in vitro loading of apo-transferrin with increasing concentrations of ferric ammonium citrate (FAC), the TFS was assayed by urea-PAGE and Western blot (see Materials and Methods). This method gives accurate results also in the presence of iv.Fe.

 


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  		Figure 4. Effect of HD and iv.Fe on TFS. Serum samples (see Figure 1) were analyzed by urea-PAGE and Western blot as described in Materials and Methods. The relative amount of apo-, holo-, and monoferric transferrin is represented as a percentage of total transferrin on the blot. (A) Apo-transferrin. (B) Monoferric transferrin. (C) Holo-transferrin. TFS significantly increases only in the iv.Fe group (in sample B and to a lesser extent in sample C), but no oversaturation (complete disappearance of apo-transferrin or monoferric transferrin) can be detected. There is only a shift from apo- to monoferric transferrin.

 
Detection of NTBI after Iv.Fe Administration
Although Figure 4 clearly shows that there is no "oversaturation" of transferrin in the iv.Fe patient group, NTBI could be detected in ESRD serum after iv.Fe administration by both methods used (Figure 5). In method A (Figure 5A), NTBI was mobilized with 100 mmol/L oxalate, whereas in method B (Figure 5B), the oxalate concentration was increased to 200 mmol/L, resulting in much higher maximal NTBI values. In the time-control group, NTBI remained at the baseline level during the study, whereas in the iv.Fe group, NTBI significantly increased after iv.Fe administration and remained high during the first hour. NTBI concentration returned back to the baseline level before the next HD session.



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  		Figure 5. Detection of non–transferrin-bound (NTBI) iron by two variants of the NTBI assay. NTBI was measured according to the method published by Breuer et al. (12), which is based on the binding of NTBI from the sera to desferrioxamine (DFO)-coated wells in the presence of a mobilizing agent (oxalate). After removal of the incubation mixture, the binding of NTBI to DFO in the wells is detected with a nonfluorescence iron-calcein complex, which can donate iron to DFO not occupied by NTBI from the serum sample. Because calcein has a lower affinity to iron than DFO, calcein is rendered iron-free and fluorescence of free calcein can be measured. In A, when 100 mmol/L oxalate as mobilizing reagent was used in the assay (method A), NTBI can be detected only in the iv.Fe group after iv.Fe administration (in samples B and C) and disappears before the next HD session (sample D). (B) Using a high concentration of mobilizing agent (200 mmol/L oxalate) in the assay (method B), NTBI is detectable in all samples, but only in the iv.Fe group is there a significant increase of NTBI concentration after iv.Fe treatment (samples B and C).

 
DFO-Chelatable Iron in the iv.Fe Preparation Venofer
Recently, Esposito et al. (13) assessed the presence of chelatable iron in different polymeric iron formulations directly in a fluorescence-based one-step assay with the metallosensing probes fluorescein-transferrin and fluorescein-DFO in HBS. However, the assay system used by Esposito et al. is limited to concentrations up to 200 µmol/L iron of the formulations, which is only a low-range approximation of the levels used therapeutically. Higher concentrations cannot be analyzed by fluorescence in a one-step assay because of inner-filter effects caused by the dark color of the iv.Fe preparations (13). As concentrations up to 600 µmol/L iv.Fe can easily be achieved in patients who receive a normal therapeutic dose in the range of 40 to 100 mg iv.Fe, it was interesting to assay the amount of chelatable iron in ferric saccharate at concentrations up to 600 µmol/L iron using a two-step assay, with removal of the colored iv.Fe preparation before the fluorescence measurement.

In the two-step assay, DFO-chelatable iron in the iv.Fe preparation Venofer could be assayed in a serial dilution with up to 600 µmol/L iron in HBS (pH 7.3). The samples were incubated with two concentrations of mobilizing reagent (method A, 100 mmol/L oxalate; method B, 200 mmol/L oxalate) in DFO-coated wells. Chelatable iron binds to DFO in the well, and after washings and therefore removal of the colored iv.Fe-preparation, iron bound to DFO was detected with the nonfluorescence iron-calcein complex, which is rendered iron-free as a result of its lower affinity than DFO. Then fluorescence of free calcein was measured (Figure 6). After incubation with 100 mmol/L oxalate (method A) for 2 h at 37°C, a mean of ~1.5% of total iv.Fe in the samples was DFO-chelatable iron; with 200 mmol/L oxalate, a mean of ~7.5% of total iv.Fe in the sample was DFO-chelatable iron. These results are similar to the results recently reported by Esposito et al. (13) using the one-step assay system.



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  		Figure 6. DFO-chelatable iron in the iv.Fe preparation. A serial dilution of ferric saccharate (Venofer) was prepared in HEPES-buffered saline (HBS) (pH 7.3). The samples were incubated for 2 h at 37°C in the presence of a mobilizing agent (oxalate) in DFO-coated wells. After removal of the incubation mixture, the binding of iron to DFO in the wells was detected with a nonfluorescence iron-calcein complex, which can donate iron to DFO not occupied by iron from the iv.Fe. Because calcein has a lower affinity to iron than DFO, calcein is rendered iron-free and fluorescence of free calcein can be measured. In method A, 100 mmol/L oxalate, and in method B 200 mmol/L oxalate was used as mobilizing agent. Shown are the means ± SEM of duplicates of two different experiments. DFO-chelatable iron is represented as µmol/L (left axes) and % of total iv.Fe in the samples (right axes).

 
Bioavailable Iron in ESRD Serum
In a cell culture model with HepG2 cells, the bioavailable iron from ESRD serum was assayed by the fluorescence-calcein method. Recently, we used this model to show that iv.Fe preparations transiently increased the LIP of HepG2 cells (33). Elevations of the LIP are implicated in the generation of oxidative cell injury (34). We therefore were interested in whether ESRD serum (containing in vivo administered iv.Fe) shows the same effect on the LIP. As dialysis treatment itself is also likely to contribute to iron deficiency in ESRD patients, we were also interested in whether HD treatment reduces the amount of biologically available iron.

Compared with normal tissue culture conditions (control), in which the cells are relatively iron poor, the LIP was increased when the cells were incubated with serum from a healthy person (control serum; Figure 7). The LIP decreased with ESRD serum collected after HD, compared with predialysis serum, which is likely due to removal of iron by the dialysis treatment. Iv.Fe administration of 100 mg of ferric saccharate significantly increased the LIP, and the LIP returned to a lower level after 2 d (before the next HD session) but not back to the baseline predialysis value. These findings suggest that the 1-wk washout phase without iron administration (before the first HD session) could effectively reduce the bioavailable iron in ESRD serum.



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  		Figure 7. Effect of ESRD serum on the labile iron pool in HepG2 cells. After a 1-wk washout phase without iv.Fe administration, serum samples from five different ESRD patients were collected: before the first dialysis (predialysis); at the end of the first dialysis (after HD; –iv.Fe); directly after Fe infusion at the end of the first dialysis session (after HD; +iv.Fe); and after 2 d (before next HD). HepG2 cells were exposed to these sera for 2.5 h at 37°C. Then the cells were washed and incubated with 0.25 µmol/L calcein-AM in DMEM, buffered with 20 mmol/L Hepes (pH 7.4) for 15 min at 37°C. After washing, the cells were incubated with DMEM, containing 20 mmol/L Hepes and anti-calcein antibody. After registration of the baseline fluorescence, the amount of intracellular metal bound to calcein (Ca-Fe) was assessed by addition of 100 µmol/L of the fast permeating chelator salicylaldehyde hydrazone. Calcein fluorescence was measured when the signal reached full fluorescence and remained stable (after 2 min). Control cells were incubated with cell culture medium alone (control) and were set as 100%. Control serum was collected from a healthy person. Shown are the means ± SEM from five patients, measured in duplicate.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed a significant shift from apo- to unsaturated monoferric transferrin in HD patients who received a single infusion of 100 mg ferric saccharate during 10 min at the end of the first dialysis session, but we could not find any oversaturation of transferrin. This is in contrast to several recent reports on an apparent oversaturation of transferrin during iv.Fe therapy (14,15,38,50). One possible explanation for the opposite findings could arise from the study design: Before iv.Fe administration, our patients received no iv.Fe for 1 wk (washout phase). Moreover, these studies used routine laboratory assessments to calculate TFS from serum iron and transferrin content.

Although iv.Fe preparations are very stable polynuclear iron-(III)-hydroxide carbohydrate complexes of large mass, depending on the method used for serum iron determination, large amounts of iron from the preparations can be reduced and form complexes with iron(II)-chelators such as ferrozine. Calculated TFS in the presence of iv.Fe therefore can lead to overestimation of TFS. Urea-PAGE can be an alternative to obtain more accurate informations about TFS in the presence of iv.Fe.

One of the major concerns about increasing the efficacy of r-huEPO in ESRD patients by higher doses of iv.Fe is related to the possible generation of toxic oxygen radicals by iron (23,24). In the present study, we observed that HD significantly increased total peroxide concentration in both groups, whether they received iv.Fe or not. Peroxide generation in our ESRD patients therefore seemed not to be correlated to iv.Fe administration; the observed increase is more likely to result from other sources of oxidative stress related to HD (22).

NTBI was first described in conditions of iron overload, when TFS exceeded 100%. This led to the concept that NTBI represents a heterogeneous fraction of iron not bound to transferrin or ferritin, and it was thought to be composed of iron bound to serum albumin, citrate, and other, undefined, negatively charged ligands (29). Using this concept, iv.Fe therapy would add large amounts of NTBI to the plasma.

Iron that is not tightly bound to transferrin or other molecules can be mobilized by iron chelators such as oxalate and then can be bound by DFO (12). Using this method, we found appreciable concentrations of mobilizable "labile iron." NTBI apparently occurs despite sufficient iron-binding capacity of transferrin. The amount of detectable NTBI varied considerably, depending on the concentration of mobilizing agent used. Different concentrations of oxalate (methods A and B) apparently mobilized different pools of NTBI. Although both concentrations significantly increased NTBI after iv.Fe administration, 100 mmol/L oxalate could apparently only mobilize that part of labile iron from the preparation that could also be chelated in vitro. However, using 200 mmol/L oxalate, the increase in NTBI concentration after iv.Fe administration was not only due to mobilizing iron from ferric saccharate, as the amount of mobilizable iron from the preparations in vitro was much lower (13).

It is interesting that with 200 mmol/L oxalate (method B), very high NTBI concentrations could be obtained in the iv.Fe group after iron infusion, but also the baseline values were much higher. This suggests that large amounts of iron from the preparations can be mobilized by 200 mmol/L oxalate. From pharmacokinetic studies in healthy volunteers, it is known that infusion of ferric saccharate leads to rapid high serum iron levels and that the mean volume of distribution of the central compartment is ~3 L, hence close to the volume of serum (37). The expected serum concentration of iv.Fe after infusion of 100 mg ferric saccharate therefore is close to 600 µmol/L serum. This means that after in vivo administration, a considerable amount of ~25% of the applied dose was mobilized by 200 mmol/L oxalate and therefore was detected as NTBI by this assay. In a solution of 600 µM iv.Fe, only 5% of total iv.Fe was DFO-chelatable iron in vitro. This means that the resting 20% of NTBI measured in the serum samples are likely to arise from another LIP. Recently, we demonstrated that parenteral iron preparations increase the intracellular LIP of the human hepatoma HepG2 cells (33). Using this model system, we show that dialysis reduced the amount of biologically available iron. This suggests that dialysis treatment itself is also likely to contribute to iron deficiency reported in ESRD patients. ESRD serum (containing in vivo–administered iv.Fe) significantly increased the amount of bioavailable iron. Two days after iv.Fe administration, the amount of bioavailable iron decreased but remained high compared with the predialysis sample. This means that the 1-wk washout phase before the first dialysis effectively reduced bioavailable iron.

In conclusion, because preparations such as ferric saccharate do not yield iron effectively to the part of iron that is termed NTBI in vitro, processing by reticuloendothelial cells or by the liver presumably precedes iron release into the labile plasma iron pool.


   Acknowledgments
 
This work was supported by the Austrian Research Found (FWF) # P14842-PAT and Hochschuljubilaeumsstiftung der Stadt Wien # H-83/2000.

This work was presented in part at the 3rd International Biometals Symposium, Biometals 2002, Kings College, London, UK, April 10–13, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McGonigle RW, Wallin JD, Shadduck RK, Fisher JW: Erythropoietin deficiency and inhibition of erythropoiesis in renal insufficiency. Kidney Int 25: 437–444, 1984[Medline]
  2. Eschbach JW, Kelly MR, Haley NR: Treatment of anemia of progressive renal failure with recombinant human erythropoietin. N Engl J Med 321: 158–163, 1989[Abstract]
  3. Sunder-Plassmann G, Hörl WH: Importance of iron supply for erythropoietin therapy. Nephrol Dial Transplant 10: 2070–2076, 1995[Abstract/Free Full Text]
  4. Hörl WH: Consensus statement. How to diagnose and correct iron deficiency during rHuEpo therapy: A consensus report. Nephrol Dial Transplant 11: 246–250, 1996[Free Full Text]
  5. Fishbane S, Imbriano LJ, Kowalski EA, Maesaka JK: The evaluation of iron status in hemodialysis patients. J Am Soc Nephrol 7: 2654–2657, 1996[Abstract]
  6. Nissenson AR, Strobos J: Iron deficiency in patients with renal failure. Kidney Int 69S: 107–118, 1999[CrossRef]
  7. Sunder-Plassmann G, Horl WH: Safety aspects of parenteral iron in patients with end-stage renal disease. Drug Saf 17: 241–250, 1997[Medline]
  8. Fishbane S, Kowalski EA: The comparative safety of intravenous iron dextran, iron saccharate, and sodium ferric gluconate. Semin Dial 13: 381–384, 2000[CrossRef][Medline]
  9. Chandler G, Harchoval J, McDougall IC: Intravenous iron sucrose: Establishing a safe dose. Am J Kidney Dis 38: 988–991, 2001[Medline]
  10. Banyai S, Rainer V, Derfler K, Druml W, Hörl W, Sunder-Plassmann G: Bleomycin-detectable free iron (BDI) is present in patients on intravenous (IV) iron therapy [Abstract]. J Am Soc Nephrol 9: 199, 1998[CrossRef]
  11. Parkkinen J, von Bonsdorff L, Peltonen S, Gronhagen-Riska C, Rosenlof K: Catalytically active iron and bacterial growth in serum of hemodialysis patients after i.v. iron saccharate administration. Nephrol Dial Transplant 15: 1827–1834, 2000[Abstract/Free Full Text]
  12. Breuer W, Ronson A, Slotki IN, Abramov A, Hershko C, Cabantchik ZI: The assessment of serum nontransferrin-bound iron in chelation therapy and iron supplementation. Blood 95: 2975–2982, 2000[Abstract/Free Full Text]
  13. Esposito BP, Breuer W, Slotki I, Cabantchik ZI: Labile iron in parenteral iron formulations and its potential for generating plasma nontransferrin-bound iron in dialysis patients. Eur J Clin Invest 32S: 42–49, 2002[CrossRef]
  14. Kooistzra MP, Kersting S, Gosriwatana I, Lu S, Nijhoff-Schutte J, Hider RC, Marx JJM: Nontransferrin-bound iron in the plasma of hemodialysis patients after intravenous iron saccharate infusion. Eur J Clin Invest 32S: 36–41, 2002[CrossRef]
  15. Roob JM, Khoschsorur G, Tiran A, Horina JH, Holzer H, Winklhofer-Roob BM: Vitamin E attenuates oxidative stress induced by intravenous iron in patients on hemodialysis. J Am Soc Nephrol 11: 539–549, 2000[Abstract/Free Full Text]
  16. Herrera J, Nava M, Romero F, Rodriguez-Iturbe B: Melatonin prevents oxidative stress resulting from iron and erythropoietin administration. Am J Kidney Dis 37: 750–757, 2001[Medline]
  17. Lim P-S, Wei Y-H, Yu YL, Kho B: Enhanced oxidative stress in hemodialysis patients receiving intravenous iron therapy. Nephrol Dial Transplant 14: 2680–2687, 1999[Abstract/Free Full Text]
  18. Cavdar C, Temiz A, Yenicerioglu Y, Caliskn S, Celik A, Sifil A, Onvural B, Camsari T: The effects of intravenous iron treatment on oxidant stress and erythrocyte deformability in hemodialysis patients. Scand J Urol Nephrol 37: 77–82, 2003[CrossRef][Medline]
  19. Patruta SI, Edlinger R, Sunder-Plassmann G, Hörl WH: Neutrophil impairment associated with iron therapy in hemodialysis patients with functional iron deficiency. J Am Soc Nephrol 9: 655–663, 1998[Abstract]
  20. Collins A, Ebben J, Ma J: Frequent IV iron dosing is associated with higher infectious deaths [Abstract]. J Am Soc Nephrol 8: 190, 1997
  21. Canziani ME, Yumiya ST, Rangel EB, Manfredi SR, Neto MC, Draibe SA: Risk of bacterial infection in patients under intravenous iron therapy: Dose versus length of treatment. Artif Organs 25: 866–869, 2001[CrossRef][Medline]
  22. Locatelli F, Canaud B, Eckardt KU, Stenvinkel P, Wanner C, Zoccali C: Oxidative stress in end-stage renal disease: an emerging threat to patient outcome. Nephrol Dial Transplant 18: 1272–1280, 2003[Abstract/Free Full Text]
  23. Halliwell G, Gutteridge JM: Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol 186: 1–85, 1990[CrossRef][Medline]
  24. Gutteridge LM, Halliwell B: Free radicals and antioxidants in the year 2000. A historical look to the future. Ann N Y Acad Sci 899: 136–147, 2000[Medline]
  25. Hider RC: Nature of nontransferrin-bound iron. Eur J Clin Invest 32S: 50–54, 2002[CrossRef]
  26. Grootveld M, Bell JD, Halliwell B, Aruoma OI, Bromford A, Sadler PJ: Non-transferrin-bound iron in plasma or serum from patients from patients with idiopathic hemochromatosis. J Biol Chem 264: 4417–4422, 1989[Abstract/Free Full Text]
  27. Hershko CA, Graham G, Bates GW, Rachmilewitz EA: Non-specific serum iron in thalassaemia: An abnormal serum iron fraction of potential toxicity. Br J Haematol 40: 255–263, 1978[Medline]
  28. Grootveld M, Bell JD, Halliwell B, Aruoma OJ, Bomford A, Sadler PJ: Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis: Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy. J Biol Chem 264: 4417–4422, 1989
  29. Breuer W, Hershko C, Cabantchik ZI: The importance of non-transferrin bound iron in disorders of iron metabolism. Transfus Sci 23: 185–192, 2000[CrossRef][Medline]
  30. Seligman PA, Schleicher RB: Comparison of methods used to measure serum iron in the presence of iron gluconate or iron dextran. Clin Chem 45: 898–901, 1999[Free Full Text]
  31. Besarab A: Parenteral iron therapy: Safety and efficacy. Semin Dial 12: 237–242, 1999[CrossRef]
  32. Scheiber-Mojdehkar B, Sturm B, Plank L, Kryzer I, Goldenberg H: Influence of parenteral iron preparations on non-transferrin bound iron uptake, the iron regulatory protein and the expression of ferritin and the divalent metal transporter DMT-1 in HepG2 human hepatoma cells. Biochem Pharmacol 65: 1973–1978, 2003[CrossRef][Medline]
  33. Sturm B, Goldenberg H, Scheiber-Mojdehkar B: Transient increase of the labile iron pool in HepG2 cells by intravenous iron preparations. Eur J Biochem 270: 3731–3738, 2003[Medline]
  34. Rothman RJ, Serroni A, Farber JL: Cellular pool of transient ferric iron, chelatable by deferoxamine and distinct from ferritin, that is involved in oxidative cell injury. Mol Pharmacol 42: 703–710, 1992[Abstract]
  35. Kirschbaum B: Hypotransferrinemia of chronically hemodialyzed patients. Artif Organs 23: 1047–1054, 1999[CrossRef][Medline]
  36. Geisser P, Baer M, Schaub E: Structure/histotoxicity relations of parenteral iron. Drug Res 42: 1439–1452, 1992[Medline]
  37. Danielson BG, Salmonson T, Derendorf H, Geisser P: Pharmacokinetics of Iron(III)-sucrose complex after a single intravenous dose in healthy volunteers. Drug Res 46: 615–621, 1996[Medline]
  38. Sunder-Plassmann G, Hoerl WH: Safety of intravenous injection of iron saccharate in haemodialysis patients. Nephrol Dial Transplant 11: 1797–1802, 1996[Abstract/Free Full Text]
  39. Gutteridge JMC, Rowley DA, Halliwell B: Superoxide-dependent formation of hydroxyl radicals in the presence of iron salts. Biochem J 199: 263–265, 1981[Medline]
  40. Evans PJ, Halliwell B: Measurement of iron and copper in biological systems: Bleomycin and copper-phenantroline assays. Methods Enzymol 233: 82–92, 1994[CrossRef][Medline]
  41. Esposito BP, Breuer W, Sirankapracha P, Pootrakul P, Hershko C, Cabantchik ZI: Labile plasma iron in iron overload: Redox activity and susceptibility to chelation. Blood 102: 2670–2677, 2003[Abstract/Free Full Text]
  42. Singh S, Hider RC, Porter JB: A direct method for quantification of non-transferrin-bound iron. Anal Biochem 186: 320–323, 1990[CrossRef][Medline]
  43. Esposito BP, Epsztejn S, Breuer W, Cabantchik ZI: A review of fluorescence methods for assessing labile iron in cells and biological fluids. Anal Biochem 304: 1–18, 2002[CrossRef][Medline]
  44. Gosriwatana I, Loreal O, Lu S, Brissot P, Porter JB, Hider RC: Quantification of non-transferrin-bound iron in the presence of unsaturated transferrin. Anal Biochem 273: 212–220, 1999[CrossRef][Medline]
  45. Makey DG, Seal US. The detection of four molecular forms of human transferrin during the iron binding process. Biochim Biophys Acta 453: 250–256, 1976[Medline]
  46. Pollack S, Vanderhoff G, Lasky F: Iron removal from transferrin. An experimental study. Biochim Biophys Acta 497: 481–487, 1977[Medline]
  47. Baldwin DA: The kinetics of iron release from human transferrin by EDTA. Effect of salts and detergents. Biochim Biophys Acta 623: 183–198, 1980[Medline]
  48. Plekhanov AY: Immunoreplica from the gel surface: Rapid and sensitive blot plus intact gel. Anal Biochem 239: 110–111, 1996[CrossRef][Medline]
  49. Breuer W, Epsztejn S, Cabantchik ZI: Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron. J Biol Chem 270: 24209–24215, 1995[Abstract/Free Full Text]
  50. Zanen AL, Adriaansen HJ, Bommel EHF, Posthuma R, de Jong GMTh: "Oversaturation" of transferrin after intravenous ferric gluconate (Ferrlecit®) in hemodialysis patients. Nephrol Dial Transplant 11: 820–824, 1996[Abstract/Free Full Text]
Received for publication October 7, 2003. Accepted for publication March 2, 2004.




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