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Comparative toxicity and cell-tissue distribution study on nanoparticular iron complexes using avian embryos and HepG2-cells
Comparative toxicity and cell-tissue distribution study on nanoparticular iron complexes using avian embryos and HepG2-cells SUSANNE ROTH, PETER LANGGUTH, KARSTEN SPICHER, and HARALD ENZMANN BONN AND MAINZ, GERMANY
In this study the toxicity and intracellular availability of iron from iron dextran (FeD), iron sucrose (FeS), and iron gluconate (FeG) was compared in organs of avian (turkey) embryos and in isolated cells (HepG2) in cell culture. Iron uptake was more pronounced in embryonic liver than in renal tissue. Cellular iron uptake in liver and kidney was more or less similar for the different compounds. Only some experiments showed slightly greater iron concentrations in liver and kidney with FeG compared with FeD and FeS. Significant differences were found in the survival ratios of the eggs and the embryo weights depending on the type of iron complex administered. The rank order of toxicities was FeG > FeS > FeD. Iron accumulation in HepG2-cells was extremely high with FeS and FeG, whereas FeD did not lead to a relevant iron uptake by HepG2 cells. The excessively high iron content of the cells is an in vitro phenomenon found neither in the in ovo model with the turkey embryos nor in the clinical use of the compounds. The rank order of toxicities in HepG2 cells was FeS > FeG > FeD. Iron uptake in cell culture does not reflect the in vivo situation. The in ovo model is more suitable to assess the cellular iron uptake and iron toxicity in cells and tissues than the in vitro model. In both in ovo and in vitro experiments, FeD seemed to be superior in terms of toxicity. (Translational Research 2008;151:36-44) Abbreviations: CAM ⫽ chorio-allantoic-membrane; DMT-1 ⫽ divalent metal transporter-1; FeD ⫽ iron dextran; FeG ⫽ iron gluconate; FeS ⫽ iron sucrose; PBS ⫽ phosphate-buffered saline; r/min ⫽ rotations per minute; RPMI ⫽ Roswell Park Memorial Institute (cell culture medium); RT ⫽ room temperature; SEM ⫽ standard error of the mean
P
arenteral iron therapy is used widely for the treatment of anemia in patients with various underlying diseases. Intravenous iron therapy is the most effective regimen for the treatment of anemia in patients with chronic kidney disease. This outcome is to be expected as a dialysis patient encounters an average yearly blood From the Federal Institute for Drugs and Medical Devices, Bonn, and the Institute of Pharmacy, Johannes Gutenberg-University, Mainz, Germany. Submitted for publication April 16, 2007; revision submitted August 20, 2007; accepted for publication September 4, 2007. Reprint requests: Susanne Roth, University of Bonn, Institute of Pharmacy, Clinical Pharmacy, An der Immenburg 4, 53121 Bonn, Germany; e-mail: [email protected]. 1931-5244/$ – see front matter © 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.trsl.2007.09.001
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loss equivalent to 1–1.5 g of elementary iron.1 The treatment of anemia with intravenous iron leads to improvement in morbidity, mortality, and quality of life.2 Parenteral iron has been shown to reduce the erythropoietin dose requirements in patients with chronic kidney disease up to 40%, which seems to be of special importance in the context of the potential adverse effects of high doses of erythropoietin that reduces the survival of cancer patients.3,4 The use of intravenous supplemental iron therapy rather than oral treatment has been proposed for cancer patients.5 Generally, the parenteral iron formulations are regarded as safe. Nevertheless, all intravenous iron complexes in use have the potential to induce oxidant stress by the formation of very strong oxidants such as the hydroxylradical in the Haber–Weiss reaction.6 Concerning the long-term toxicity of parenteral iron preparations, several reports describe a connection between increased
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AT A GLANCE COMMENTARY Background
Intravenous iron complexes currently used for the treatment of anemia have potential side effects and associated risks that might depend on the type of carbohydrate complexing the iron core in the different iron preparations. Translational Significance
The toxicity and iron uptake of 3 parenteral iron complexes were tested using 2 different models. Iron uptake in the in ovo model with the more complex interaction of cells and tissues better reflected the clinical situation than HepG2-cells, whereas toxicity was similar in both models. Therefore, the in ovo model may contribute to detect clinically relevant differences between available or newly developed iron preparations with different carbohydrate complexes.
body iron stores and an increased risk of cancer and detail how an iron overload leads to an increased risk of infection.7,8 Widely different models for in vitro studies on iron toxicity are available, and they may be used for different purposes. The goal of this study was to compare cellular iron uptake and iron induced cytotoxicity of 3 different nanoparticular iron complexes [iron dextran (FeD), iron sucrose (FeS), iron gluconate (FeG)] in a well-defined in vitro model with HepG2-cells and a new, more complex avian embryo model to the wellknown effects of parenteral iron in humans. In the in ovo experiments, 2 different routes of administration were studied: injection in the egg white and application on the chorio-allantoic-membrane (CAM). The availability of iron from the different complexes was measured as cell-bound iron in HepG2-cells and as hepatic and renal iron storage in the turkey embryos. The toxicity of the different iron complexes was reflected in the effects on embryo survival and embryo weight and in the cytotoxic effects on HepG2-cells. METHODS Iron complexes. FeD (CosmoFer; GRY-Pharma GmbH, Kirchzarten, Germany, batch number 0343110-4), FeS (Venofer; Fresenius Medical Care, Bad Homburg, Germany, batch number 397100), and FeG (Ferrlecit; Aventis-Pharma Deutschland GmbH, Frankfurt am Main, Germany, batch number D4A708) were purchased from a pharmacy. CosmoFer is an iron(III)– hydroxide– dextran complex containing 100 mg Fe3⫹ in a 2-mL ampoule in aqua ad injectabilia with NaOH and HCl for pH-adjustment. Venofer is an
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iron(III)– hydroxide–sucrose complex containing 100 mg Fe3⫹ in a 5-mL ampoule dissolved in aqua ad injectabilia and adjusted to pH with NaOH. The resulting solution has a pH of approximately 10.5. Ferrlecit is an iron(III)–sodium– gluconate complex containing 40 mg Fe3⫹ in a 3.2-mL ampoule in sucrose and aqua ad injectabilia. Ferrlecit also contains 0.9% benzylalcohol as a preservative. In ovo model. The fertilized turkey eggs were purchased from the Schabrockerhof hatchery (Wachtendonk, Germany). Turkey eggs have a hatching period of 28 days. In this series of experiments, the dose for each egg was set to 8 mg of Fe3⫹. The resulting injection volumes were FeD 160 L, FeS 400 L, and FeG 640 L. Control eggs received 160 L, 400 L, or 640 L of physiologic sodium chloride solution (Serumwerk Bernburg, Bernburg, Germany). Different injection volumes had no effect on egg survival. Egg white injection. Before incubation, fertilized turkey eggs were separated into intervention and control groups. The resulting average egg weights of each group differed by 1% to 2%. The eggs were positioned with the large end pointing upward in an egg carton. A cross was marked 1–2 cm above the egg point with a pencil, and the area was disinfected with a piece of cloth soaked with ethanol 70%. With a pointed pair of scissors, the egg shell was pierced in the middle of the marked cross. The iron complex solutions were drawn up into a syringe and injected into the white of the egg through the hole. Control eggs received the corresponding volume of physiologic sodium chloride solution. After the treatment, the injection site was sealed with adhesive tape and the eggs were placed in an incubator at 37°C and 65% humidity (BSS 420; Ehret Labor-und Pharmatechnik, Emmendingen, Germany). After an incubation period of 22 days, the experiment was terminated. The vitality and weights of each embryo were determined. Livers and kidneys were removed and frozen at – 80°C until iron analytics were performed. Egg white injection of benzylalcohol. A 0.9% solution of benzylalcohol in physiologic sodium chloride solution (solution A) and a solution that contained FeD and 0.9% benzylalcohol in physiologic sodium chloride solution (solution B) were injected at volumes of 640 L each to study the impact of the preservative on the resulting toxicity. Overall, 640 L of solution B contained 8 mg of Fe3⫹. The incubation time was 22 days. CAM application. Preparation of the chorioallantoic membrane was by a modification of a described method by Kunzi-Rapp et al.9 Fertilized turkey eggs, incubated for 12 days at 37°C and 65% humidity, were incubated with the pointed end downward until day 13. During this time, the embryos moved to the blunt end of the egg. On day 13, the eggs were turned again and a hole was drilled through the pointed end of the shell. The hole was sealed with adhesive tape, and the eggs were incubated again until day 14. Because of the hole in the pointed end of the egg, the air pocket moved from the blunt end to the pointed end. On day 14, the air pocket was positioned between the CAM and the inner egg shell. The hole was enlarged by peeling a round, 5-mm window. The iron complex solutions were applied carefully drop by drop.
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Control eggs received physiologic sodium chloride solution in the same manner. After the treatment, the application site was sealed with adhesive tape and the incubation was carried on for 2 or 4 more days. After a total of 16 or 18 days in the incubator, the experiment was terminated. The vitality and the weights of the embryos were determined. Livers and kidneys were removed and frozen at – 80°C until iron analysis was performed. In vitro model. HepG2-cells are human hepatoma cells, which have epithelial appearance and grow as monolayer in small cell associations. Cells were grown in RPMI (Roswell Park Memorial Institute cell culture medium) and were supplemented with 10% fetal calf serum, penicillin/streptomycin, and glutamine under standard tissue culture conditions (37°C, 5% CO2). The cells were subcultured every few days. The medium was then removed with a pump, and the flask with the cells was washed with about 10 mL of phosphate-buffered saline (PBS). PBS was removed, and 2 mL of trypsin/ethylenediamine tetraacetic acid was added. The flask was incubated for 5–10 min at 37°C until the cells detached. A total of 5–10 mL of RPMI was added to stop trypsin activity. The medium that contained the cells was put into a centrifuge tube and centrifuged at 1000 rotations per minute (r/min) for 5 min at room temperature (RT). The supernatant was discarded, and the cells were resuspended in 5–10 mL of fresh RPMI with a needle and a syringe. The resuspended cells were then dispensed into new culture flasks, which contained 5 mL of RPMI, and put back in the incubator. Iron loading of the cells. Each confluent HepG2-cell culture flask was subcultured on 5 cell plates, 15 mL of RPMI was added, and the cells were incubated for 1 day at 37°C and 5% CO2. One day later, solutions of the iron complexes were prepared in RPMI. At this time, 15 mL of medium was put in a sterile flask and 7.5 or 75 mg of Fe3⫹ was added, respectively. Depending on the iron concentration in each iron complex solution, the added volumes differed slightly (eg, for FeD, 150 L vs FeG600 L). After adding the iron complexes to the medium, the tubes were shaken to form a homogenous solution. The old medium was then removed from the cell plates, and the new medium that contained 7.5 or 75 mg of Fe3⫹, respectively, was added to the cells. Control plates received the medium supplemented with the corresponding volume of physiologic sodium chloride solution. After iron loading, the cell plates were incubated at 37°C, 5% CO2, and were harvested after an incubation time of 1 or 4 days, respectively, which resulted in a total of 4 different intervention groups and 4 corresponding control groups. Each group contained at least n ⫽ 3 cell plates. Cell harvest. Before harvesting the cells, the morphologic appearance of the cells was assessed with a microscope (Leica Microsystems, Wetzlar, Germany) and was enlarged 100-fold and 200-fold. Before removing the old medium with a pump, 10 mL of medium was added to the cells so that the cells were not damaged while removing the medium. After the medium was removed, the cells were washed 5 times with 15 mL of PBS to remove the iron remnants. Then, 5 mL of PBS was added and the cells were recovered by scraping with a cell scraper, washed again with 5 mL of PBS, and centri-
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fuged (2000 r/min for 10 min) at RT. The supernatant was discarded, and the cell pellet was resuspended in 200 L of PBS. The cell suspension was transferred into a tared 1.5-mL centrifuge tube and centrifuged for 13,200 r/min for 10 min at 4°C. The supernatant was discarded, and the pellet was dried completely with compressed air. The tube and pellet was weighed and frozen at ⫺80°C until iron analysis was performed. Microanalysis of iron in tissue. The quantification of iron in liver tissue and in kidney tissue was carried out in accordance with the method of Rebouche et al.10 The following reagents were obtained from Sigma-Aldrich (Steinheim, Germany): sodium 3-(2-pyridyl)-5,6-bis(4-phenylsulfonate)1,2,4-triazine (ferrozine), thioglycolic acid, and trichloroacetic acid. Iron standard solution (989-g Fe/mL in 1.1% HCl) was purchased from Aldrich (Milwaukee, Minn). HCL 37% was obtained from Merck (Darmstadt, Germany). Sodium acetate was obtained from Roth (Karlsruhe, Germany). Liver and kidney tissue were homogenized after dilution of the tissue 1:10 (wt/vol) with deionized water, using a Potter homogenizer (Braun Biotech International, Melsungen, Germany) at 1100 RPM for 2–3 min (liver) and 1–2 min (kidney), under cooling in an ice water mixture. Equal volumes of tissue homogenates and protein precipitation solution (1N HCL that contained 10% trichloroacetic acid in deionized water) were combined in 1.5-mL centrifuge tubes (Sarstedt, Nümbrecht, Germany), mixed by vortex, and placed in a 95°C heating block for 1 h. Tubes and contents were cooled in water at RT for 2 min, vortex mixed, and then centrifuged at 16.000 ⫻ g for 10 min at RT (Digifuge, Heraeus; Kendro Laboratory Products, Langenselbold, Germany). The supernatant was divided into three 1.5-mL centrifuge tubes (50 L each). Two centrifuge tubes, 50 L of chromogen solution (0.508 mmol/L ferrozine, 1.5 M sodium acetate, and 0.1% (vol/vol) in deionized water) was added. Sample blanks consisted of 50 L of tissue extract supernatant and 50 L of 1.5 M sodium acetate that contained 0.1% (vol/vol) thioglycolic acid. The mixtures were vortexed, and after 30 min at RT, absorbance was measured at 562 nm in microcuvettes (Plastibrand; Brand, Wertheim, Germany) in a photometer (MBA 2000; Perkin Elmer, Wellesley, Mass). Standard curves were prepared with each measurement using iron standards that contained 0-, 1-, 2-, 4-, 6-, 8-, and 10-g Fe3⫹/mL of the iron standard solution and diluted with an equal volume of protein precipitation solution. The equation of the regression line was used to calculate the iron contents of the sample solutions. Microanalysis of iron in cells. The quantification of iron in HepG2 cells was carried out in accordance with the method of Rebouche et al.10 The cell pellet was diluted with water 1:10 (wt/vol) and resuspended with a needle and a syringe. Afterward, the cell suspension was put into an ultrasound bath (Bandelin Electronic, Berlin, Germany) for 10 min. For the next procedure, see “Microanalysis of iron in tissue,” which differs from the first protocol only by the handling of the precipitated protein pellet. The precipitated protein pellet was dried and frozen at ⫺80°C until protein analysis was performed. Protein analysis. The BCA-assay (BCA protein assay kit; Pierce, Rockford, Ill) was used for protein analysis that is
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based on the known biuret-reaction.11 The precipitated protein pellets were defrosted and dissolved in 500 L of 1 M NaOH. In total, 1 M HCl was added to adjust pH to pH 7. The samples were diluted with H2O 1:2 (wt/vol). Protein standards were prepared from a BSA-standard solution (Bovine serum albumin 2000 g/mL), which contained 1500 g/mL, 1000 g/mL, 750 g/mL, 500 g/mL, 250 g/mL, 125 g/mL, 25 g/mL, and 0 g/mL of BSA in H2O. The microplate protocol was used, and absorption was measured at 570 nm with a microplate reader (Dynex Technologies, Chantilly, Va). A standard curve was prepared with the protein standards. The equation of the regression line was used to calculate the protein contents of the sample solutions. The iron content of the HepG2-cells was assessed before it was applied to the protein content of the cells. Statistical analysis. Statistical computations were performed using S-Plus 2000 (Insightful Corporation, Seattle, Wash). Results are presented as means ⫾ standard error of the mean (SEM). A comparison of the embryo weights and liver and kidney iron contents of the intervention group with the parameters of the control group was performed by applying the Wilcoxon rank sum test. This test was also used to compare different intervention groups with embryo weights and liver and kidney iron contents. Group sizes ranged from 4 to 17 (control group, embryo weight), 6 to 17 (intervention group, embryo weight), 4 to 12 (control group iron content in liver/kidney), and 6 to 16 (intervention group, iron content in liver/kidney). The t test was applied to analyze the iron content in g/mg protein of the HepG2-cells (confidence-interval 95%). For iron analysis in the cells, n ⫽ 3 cell pellets were used. Ethical statement. This article conforms to the relevant ethical guidelines for animal research. RESULTS In ovo model. Survival ratios and embryo weights. Pronounced differences existed between the survival ratios of the various iron complexes (Table I). All experiments showed the same tendency, the survival ratio rank list was as follows: FeD ⬎ FeS ⬎ FeG. The same rank order as in the survival ratios was observed for the embryo weights (Fig 1). Statistical significance existed in embryo weights after egg white injection of FeG in comparison with control group (P ⬍ 0.05), after egg white injection of FeD in comparison with FeG (P ⬍ 0.01), after egg white injection of FeD in comparison with FeS (P ⬍ 0.05), and after CAM application of FeD in comparison with FeS with an incubation time of 4 days (P ⬍ 0.01). At variance with the other iron complexes, the iron gluconate preparation Ferrlecit contains 0.9% benzylalcohol as preservative. To conclude that the observed effects are caused not by the iron complex but are caused by benzylalcohol, the 0.9% benzylalcohol control group was included. To conclude additionally that the observed effects of Ferrlicit are caused not by the iron complex but also by a combination of iron
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Table I. In ovo experiments with survival ratios of the eggs in % of control after administration of FeD, FeS, and FeG containing 8-mg Fe3⫹ Experiment
Egg-white injection, 22 d FeD, 8-mg Fe3⫹ FeS, 8-mg Fe3⫹ FeG, 8-mg Fe3⫹ CAM application, 2 d FeD, 8-mg Fe3⫹ FeS, 8-mg Fe3⫹ FeG, 8-mg Fe3⫹ CAM application, 4 d FeD, 8-mg Fe3⫹ FeS, 8-mg Fe3⫹ FeG, 8-mg Fe3⫹
Eggs n ⴝ
Survival ratio in % of control
10 25 20
100 95 47.1
18 15 15
166.7 71.8 53.8
31 30 12
111.2 67.9 55
Abbreviations: 22 d, 2 d, 4 d, incubation time 22, 2, or 4 days, respectively.
and benzylalcohol, a control group exposed to 0.9% benzylalcohol and another iron complex was included. Neither benzylalcohol alone nor benzylalcohol in combination with another iron complex decreased the survival of the embryos. Iron content in embryonic turkey liver and kidney. Endogenous liver iron content. Livers of different ages
were examined for their iron content (16, 18, or 22 days old). Mean liver iron content decreased with embryo age, from 5.19 g/mL (⫾SEM ⫽ 0.706 g/mL) at 16 days to 5.145 g/mL (⫾SEM ⫽ 0.655 g/mL) at 18 days to 2.442 g/mL (⫾SEM ⫽ 0.376 g/mL) at 22 days. The differences between the liver iron content of a 22-day-old embryo and the 2 other age groups were statistically significant (P ⬍ 0.001). To compare the different experiments, the control livers were set to 100% regarding their iron content. Liver iron content in intervention group. In both methods of administration, all administered iron complexes resulted in an increase in liver iron content in comparison with the control group (Fig 2). The egg white injection of FeG led to an approximately 8-fold increase in liver iron content; FeD and FeS induced an about a 4-fold increase. The liver iron content after egg white injection of all iron complexes led to a statistically significant difference in comparison with the control group (FeD, FeS: P ⬍ 0.01; FeG: P ⬍ 0.001). The same tendency was observed after CAM application with an incubation time of 2 days. The increase in iron storage was much less pronounced after CAM application, and the difference in liver iron content reached statistical significance (P ⬍ 0.01) only for FeG. No differences existed between the iron complexes after CAM application with an incubation time of 4 days.
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Fig 1. Mean embryo weights (in % of control ⫾ SEM) after administration of FeD, FeS, and FeG that contains 8-mg Fe3⫹ to fertilized turkey eggs. (A) Egg white injection, incubation time was 22 days. (B) CAM application, incubation time was 2 days. (C) CAM application, incubation time was 4 days. Statistical significance in comparison with control is shown.
Fig 2. Mean liver iron content (in % of control ⫾ SEM) after administration of FeD, FeS, and FeG that contains 8-mg Fe3⫹ to fertilized turkey eggs. Egg white injection, incubation time was 22 days. Statistical significance in comparison with control is shown.
In contrast to the endogenous liver iron content, no age-dependent kidney iron content existed. Kidney iron content in intervention group. In both methods of administration, all administered iron complexes resulted in an increase in kidney iron content in comparison with the control group. As in the liver iron content, a similar tendency could be observed. The egg white injection of FeG led to the highest kidney iron content (Fig 3). The differences in kidney iron content after egg white injection of the iron complexes in comparison with the control group were statistically significant (FeD: P ⬍ 0.001, FeS; FeG: P ⬍ 0.01). After CAM application of FeG with an incubation time of 2 days, the greatest increase in kidney iron content was induced, which was statistically significant in comparison with FeD, FeS and with the control group (P ⬍ 0.01). No differences were found in the kidney iron content between the iron complexes after CAM application with an incubation time of 4 days. Endogenous kidney iron content.
Fig 3. Mean kidney iron content (in % of control ⫾ SEM) after administration of FeD, FeS, and FeG that contains 8-mg Fe3⫹ to fertilized turkey eggs. Egg white injection, incubation time was 22 days. Statistical significance in comparison with control is shown.
Liver iron content after egg white injection and after CAM applications. The increase in liver iron content
after the egg white injection of the complexes was greater than after the CAM applications, as demonstrated in Fig 4. The difference was statistically significant (P ⬍ 0.001). The same tendency could be observed with the increase in kidney iron content. In vitro model. Morphologic and gross appearance of HepG2-cells after incubation with iron complexes. The
morphologic and gross appearances of the cells after incubation with FeD were identical to those of the control cells, independent of the iron dose administered or the incubation time. The cells were adherent to the plate and had a rhombic shape. A cell pellet could be harvested in all experiments. FeD caused no color change of the cell pellets. After incubation with FeG, iron dose 7.5-mg Fe3⫹, and incubation time 1 day, the cells had a rhombic shape and were adherent to the plate. A cell pellet could be harvested. After incubation with FeG, iron dose 75-mg Fe3⫹, incubation time 1 day, and iron dose 7.5-mg Fe3⫹, incubation time 4
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Fig 4. Egg white injection versus CAM application. Comparison of mean liver iron content (in % of control ⫾ SEM) after egg white injection of FeD, FeS, and FeG that contains 8-mg Fe3⫹ with an incubation time of 22 days and CAM applications of FeD, FeS, and FeG containing 8-mg Fe3⫹ with an incubation time of 2 and 4 days to fertilized turkey eggs. o Egg white injection, d CAM 2 days, □ CAM 4 days.
days, the cells appeared apoptotic and swimming cells could be observed in the medium. Cell pellets could be harvested. All cell pellets showed a distinct, dark brown discoloration. After incubation with FeG, iron dose 75-mg Fe3⫹, incubation time 4 days, all cells appeared apoptotic, with most of them were swimming in the medium. No cell pellet could be harvested. After incubation with FeS, iron dose 7.5-mg Fe3⫹, incubation time 1 day, the cells had a rhombic shape and were adherent to the plate. With an incubation time of 4 days, same iron dose, the cells appeared apoptotic and some were swimming in the medium. The harvested cell pellets of both incubation times showed an even more profound color change than induced by FeG. After incubation for 1 day or 4 days with the high iron dose of 75-mg Fe3⫹ of FeS, all cells appeared apoptotic and most of them were swimming in the medium. No cell pellet could be harvested. According to the observed and described appearance of the cells, the toxicity was as follows: FeS ⬎ FeG ⬎ FeD. Iron content in HepG2-cells. Distinct differences in iron content (g/mg protein) in HepG2-cells could be detected depending on the iron complex and the incubation time. Figure 5 shows the mean iron content in g/mg protein (⫾SEM) in HepG2-cells after incubation of 1 and 4 days with FeD, FeS, and FeG and an iron dose of 7.5-mg Fe3⫹. The iron concentrations in the cells increased as incubation time increased. FeD did not lead to a considerable iron uptake in the cells, independent of the incubation time: 0.03-g/mg protein could be measured after 1 day and 0.22-g/mg protein after 4 days. The cells showed a greater iron content
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after incubation with FeG: 27.55-g/mg protein after 1 day and 188.65-g/mg protein after 4 days. FeS led to the greatest iron content in the cells: 79.80-g/mg protein after 1 day and 305.37-g/mg protein after 4 days. All measured iron concentrations were significant (P ⱕ 0.001) in comparison with control cells except for the iron concentration measured after incubation with FeD and an incubation time of 1 day. The differences in iron concentrations caused by the different iron complexes were also significant (P ⱕ 0.001; FeS compared with FeG, incubation time 4 days: P ⬍ 0.05). Figure 6 shows the mean iron content in g/mg protein (⫾SEM) in HepG2-cells after incubation with FeD with an iron dose of 7.5-mg Fe3⫹ and 75-mg Fe3⫹ and an incubation time of 1 day and 4 days. It was observed that the iron content in the cells increased as incubation time increased and as iron dose increased. By prolonging the incubation time from 1 day to 4 days, iron content increased from 0.03-g/mg protein to 0.22-g/mg protein with the iron dose of 7.5-mg Fe3⫹(P ⬍ 0.01) and from 0.84-g/mg protein to 1.46g/mg protein with the iron dose of 75-mg Fe3⫹ (P ⬍ 0.05). Even the iron content after the 10-fold greater iron dose of FeD remained different from the results with FeS (305.37 g/mg) and FeG (188.65 g/mg). DISCUSSION
Preclinical experiments on iron-induced renal damage may not predict directly or reflect the clinically relevant adverse effects. When deciding between oral and intravenous iron therapy, the potential consequences of intravenous iron on various tissues should be taken into account.12-14 Recent reports suggest that pronounced differences exist in the local and systemic tolerability and toxicity of the intravenous iron complexes.15,16 It is suggested that these differences derive from the variable carbohydrate structure that complexes the Fe3⫹- core, and thus, it protects the surrounding tissues from the highly reactive free iron.17 The size and the molecular weight of the iron nanoparticle complexes vary distinctly as reviewed recently.18 In the in ovo model, we observed a toxicity profile of the different iron complexes as follows: FeD ⬍ FeS ⬍ FeG. FeD showed the best tolerability in all experiments. The survival ratio, as well as the embryo weights, compared with the control group, were the greatest after administration of FeD. No reduction of survival ratio or embryo weight existed after administration of FeD compared with the control group. It even seemed that FeD supported the embryo survival as well as the embryo growth, as demonstrated by the greater survival ratio and the greater embryo weight in comparison with the control group. The greatest toxicity was observed after administration of FeG.
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Fig 5. Mean iron content (g/mg protein ⫾ SEM) in HepG2-cells after incubation with FeD, FeS, and FeG (iron dose: 7.5-mg Fe3⫹). (A) Incubation time 1 day. (B) Incubation time 4 days. Statistical significance in comparison with control is shown.
Fig 6. Mean iron content (g/mg protein ⫾ SEM) in HepG2-cells after incubation with FeD with an iron dose of 7.5-mg Fe3⫹ and 75-mg Fe3⫹ and an incubation time of 1 day and 4 days. Statistical significance in comparison with control is shown.
Studies on the particle size in preparations of iron gluconate and iron sucrose found that the particle size was dependent on the solution. Under similar conditions, however, iron gluconate particles were smaller than iron sucrose particles.19 The differences in particle size correspond to the differences in stability of the complexes. The transfer of iron from the complexes to transferrin has been used as a stability parameter of the iron carbohydrate complexes. Van Wyck et al20 found that the percentage of labile iron increased according to this study in the order FeD (Dexferrum; American Regent, Inc., Shirley, NY) ⬍ FeD (InFed; Watson Pharma, Inc.. Corona, Calif, which corresponds to CosmoFer) ⬍ FeS (Venofer) ⬍ FeG (Ferrlecit). In vitro studies by Agarwal et al21 also described a more rapid transferrin saturation with FeG compared with FeS. Unpublished investigations into the stability of FeD, FeS, and FeG in serum done by the working group of Langguth et al point in the same direction, which shows that FeD is more stable than FeS and FeG. In this study, the analysis of iron in the liver and
kidney showed a distinct increase in iron concentration for all complexes, in both the egg white injection and the CAM application compared with the control. Differences that were found in iron concentration were determined by the organ (liver or kidney), the method of application, and—to a lesser extent—the iron complex used. After the egg white injection and the CAM application with an incubation time of 2 days, FeG caused a greater increase in liver iron concentration than the other 2 complexes. The same was true for the iron increase in kidney but less pronounced. All preparations enhanced the iron content clearly in liver and kidney, and all preparations have been used successfully in patients. Therefore, the minor differences between the various iron preparations with regard to the hepatic and renal iron may not be relevant clinically. After the egg white injection of the iron complexes, the increase in liver iron content was greater than the increase in kidney iron content. Whereas under physiologic conditions most excess iron is stored in the hepatic parenchyma, increased loading of the plasma transferrin with iron has been shown to increase the uptake in other parenchymal tissues.22 In our case, this was demonstrated with the kidney parenchyma. The increase in liver iron content after the egg white injection turned out to be significantly greater than after the CAM applications. The same tendency could be observed with the increase in kidney iron content, although to a lesser extent. The most important reason for this difference was probably the longer incubation time of the egg white injection with 22 days, compared with 2 or 4 days of the CAM applications. The iron complexes had more time to be absorbed by the embryo and to be distributed into the liver and kidney afterward. Furthermore, the iron complexes could be absorbed in more ways after the egg white injection than after the CAM application. The iron could have been absorbed via the skin of the embryo, and the embryo could have swallowed the iron complexes and could have absorbed the iron from its guts afterward. A third possibility is
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that iron was absorbed via the CAM. After the CAM application, only the absorption via the CAM was possible. The in vitro experiments suggest a different toxicity profile than observed in the in ovo experiments because of the morphologic changes of the cells after incubation with the iron solutions. The in vitro toxicity rank order in HepG2-cells might be FeS ⬎ FeG ⬎ FeD as has been suggested by Zager et al16 in human kidney cells. The iron concentrations in the cells decreased in the same rank order FeS ⬎ FeG ⬎ FeD. In experiments by Scheiber-Mojdehkar et al, 23 it could be demonstrated that FeS and FeG donate iron to HepG2-cells as efficiently as low-molecular-weight iron. They induced an increase in the expression of ferritin and of divalent metal transporter-1 (DMT-1), the divalent metal transporter. The incubation of the cells with FeD did not lead to a considerable iron uptake and was only a weak inductor of the expression of ferritin and DMT-1. These findings are in agreement with the results of our experiments, but we cannot determine distinctly whether the iron we measured was simply adherent iron on the plasma membrane of the cells or iron that had been taken up by the cells, via the DMT-1, or both. The apparent iron content of the HepG2-cells after treatment with FeS and FeG was excessively high. The values exceeded clearly the iron contents from liver tissue from patients with pathologically increased iron storage.24 The striking, dark brown discoloration of the cells after incubation with FeS and FeG suggests that at least some iron was attached to the cell membrane. Therefore, the interpretation of the excessively high iron concentrations in the cells is difficult. The fact that FeD did not lead to a considerable iron uptake in the cells also shows that the in vitro experiment is not suitable to answer the question of iron uptake in cells and is therefore less relevant biologically. We do know from clinical experience that FeD as well as FeS and FeG does increase hemoglobin levels as well as the serum ferritin concentration and transferrin saturation; therefore, the FeD iron must be able to overcome cellular barriers and be internalized within cells. Different models for in vitro studies on iron toxicity are available that may be used to address different questions. Experimental studies can be helpful if they reflect the clinical reality sufficiently. Cell culture models are well standardized and include less potentially variable parameters than do the more complex in vivo experiments with laboratory animals or similarly complex models of avian embryos. Other studies of parenteral iron complexes have used HepG2-cells as well.23 In our experiments, significant cellular iron uptake in the in ovo model in the embryonic liver and kidney was in line with the known clinical efficacy of the different
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iron complexes. At variance, in HepG2-cells we observed either excessively high levels of cell-bound iron (FeS and FeG) or no relevant iron uptake at all (FeD). We found that the in ovo model as a multitissue system may better reflect the clinically relevant effects on intracellular iron storage and the toxicity of the compounds than an in vitro model with HepG2 cells. We conclude that the avian embryonic model seems interesting for preclinical studies on the balance of efficacy and safety, which may be useful for the comparison of different products of this group in a regulatory context. In addition, the in ovo model might be used to investigate the efficacy of chelators in removing iron from tissues similar to experiments that have been described in rodents.25 CONCLUSION
In this study, 3 different approved parenteral iron complexes were analyzed in an in ovo model as well as in in vitro experiments. The results were compared with their known clinical effectiveness. The impact of the type of carbohydrate used in the formulation of parenteral iron on toxicity could be demonstrated. The most stable form of intravenous iron—low-molecular-weight iron dextran—seemed to be less toxic in both the in ovo model and the in vitro experiments. Furthermore, this study shows that in respect of iron uptake the in ovo model with the more complex interaction of cells and tissues can present results that reflect the clinical situation in humans better than in experiments with isolated cells. The assistance of Ms. Claudia Götze, Ms. Bianka Keil, and Ms. Ute Hartung is gratefully acknowledged. REFERENCES
1. Chertow GM, Mason PD, Vaage-Nilsen O, Ahlmen J. On the relative safety of parenteral iron formulations. Nephrol Dial Transplant 2004;19:1571–5. 2. Valderrabano F, Jofre R, Lopez-Gomez JM. Quality of life in end-stage renal disease patients. Am J Kidney Dis 2001;38:443– 64. 3. Besarab A, Amin N, Ahsan M, et al. Optimization of epoetin therapy with intravenous iron therapy in hemodialysis patients. J Am Soc Nephrol 2000;11:530 – 8. 4. Lappin TR, Maxwell AP, Johnston PG. Warning flags for erythropoiesis-stimulating agents and cancer-associated anemia. Oncologist 2007;12:362–5. 5. Henry DH. The role of intravenous iron in cancer-related anemia. Oncology 2006;20(8 suppl 6):21– 4. 6. Shah SV, Alam MG. Role of iron in atherosclerosis. Am J Kidney Dis 2003;41(3 suppl 1):S80 –3. 7. Macdougall IC. Strategies for iron supplementation: oral versus intravenous. Kidney Int 1999; 69(suppl):S61– 6. 8. Afzali B, Goldsmith DJ. Intravenous iron therapy in renal failure: friend and foe? J Nephrol 2004;17:487–95. 9. Kunzi-Rapp K, Genze F, Kufer R, Reich E, Hautmann RE, Gschwend JE. Chorioallantoic membrane assay: vascularized
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3-dimensional cell culture system for human prostate cancer cells as an animal substitute model. J Urol 2001;166:1502–7. Rebouche CJ, Wilcox CL, Widness JA. Microanalysis of nonheme iron in animal tissues. J Biochem Biophys Methods 2004; 58:239 –51. Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76 – 85. Agarwal R, Rizkala AR, Bastani B, Kaskas MO, Leehey DJ, Besarab A. A randomized controlled trial of oral versus intravenous iron in chronic kidney disease. Am J Nephrol 2006;26:445– 54. Sengolge G, Horl WH, Sunder-Plassmann G. Intravenous iron therapy: well-tolerated, yet not harmless. Eur J Clin Invest 2005; 35(suppl 3):46 –51. Horl WH. Iron therapy for renal anemia: how much needed, how much harmful? Pediatr Nephrol 2007;22:480 –9. Zager RA, Johnson AC, Hanson SY, Wasse H. Parenteral iron formulations: a comparative toxicologic analysis and mechanisms of cell injury. Am J Kidney Dis 2002;40:90 –103. Zager RA, Johnson AC, Hanson SY. Parenteral iron nephrotoxicity: potential mechanisms and consequences. Kidney Int 2004; 66:144 –56. Boettcher-Buehler E, Gethins M. Safety and tolerability of parenteral iron formulations. Fe Report, XXXIX Congress of the European Renal Association (ERA)—European Dialysis and Transplant Association (EDTA), Copenhagen, 2002.
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18. Jahn M, Roth S, Langguth P. Parenterale kolloidale Eisenkomplexpräparate. Deutsche Apotheker Zeitung 2006;146:15110 –9. 19. Kudasheva DS, Lai J, Ulman A, Cowman MK. Structure of carbohydrate-bound polynuclear iron oxyhydroxide nanoparticles in parenteral formulations. J Inorg Biochem 2004; 98:1757– 69. 20. Van Wyck D, Anderson J, Johnson K. Labile iron in parenteral iron formulations: a quantitative and comparative study. Nephrol Dial Transplant 2004;19:561–5. 21. Agarwal R. Transferrin saturation with intravenous irons: an in vitro study. Kidney Int 2004;66:1139 – 44. 22. Torrance JD, Bothwell TH. Tissue iron stores. In: Cook JD, ed. Iron. Methods in hematology, 1st ed. New York: Churchill Livingstone; 1980:90 –115. 23. 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 2003;65:1973– 8. 24. Rose C, Vandevenne P, Bourgeois E, Cambier N, Ernst O. Liver iron content assessment by routine and simple magnetic resonance imaging procedure in highly transfused patients. Eur J Haematol 2006;77:145–9. 25. Hodges YK, Weinberger HD, Stephens J, Horwitz MA, Horwitz LD. Desferri-Exochelin, a lipid-soluble, hexadentate iron chelator, effectively removes tissue iron. Transl Res 2006;148:63–71.
In this study the toxicity and intracellular availability of iron from iron dextran (FeD), iron sucrose (FeS), and iron gluconate (FeG) was compared in organs of avian (turkey) embryos and in isolated cells (HepG2) in cell culture. Iron uptake was more pronounced in embryonic liver than in renal tissue. Cellular iron uptake in liver and kidney was more or less similar for the different compounds. Only some experiments showed slightly greater iron concentrations in liver and kidney with FeG compared with FeD and FeS. Significant differences were found in the survival ratios of the eggs and the embryo weights depending on the type of iron complex administered. The rank order of toxicities was FeG > FeS > FeD. Iron accumulation in HepG2-cells was extremely high with FeS and FeG, whereas FeD did not lead to a relevant iron uptake by HepG2 cells. The excessively high iron content of the cells is an in vitro phenomenon found neither in the in ovo model with the turkey embryos nor in the clinical use of the compounds. The rank order of toxicities in HepG2 cells was FeS > FeG > FeD. Iron uptake in cell culture does not reflect the in vivo situation. The in ovo model is more suitable to assess the cellular iron uptake and iron toxicity in cells and tissues than the in vitro model. In both in ovo and in vitro experiments, FeD seemed to be superior in terms of toxicity. (Translational Research 2008;151:36-44) Abbreviations: CAM ⫽ chorio-allantoic-membrane; DMT-1 ⫽ divalent metal transporter-1; FeD ⫽ iron dextran; FeG ⫽ iron gluconate; FeS ⫽ iron sucrose; PBS ⫽ phosphate-buffered saline; r/min ⫽ rotations per minute; RPMI ⫽ Roswell Park Memorial Institute (cell culture medium); RT ⫽ room temperature; SEM ⫽ standard error of the mean
P
arenteral iron therapy is used widely for the treatment of anemia in patients with various underlying diseases. Intravenous iron therapy is the most effective regimen for the treatment of anemia in patients with chronic kidney disease. This outcome is to be expected as a dialysis patient encounters an average yearly blood From the Federal Institute for Drugs and Medical Devices, Bonn, and the Institute of Pharmacy, Johannes Gutenberg-University, Mainz, Germany. Submitted for publication April 16, 2007; revision submitted August 20, 2007; accepted for publication September 4, 2007. Reprint requests: Susanne Roth, University of Bonn, Institute of Pharmacy, Clinical Pharmacy, An der Immenburg 4, 53121 Bonn, Germany; e-mail: [email protected]. 1931-5244/$ – see front matter © 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.trsl.2007.09.001
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loss equivalent to 1–1.5 g of elementary iron.1 The treatment of anemia with intravenous iron leads to improvement in morbidity, mortality, and quality of life.2 Parenteral iron has been shown to reduce the erythropoietin dose requirements in patients with chronic kidney disease up to 40%, which seems to be of special importance in the context of the potential adverse effects of high doses of erythropoietin that reduces the survival of cancer patients.3,4 The use of intravenous supplemental iron therapy rather than oral treatment has been proposed for cancer patients.5 Generally, the parenteral iron formulations are regarded as safe. Nevertheless, all intravenous iron complexes in use have the potential to induce oxidant stress by the formation of very strong oxidants such as the hydroxylradical in the Haber–Weiss reaction.6 Concerning the long-term toxicity of parenteral iron preparations, several reports describe a connection between increased
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AT A GLANCE COMMENTARY Background
Intravenous iron complexes currently used for the treatment of anemia have potential side effects and associated risks that might depend on the type of carbohydrate complexing the iron core in the different iron preparations. Translational Significance
The toxicity and iron uptake of 3 parenteral iron complexes were tested using 2 different models. Iron uptake in the in ovo model with the more complex interaction of cells and tissues better reflected the clinical situation than HepG2-cells, whereas toxicity was similar in both models. Therefore, the in ovo model may contribute to detect clinically relevant differences between available or newly developed iron preparations with different carbohydrate complexes.
body iron stores and an increased risk of cancer and detail how an iron overload leads to an increased risk of infection.7,8 Widely different models for in vitro studies on iron toxicity are available, and they may be used for different purposes. The goal of this study was to compare cellular iron uptake and iron induced cytotoxicity of 3 different nanoparticular iron complexes [iron dextran (FeD), iron sucrose (FeS), iron gluconate (FeG)] in a well-defined in vitro model with HepG2-cells and a new, more complex avian embryo model to the wellknown effects of parenteral iron in humans. In the in ovo experiments, 2 different routes of administration were studied: injection in the egg white and application on the chorio-allantoic-membrane (CAM). The availability of iron from the different complexes was measured as cell-bound iron in HepG2-cells and as hepatic and renal iron storage in the turkey embryos. The toxicity of the different iron complexes was reflected in the effects on embryo survival and embryo weight and in the cytotoxic effects on HepG2-cells. METHODS Iron complexes. FeD (CosmoFer; GRY-Pharma GmbH, Kirchzarten, Germany, batch number 0343110-4), FeS (Venofer; Fresenius Medical Care, Bad Homburg, Germany, batch number 397100), and FeG (Ferrlecit; Aventis-Pharma Deutschland GmbH, Frankfurt am Main, Germany, batch number D4A708) were purchased from a pharmacy. CosmoFer is an iron(III)– hydroxide– dextran complex containing 100 mg Fe3⫹ in a 2-mL ampoule in aqua ad injectabilia with NaOH and HCl for pH-adjustment. Venofer is an
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iron(III)– hydroxide–sucrose complex containing 100 mg Fe3⫹ in a 5-mL ampoule dissolved in aqua ad injectabilia and adjusted to pH with NaOH. The resulting solution has a pH of approximately 10.5. Ferrlecit is an iron(III)–sodium– gluconate complex containing 40 mg Fe3⫹ in a 3.2-mL ampoule in sucrose and aqua ad injectabilia. Ferrlecit also contains 0.9% benzylalcohol as a preservative. In ovo model. The fertilized turkey eggs were purchased from the Schabrockerhof hatchery (Wachtendonk, Germany). Turkey eggs have a hatching period of 28 days. In this series of experiments, the dose for each egg was set to 8 mg of Fe3⫹. The resulting injection volumes were FeD 160 L, FeS 400 L, and FeG 640 L. Control eggs received 160 L, 400 L, or 640 L of physiologic sodium chloride solution (Serumwerk Bernburg, Bernburg, Germany). Different injection volumes had no effect on egg survival. Egg white injection. Before incubation, fertilized turkey eggs were separated into intervention and control groups. The resulting average egg weights of each group differed by 1% to 2%. The eggs were positioned with the large end pointing upward in an egg carton. A cross was marked 1–2 cm above the egg point with a pencil, and the area was disinfected with a piece of cloth soaked with ethanol 70%. With a pointed pair of scissors, the egg shell was pierced in the middle of the marked cross. The iron complex solutions were drawn up into a syringe and injected into the white of the egg through the hole. Control eggs received the corresponding volume of physiologic sodium chloride solution. After the treatment, the injection site was sealed with adhesive tape and the eggs were placed in an incubator at 37°C and 65% humidity (BSS 420; Ehret Labor-und Pharmatechnik, Emmendingen, Germany). After an incubation period of 22 days, the experiment was terminated. The vitality and weights of each embryo were determined. Livers and kidneys were removed and frozen at – 80°C until iron analytics were performed. Egg white injection of benzylalcohol. A 0.9% solution of benzylalcohol in physiologic sodium chloride solution (solution A) and a solution that contained FeD and 0.9% benzylalcohol in physiologic sodium chloride solution (solution B) were injected at volumes of 640 L each to study the impact of the preservative on the resulting toxicity. Overall, 640 L of solution B contained 8 mg of Fe3⫹. The incubation time was 22 days. CAM application. Preparation of the chorioallantoic membrane was by a modification of a described method by Kunzi-Rapp et al.9 Fertilized turkey eggs, incubated for 12 days at 37°C and 65% humidity, were incubated with the pointed end downward until day 13. During this time, the embryos moved to the blunt end of the egg. On day 13, the eggs were turned again and a hole was drilled through the pointed end of the shell. The hole was sealed with adhesive tape, and the eggs were incubated again until day 14. Because of the hole in the pointed end of the egg, the air pocket moved from the blunt end to the pointed end. On day 14, the air pocket was positioned between the CAM and the inner egg shell. The hole was enlarged by peeling a round, 5-mm window. The iron complex solutions were applied carefully drop by drop.
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Control eggs received physiologic sodium chloride solution in the same manner. After the treatment, the application site was sealed with adhesive tape and the incubation was carried on for 2 or 4 more days. After a total of 16 or 18 days in the incubator, the experiment was terminated. The vitality and the weights of the embryos were determined. Livers and kidneys were removed and frozen at – 80°C until iron analysis was performed. In vitro model. HepG2-cells are human hepatoma cells, which have epithelial appearance and grow as monolayer in small cell associations. Cells were grown in RPMI (Roswell Park Memorial Institute cell culture medium) and were supplemented with 10% fetal calf serum, penicillin/streptomycin, and glutamine under standard tissue culture conditions (37°C, 5% CO2). The cells were subcultured every few days. The medium was then removed with a pump, and the flask with the cells was washed with about 10 mL of phosphate-buffered saline (PBS). PBS was removed, and 2 mL of trypsin/ethylenediamine tetraacetic acid was added. The flask was incubated for 5–10 min at 37°C until the cells detached. A total of 5–10 mL of RPMI was added to stop trypsin activity. The medium that contained the cells was put into a centrifuge tube and centrifuged at 1000 rotations per minute (r/min) for 5 min at room temperature (RT). The supernatant was discarded, and the cells were resuspended in 5–10 mL of fresh RPMI with a needle and a syringe. The resuspended cells were then dispensed into new culture flasks, which contained 5 mL of RPMI, and put back in the incubator. Iron loading of the cells. Each confluent HepG2-cell culture flask was subcultured on 5 cell plates, 15 mL of RPMI was added, and the cells were incubated for 1 day at 37°C and 5% CO2. One day later, solutions of the iron complexes were prepared in RPMI. At this time, 15 mL of medium was put in a sterile flask and 7.5 or 75 mg of Fe3⫹ was added, respectively. Depending on the iron concentration in each iron complex solution, the added volumes differed slightly (eg, for FeD, 150 L vs FeG600 L). After adding the iron complexes to the medium, the tubes were shaken to form a homogenous solution. The old medium was then removed from the cell plates, and the new medium that contained 7.5 or 75 mg of Fe3⫹, respectively, was added to the cells. Control plates received the medium supplemented with the corresponding volume of physiologic sodium chloride solution. After iron loading, the cell plates were incubated at 37°C, 5% CO2, and were harvested after an incubation time of 1 or 4 days, respectively, which resulted in a total of 4 different intervention groups and 4 corresponding control groups. Each group contained at least n ⫽ 3 cell plates. Cell harvest. Before harvesting the cells, the morphologic appearance of the cells was assessed with a microscope (Leica Microsystems, Wetzlar, Germany) and was enlarged 100-fold and 200-fold. Before removing the old medium with a pump, 10 mL of medium was added to the cells so that the cells were not damaged while removing the medium. After the medium was removed, the cells were washed 5 times with 15 mL of PBS to remove the iron remnants. Then, 5 mL of PBS was added and the cells were recovered by scraping with a cell scraper, washed again with 5 mL of PBS, and centri-
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fuged (2000 r/min for 10 min) at RT. The supernatant was discarded, and the cell pellet was resuspended in 200 L of PBS. The cell suspension was transferred into a tared 1.5-mL centrifuge tube and centrifuged for 13,200 r/min for 10 min at 4°C. The supernatant was discarded, and the pellet was dried completely with compressed air. The tube and pellet was weighed and frozen at ⫺80°C until iron analysis was performed. Microanalysis of iron in tissue. The quantification of iron in liver tissue and in kidney tissue was carried out in accordance with the method of Rebouche et al.10 The following reagents were obtained from Sigma-Aldrich (Steinheim, Germany): sodium 3-(2-pyridyl)-5,6-bis(4-phenylsulfonate)1,2,4-triazine (ferrozine), thioglycolic acid, and trichloroacetic acid. Iron standard solution (989-g Fe/mL in 1.1% HCl) was purchased from Aldrich (Milwaukee, Minn). HCL 37% was obtained from Merck (Darmstadt, Germany). Sodium acetate was obtained from Roth (Karlsruhe, Germany). Liver and kidney tissue were homogenized after dilution of the tissue 1:10 (wt/vol) with deionized water, using a Potter homogenizer (Braun Biotech International, Melsungen, Germany) at 1100 RPM for 2–3 min (liver) and 1–2 min (kidney), under cooling in an ice water mixture. Equal volumes of tissue homogenates and protein precipitation solution (1N HCL that contained 10% trichloroacetic acid in deionized water) were combined in 1.5-mL centrifuge tubes (Sarstedt, Nümbrecht, Germany), mixed by vortex, and placed in a 95°C heating block for 1 h. Tubes and contents were cooled in water at RT for 2 min, vortex mixed, and then centrifuged at 16.000 ⫻ g for 10 min at RT (Digifuge, Heraeus; Kendro Laboratory Products, Langenselbold, Germany). The supernatant was divided into three 1.5-mL centrifuge tubes (50 L each). Two centrifuge tubes, 50 L of chromogen solution (0.508 mmol/L ferrozine, 1.5 M sodium acetate, and 0.1% (vol/vol) in deionized water) was added. Sample blanks consisted of 50 L of tissue extract supernatant and 50 L of 1.5 M sodium acetate that contained 0.1% (vol/vol) thioglycolic acid. The mixtures were vortexed, and after 30 min at RT, absorbance was measured at 562 nm in microcuvettes (Plastibrand; Brand, Wertheim, Germany) in a photometer (MBA 2000; Perkin Elmer, Wellesley, Mass). Standard curves were prepared with each measurement using iron standards that contained 0-, 1-, 2-, 4-, 6-, 8-, and 10-g Fe3⫹/mL of the iron standard solution and diluted with an equal volume of protein precipitation solution. The equation of the regression line was used to calculate the iron contents of the sample solutions. Microanalysis of iron in cells. The quantification of iron in HepG2 cells was carried out in accordance with the method of Rebouche et al.10 The cell pellet was diluted with water 1:10 (wt/vol) and resuspended with a needle and a syringe. Afterward, the cell suspension was put into an ultrasound bath (Bandelin Electronic, Berlin, Germany) for 10 min. For the next procedure, see “Microanalysis of iron in tissue,” which differs from the first protocol only by the handling of the precipitated protein pellet. The precipitated protein pellet was dried and frozen at ⫺80°C until protein analysis was performed. Protein analysis. The BCA-assay (BCA protein assay kit; Pierce, Rockford, Ill) was used for protein analysis that is
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based on the known biuret-reaction.11 The precipitated protein pellets were defrosted and dissolved in 500 L of 1 M NaOH. In total, 1 M HCl was added to adjust pH to pH 7. The samples were diluted with H2O 1:2 (wt/vol). Protein standards were prepared from a BSA-standard solution (Bovine serum albumin 2000 g/mL), which contained 1500 g/mL, 1000 g/mL, 750 g/mL, 500 g/mL, 250 g/mL, 125 g/mL, 25 g/mL, and 0 g/mL of BSA in H2O. The microplate protocol was used, and absorption was measured at 570 nm with a microplate reader (Dynex Technologies, Chantilly, Va). A standard curve was prepared with the protein standards. The equation of the regression line was used to calculate the protein contents of the sample solutions. The iron content of the HepG2-cells was assessed before it was applied to the protein content of the cells. Statistical analysis. Statistical computations were performed using S-Plus 2000 (Insightful Corporation, Seattle, Wash). Results are presented as means ⫾ standard error of the mean (SEM). A comparison of the embryo weights and liver and kidney iron contents of the intervention group with the parameters of the control group was performed by applying the Wilcoxon rank sum test. This test was also used to compare different intervention groups with embryo weights and liver and kidney iron contents. Group sizes ranged from 4 to 17 (control group, embryo weight), 6 to 17 (intervention group, embryo weight), 4 to 12 (control group iron content in liver/kidney), and 6 to 16 (intervention group, iron content in liver/kidney). The t test was applied to analyze the iron content in g/mg protein of the HepG2-cells (confidence-interval 95%). For iron analysis in the cells, n ⫽ 3 cell pellets were used. Ethical statement. This article conforms to the relevant ethical guidelines for animal research. RESULTS In ovo model. Survival ratios and embryo weights. Pronounced differences existed between the survival ratios of the various iron complexes (Table I). All experiments showed the same tendency, the survival ratio rank list was as follows: FeD ⬎ FeS ⬎ FeG. The same rank order as in the survival ratios was observed for the embryo weights (Fig 1). Statistical significance existed in embryo weights after egg white injection of FeG in comparison with control group (P ⬍ 0.05), after egg white injection of FeD in comparison with FeG (P ⬍ 0.01), after egg white injection of FeD in comparison with FeS (P ⬍ 0.05), and after CAM application of FeD in comparison with FeS with an incubation time of 4 days (P ⬍ 0.01). At variance with the other iron complexes, the iron gluconate preparation Ferrlecit contains 0.9% benzylalcohol as preservative. To conclude that the observed effects are caused not by the iron complex but are caused by benzylalcohol, the 0.9% benzylalcohol control group was included. To conclude additionally that the observed effects of Ferrlicit are caused not by the iron complex but also by a combination of iron
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Table I. In ovo experiments with survival ratios of the eggs in % of control after administration of FeD, FeS, and FeG containing 8-mg Fe3⫹ Experiment
Egg-white injection, 22 d FeD, 8-mg Fe3⫹ FeS, 8-mg Fe3⫹ FeG, 8-mg Fe3⫹ CAM application, 2 d FeD, 8-mg Fe3⫹ FeS, 8-mg Fe3⫹ FeG, 8-mg Fe3⫹ CAM application, 4 d FeD, 8-mg Fe3⫹ FeS, 8-mg Fe3⫹ FeG, 8-mg Fe3⫹
Eggs n ⴝ
Survival ratio in % of control
10 25 20
100 95 47.1
18 15 15
166.7 71.8 53.8
31 30 12
111.2 67.9 55
Abbreviations: 22 d, 2 d, 4 d, incubation time 22, 2, or 4 days, respectively.
and benzylalcohol, a control group exposed to 0.9% benzylalcohol and another iron complex was included. Neither benzylalcohol alone nor benzylalcohol in combination with another iron complex decreased the survival of the embryos. Iron content in embryonic turkey liver and kidney. Endogenous liver iron content. Livers of different ages
were examined for their iron content (16, 18, or 22 days old). Mean liver iron content decreased with embryo age, from 5.19 g/mL (⫾SEM ⫽ 0.706 g/mL) at 16 days to 5.145 g/mL (⫾SEM ⫽ 0.655 g/mL) at 18 days to 2.442 g/mL (⫾SEM ⫽ 0.376 g/mL) at 22 days. The differences between the liver iron content of a 22-day-old embryo and the 2 other age groups were statistically significant (P ⬍ 0.001). To compare the different experiments, the control livers were set to 100% regarding their iron content. Liver iron content in intervention group. In both methods of administration, all administered iron complexes resulted in an increase in liver iron content in comparison with the control group (Fig 2). The egg white injection of FeG led to an approximately 8-fold increase in liver iron content; FeD and FeS induced an about a 4-fold increase. The liver iron content after egg white injection of all iron complexes led to a statistically significant difference in comparison with the control group (FeD, FeS: P ⬍ 0.01; FeG: P ⬍ 0.001). The same tendency was observed after CAM application with an incubation time of 2 days. The increase in iron storage was much less pronounced after CAM application, and the difference in liver iron content reached statistical significance (P ⬍ 0.01) only for FeG. No differences existed between the iron complexes after CAM application with an incubation time of 4 days.
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Fig 1. Mean embryo weights (in % of control ⫾ SEM) after administration of FeD, FeS, and FeG that contains 8-mg Fe3⫹ to fertilized turkey eggs. (A) Egg white injection, incubation time was 22 days. (B) CAM application, incubation time was 2 days. (C) CAM application, incubation time was 4 days. Statistical significance in comparison with control is shown.
Fig 2. Mean liver iron content (in % of control ⫾ SEM) after administration of FeD, FeS, and FeG that contains 8-mg Fe3⫹ to fertilized turkey eggs. Egg white injection, incubation time was 22 days. Statistical significance in comparison with control is shown.
In contrast to the endogenous liver iron content, no age-dependent kidney iron content existed. Kidney iron content in intervention group. In both methods of administration, all administered iron complexes resulted in an increase in kidney iron content in comparison with the control group. As in the liver iron content, a similar tendency could be observed. The egg white injection of FeG led to the highest kidney iron content (Fig 3). The differences in kidney iron content after egg white injection of the iron complexes in comparison with the control group were statistically significant (FeD: P ⬍ 0.001, FeS; FeG: P ⬍ 0.01). After CAM application of FeG with an incubation time of 2 days, the greatest increase in kidney iron content was induced, which was statistically significant in comparison with FeD, FeS and with the control group (P ⬍ 0.01). No differences were found in the kidney iron content between the iron complexes after CAM application with an incubation time of 4 days. Endogenous kidney iron content.
Fig 3. Mean kidney iron content (in % of control ⫾ SEM) after administration of FeD, FeS, and FeG that contains 8-mg Fe3⫹ to fertilized turkey eggs. Egg white injection, incubation time was 22 days. Statistical significance in comparison with control is shown.
Liver iron content after egg white injection and after CAM applications. The increase in liver iron content
after the egg white injection of the complexes was greater than after the CAM applications, as demonstrated in Fig 4. The difference was statistically significant (P ⬍ 0.001). The same tendency could be observed with the increase in kidney iron content. In vitro model. Morphologic and gross appearance of HepG2-cells after incubation with iron complexes. The
morphologic and gross appearances of the cells after incubation with FeD were identical to those of the control cells, independent of the iron dose administered or the incubation time. The cells were adherent to the plate and had a rhombic shape. A cell pellet could be harvested in all experiments. FeD caused no color change of the cell pellets. After incubation with FeG, iron dose 7.5-mg Fe3⫹, and incubation time 1 day, the cells had a rhombic shape and were adherent to the plate. A cell pellet could be harvested. After incubation with FeG, iron dose 75-mg Fe3⫹, incubation time 1 day, and iron dose 7.5-mg Fe3⫹, incubation time 4
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Fig 4. Egg white injection versus CAM application. Comparison of mean liver iron content (in % of control ⫾ SEM) after egg white injection of FeD, FeS, and FeG that contains 8-mg Fe3⫹ with an incubation time of 22 days and CAM applications of FeD, FeS, and FeG containing 8-mg Fe3⫹ with an incubation time of 2 and 4 days to fertilized turkey eggs. o Egg white injection, d CAM 2 days, □ CAM 4 days.
days, the cells appeared apoptotic and swimming cells could be observed in the medium. Cell pellets could be harvested. All cell pellets showed a distinct, dark brown discoloration. After incubation with FeG, iron dose 75-mg Fe3⫹, incubation time 4 days, all cells appeared apoptotic, with most of them were swimming in the medium. No cell pellet could be harvested. After incubation with FeS, iron dose 7.5-mg Fe3⫹, incubation time 1 day, the cells had a rhombic shape and were adherent to the plate. With an incubation time of 4 days, same iron dose, the cells appeared apoptotic and some were swimming in the medium. The harvested cell pellets of both incubation times showed an even more profound color change than induced by FeG. After incubation for 1 day or 4 days with the high iron dose of 75-mg Fe3⫹ of FeS, all cells appeared apoptotic and most of them were swimming in the medium. No cell pellet could be harvested. According to the observed and described appearance of the cells, the toxicity was as follows: FeS ⬎ FeG ⬎ FeD. Iron content in HepG2-cells. Distinct differences in iron content (g/mg protein) in HepG2-cells could be detected depending on the iron complex and the incubation time. Figure 5 shows the mean iron content in g/mg protein (⫾SEM) in HepG2-cells after incubation of 1 and 4 days with FeD, FeS, and FeG and an iron dose of 7.5-mg Fe3⫹. The iron concentrations in the cells increased as incubation time increased. FeD did not lead to a considerable iron uptake in the cells, independent of the incubation time: 0.03-g/mg protein could be measured after 1 day and 0.22-g/mg protein after 4 days. The cells showed a greater iron content
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after incubation with FeG: 27.55-g/mg protein after 1 day and 188.65-g/mg protein after 4 days. FeS led to the greatest iron content in the cells: 79.80-g/mg protein after 1 day and 305.37-g/mg protein after 4 days. All measured iron concentrations were significant (P ⱕ 0.001) in comparison with control cells except for the iron concentration measured after incubation with FeD and an incubation time of 1 day. The differences in iron concentrations caused by the different iron complexes were also significant (P ⱕ 0.001; FeS compared with FeG, incubation time 4 days: P ⬍ 0.05). Figure 6 shows the mean iron content in g/mg protein (⫾SEM) in HepG2-cells after incubation with FeD with an iron dose of 7.5-mg Fe3⫹ and 75-mg Fe3⫹ and an incubation time of 1 day and 4 days. It was observed that the iron content in the cells increased as incubation time increased and as iron dose increased. By prolonging the incubation time from 1 day to 4 days, iron content increased from 0.03-g/mg protein to 0.22-g/mg protein with the iron dose of 7.5-mg Fe3⫹(P ⬍ 0.01) and from 0.84-g/mg protein to 1.46g/mg protein with the iron dose of 75-mg Fe3⫹ (P ⬍ 0.05). Even the iron content after the 10-fold greater iron dose of FeD remained different from the results with FeS (305.37 g/mg) and FeG (188.65 g/mg). DISCUSSION
Preclinical experiments on iron-induced renal damage may not predict directly or reflect the clinically relevant adverse effects. When deciding between oral and intravenous iron therapy, the potential consequences of intravenous iron on various tissues should be taken into account.12-14 Recent reports suggest that pronounced differences exist in the local and systemic tolerability and toxicity of the intravenous iron complexes.15,16 It is suggested that these differences derive from the variable carbohydrate structure that complexes the Fe3⫹- core, and thus, it protects the surrounding tissues from the highly reactive free iron.17 The size and the molecular weight of the iron nanoparticle complexes vary distinctly as reviewed recently.18 In the in ovo model, we observed a toxicity profile of the different iron complexes as follows: FeD ⬍ FeS ⬍ FeG. FeD showed the best tolerability in all experiments. The survival ratio, as well as the embryo weights, compared with the control group, were the greatest after administration of FeD. No reduction of survival ratio or embryo weight existed after administration of FeD compared with the control group. It even seemed that FeD supported the embryo survival as well as the embryo growth, as demonstrated by the greater survival ratio and the greater embryo weight in comparison with the control group. The greatest toxicity was observed after administration of FeG.
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Fig 5. Mean iron content (g/mg protein ⫾ SEM) in HepG2-cells after incubation with FeD, FeS, and FeG (iron dose: 7.5-mg Fe3⫹). (A) Incubation time 1 day. (B) Incubation time 4 days. Statistical significance in comparison with control is shown.
Fig 6. Mean iron content (g/mg protein ⫾ SEM) in HepG2-cells after incubation with FeD with an iron dose of 7.5-mg Fe3⫹ and 75-mg Fe3⫹ and an incubation time of 1 day and 4 days. Statistical significance in comparison with control is shown.
Studies on the particle size in preparations of iron gluconate and iron sucrose found that the particle size was dependent on the solution. Under similar conditions, however, iron gluconate particles were smaller than iron sucrose particles.19 The differences in particle size correspond to the differences in stability of the complexes. The transfer of iron from the complexes to transferrin has been used as a stability parameter of the iron carbohydrate complexes. Van Wyck et al20 found that the percentage of labile iron increased according to this study in the order FeD (Dexferrum; American Regent, Inc., Shirley, NY) ⬍ FeD (InFed; Watson Pharma, Inc.. Corona, Calif, which corresponds to CosmoFer) ⬍ FeS (Venofer) ⬍ FeG (Ferrlecit). In vitro studies by Agarwal et al21 also described a more rapid transferrin saturation with FeG compared with FeS. Unpublished investigations into the stability of FeD, FeS, and FeG in serum done by the working group of Langguth et al point in the same direction, which shows that FeD is more stable than FeS and FeG. In this study, the analysis of iron in the liver and
kidney showed a distinct increase in iron concentration for all complexes, in both the egg white injection and the CAM application compared with the control. Differences that were found in iron concentration were determined by the organ (liver or kidney), the method of application, and—to a lesser extent—the iron complex used. After the egg white injection and the CAM application with an incubation time of 2 days, FeG caused a greater increase in liver iron concentration than the other 2 complexes. The same was true for the iron increase in kidney but less pronounced. All preparations enhanced the iron content clearly in liver and kidney, and all preparations have been used successfully in patients. Therefore, the minor differences between the various iron preparations with regard to the hepatic and renal iron may not be relevant clinically. After the egg white injection of the iron complexes, the increase in liver iron content was greater than the increase in kidney iron content. Whereas under physiologic conditions most excess iron is stored in the hepatic parenchyma, increased loading of the plasma transferrin with iron has been shown to increase the uptake in other parenchymal tissues.22 In our case, this was demonstrated with the kidney parenchyma. The increase in liver iron content after the egg white injection turned out to be significantly greater than after the CAM applications. The same tendency could be observed with the increase in kidney iron content, although to a lesser extent. The most important reason for this difference was probably the longer incubation time of the egg white injection with 22 days, compared with 2 or 4 days of the CAM applications. The iron complexes had more time to be absorbed by the embryo and to be distributed into the liver and kidney afterward. Furthermore, the iron complexes could be absorbed in more ways after the egg white injection than after the CAM application. The iron could have been absorbed via the skin of the embryo, and the embryo could have swallowed the iron complexes and could have absorbed the iron from its guts afterward. A third possibility is
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that iron was absorbed via the CAM. After the CAM application, only the absorption via the CAM was possible. The in vitro experiments suggest a different toxicity profile than observed in the in ovo experiments because of the morphologic changes of the cells after incubation with the iron solutions. The in vitro toxicity rank order in HepG2-cells might be FeS ⬎ FeG ⬎ FeD as has been suggested by Zager et al16 in human kidney cells. The iron concentrations in the cells decreased in the same rank order FeS ⬎ FeG ⬎ FeD. In experiments by Scheiber-Mojdehkar et al, 23 it could be demonstrated that FeS and FeG donate iron to HepG2-cells as efficiently as low-molecular-weight iron. They induced an increase in the expression of ferritin and of divalent metal transporter-1 (DMT-1), the divalent metal transporter. The incubation of the cells with FeD did not lead to a considerable iron uptake and was only a weak inductor of the expression of ferritin and DMT-1. These findings are in agreement with the results of our experiments, but we cannot determine distinctly whether the iron we measured was simply adherent iron on the plasma membrane of the cells or iron that had been taken up by the cells, via the DMT-1, or both. The apparent iron content of the HepG2-cells after treatment with FeS and FeG was excessively high. The values exceeded clearly the iron contents from liver tissue from patients with pathologically increased iron storage.24 The striking, dark brown discoloration of the cells after incubation with FeS and FeG suggests that at least some iron was attached to the cell membrane. Therefore, the interpretation of the excessively high iron concentrations in the cells is difficult. The fact that FeD did not lead to a considerable iron uptake in the cells also shows that the in vitro experiment is not suitable to answer the question of iron uptake in cells and is therefore less relevant biologically. We do know from clinical experience that FeD as well as FeS and FeG does increase hemoglobin levels as well as the serum ferritin concentration and transferrin saturation; therefore, the FeD iron must be able to overcome cellular barriers and be internalized within cells. Different models for in vitro studies on iron toxicity are available that may be used to address different questions. Experimental studies can be helpful if they reflect the clinical reality sufficiently. Cell culture models are well standardized and include less potentially variable parameters than do the more complex in vivo experiments with laboratory animals or similarly complex models of avian embryos. Other studies of parenteral iron complexes have used HepG2-cells as well.23 In our experiments, significant cellular iron uptake in the in ovo model in the embryonic liver and kidney was in line with the known clinical efficacy of the different
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iron complexes. At variance, in HepG2-cells we observed either excessively high levels of cell-bound iron (FeS and FeG) or no relevant iron uptake at all (FeD). We found that the in ovo model as a multitissue system may better reflect the clinically relevant effects on intracellular iron storage and the toxicity of the compounds than an in vitro model with HepG2 cells. We conclude that the avian embryonic model seems interesting for preclinical studies on the balance of efficacy and safety, which may be useful for the comparison of different products of this group in a regulatory context. In addition, the in ovo model might be used to investigate the efficacy of chelators in removing iron from tissues similar to experiments that have been described in rodents.25 CONCLUSION
In this study, 3 different approved parenteral iron complexes were analyzed in an in ovo model as well as in in vitro experiments. The results were compared with their known clinical effectiveness. The impact of the type of carbohydrate used in the formulation of parenteral iron on toxicity could be demonstrated. The most stable form of intravenous iron—low-molecular-weight iron dextran—seemed to be less toxic in both the in ovo model and the in vitro experiments. Furthermore, this study shows that in respect of iron uptake the in ovo model with the more complex interaction of cells and tissues can present results that reflect the clinical situation in humans better than in experiments with isolated cells. The assistance of Ms. Claudia Götze, Ms. Bianka Keil, and Ms. Ute Hartung is gratefully acknowledged. REFERENCES
1. Chertow GM, Mason PD, Vaage-Nilsen O, Ahlmen J. On the relative safety of parenteral iron formulations. Nephrol Dial Transplant 2004;19:1571–5. 2. Valderrabano F, Jofre R, Lopez-Gomez JM. Quality of life in end-stage renal disease patients. Am J Kidney Dis 2001;38:443– 64. 3. Besarab A, Amin N, Ahsan M, et al. Optimization of epoetin therapy with intravenous iron therapy in hemodialysis patients. J Am Soc Nephrol 2000;11:530 – 8. 4. Lappin TR, Maxwell AP, Johnston PG. Warning flags for erythropoiesis-stimulating agents and cancer-associated anemia. Oncologist 2007;12:362–5. 5. Henry DH. The role of intravenous iron in cancer-related anemia. Oncology 2006;20(8 suppl 6):21– 4. 6. Shah SV, Alam MG. Role of iron in atherosclerosis. Am J Kidney Dis 2003;41(3 suppl 1):S80 –3. 7. Macdougall IC. Strategies for iron supplementation: oral versus intravenous. Kidney Int 1999; 69(suppl):S61– 6. 8. Afzali B, Goldsmith DJ. Intravenous iron therapy in renal failure: friend and foe? J Nephrol 2004;17:487–95. 9. Kunzi-Rapp K, Genze F, Kufer R, Reich E, Hautmann RE, Gschwend JE. Chorioallantoic membrane assay: vascularized
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18. Jahn M, Roth S, Langguth P. Parenterale kolloidale Eisenkomplexpräparate. Deutsche Apotheker Zeitung 2006;146:15110 –9. 19. Kudasheva DS, Lai J, Ulman A, Cowman MK. Structure of carbohydrate-bound polynuclear iron oxyhydroxide nanoparticles in parenteral formulations. J Inorg Biochem 2004; 98:1757– 69. 20. Van Wyck D, Anderson J, Johnson K. Labile iron in parenteral iron formulations: a quantitative and comparative study. Nephrol Dial Transplant 2004;19:561–5. 21. Agarwal R. Transferrin saturation with intravenous irons: an in vitro study. Kidney Int 2004;66:1139 – 44. 22. Torrance JD, Bothwell TH. Tissue iron stores. In: Cook JD, ed. Iron. Methods in hematology, 1st ed. New York: Churchill Livingstone; 1980:90 –115. 23. 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 2003;65:1973– 8. 24. Rose C, Vandevenne P, Bourgeois E, Cambier N, Ernst O. Liver iron content assessment by routine and simple magnetic resonance imaging procedure in highly transfused patients. Eur J Haematol 2006;77:145–9. 25. Hodges YK, Weinberger HD, Stephens J, Horwitz MA, Horwitz LD. Desferri-Exochelin, a lipid-soluble, hexadentate iron chelator, effectively removes tissue iron. Transl Res 2006;148:63–71.