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Blood 2008-sohn-1690-9

  1. 1. From by guest on September 28, 2011. For personal use only. 2008 111: 1690-1699 Prepublished online November 1, 2007; doi:10.1182/blood-2007-07-102335Redistribution of accumulated cell iron: a modality of chelation withtherapeutic implicationsYang-Sung Sohn, William Breuer, Arnold Munnich and Z. Ioav CabantchikUpdated information and services can be found at: on similar topics can be found in the following Blood collections Red Cells (1174 articles)Information about reproducing this article in parts or in its entirety may be found online at: about ordering reprints may be found online at: about subscriptions and ASH membership may be found online at: (print ISSN 0006-4971, online ISSN 1528-0020), is published weeklyby the American Society of Hematology, 2021 L St, NW, Suite 900,Washington DC 20036.Copyright 2011 by The American Society of Hematology; all rights reserved.
  2. 2. From by guest on September 28, 2011. For personal use only.RED CELLSRedistribution of accumulated cell iron: a modality of chelation withtherapeutic implicationsYang-Sung Sohn,1 William Breuer,1 Arnold Munnich,2 and Z. Ioav Cabantchik11Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Safra Campus at Givat Ram, Jerusalem,Israel; and 2Clinical Research Unit, Medical Genetic Clinic and Research Unit, Inserm Unite 781, Hopital Necker-Enfants-Malades and Universite Paris V Rene ˆ ´ ´Descartes, Paris, FranceVarious pathologies are characterized by feriprone’s capacity for shuttling iron be- absence of transferrin. These uniquethe accumulation of toxic iron in cell tween cellular organelles was assessed properties of deferiprone underlie mecha-compartments. In anemia of chronic dis- with organelle-targeted fluorescent iron nistically its capacity to alleviate ironease, iron is withheld by macrophages, sensors in conjunction with time-lapse accumulation in dentate nuclei of Fried-leaving extracellular fluids iron-depleted. fluorescence microscopy imaging. De- reich ataxia patients and to donate tissue-In Friedreich ataxia, iron levels rise in the feriprone facilitated transfer of iron from chelated iron to plasma transferrin inmitochondria of excitable cells but de- extracellular media into nuclei and mito- thalassemia intermedia patients. De-crease in the cytosol. We explored the chondria, from nuclei to mitochondria, feriprone’s shuttling properties could bepossibility of using deferiprone, a from endosomes to nuclei, and from intra- exploited clinically for treating diseasesmembrane-permeant iron chelator in clini- cellular compartments to extracellular involving regional iron use, to capture labile iron accumu- apotransferrin. Furthermore, it mobilized (Blood. 2008;111:1690-1699)lated in specific organelles of cardiomyo- iron from iron-loaded cells and donated itcytes and macrophages and convey it to to preerythroid cells for hemoglobin syn-other locations for physiologic reuse. De- thesis, both in the presence and in the © 2008 by The American Society of HematologyIntroductionThe pathologic effects of iron accumulation in tissue are recog- ferroportin in the plasma membranes of macrophages in ACD isnized in diseases of systemic iron overload, in which the liver, thought to be triggered posttranslationally by the circulatingheart, and endocrine glands are the principal affected organs.1 At iron-regulatory peptide hepcidin, which is induced by inflamma-the cellular level, labile iron begins to rise once the intracellular tion8 and transcriptionally by certain cytokines.7,8 Thus, macro-capacity for iron storage is surpassed, leading to catalytic phage iron accumulation in ACD not only coexists with anemia butformation of reactive oxygen species (ROS) that ultimately also is largely responsible for it.9overwhelm the cellular antioxidant defense mechanisms and Here we explore the concept of relocating iron from areas oflead to cell damage.2 accumulation to areas of deprivation using agents capable of In recent years, several pathologies have been shown to be permeating membranes, chelating cell labile iron and donating ironassociated with specific defects in cellular iron metabolism that do to physiologic acceptors. Most agents in clinical use for managingnot give rise to conspicuous systemic iron overload.3 In the systemic iron overload1 were designed and/or are applied forneuromuscular disorder Friedreich ataxia, the deficiency of the correcting or preventing hepatic and cardiac siderosis, the majoriron-chaperone protein frataxin is thought to lead to mitochondrial causes of premature death in thalassemia.2 Those chelators demon-iron accumulation due to improper processing of iron for heme and strably reduce labile iron levels in plasma, but a few, by virtue ofiron-sulfur-cluster formation.4 In the group of neuromuscular their membrane-crossing ability, also do so in cells.1,10 However,disorders termed NBIA (neurodegeneration with brain iron accumu- whether such agents could or should be used in diseases in whichlation), a deficiency in pantothenate kinase (PKAN-2), a key regional iron accumulation is not accompanied by hyperferremia isenzyme in coenzyme A synthesis,5 leads to iron deposition and questionable. In the present case, the goal of chelation is not merelyensuing brain damage by still-unresolved mechanisms.6 In heredi- systemic depletion, but redistribution of iron.tary X-linked sideroblastic anemia, impaired heme synthesis The selection of the orally active chelator deferiprone (DFP) ascauses mitochondrial iron accumulation and siderosis in erythroid a prototype iron-relocating agent is based on several observations.cells.3 However, an extreme example of systemic misdistribution of DFP has been shown to shuttle tissue iron into the circulation toiron is encountered in anemia of chronic disease (ACD), where iron higher-affinity chelators such as desferrioxamine (DFO)11 and toaccumulates in reticuloendothelial cells responsible for recycling increase transferrin (Tf) saturation in individuals with unimpairedaged erythrocytes, presumably due to decreased activity of the serum iron–binding capacity.12 Donation of iron by DFP to DFOiron-efflux protein ferroportin.7 The marked down-regulation of and to transferrin was also observed in vitro.11 Recently, DFP wasSubmitted July 19, 2007; accepted October 25, 2007. Prepublished online as The publication costs of this article were defrayed in part by page chargeBlood First Edition paper, November 1, 2007; DOI 10.1182/blood-2007- payment. Therefore, and solely to indicate this fact, this article is hereby07-102335. marked ‘‘advertisement’’ in accordance with 18 USC section 1734.An Inside Blood analysis of this article appears at the front of this issue. © 2008 by The American Society of Hematology1690 BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3
  3. 3. From by guest on September 28, 2011. For personal use only.BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1691Table 1. Iron binding parameters of agents used in this study tion onto 96-well plates, or onto microscopic slides attached to perforatedIron ligands (abbreviations) pFe(III)* tissue culture 3-cm plates. Probe loading into cells. For cytosolic loading of CALG, cells inCalcein green (CALG) 20.3† DMEM plus 10mM Na-HEPES (DMEM-HEPES) at 37°C were exposedDeferrioxamine (DFO) 26.6 for 10 minutes to 0.25 M CALG-AM, washed and incubated inDeferiprone (DFP) 19.3 DMEM-HEPES containing 0.5 mM probenecid (to minimize probe leak-Diethylene-triamine-pentaacetic acid (DTPA) 28.2 age). For CALG-Fe(III) (1:1) complexes and for sulforhodamine loadingNitrilotriacetic acid (NTA) 18.1 into endosomes, cells were exposed to 50 M to 100 M complex inTransferrin (Tf) 22.3 DMEM-HEPES for 30 minutes at 37°C and washed extensively with theRhodamine B- (1, 10-phenanthrolin-5-yl) 7.7 (11.5 for Fe(II))‡ same probe-free medium and subsequently with HEPES-buffered saline aminocarbonyl benzyl ester (RPA) (HBS; 20 mM HEPES, 135 mM NaCl; pH 7.4).20,21 RPA was loaded into *pFe(III) values are log M , where M is the concentration of the free metal ion mitochondria as described in earlier studies.21-23when ligand 10 M and Fe(III) 1 M at pH 7.4. Data from Harris.17 Histone-CALG. For loading into the nucleus, CALG was coupled to †Data from Thomas et al.18 core histones from calf thymus (H) 7.2 mg/mL, in 50 mM Na-MES, ‡Values shown are for 1,10-phenanthroline. 100 mM NaCl, pH 5.5, containing 1.25 mM CALG:Cobalt (Co protects CALG metal-binding groups). Coupling was initiated by adding 12 mM EDC, followed by incubation for 1 hour at room temperature and overnightfound to reduce iron accumulation in the dentate nuclei of at 5°C. After exhaustive dialysis, 11 mM DTPA was added (to removeFriedreich ataxia patients,13 indicating its ability to cross the Co(II)) and histone-CALG (H-CALG) was further dialyzed against HBS.blood-brain barrier in humans, which is consistent with previous CALG coupling to H was estimated at 1.1:1 by fluorescence ( excitationfindings in animals.14-16 480 nm, emission 520 nm). H-CALG-Fe was prepared by titrating CALG In the present study we assess DFP’s capacity to act as an with FAS. Loading 10 M H-CALG or H-CALG-Fe into cells was done iniron-relocating agent at the cellular level. To trace labile iron DMEM-HEPES containing 100 M chloroquine (for improved FMSmobilized by chelators, we used model cardiac and macrophage targeting to cell nuclei),24 followed by HBS washes.cells in culture and fluorescent metal sensors (FMS) targeted to Fluorescein-DFO-histone. The probe was prepared by coupling ofcellular and extracellular compartments. We show that DFP can N-(fluorescein-5-thiocarbamoyl)-desferrioxamine (Evrogen, Moscow, Rus- sia) to H using fluorescein-DFO (FlDFO)–Fe (1:1) complex generated byrelocate iron accumulated in cell compartments within and across adding 1.1 mM FeSO4 to 1 mM FlDFO in HBS. To 2.64 mL of FlDFO-Fethe cell, and we demonstrate that iron withdrawn from sites of cell complex was added 8.5 mg H, followed by 0.18 mL of 1 M MES (Na form),accumulation can be safely transferred to extracellular transferrin pH 6.5, and 15 mg of EDC. After overnight agitation at 5°C, the mixturefor physiologic reuse. was dialyzed (3.5 kDa cut-off tubing) against 10 mM acetic acid; 1 mM EDTA; 150 mM NaCl, pH 4.5 (to remove bound iron); and finally HBS. The fluorescein-DFO-histone (H-FlDFO) ratio was 1:1, based on stoichiometric iron-quenching of H-FlDFO fluorescence ( excitationMethods 494 nm, emission 520nm). H-FlDFO, like H-CALG, was loaded into cells in DMEM-HEPES.Materials Rhodamine-labeled apotransferrin. Human apotransferrin (4 mg/mLCalcein green (CALG) 3,3 -bis[N,N-bis(Carboxymethyl) aminomethyl]fluo- in 25 mM Na2CO3, 75 mM NaHCO3; pH 9.8), was incubated at 5°Crescein and its acetomethoxy (AM) precursors CALG-AM, lissamine overnight with 1 mM lissamine rhodamine sulfonyl chloride and the labeledrhodamine sulfonyl chloride, and sulforhodamine were from Molecular protein isolated by gel filtration on Sepharose G25 (Sigma-Aldrich)Probes (Eugene, OR). Human serum holotransferrins and apotransferrins preequilibrated with 150 mM NaCl, 20 mM MES; pH 5.3.(aTf) were from Kamada (Rehovot, Israel). Diethylene-triamine- Transfer of iron from DFP-Fe to apotransferrin. DFP-Fe complexespentaacetic acid (DTPA), nitrilotriacetate (NTA), EDC (1-ethyl-3-(3- were generated by incubating Fe-NTA (50:150 M) with either 150 M ordimethylaminopropyl) carbodiimide), ferric ammonium citrate (FAC), 250 M DFP in HBS for 20 minutes, and completion of complex formationferrous ammonium sulfate (FAS), succinyl acetone, HEPES (N-2- was assessed by absorption at 455 nm. The complexes were mixed withhydroxyethylpiperazine-N -2-ethanesulfonic acid), 4-morpholine ethanesul- 50 M aTf and 25 mM NaHCO3 and incubated for 1.5 hours in a 5% CO2fonic acid (MES), core histone mixture from calf thymus (H), N,N - incubator. Because of the large spectral overlap of DFP-Fe and transferrin-hexamethylene-bis-acetamide, and octyl-glucoside were from Sigma- Fe, we added DTPA (10 mM) to selectively scavenge iron from DFP-Fe.Aldrich (St Louis, MO). Rhodamine isothiocyanate was from Fluka (Buchs, Transferrin that was free of low-molecular components was obtained bySwitzerland). DFP (1,2-dimethyl-3-hydroxypyridin-4-one) was from Apo- either filtering 0.5 mL through 30-kDa cut-off spin filters (Pall, East Hills,Pharma (Toronto, ON). DFO was from Novartis (Basel, Switzerland). The NY) via centrifugation (2900g for 20 minutes), or by use of a 5-mLred fluorescent mitochondrial metal–sensor rhodamine B-[(1, 10-phenanth- dry-spin–gel filtration-centrifugation at 1100g over precentrifuged Seph-rolin-5-yl aminocarbonyl] benzyl ester (RPA), was a kind gift from U. adex G-50 medium columns (Sigma-Aldrich). The complex-free transferrinRauen and R. Sustmann, University of Duisburg-Essen, Essen, Germany. was diluted in HBS, pH 7.4. Tf-Fe was analyzed by absorbance at 465 nm,A summary of the probes used and their properties is given in Table 1. and aTf by tryptophan fluorescence (280 nm excitation, 306 nm emission; Felix spectrofluorimeter station version 2.5; Photon Technology Interna- tional, Lawrenceville, NJ).Methods Iron-loading of cells was done by overnight incubation of cells withComplexes of iron with NTA (Fe-NTA) were generated by mixing FAS and 100 M FAC in culture conditions followed by washing with HBSNTA (1:3 molar ratio) in water and allowing iron to oxidize in ambient containing 100 M DFO, and with HBS alone. To achieve selectiveconditions. The DFP-Fe(III) complexes were generated by mixing Fe-NTA accumulation of iron in mitochondria, cells were treated with 1 mMwith DFP in water; formation of fully substituted DFP-Fe complexes was succinyl acetone for 3 hours at 37°C.confirmed by measuring absorbance at 455 nm.19 Cell hemoglobin synthesis. MEL cells cultured for 2 days in media Cell-culture lines. Rat H9C2 cardiomyocytes, J774 mouse macro- supplemented with 5mM hexamethylene-bis-acetamide were washed andphages, and erythroleukemia (MEL) were grown in 5% CO2 Dulbecco- lysed with octyl-glucoside (1.5%). After 2 minutes centrifugation atmodified Eagle medium (DMEM) supplemented with 10% fetal calf serum, 12 000g, 100 L of lysate supernatants was transferred to a 96-well4.5 g/L D-glucose, glutamine, and antibiotics (Biological Industries, microplate for estimating hemoglobin (by measuring absorbance atKibbutz Bet Haemek, Israel). Cells were plated 1 day before experimenta- 410 nm). Alternatively, lysates were mixed with 100 L freshly prepared
  4. 4. From by guest on September 28, 2011. For personal use only.1692 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 Figure 1. Transfer of iron from extracellular DFP-iron complexes to mitochondria. H9C2 cells loaded with the mitochondrial iron-sensor RPA were incubated with or without DFP-Fe (15:5 M) and epifluorescence micro- scopic images were recorded every 5 minutes under settings for rhodamine. Representative fields of initial cell fluorescence at time 0 (A,C) and after incubation for 1 hour in the presence (B) or absence (D) of 5 M DFP-Fe complex. Magnification was 600; oil objective was a (Plan Apo) 60 /1.40 NA. (E) Mean fluorescence values in relative units (r.u.) plus or minus SD of 5 cells per field calculated for each time-point image and normalized to the initial fluorescence, representing cells incubated without (None) and with 5 M Fe-complex (DFP-Fe or NTA-Fe); the lines denoted as DFP-Fe and NTA-Fe were cor- rected for spontaneous decay given by the control (None). Arrow indicates time the Fe complex was added. (F) Effect of various iron chelates on the fluorescence (f normalized to initial value, f0, in r.u.) of 0.5 M RPA in solution (HBS buffer). The iron chelates all contained 5 M Fe complexed to NTA (1:3 ratio) in the absence (f) and presence ( ) of 100 M ascorbate (Fe:NTA, Fe:NTA ASC) or to DFP, with 100 M ascorbate, at DFP:Fe ratios of 3:1 (F), 5:1 (E), and 7:1 (‚). The values of fluorescence intensity are means of triplicate samples run in parallel, plus or minus SEM.tetramethylbenzidine (0.5 mg/mL in 10% acetic acid) and finally 8 L of endogenous labile iron and/or to probe photobleaching. On the30% H2O2. Absorption (604 nm) was read after 90 seconds. other hand, addition of preformed DFP-Fe(III) complexes (DFP- Data acquisition and analysis. Epifluorescence imaging (Nikon TE Fe, ratio 3:1) evoked a time-dependent (t ⁄ 15 minutes) quenching 122000 microscope; Melville, NY; and Orca-Era CCD camera; Hamamatsu, of mitochondrial RPA fluorescence (Figure 1E). Consistent withBridgewater, NJ) coupled to a Volocity 4 system (Improvision, Coventry,United Kingdom) was used for image data acquisition and analysis.20 For RPA’s selectivity for Fe(II), no quenching of RPA by Fe-NTAhigh-throughput fluorescence monitoring we used fluorescence plate read- occurred in solution unless ascorbate was added. Transfer of ironing (Tecan-Safire; Neotec, Mannedorf, Austria) essentially as described.20 ¨ from DFP-Fe complexes to RPA in the presence of ascorbate was detected over a wide range of DFP to Fe ratios (1:1 to 7:1). Transfer of iron from extracellular DFP-Fe complexes toResults nuclei. With the view of targeting a fluorescent iron sensor into the cell nucleus, we constructed a conjugate of H with theTo follow DFP-mediated translocation of iron from the medium to high-affinity iron chelator and iron sensor H-FlDFO.11 To minimizecells, within cells, and from cells to the medium, we used uptake via the endocytic pathway, incubation was carried out atfluorescent acceptors and donors of iron that undergo quenching or room temperature and in the presence of chloroquine, as describeddequenching upon metal binding or removal. An additional feature in “Histone-CALG.” H-FlDFO within nuclei was rapidlyof these probes was their localization in specific cell compartments (t ⁄ 12 5 minutes) quenched by DFP-Fe (3:1) added to the extracel-or in the extracellular medium. lular medium (Figure 2E), consistent with the high cell permeabil- ity of DFP-Fe complexes shown in Figure 1. In solution, H-FlDFOTransfer of iron from extracellular DFP-iron complexes to is efficiently and rapidly (t ⁄12 1 minute) quenched by DFP-Femitochondria complexes even at a 9:1 DFP:Fe ratio (Figure 2F), as expected fromIngress of ionic iron from the extracellular medium to the DFP’s capacity to shuttle iron to DFO both in vivo and in vitro.11mitochondria of H9C2 cardiomyocytes was followed with the DFP-mediated redistribution of iron between cellular compart-high-affinity Fe(II)–acceptor probe, RPA22,23 (Figure 1). The hydro- ments: from nuclei to mitochondria. DFP’s facilitation of ironphobic cationic rhodamine B is responsible for RPA’s potentiomet- movement between discrete cell compartments was followed inric partitioning in the mitochondria, and phenanthroline is respon- H9C2 cells sequentially labeled in cell nuclei with the iron-donorsible for detection of Fe(II).22 Exposure of cells to Fe(III)-NTA probe H-CALG-Fe(III) and in mitochondria with the Fe(II)-evoked no significant changes in RPA fluorescence relative to the acceptor probe RPA. H-CALG-Fe penetrates the plasma membranespontaneous decrease (t ⁄ 60 minutes), probably due to binding of 12 and accumulates initially in the cytosol and subsequently in the
  5. 5. From by guest on September 28, 2011. For personal use only.BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1693Figure 2. Transfer of iron from extracellular DFP-iron complexes to nuclei.H9C2 cells loaded with the nuclear iron sensor H-FlDFO were incubated with or Figure 3. DFP-mediated relocation of iron between cellular compartments:without 5 M DFP-Fe complex (DFP:Fe ratio 3:1) and epifluorescent microscopic from nuclei to mitochondria. H9C2 cells were loaded with the nuclear iron sensorimages were recorded every 5 minutes under settings for fluorescein. Representative H-CALG that had been precomplexed to iron (H-CALG-Fe), followed by loading withfields of initial cell fluorescence at time 0 (A,C) and after incubation for 1 hour in the the mitochondrial iron sensor RPA. The double-labeled cells were then exposed toabsence (B) or presence (D) of 5 M DFP-Fe complex. (E) Mean fluorescence values 50 M DFP and epifluorescence images were recorded every 5 minutes. Representa-in r.u. plus or minus SD of 5 cells per field, calculated for each time-point image and tive fields of cell fluorescence observed under settings for fluorescein (A,B) andnormalized to the initial fluorescence (f/f0), representing cells incubated without rhodamine (C,D). Images are shown at time 0 (A,C) and after incubation for 1 hour in(None) and with 5 M DFP-Fe complex (DFP-Fe). Arrow indicates time the Fe the presence of 50 M DFP (B,D). (E) Mean fluorescence values plus or minus SD incomplex was added. (F) Effect of DFP-Fe chelates (DFP:Fe at ratios of 1:1, 3:1, 5:1, r.u. of 5 cells per field, calculated for each time-point image and normalized to the9:1) on the fluorescence (normalized to initial value, f0, in r.u.) of 0.5 M H-FlDFO in initial fluorescence (f/f0), representing cells incubated with 50 M DFP (circles) andsolution (HBS buffer). The values of fluorescence intensity are means of triplicate with no addition (squares). Fluorescence of H-CALG is indicated by filled symbolssamples run in parallel, plus or minus SEM. (F and f), and fluorescence of RPA is indicated by open symbols (E and ). The scheme illustrates entry of DFP into the cytosol (C), nuclei (N) containing H-CALG-Fe (iron donor) and mitochondria (M) containing RPA (iron acceptor), and transfer of iron from nuclei to mitochondria. E refers to endosomes.nucleus, particularly in nucleolus-like sites.21 H-CALG-Fe wasused subsaturated with iron so that its accumulation in the nucleicould be followed via changes in the residual fluorescence of the CALG-Fe into endosome/pinosomes and its accessibility to DFP, asunquenched fraction of the probe (revealed by chelation; Figure evidenced by dequenching, in a previous study.20 Moreover, CALG-Fe3A). Loading of RPA into mitochondria was apparent from the coendocytosed with the classical fluid phase markers sulforhodaminecharacteristic perinuclear staining pattern (Figure 3C), from colo- and rhodaminated dextran, both of which showed less than 65% cellcalization with other potentiometric probes and sensitivity to colocalization as obtained with the Volocity program (not shown).protonophores.22,23 Addition of 50 M DFP produced an increase inthe fluorescence of H-CALG-Fe in the nuclei and a concomitant H-FlDFO was detectable in the nuclear area, while CALG-Fe wasdecrease in the fluorescence of RPA in the mitochondria, both of concentrated in punctuate, perinuclear spots, attributable to endosome-which occurred within 5 to 8 minutes and were complete within trapped CALG-Fe (Figure 4). However, after incubation with 50 M20 minutes (Figure 3E). The changes are attributable to dequench- DFP, the fluorescence in the endosomes increased significantly, whileing of H-CALG-Fe due to removal of iron by DFP and to that in the nuclei decreased relative to the respective untreated controls.quenching of RPA due to transfer of iron from DFP-Fe. Only minor The kinetics of the changes in the 2 compartments followed similarchanges in fluorescence were observed in untreated control cells. It patterns, although in opposite directions. To ascertain whether the ironis likely that nuclear H-CALG-Fe was not the only source of iron acquired by DFP was exclusively intracellular, the cells were washeddelivered to RPA by DFP, because the addition of DFP to with 0.1 mM DFO to remove all extracellular iron before the addition ofRPA-loaded cells also led to quenching of mitochondrial probe DFP. Furthermore, as the addition of DFP to cells loaded with H-FlDFO(data not shown), although to a much lesser extent than in the alone failed to produce a decrease in its fluorescence (data not shown),presence of H-CALG-Fe. the quenching of H-FlDFO in cells coloaded with CALG-Fe is most DFP-mediated relocation of iron between cellular compart- likely due to DFP-mediated mobilization of iron from endosomes.ments: from endosomes to nuclei. H9C2 cells were labeled However, the possibility that in the course of the experiment somesequentially in the nuclei with the iron-acceptor probe H-FlDFO and in iron might have spontaneously leaked from endosomes to cytosolthe endosomes with the iron-donor probe CALG-Fe (Figure 4). We and rendered it transferable to mitochondria by added DFP cannotshowed the bulk-phase pinocytic uptake of the iron-quenched probe be ignored.
  6. 6. From by guest on September 28, 2011. For personal use only.1694 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 Figure 4. DFP-mediated relocation of iron between cellular compartments: from endosomes to nuclei. H9C2 cells were loaded with the nuclear iron-sensor H-FlDFO and subsequently exposed to preformed complexes of CALG and iron (CALG-Fe, 1:1 ratio) that were taken up by adsorptive pinocytosis into endo- somes. The cells containing both labels were then exposed to 50 M DFP and epifluorescent images were recorded every 5 minutes under fluorescein set- tings. Representative fields of cell fluorescence are shown at time 0 (A) and after incubation for 20 minutes (B) in the presence of 50 M DFP. (C) The nuclei (N) and endosomes (E) are indicated by arrows. (D) Mean fluorescence values in r.u. within selected subcellular areas corresponding to endosomes (Di) and nuclei (Dii), using 5 cells per field, calculated for each time-point image and normalized to the initial fluorescence (f/f0). Transfer of iron from DFP-Fe complexes to transferrin. We The rationale was that intracellular iron mobilized to the extracellu-followed iron complexation by transferrin using DFP-Fe com- lar medium by DFP would be transferred to the fluorescent Tf,plexes (3:1 and 5:1, 50 M in Fe) and NTA-Fe complexes (3:1, which, upon binding iron, would then bind to cell-surface trans-50 M in Fe) as iron sources and apotransferrin (50 M) as ferrin, and after receptor-mediated endocytosis, would becomeacceptor. To remove from the reaction mixture any traces of iron concentrated in endosomes. To increase the level of mitochondrialassociated with low molecular weight complexes or free ligands, labile iron, the cells were pretreated with the heme-synthesiswe added DTPA after 1 hour of reaction and followed it by size inhibitor succinylacetone, which causes selective accumulation offiltration. Filtration and differential chelation allowed the assess- unused, chelatable iron in the mitochondria.22 After 1 hour ofment of DFP-Fe transfer of iron to aTf both by light absorption of incubation with DFP, the level of cell-bound fluorescent aTf roseTf-Fe complexes at 465 nm (Figure 5) and by tryptophan fluores- by 2.3-fold over controls without DFP (Figure 6A,B). The bindingcence of aTf that is quenched after specific binding of iron25 was specific and iron dependent, as it was fully blocked by excess(Figure 5). While the 2 analytical methods monitored binding of holotransferrin (Figure 6C,D) and enhanced by addition of saturat-iron to transferrin, the optical changes are mirror images of each ing concentrations of Fe-NTA (Figure 6E,F), both in the presenceother. Transfer of iron from DFP-Fe to transferrin ensued at both and in the absence of DFP. In control cells not treated with3:1 and 5:1 stoichiometries of DFP:Fe and was of a stable nature: it succinylacetone, DFP failed to enhance fluorescent-Tf bindingpersisted after addition of chelators that differentially dissociate (data not shown), indicating that under normal conditions whereDFP-Fe complexes (but not Fe-transferrin). On the other hand, iron incorporation into heme is not blocked, the concentration ofDFP alone and NTA alone evoked only minor changes that were DFP-mobilized iron is insufficient to be detectable in this system.eliminated by addition of DTPA before the reaction with aTf, DFP-mediated iron delivery for hemoglobin synthesis. Theindicating that the changes were associated with traces of iron potential of DFP-Fe complexes as donors of iron for physiologicpresent in the medium. Essentially similar results were obtained by use was assessed using a model system of cellular iron incorpora-assessing the transfer of iron to apotransferrin using fluorescein- tion, synthesis of hemoglobin (Hb) by differentiating preerythroidlabeled aTf (not shown). However, with lissamine rhodamine- cells. The well-documented induction of Hb synthesis in MEL cellslabeled aTf (R-aTf), the acquisition of iron evoked insignificant by hexamethylene bisacetamide (HMBA) is highly dependent onchanges in fluorescence. the supply of iron.26 It is repressed in serum-free medium lacking transferrin, but it is significantly enhanced by the addition ofDFP-mediated mobilization of iron from H9C2 cells to Fe-NTA irrespective of the presence of Tf (Figure 7). DFP-Fe (3:1)extracellular transferrin: receptor-mediated uptake of supported Hb synthesis to a degree similar to that seen withholotransferrin as an index of iron shuttling Fe-NTA, both in the absence and presence of added Tf. FullyMobilization of cellular iron by DFP and its transfer to extracellular saturated holotransferrin (15 M) was equally effective as Fe-NTAtransferrin are critical features of DFP’s potential function as an and DFP-Fe (data not shown), indicating that various forms of ironiron shuttle. This capability was assessed using H9C2 cells can equally support Hb synthesis in this experimental system. Onincubated with lissamine R-aTf in the presence of DFP (Figure 6). the other hand, DFP without added iron depressed Hb synthesis
  7. 7. From by guest on September 28, 2011. For personal use only.BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1695Figure 5. Transfer of iron from DFP-Fe complexesto transferrin. DFP-Fe complexes (1:3 and 1:5 ratios;50 M Fe(III)) were mixed with apotransferrin aTf(50 M) and incubated for 1.5 hours in a humidified 5%CO2 incubator at 37°C. At the conclusion of the reac-tion, 10 mM DTPA was added and the low–molecularweight material was removed by filtration as describedin “Histone-CALG.” The tryptophan (trp) fluorescenceat 280 nm excitation/306 nm emission (top graph) wasobtained from the emission spectra (inset). The high–molecular weight fractions were diluted in HBS, pH 7.4,and the absorbance was read at 465nm (bottom graph).Data are given as means plus or minus SD of3 independent experiments. The labels below the barsindicate the complexes tested and their concentrations.The values above the bars represent the percentagechange in absorbance or fluorescence.below control levels, indicating that the chelator can have an (data not shown), an effect similar to DFO-induced iron depriva-iron-withdrawing effect under conditions of limited iron supply. tion in other hematopoietic cells.27 To demonstrate that transfer ofIn the serum-free medium used in these experiments, DFP iron from DFP-Fe to aTf gave rise to functionally active holotrans-( 100 M) without added iron, as well as DFO (100 M), ferrin, DFP-Fe was allowed to interact with aTf for 1 hour, and thenprevented differentiation and ultimately caused massive cell death the mixture was exhaustively dialyzed (cut-off 12 kDa) against Figure 6. DFP-mediated mobilization of iron from H9C2 cells to extracellular apotransferrin: receptor- mediated uptake of holotransferrin as an index of iron transfer. H9C2 cells were pretreated with succinylac- etone as described in “Methods” to increase mitochon- drial labile iron levels. They were then incubated at 37°C in DMEM-HEPES medium containing 20 M lissamine R-aTf with various additions, and epifluores- cence microscopy images were obtained under rhodamine settings after 60 minutes of incubation. Representative images of cells with no addition (A), 30 M DFP (B), 100 M unlabeled holotransferrin (C), 100 M unlabeled holotransferrin with 30 M DFP (D), 20 M Fe-NTA, 1:3 ratio (E), and 20 M Fe-NTA with 30 M DFP (F). (G) The illustration represents entry of DFP into cells, mobilization of iron from the cytosol (C), nuclei (N) and mitochondria (M), followed by exit of DFP-Fe complexes from the cells (step 1). This is followed by transfer of iron from DFP-Fe to R*-aTf to form R*-Tf-Fe (step 2), which then binds to transferrin receptors on the cell surface (step 3), concen- trates in the endosomes, and is detected by fluorescence microscopy as punctuate fluorescence typical of mi- crovesicles. (H) Mean cell-associated fluorescence values in r.u. of 5 cells per field ( SD, from 3 separate experi- ments), calculated from snapshots such as shown in panels A through F, obtained after 1 hour of incubation.
  8. 8. From by guest on September 28, 2011. For personal use only.1696 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3Figure 7. Stimulation of Hb synthesis in murine erythroleukemia cells. (A) DFP-mediated iron delivery to murine erythroleukemia (MEL) cells synthesizing hemoglobin isschematically depicted. (B) MEL cells suspended in serum-free DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA were supplemented with various ironcomplexes (step 1 in panel A) and cultured for 48 hours. Hemoglobin synthesis (step 2 in panel A) was assessed in terms of hemoglobin content in cell lysates as described in“Cell hemoglobin synthesis” and is expressed relative to the hemoglobin content in control cells with no additions (set as 100%). The additions included 10 M Fe complexed to30 M NTA without (Fe-NTA) and with 15 M human apotransferrin (Fe:NTA aTf); 10 M Fe complexed to 30 M DFP without (DFP-Fe) and with 15 M humanapotransferrin (DFP-Fe aTf); 30 M DFP alone (DFP); 15 M human apotransferrin alone (aTf). Generation of holotransferrin from DFP and apotransferrin (DFP-Fe aTfDial.): 10 M Fe:30 M DFP complex was preincubated with 15 M human for 1 hour and dialyzed. Results shown are averages of 3 separate experiments plus or minus SD;the average Abs604 of control cells was 0.39. (C) DFP-mediated mobilization of iron from J774 (Fe donor; step 1a in panel A) cells to the extracellular medium (step 1b in panelA), followed by entry of DFP-Fe complexes into MEL (Fe acceptor) cells (step 1c in panel A) and intracellular donation of iron for hemoglobin (Hb) synthesis (step 2 in panel A).MEL cells were cultured for 48 hours in DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA without (Con) or with 10 M Fe-NTA (NTA-Fe), or with varioussupernatants of J774 cell lysates that were supplemented with 5 mM HMBA, and lysates were assayed for hemoglobin content. To obtain J774 mouse macrophagesupernatants, J774 cells were cultured overnight without (untreated J774) or with 100 M FAC (Fe-loaded J774), washed with 100 M DFO to remove all extracellular iron, andincubated for 2 hours at 37°C in serum-free DMEM containing 1.25 mg/mL bovine serum albumin, without ( DFP) or with 30 M DFP ( DFP). The cell supernatants werecollected and centrifuged to remove detached cells, and HMBA (5 mM) was added to MEL cells. Shown are values of hemoglobin (Hb; from a representative experiment)obtained in MEL cells exposed for 48 hours to the various conditions.buffer containing 80 mg/L aTf to remove DFP-Fe without gaining support for the proposed use of DFP as an iron shuttle even withoutcontaminant iron from the solution. When added to differentiating the mediation of transferrin, such as might occur in the centralMEL cells, this preparation, generated from DFP-Fe and aTf, fully nervous system.supported Hb synthesis (Figure 7B). Finally, we assessed DFP’s ability to shuttle iron from cell tocell (Figure 7C). In vivo, direct intercellular transfer of iron via DiscussionDFP is not likely, due to the presence of more than 50 M due tothe presence of more than 25 M transferrin in plasma.28 However, The recognition that intracellular iron accumulation is one of theintercellular transfer of iron via DFP could occur in the brain where underlying causes of some clinical syndromes has led to thethe iron-binding capacity of transferrin in cerebrospinal fluid (CSF) suggestion that it might be amenable to treatment with ironis estimated to be in the submicromolar range.29,30 In the experi- chelators.3,33,34 These syndromes include Parkinson disease, neuro-ment illustrated in Figure 7A, J774 mouse macrophages were degeneration with NBIA, and Friedreich ataxia, where iron depos-chosen as the iron-donating cells because of their high capacity for its in specific areas of the brain have been identified by histologic oriron accumulation.31 DFP at a concentration of 30 M, achieved in imaging techniques34 and are thought to be a central factor in thevivo with moderate oral doses,1,32 mobilized sufficient iron from development of the diseases.35 Similarly, in anemia of chroniciron-loaded J774 cells, but not from untreated ones, to significantly disease, iron is retained within erythrocyte-phagocytosing macro-enhance Hb synthesis in MEL cells (Figure 7C). Spontaneous phages, presumably to prevent access by invading pathogens toefflux of iron from iron-loaded J774 cells took place, as evidenced iron from the circulation.7-9,31 While regional iron mobilizationby the increased MEL cell Hb levels even in the absence of might be essential for stabilizing or reversing the toxic effects ofDFP (although increased, these levels were significantly lower in labile iron, in some pathologies it might be equally important tothe absence of DFP than in its presence). This result provides render the mobilized iron available for metabolic reuse. This is
  9. 9. From by guest on September 28, 2011. For personal use only.BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1697especially important in those conditions in which regional iron hemoglobin synthesis when presented together with DFP or afteraccumulation is accompanied by regional or systemic deprivation. DFP’s removal (Figure 7).All the chelators in clinical use have been designed for massive The direct bioavailability of DFP-bound iron was investigatedremoval and excretion of iron from organs of iron-overloaded using hemoglobin synthesis by cells in culture as a model. DFP-Fepatients.2,10,33 Here we assessed the potential application of an iron supported hemoglobin synthesis even in the absence of transferrinchelator in clinical use (DFP) as a shuttling agent capable of (Figure 7), consistent with a similar activity of 1:1 complexes ofredistributing iron within and among cells, while satisfying certain iron with salicylaldehyde isonicotinoyl hydrazone.43 More impor-basic requirements. tant, DFP enhanced the transfer of iron from iron-loaded macro- Permeability across cell membranes in the free and iron- phages to preerythroid cells for hemoglobin synthesis, without thebound form. The high membrane permeability of the iron-free intermediary presence of transferrin (Figure 7). Such direct dona-form of DFP is well documented, as shown by its capacity to tion to intracellular iron-requiring or iron-metabolizing enzymesrapidly access and deplete intracellular labile iron pools.36,37 On the may not be a necessary feature if iron is efficiently transferred fromother hand, the ability of DFP-Fe complexes to traverse intracellu- the shuttle to circulating transferrin. However, it may be desirablelar and plasma membranes of cells has not been studied in or even essential in the brain, where the iron-binding capacity ofdetail.20,21,36,37 One indication is the capacity of orally administered transferrin in the CSF is negligible and has been estimated in theDFP to increase serum transferrin saturation12 and labile iron11 submicromolar range.29,30within an hour after intake in thalassemia patients, which is Low toxic potential of iron-shuttling agent complexes.attributable to the rapid exit of DFP-Fe complexes from cells into A major concern in the use of chelators is the formation of ironthe circulation. At the intracellular level, it is shown in this work complexes that undergo redox cycling and thereby catalyze ROSthat DFP can facilitate the removal of labile iron from nuclei generation. This tendency is present in chelators with relatively low(Figure 3), from endosomes (Figure 4), and from mitochondria redox potentials, such as those based on amine- and carboxyl-(Figure 6), and can transfer iron to acceptors in the mitochondria liganding groups but not with the bidentate 3:1 complexing(Figure 3), nuclei (Figure 4), and extracellular medium (Figure 6). hydroxypyridone DFP.20,21,44,45 However, speciation studies10 andThe transfer might involve dissociation of the DFP-Fe complexes recent examination of ROS production42 indicated that complete abolition of labile iron by DFP is attained only at DFP:Fe(III) ratiosand/or formation of ternary complexes with other ligands. close to 5:1. Whether DFP levels reached therapeutically are Ability to compete effectively for intracellular labile iron with consistently high enough to avoid formation of substoichiometricother intermediate affinity species. Labile iron that is likely to be redox-active complexes is difficult to predict. This is particularlyaccessible to DFP is assumed to be bound to several possible important in susceptible, DNA-containing organelles such asligands (eg, citrate, ATP) and to be in dynamic equilibrium between nuclei and mitochondria. The same applies to intracellular signal-Fe(II) and Fe(III) as dictated by the redox status of the intracellular ing pathways activated by oxidative stress that could be generatedenvironment.38,39 DFP competes effectively for labile cell iron both by DFP-mediated redistribution of cell vitro and in vivo,32 and in individuals with normal iron balance it Permeability across the blood-brain barrier (BBB) for thera-raises serum iron levels, indicating mobilization of tissue iron.12 peutic applications to neurodegenerative diseases. The capacityThis was also observed with isolated cells (Figures 3,4), where of an iron-shuttling agent to redistribute iron in the central nervousDFP readily chelated iron bound to the EDTA analog CALG in system (CNS) may be an essential requirement for iron redistribu-endosomes and nuclei and was indicated in other systems.40 An tion in Parkinson disease, Friedreich ataxia, or NBIA. A high rate ofundesirable property of a chelating molecule would be interference BBB penetration is expected for free DFP, based on its lowwith normal cellular iron metabolism by overchelation. In this molecular weight (below 300) and lipophilicity. This was con-respect, DFP ought to be used at restricted doses, as at high levels it firmed by direct measurement of DFP accumulation in the perfusedappears to inhibit iron-requiring tyrosine and tryptophan rat brain.15,16 In rats given DFP doses several times higher thanhydroxylases.10,14 analogous doses given to thalassemia patients, brain tyrosine and Ability to donate iron to physiologic acceptors. The rationale tryptophan hydroxylase activities were significantly inhibited, asbehind DFP-mediated redistribution of iron leans on DFP’s ability measured by the accumulation of 3,4-dihydroxyphenylalanineto transfer the metal to extracellular transferrin, thereby minimizing (DOPA) and 5-hydroxytryptophan, respectively, presumably viaundesired iron loss by excretion via the urinary or biliary pathways. coordination to iron bound by these enzymes.14 Yet, despiteWe surmised that under physiologic conditions iron transfer to aTf DFP’s brain accessibility, major effects on CNS function have notis likely to occur (1) spontaneously, as the pFe value of Fe(III) for been reported.32,44transferrin is 3 orders of magnitude higher than for DFP41 (Table 1), Whether DFP can alter the iron balance in the brain by shuttlingand (2) directly as Fe(III), because the thermodynamically favored iron across the BBB remains an open question. The absence of ironDFP-Fe complexes are (DFP)2-Fe(III) and (DFP)3-Fe(III).10,25,42 In loading of the CNS in iron-overload conditions indicates thatvitro studies based on an indirect analytical method11 corroborated neither transferrin-bound nor non–transferrin-bound iron is able toearlier observations that a single oral dose of 3 g DFP raised within freely cross the BBB.35 Because DFP treatment has not been6 hours the transferrin saturation (determined by urea-gel electro- reported to be associated with increased iron loading of the CNS inphoresis) of a healthy individual from a normal level of 20% to thalassemia patients, it is conceivable that DFP-Fe complexes do80%.12 In this work we provided direct demonstration of iron not readily permeate the BBB. The permeabilities of free andtransfer from DFP-Fe to aTf at physiologic concentrations (Figure iron-bound DFP may differ due to differences in their molecular5). Using spectrophotometric and fluorimetric methods, we showed weights (473 for 3DFP:1Fe compared with 139 for DFP). In athat efficient transfer occurs at various DFP:Fe ratios and within recent study, 6 months of treatment with DFP was shown to result1 to 2 hours in culture conditions. The resulting holotransferrin in a decrease in the iron-associated magnetic resonance imagingformed from DFP-Fe was biologically active, as it was recognized (MRI) transverse relaxation rate (R2*) signals in dentate nuclei ofby the transferrin receptor (Figure 6) and provided iron to cells for Friedreich ataxia patients.13 Whether this decrease was caused by
  10. 10. From by guest on September 28, 2011. For personal use only.1698 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3egress of DFP-Fe complexes from the brain or by redistribution of iron loss.48,49 Optimization of such strategies with novel chelatorsiron from an area of accumulation to areas of lower concentration for treating the various conditions of regional iron accumulationwithin the brain is unclear. demand further laboratory and clinical work. We show that redistribution of iron in multiple directions and tomultiple recipients is experimentally feasible, particularly fromareas of accumulation to areas of iron need, via donation to Acknowledgmentstransferrin or by direct metal donation. A modality of iron chelationbased on iron redistribution has several therapeutic implications. In This work was supported by the Association Française contre lesthe CNS, it could potentially be used to relocate local iron deposits Myopathies, the Israel Science Foundation (ISF), the EEC Frame- work 6 (LSHM-CT-2006-037296 Euroiron1) and French-Israelithat may be an important factor in the etiology of several Organization for Research in Neuroscience (AFIRN). Z.I.C. is theneurodegenerative diseases. In ACD, it could be used to release Sergio and Adelina Della Pergolla Professor of Life Sciences.iron trapped within macrophages. Current experimental approachesto alleviation of ACD tend to be directed at depressing the levels ofhepcidin, the iron-regulator protein responsible for macrophage Authorshipiron retention. However, recent evidence for hepcidin-independentdown-regulation of the iron exporter ferroportin by the inflamma- Contribution: Y.-S.S. and W.B. performed experimentation andtory mediators46 could obviate such an approach. As shown in this analysis; A.M. and Z.I.C. designed and supervised the research. Allwork, an alternate or complementary approach could be based on authors contributed to the writing of the paper.iron-shuttling agents that relocate misdistributed iron and safely Conflict-of-interest disclosure: The authors declare no compet-bypass impaired internal routes of iron trafficking. Early pilot ing financial interests.studies with iron chelators in rheumatoid arthritis patients with Correspondence: Z. I. Cabantchik, Alexander Silberman Insti-ACD47 provided clinical evidence that agents like DFP might be tute of Life Sciences, Hebrew University of Jerusalem, Safraadopted as part of a short-term strategy of regional metal detoxifi- Campus at Givat Ram, Jerusalem, Israel 91904; e-mail:cation and systemic relocation that generates no significant urinary 1. Hershko C, Link G, Konijn AM, Cabantchik ZI. of catechol-O-methyltransferase (COMT) as well 24. Haberland A, Bottger M. Nuclear proteins as Objectives and mechanism of iron chelation as tyrosine and tryptophan hydroxylase by the gene-transfer vectors. Biotechnol Appl Biochem. therapy. Ann N Y Acad Sci. 2005;1054:124-135. orally active iron chelator, 1,2-dimethyl-3-hy- 2005;42:97-106. 2. Hershko C, Cappellini MD, Galanello R, Piga A, droxypyridin-4-one (L1, CP20), in rat brain in vivo. 25. He QY, Mason AB, Lyons BA, et al. Spectral and Tognoni G, Masera G. Purging iron from the Biochem Pharmacol. 1993;45:2417-2424. metal binding properties of three single-point tryp- heart. Br J Haematol. 2004;125:545-551. 15. Habgood MD, Liu ZD, Dehkordi LS, Khodr HH, tophan mutants of the human transferring N-lobe. Abbott J, Hider RC. Investigation into the correla- Biochem J. 2001;354:423-429. 3. Napier I, Ponka P, Richardson DR. Iron trafficking in the mitochondrion: novel pathways revealed by tion between the structure of hydroxypyridinones 26. Marks PA, Rifkind RA. Hexamethylene bis-ac- disease. Blood. 2005;105:1867-1874. and blood-brain barrier permeability. Biochem etamide-induced differentiation of transformed Pharmacol. 1999;57:1305-1310. cells: molecular and cellular effects and thera- 4. Wilson RB. Iron dysregulation in Friedreich 16. Fredenburg AM, Sethi RK, Allen DD, Yokel RA. peutic application. Int J Cell Cloning. 1988;6: ataxia. Semin Pediatr Neurol. 2006;13:166-175. The pharmacokinetics and blood-brain barrier 230-240. 5. Hayflick SJ. Neurodegeneration with brain iron 27. Alcantara O, Boldt DH. Iron deprivation blocks permeation of the chelators 1,2 dimethly-, 1,2 di- accumulation: from genes to pathogenesis. Se- multilineage haematopoietic differentiation by in- ethyl-, and 1-[ethan-1 ol]-2-methyl-3-hydroxypyri- min Pediatr Neurol. 2006;13:182-185. hibiting induction of p21(WAF1/CIP1). Br J din-4-one in the rat. Toxicology. 1996;108:191- 6. Gregory A, Hayflick SJ. Neurodegeneration with 199. Haematol. 2007;137:252-261. brain iron accumulation. Folia Neuropathol. 2005; 28. Huebers HA, Eng MJ, Josephson BM, et al. 17. Harris WR. Iron chemistry. In Templeton DM, ed. 43:286-296. Plasma iron and transferrin iron-binding capac- Molecular and Cellular Iron Transport. New York, 7. Ganz T. Molecular pathogenesis of anemia of NY: Marcel Dekker; 2002;1-40. ity evaluated by colorimetric and immunopre- chronic disease. Pediatr Blood Cancer. 2006;46: cipitation methods. Clin Chem. 1987;33:273- 18. Thomas F, Serratrice G, Beguin C, et al. Calcein 277. 554-557. as a fluorescent probe for ferric iron. Application 8. Ganz T. Hepcidin, a key regulator of iron metabo- 29. Bradbury MW. Transport of iron in the blood- to iron nutrition in plant cells. J Biol Chem. 1999; lism and mediator of anemia of inflammation. brain-cerebrospinal fluid system. J Neurochem. 274:13375- 13383. Blood. 2003;102:783-788. 1997;69:443-454. 19. Singh S, Hider RC, Porter JB. A direct method for 9. Weiss G, Goodnough LT. Anemia of chronic dis- 30. Moos T, Morgan EH. 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