Papers by Ioav Cabantchik
Nephron Clinical Practice, Jan 20, 2005
infusion and NTBI transiently appeared in some patients but markers of oxidative stress were not ... more infusion and NTBI transiently appeared in some patients but markers of oxidative stress were not signifi cantly affected. Conclusion: Although ESRD patients have a high prevalence of NTBI in their serum, no correlation could be established between the presence of NTBI and an increased oxidative stress. The slow infusion of maltofer does not promote a signifi cant increase in the plasma concentration of oxidative stress markers. It may therefore be considered as a safe complement to erythropoietin therapy.
Analytical Biochemistry, Jun 1, 2005
Non-transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. Vari... more Non-transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. Various methods to measure NTBI were comparatively assessed as part of an international interlaboratory study. Six laboratories participated in the study, using methods based on iron mobilization and detection with iron chelators or on reactivity with bleomycin. Serum samples of 12 patients with hereditary (n D 11) and secondary (n D 1) hemochromatosis were measured during a 3-day analysis using 4 determinations per sample per day, making a total of 144 measurements per laboratory. Bland-Altman plots for repeated measurements are presented. The methods diVered widely in mean serum NTBI level (range 0.12-4.32 mol/L), between-sample variation (SD range 0.20-2.13 mol/L and CV range 49.3-391.3%), and within-sample variation (SD range 0.02-0.45 mol/L and CV range 4.4-193.2%). The results obtained with methods based on chelators correlated signiWcantly (R 2 range 0.86-0.99). On the other hand, NTBI values obtained by the various methods related diVerently from those of serum transferrin saturation (TS) when expressed in terms of both regression coeYcients and NTBI levels at TS of 50%. Recent studies underscore the clinical relevance of NTBI in the management of iron-overloaded patients. However, before measurement of NTBI can be introduced into clinical practice, there is a need for more reproducible protocols as well as information on which method best represents the pathophysiological phenomenon and is most pertinent for diagnostic and therapeutic purposes.
Journal of Biological Chemistry, Jul 1, 1975
Pyridoxal phosphate is a potent probe for exploring the "sidedness" of proteins... more Pyridoxal phosphate is a potent probe for exploring the "sidedness" of proteins in the membrane of the intact red blood cell. It reacts with amino groups with a high degree of specificity, forming a Schiff's base that can be fixed as an irreversible bond upon reduction with NaBH4; its binding site can be identified by use of [3-H]pyridoxal phosphate or NaB3-H4; it can be used as a surface probe under conditions of minimal penetration, or it can be used as a probe for proteins on the inside of the membrane under conditions of substantial uptake. Pyridoxal phosphate uptake involves a rapid and a slow component. The former represents the binding to the outer surface of the membrane; it is not substantially affected by pH and temperature, but is reduced considerably by pretreatment of cells by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid, a specific inhibitor of anion transport. The slow component represents penetration into the cell; it is blocked by high pH, low temperature, or pretreatment with the disulfonic stilbene. Pyridoxal phosphate itself is also an effective and specific inhibitor of the permeation of other anions. Under conditions of minimal uptake, the only labeled proteins are three glycoproteins and a protein of apparent molecular weight 95,000. Under conditions of substantial uptake into the cell, the other major protein bands seen by staining on acrylamide gels after electrophoresis are labeled. It is concluded that virtually all of the major membrane proteins interact with pyridoxal phosphate from one side of the membrane or the other. The differences in labeling under conditions of minimal or maximal uptake can, therefore, be attributed to the sidedness in the distribution of the membrane proteins rather than to differences in their reactivity.
Journal of Biological Chemistry, Oct 1, 1995
The release of iron from transferrin (Tf) in the acidic milieu of endosomes and its translocation... more The release of iron from transferrin (Tf) in the acidic milieu of endosomes and its translocation into the cytosol are integral steps in the process of iron acquisition via receptor-mediated endocytosis (RME). The translocated metal is thought to enter a low molecular weight cytoplasmic pool, presumed to contain the form of iron which is apparently sensed by iron responsive proteins and is the direct target of iron chelators. The process of iron delivery into the cytoplasmic chelatable pool of K562 cells was studied in situ by continuous monitoring of the fluorescence of cells loaded with the metal-sensitive probe calcein. Upon exposure to Tf at 37°C, intracellular fluorescence decayed, corresponding to an initial iron uptake of 40 nM/min. The Tf-mediated iron uptake was profoundly inhibited by weak bases, the protonophore monensin, energy depletion, or low temperatures (<25°C), all properties characteristic of RME. Cell iron levels were affected by the slowly permeating chelator desferrioxamine only after prolonged incubations. Conversely, rapidly penetrating, lipophilic iron-(II) chelators such as 2,2-bipyridyl, evoked swift increases in cell calcein fluorescence, equivalent to sequestration of 0.2-0.5 M cytosolic iron, depending on the degree of pre-exposure to Tf. Addition of iron(III) chelators to permeabilized 2,2-bipyridyl-treated cells, failed to reveal significant levels of chelatable iron(III). The finding that the bulk of the in situ cell chelatable pool is comprised of iron(II) was corroborated by pulsing K562 cells with Tf-55 Fe, followed by addition of iron-(II) and/or iron(III) chelators and extraction of chelator-55
Blood, Nov 19, 2010
Abstract 4283 Beta-thalassemia results from a mutation in the beta-globin gene which leads to ine... more Abstract 4283 Beta-thalassemia results from a mutation in the beta-globin gene which leads to ineffective erythropoiesis (IE), shortened red blood cell (RBC) survival, and anemia requiring varying degrees of transfusion-dependence. Our previous data demonstrate that thalassemic (Hbbth1/th1) mice treated with apo-transferrin have more circulating RBCs, each with lower mean corpuscular hemoglobin (MCH), increased hemoglobin, and reversal of splenomegaly. These results suggest that anemia in beta-thalassemia is a consequence of excess intracellular heme in developing erythroblasts. Because iron in erythroid precursors is predominantly used for heme synthesis and iron uptake is limited to transferrin-bound iron through transferrin receptor 1 (TfR1), we hypothesized that exogenous apo-transferrin injections reduce MCH by decreasing surface TfR1 expression. To clarify the direct effect of splenic erythropoiesis in this experimental system, we analyzed splenectomized thalassemic mice for the effect of apo-transferrin injections on erythroid precursor differentiation and expression of TfR1 in the bone marrow as well as all RBC parameters, serum erythropoietin levels, and degree of extramedullary erythropoiesis (EMH) in the liver. We used intraperitoneal human apo-transferrin injection, 10 mg daily for 20 days. Apo-transferrin treated splenectomized thalassemic mice were compared with age and gender matched untreated splenectomized mice. A novel flow cytometry analysis using CD44 and TER119 in the bone marrow allowed us to measure the expression levels (using mean fluorescence intensity) of TfR1 at different stages of erythroid differentiation. Splenectomy in thalassemic mice resulted in decreased reticulocyte count…
Blood Cells Molecules and Diseases, Jul 1, 2018
Frontiers in Pharmacology, Aug 3, 2016
Blood, Nov 15, 1999
The labile iron pool (LIP) of animal cells has been implicated in cell iron regulation and as a k... more The labile iron pool (LIP) of animal cells has been implicated in cell iron regulation and as a key component of the oxidative-stress response. A major mechanism commonly implied in the downregulation of LIP has been the induced expression of ferritin (FT), particularly the heavy subunits (H-FT) that display ferroxidase activity. The effects of H-FT on LIP and other physiological parameters were studied in murine erythroleukemia (MEL) cells stably transfected with H-FT subunits. Clones expressing different levels of H-FT displayed similar concentrations of total cell iron (0.3 ؎ 0.1 mmol/L) and of reduced/total glutathione. However, with increasing H-FT levels the cells expressed lower levels of LIP and reactive oxygen species (ROS) and ensuing cell death after iron loads and oxidative challenges. These results provide direct experimental support for the alleged roles of H-FT as a regulator of labile cell iron and as a possible attenuator of the oxidative cell response. H-FT overexpression was of no apparent consequence to the cellular proliferative capacity. However, concomitant with the acquisition of iron and redox regulatory capacities, the H-FT-transfectant cells commensurately acquired multidrug resistance (MDR) properties. These properties were identified as increased expression of MDR1 mRNA (by reverse transcription polymerase chain reaction [RT-PCR]), P-glycoprotein (Western immunoblotting), drug transport activity (verapamil-sensitive drug efflux), and drug cytotoxicity associated with increased MDR1 or PgP. Although enhanced MDR expression per se evoked no significant changes in either LIP levels or ROS production, it might be essential for the survival of H-FT transfectants, possibly by expediting the export of cellgenerated metabolites.
Best Practice & Research Clinical Haematology, Jun 1, 2005
Labile plasma iron (LPI) represents a component of non-transferrin-bound iron (NTBI) that is both... more Labile plasma iron (LPI) represents a component of non-transferrin-bound iron (NTBI) that is both redox-active and chelatable, capable of permeating into organs and inducing tissue iron overload. It appears in various types of hemosiderosis (transfusional and non-transfusional) and in other iron-overload conditions. Sustained levels of LPI could over time compromise organ (e.g. heart) function and patient survival. With the advent of methods for measuring LPI in the clinical setting, it has become possible to assess the implications of LPI in the management of iron overload based on regimens of iron chelation. As LPI is detected primarily in patients with transfusional iron overload and other forms of hemosiderosis, we review here regimens of iron chelation with deferrioxamine and deferiprone (separately or combined) in terms of their efficacy in minimizing daily exposure to LPI in thalassemia major and thalassemia intermedia patients.
Human erythrocyte glycophorin, a putative receptor to Plasmodiumfalciparum malaria parasites, was... more Human erythrocyte glycophorin, a putative receptor to Plasmodiumfalciparum malaria parasites, was studied in terms of its structural domains involved in mediating invasion. These domains were isolated from purified glycophorin A and from supernatants and membranes obtained from protease-treated erythrocytes. They were tested for invasion blocking capacity by using an in vitro assay system. The role of carbohydrate-rich domains was assessed with the following compounds: (i) sialoglycopeptides released by proteases either from whole cells or isolated glycophorin A; (ii) the sialoglycoproteins fetuin and a1 acid glycoprotein and the N-acetylglucosamine-rich ovomucoid; and (iii) the saccharides N-acetylneuraminlactose, N-acetylglucosamine, and free sialic acid. With the exception of N-acetylglucosamine, all of the compounds failed to block invasion. The role of carbohydrate-poor domains of glycophorin was assessed with peptides isolated from membranes of proteolyzed cells and with the hydrophobic fragment of glycophorin A. Glycophorin and the derived hydrophobic peptides formed highmolecular-weight aggregates in physiological solutions. They all inhibited invasion to a comparable extent. The inhibitory potency of glycophorin A increased by sixfold after reconstitution into egg lecithin vesicles. The observations reported here underscore the role played by the hydrophobic domain in the
Fems Immunology and Medical Microbiology, Dec 1, 1999
Malaria is the major life threatening parasitic disease and the cause of a global public health p... more Malaria is the major life threatening parasitic disease and the cause of a global public health problem. The failure of vector eradication programs and the appearance and spread of drug resistant parasites have posed the urgent challenge of developing effective, safe and affordable anti-malarial drugs. The design of such drugs is largely based on the targeting of agents to the parasite-based machinery for host digestion and to the products of hemoglobin catabolism. Iron chelators, by depriving intracellular parasites from essential iron, lead to selective suppression of parasite growth. However, by acting on parasiteimpaired macrophages, chelators can also expedite resumption of phagocytosis and elimination of parasites. In order to be clinically effective, chelators need to be maintained in the blood for extensive time periods. Therapeutic doses can be attained with appropriate drug combinations and formulations or delivery devices and these must be presented in a form well tolerated by the host. The early documentation that chelation therapy has activity against human malaria has paved the road for the design of novel and more efficient remedies based on short-term iron deprivation.
Journal of Biological Chemistry, Jul 1, 1993
Journal of The American Society of Nephrology, May 21, 2015
British Journal of Haematology, Dec 1, 2009
Secondary iron overload, which develops in Thalassaemia major patients as a result of multiple bl... more Secondary iron overload, which develops in Thalassaemia major patients as a result of multiple blood transfusions, eventually leads to uncontrolled iron release into the circulation, ultimately surpassing the plasma transferrin iron-binding capacity and generating a variety of complexes collectively labelled as non-transferrin bound iron (NTBI) (Hershko et al, 1978; Hider, 2002; Breuer et al, 2000). A fraction of NTBI that is redox active and chelatable is referred to as labile plasma iron (LPI)
BMC Neurology, May 6, 2015
Background: Growing body of evidence suggests that Parkinson's disease (PD) is associated with ox... more Background: Growing body of evidence suggests that Parkinson's disease (PD) is associated with oxidative damage via iron accumulation in the substantia nigra (SN). Low ceruloplasmin (CP)-ferroxidase activity has been identified in the SN and the cerebrospinal fluid (CSF) of patients with PD. The iron chelator, deferiprone, reduces the abnormally high levels of iron in the SN. In order to determine CP's involvement in iron accumulation in SN and PD progression, we aim to compare the ability of iron chelation treatment to reducing both SN iron levels and motor handicap in PD patients according to the level of ceruloplasmin activity. Methods: We used a moderate chelation protocol with deferiprone (DFP) based on a, 6-month delayed-start paradigm, randomized placebo controlled clinical trial in 40 PD patients. CP-ferroxidase activity was determined in blood and CSF together with the D544E gene polymorphism (rs701753). Iron levels were determined by R2* MRI sequence and the motor handicap by the UPDRS motor score. Results: After 6 to 12 months of DFP treatment, greater reductions in SN iron levels and UPDRS motor scores were obtained in patients with higher serum and CSF levels of CP-ferroxidase activity. After 6 months of DFP treatment, the AT genotype group displayed greater reduction of iron level in the SN with greater CSF and serum levels of CP activity than the AA genotype group. Conclusion: Although most of the DFP-treated patients displayed clinical and radiological improvements, those with the lower CP activity appeared to respond better to iron chelation. Larger RCTs are now needed to establish whether pharmacological modulation of CP activity could be an innovative neuroprotective strategy in PD. Trial registration: FAIR-PARK study (ClinicalTrials.gov reference: NCT00943748; French national reference number: 2008−006842−25). This study was approved by the French Drug Agency (ANSM) and the local institutional review board ("Comité de Protection des Personnes of Lille").
Journal of Biological Chemistry, Jun 1, 1998
In vitro studies have shown that ferritin iron incorporation is mediated by a ferroxidase activit... more In vitro studies have shown that ferritin iron incorporation is mediated by a ferroxidase activity associated with ferritin H subunits (H-Ft) and a nucleation center associated with ferritin L subunits (L-Ft). To assess the role played by the ferritin subunits in regulating intracellular iron distribution, we transfected mouse erythroleukemia cells with the H-Ft subunit gene mutated in the iron-responsive element. Stable transfectants displayed high H-Ft levels and reduced endogenous L-Ft levels, resulting in a marked change in the H:L subunit ratio from 1:1 in control cells to as high as 20:1 in some transfected clones. The effects of H-Ft overexpression on the labile iron pool were determined in intact cells by a novel method based on the fluorescent metallosensor calcein. H-Ft overexpression resulted in a significant reduction in the iron pool, from 1.3 M in control cells to 0.56 M in H-Ft transfectants, and in higher buffering capacity following iron loads. A fraction of the H-Ftassociated iron was labile, available to cell-permeant, but not cell-impermeant, chelators. The results of this study provide the first in vivo direct demonstration of the capacity of H-Ft to sequester cell iron and to regulate the levels of the labile iron pool.
Blood, Nov 1, 2005
The primary targets of iron chelators used for treating transfusional iron overload are preventio... more The primary targets of iron chelators used for treating transfusional iron overload are prevention of iron ingress into tissues and its intracellular scavenging. The present study was aimed at elucidating the capacity of clinically important iron chelators such as deferiprone (DFP), desferrioxamine, and ICL670 to (a) gain direct access to intracellular iron pools of key cells of iron accumulation (macrophages, hepatocytes, and cardiomyocyte cell lines); (b) chelate the labile iron present in discrete cell compartments/organelles; and (c) prevent labile iron involvement in the generation of reactive oxidant species. Chelation of cytosolic and organellar cell iron was visualized dynamically and quantitatively in living cells by fluorescence microscopic imaging of fluorescent metallosensors (used as ironquenched complexes of calceins) targeted to either cytosol, endosome-lysosomes, or mitochondria. The rate and extent of fluorescence recovery provided an in situ measure of the accessibility of chelators to particular cell sites/organelles. Complementary , fluorogenic redox probes associated with cell compartments enabled identification of chelator-sensitive, localized reactive oxidant production. Our studies indicate that chelation by desferrioxamine is slow and is enhanced in cells with relatively high endocytic activities, while ICL670 and DFP readily enter most cells and efficiently reach the major intracellular sites of iron accumulation.
Antioxidants, Jul 21, 2021
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Blood, Feb 1, 2008
Various pathologies are characterized by the accumulation of toxic iron in cell compartments. In ... more Various pathologies are characterized by the accumulation of toxic iron in cell compartments. In anemia of chronic disease, iron is withheld by macrophages, leaving extracellular fluids iron-depleted. In Friedreich ataxia, iron levels rise in the mitochondria of excitable cells but decrease in the cytosol. We explored the possibility of using deferiprone, a membrane-permeant iron chelator in clinical use, to capture labile iron accumulated in specific organelles of cardiomyocytes and macrophages and convey it to other locations for physiologic reuse. De-feriprone's capacity for shuttling iron between cellular organelles was assessed with organelle-targeted fluorescent iron sensors in conjunction with time-lapse fluorescence microscopy imaging. Deferiprone facilitated transfer of iron from extracellular media into nuclei and mitochondria, from nuclei to mitochondria, from endosomes to nuclei, and from intracellular compartments to extracellular apotransferrin. Furthermore, it mobilized iron from iron-loaded cells and donated it to preerythroid cells for hemoglobin synthesis, both in the presence and in the absence of transferrin. These unique properties of deferiprone underlie mechanistically its capacity to alleviate iron accumulation in dentate nuclei of Friedreich ataxia patients and to donate tissuechelated iron to plasma transferrin in thalassemia intermedia patients. Deferiprone's shuttling properties could be exploited clinically for treating diseases involving regional iron accumulation.
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Papers by Ioav Cabantchik