Plasma Non-transferrin Bound Iron Research Articles

Ultraviolet light and riboflavin accelerates red blood cell dysfunction in vitro and in a guinea pig transfusion model.

Quality assessment of modified or processed red blood cell (RBC) components, such as pathogen-reduced RBCs, using only in vitro testing may not always be predictive of in vivo performance. Mouse or rat in vivo models are limited by a lack of applicability to certain aspects of human RBC biology. Here, we used a guinea pig model to study the effects of riboflavin combined with UV light on the integrity of RBCs in vitro and following transfusion in vivo. Guinea pig RBCs were collected from whole blood (WB) treated with varying UV doses (10, 20, 40 or 80 J/mL) in the presence of riboflavin (UVR-RBCs). In vitro tests for UVR-RBCs included hemolysis, osmotic fragility, and cellular morphology by scanning electron microscopy. Guinea pigs transfused with one-day post-treatment UVR-RBCs were evaluated for plasma hemoglobin (Hb), non-transferrin bound iron (NTBI), total iron and Perls-detectable hemosiderin deposition in the spleen and kidney, and renal uptake of Hb. Acute RBC injury was dose dependently accelerated after treatment with UV light in the presence of riboflavin. Aberrant RBC morphology was evident at 20, 40, and 80 J/mL, and membrane lysis with Hb release was prominent at 80 J/mL. Guinea pigs transfused with 40 and 80 J/mL UVR-RBCs showed increased plasma Hb levels, and plasma NTBI was elevated in all UVR-RBC groups (10-80 J/mL). Total iron levels and Perls-hemosiderin staining in spleen and kidney as well as Hb uptake in renal proximal tubules were increased 8 hours post-transfusion with 40 and 80 J/mL UVR-RBCs. UVR-RBCs administered to guinea pigs increased markers of intravascular and extravascular hemolysis in a UV dose-dependent manner. This model may allow for the discrimination of RBC injury during testing of extensively processed RBCs intended for transfusion.

Plasma non-transferrin-bound iron uptake by the small intestine leads to intestinal injury and intestinal flora dysbiosis in an iron overload mouse model and Caco-2 cells.

Iron overload often occurs during blood transfusion and iron supplementation, resulting in the presence of non-transferrin-bound iron (NTBI) in host plasma and damage to multiple organs, but effects on the intestine have rarely been reported. In this study, an iron overload mouse model with plasma NTBI was established by intraperitoneal injection of iron dextran. We found that plasma NTBI damaged intestinal morphology, caused intestinal oxidative stress injury and reactive oxygen species (ROS) accumulation, and induced intestinal epithelial cell apoptosis. In addition, plasma NTBI increased the relative abundance of Ileibacterium and Desulfovibrio in the cecum, while the relative abundance of Faecalibaculum and Romboutsia was reduced. Ileibacterium may be a potential microbial biomarker of plasma NTBI. Based on the function prediction analysis, plasma NTBI led to the weakening of intestinal microbiota function, significantly reducing the function of the extracellular structure. Further investigation into the mechanism of injury showed that iron absorption in the small intestine significantly increased in the iron group. Caco-2 cell monolayers were used as a model of the intestinal epithelium to study the mechanism of iron transport. By adding ferric ammonium citrate (FAC, plasma NTBI in physiological form) to the basolateral side, the apparent permeability coefficient (Papp) values from the basolateral to the apical side were greater than 3×10-6 cm s-1. Intracellular ferritin level and apical iron concentration significantly increased, and SLC39A8 (ZIP8) and SLC39A14 (ZIP14) were highly expressed in the FAC group. Short hairpin RNA (shRNA) was used to knock down ZIP8 and ZIP14 in Caco-2 cells. Transfection with ZIP14-specific shRNA decreased intracellular ferritin level and inhibited iron uptake. These results revealed that plasma NTBI may cause intestinal injury and intestinal flora dysbiosis due to the uptake of plasma NTBI from the basolateral side into the small intestine, which is probably mediated by ZIP14.

Ferroportin inhibitor vamifeport ameliorates ineffective erythropoiesis in a mouse model of β-thalassemia with blood transfusions.

β-thalassemia is an inherited anemia characterized by ineffective erythropoiesis. Blood transfusions are required for survival in transfusion-dependent β-thalassemia and are also occasionally needed in patients with non-transfusion-dependent β-thalassemia. Patients with transfusion-dependent b-thalassemia often have elevated transferrin saturation (TSAT) and non-transferrin-bound iron (NTBI) levels, which can lead to organ iron overload, oxidative stress, and vascular damage. Vamifeport is an oral ferroportin inhibitor that was previously shown to ameliorate anemia, ineffective erythropoiesis, and dysregulated iron homeostasis in the Hbbth3/+ mouse model of β-thalassemia, under non-transfused conditions. Our study aimed to assess the effects of oral vamifeport on iron-related parameters (including plasma NTBI levels) and ineffective erythropoiesis following blood transfusions in Hbbth3/+ mice. A single dose of vamifeport prevented the transient transfusion-mediated NTBI increase in Hbbth3/+ mice. Compared with vehicle treatment, vamifeport significantly increased hemoglobin levels and red blood cell counts in transfused mice. Vamifeport treatment also significantly improved ineffective erythropoiesis in the spleens of Hbbth3/+ mice, with additive effects observed when treatment was combined with repeated transfusions. Vamifeport corrected leukocyte counts and significantly improved iron-related parameters (serum transferrin, TSAT and erythropoietin levels) versus vehicle treatment in Hbbth3/+ mice, irrespective of transfusion status. In summary, vamifeport prevented transfusion-mediated NTBI formation in Hbbth3/+ mice. When given alone or combined with blood transfusions, vamifeport also ameliorated anemia, ineffective erythropoiesis, and dysregulated iron homeostasis. Administering vamifeport together with repeated blood transfusions additively ameliorated anemia and ineffective erythropoiesis in this mouse model, providing preclinical proof-of-concept for the efficacy of combining vamifeport with blood transfusions in β-thalassemia.

The clinical relevance of detectable plasma iron species in iron overload states and subsequent to intravenous iron-carbohydrate administration.

Many disorders of iron homeostasis (e.g., iron overload) are associated with the dynamic kinetic profiles of multiple non-transferrin bound iron (NTBI) species, chronic exposure to which is associated with deleterious end-organ effects. Here we discuss the chemical nature of NTBI species, challenges with measuring NTBI in plasma, and the clinical relevance of NTBI exposure based on source (iron overload disorder vs. intravenous iron-carbohydrate complex administration). NTBI is not a single entity but consists of multiple, often poorly characterized species, some of which are kinetically non-exchangeable while others are relatively exchangeable. Prolonged presence of plasma NTBI is associated with excessive tissue iron accumulation in susceptible tissues, with consequences, such as endocrinopathy and heart failure. In contrast, intravenous iron-carbohydrate nanomedicines administration leads only to transient NTBI appearance and lacks evidence for association with adverse clinical outcomes. Assays to measure plasma NTBI are typically technically complex and remain chiefly a research tool. There have been two general approaches to estimating NTBI: capture assays and redox-activity assays. Early assays could not avoid capturing some iron from transferrin, thus overestimating NTBI. By contrast, some later assays may have promoted the donation of NTBI species to transferrin during the assay procedure, potentially underestimating NTBI levels. The levels of transferrin saturation at which NTBI species have been detectable have varied between different methodologies and between patient populations studied.

RBC Transfusion to Severe Anemic Murine Neonates Leads to Systemic Inflammation

Background: Transfusions of red blood cells (RBCT) are necessary and life-saving in premature and critically ill infants, who experience severe anemia due to physiologic and iatrogenic factors. Recently, we showed that severe anemia was associated with increased intestinal permeability, demonstrated by identifying endotoxin in the bloodstream. The risks of experiencing severe anemia during critical developmental periods must be balanced with the risks of transfusions, which can lead to the Systemic Inflammatory Response Syndrome (SIRS). In this study, we investigated the role of severe anemia in endotoxin sensitization of the liver, and of RBCT-triggered cytokine storm, in murine neonates. Methods: C57BL/6 (WT) and TLR4-KO mouse pups were studied in 4 groups (n=6 each): (1) naïve controls; (2) non-anemic RBC transfused; (3) anemic (hematocrit 20-24%); and (4) anemic RBC transfused. Pups in anemic groups underwent facial vein phlebotomy on days P2, 4, 6, 8, and 10. Leukoreduced and 7-day refrigerator-stored packed RBCs from allogeneic (FVB) adult donors were transfused intravenously into the retro-orbital plexus (20 mL/kg) of P10 pups. Blood was collected 2, 4, and 6 hours post-transfusion, and plasma was separated for quantifying inflammatory cytokines (IL-1β, TNF-α, CXCL-1, IL-6, IFN-γ) using a Luminex assay. Plasma non-transferrin bound iron (NTBI) levels were measured at 2 hours post-transfusion. To measure leaky gut associated liver endotoxin sensitization, genetically-modified E. coli, expressing green fluorescent protein (GFP; 108 colony-forming units), were administered by gavage; these bacteria were measured in the liver of anemic pups by immunofluorescence using anti-GFP antibody. To investigate the endotoxin-associated monocyte-macrophage system in the liver, single cell suspensions were prepared and flow cytometry was performed by staining with antibodies against CD45, CD11b, F4/80, CD11c, Ly6G and Ly6C. Results: RBC transfusions led to the acute release of inflammatory mediators (IL-1β, TNF-α, CXCL-1, IL-6, IFN-γ) within 2 hours, in pups with a normal hematocrit ("transfusion controls"), as well as in pups rendered anemic. However, inflammatory cytokine levels were significantly higher in RBC-transfused anemic pups as compared to transfusion controls, and remained elevated for several hours. We excluded the inflammatory response at 6 hours post-transfusion, because intestinal inflammation was observed after that time by the release of intestinal fatty acid binding protein (iFABP). No changes were found in plasma NTBI levels between transfusion control and anemic-transfused pups, indicating that the acute inflammatory response in the anemic-transfused groups is independent of liver iron accumulation due to stored RBC clearance. However, severely anemic mouse pups had elevated amounts of orally administered E. coli-GFP in the liver relative to control mice, indicating that anemia allows endotoxin delivery and sensitizes the liver by bacterial translocation across the anemic intestine. The TLR4-KO mice study showed that overall plasma levels of inflammatory cytokines (TNF-α, IL-6, IFN-γ) were reduced, as compared to anemic-transfused WT pups, but remained elevated in the plasma of anemic-transfused TLR4-KO pups, as compared to TLR4-KO transfused-control pups, indicating that this acute inflammatory response does not depend on TLR4. By flow cytometry, anemic (P10) pups showed an increased number of CD11bhiF4/80loLy6C+ hepatic monocytes as compared to control, but no changes in CD11bhiF4/80hi hepatic macrophages (i.e., Kupffer cells); qRT-PCR studies confirm that these anemic hepatic monocytes contained mRNA transcripts of IL-1β, TNF-α, CXCL-1, IL-6, and IFN-γ. In addition, the anemic mouse pups that were treated with recombinant haptoglobin (rHp, 100µg/kg) showed reduced plasma levels of IL-1β, TNF-α, CXCL-1, IL-6, and IFN-γ at 2-6 hours post-transfusion of packed-stored RBCs. Conclusions: In this mouse model, the severe anemia-associated low-grade inflammatory state in the liver leads to increased numbers of hepatic monocytes with inflammatory cytokines mRNA; in this condition, RBC transfusions activate these monocytes and cause systemic inflammation. Pre-treatment of rHp was reduce the RBCT-associated systemic inflammatory response.

Plasma Non-transferrin-Bound Iron Could Enter into Mice Duodenum and Negatively Affect Duodenal Defense Response to Virus and Immune Responses.

Plasma non-transferrin-bound iron (NTBI) exists when the plasma iron content exceeds the carrying capacity of transferrin and can be quickly cleared by the liver, pancreas, and other organs. However, whether it could enter the small intestine and its effects still remain unclear. Herein, these issues were explored. Mice were intravenously administrated of ferric citrate (treatment) or citrate acid (control) 10min after the saturation of the transferrin. Two hours later, hepatic, duodenal, and jejunal iron content and distribution were measured and duodenal transcriptome sequencing was performed. Significant increase of duodenal and hepatic iron content was detected, indicating that plasma NTBI could be absorbed by the duodenum as well as the liver. A total of 103 differentially expressed genes were identified in the duodenum of mice in the treatment group compared to the control group. Gene Ontology (GO) functional analysis of these genes showed that they were mainly involved in defense response to virus and immune response. The results of Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis revealed that there were major changes in the hematopoietic cell lineage and some virus infection pathways between the two groups. Determination of 7 cytokines in the duodenum were further conducted, which demonstrated that the anti-inflammatory factors interferon (IL)-4 and IL-10 in the duodenum were significantly decreased after NTBI uptake. Our findings revealed that NTBI in plasma can enter the duodenum, which would change the duodenal hematopoietic cell lineage and have a negative impact on defense response to the virus and immune responses.

Plasma nontransferrin bound iron-nontransferrin bound iron revisited: Implications for systemic iron overload and in iv iron supplementation.

Non-transferrin bound iron (NTBI) has been classically used to denote a potentially toxic component of plasma detected in systemic iron overload and implicated in end-organ iron accumulation and biological damage. A chelatable labile component of plasma NTBI has also served as target for novel therapeutics. However, as the term NTBI per se does not exclude non-toxic forms of iron that can be detected in plasma, its liberal use without additional pharmacological attributes might be misleading, especially in the clinical context, as for infusible iron supplements. In keeping with the historical and commonly used NTBI term, we propose here to use "labile/non-labile" plasma NTBI to indicate the toxic potential of the iron species in question. This article is protected by copyright. All rights reserved.

Non-Transferrin-Bound Iron Serves As Iron Source for Aspergillus Fumigatus Growth

The appearance of non-transferrin-bound iron (NTBI) as a consequence of saturated transferrin (Tf) levels in patients undergoing bone marrow transplantation is multifactorial. Chemotherapy-induced tissue damage on the one hand side, causing iron release from apoptotic cells, and chemotherapy-induced bone marrow suppression leading to diminished iron consumption for erythropoiesis on the other hand side as well as chronic transfusion therapy for anemia are among the major contributing factors for iron overload. The existence of NTBI is known to be associated with therapy-related complications, including infections. Nowadays invasive aspergillosis is, in spite of antifungal prophylaxis, still a serious problem with mortality rates ranging up to 70%-90%. The possible interaction of invasive aspergillosis and iron metabolism has been reported.In this study we specifically assessed the role of NTBI on the in vitro growth of Aspergillus fumigatus (A. fumigatus ; ATCC46645) in the presence of plasma. Therefore the growth of A. fumigatus in cultures containing 10% human plasma and various NTBI levels was explored. This assay revealed that the presence of NTBI, but not high Tf-saturation without NTBI, led to a considerable growth advantage. These findings suppose that A. fumigatus growth depends on the presence of NTBI rather than total iron availability. Correspondingly, addition of sufficient amounts of human apo-Tf to eliminate NTBI prevented A. fumigatus growth. These results clearly show that NTBI only exists and promotes A. fumigatus growth if Tf is saturated and disproves the widely believed notion that that Aspergillus utilizes iron from Tf. In contrast to apo-Tf, iron chelators (e.g. deferiprone or deferoxamine) tested in our in vitro system stimulated, rather than inhibited A. fumigatus growth, despite elimination of NTBI.To better understand the underlying molecular mechanisms, different A. fumigatus mutant strains with defects in iron acquisition systems are under investigation. Preliminary data indicate that the uptake of NTBI is not solely dependent on siderophore-mediated iron uptake.In conclusion our findings clearly indicate that NTBI is relevant for fungal growth, at least in vitro in the presence of plasma. Further ongoing experiments aim to clarify whether different clinical A. fumigatus isolates differ in their ability to utilize Tf- bound iron and NTBI. In addition, plasma samples from patients, who had undergone bone marrow transplantation, with already known levels of NTBI, should help to further corroborate the relevance of NTBI for fungal growth in vivo . DisclosuresPlatzbecker:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding.

Ferroportin inhibition attenuates plasma iron, oxidant stress, and renal injury following red blood cell transfusion in guinea pigs.

Red blood cell (RBC) transfusions result in the sequestration and metabolism of storage-damaged RBCs within the spleen and liver. These events are followed by increased plasma iron concentrations that can contribute to oxidant stress and cellular injury. We hypothesized that administration of a ferroportin inhibitor (FPN-INH) immediately after acute RBC exchange transfusion could attenuate posttransfusion circulatory compartment iron exposure, by retaining iron in spleen and hepatic macrophages. Donor guinea pig blood was leukoreduced, and RBCs were preserved at 4°C. Recipient guinea pigs (n = 5/group) were exchange transfused with donor RBCs after refrigerator preservation and dosed intravenously with a small-molecule FPN-INH. Groups included transfusion with vehicle (saline), 5 mg/kg or 25 mg/kg FPN-INH. A time course of RBC morphology, plasma non-transferrin-bound iron (NTBI) and plasma hemoglobin (Hb) were evaluated. End-study spleen, liver, and kidney organ iron levels, as well as renal tissue oxidation and injury, were measured acutely (24-hr after transfusion). RBC transfusion increased plasma NTBI, with maximal concentrations occurring 8 hours after transfusion. Posttransfusion iron accumulation resulted in tubule oxidation and acute kidney injury. FPN inhibition increased spleen and liver parenchymal/macrophage iron accumulation, but attenuated plasma NTBI, and subsequent renal tissue oxidation/injury. In situations of acute RBC transfusion, minimizing circulatory NTBI exposure by FPN inhibition may attenuate organ-specific adverse consequences of iron exposure.

Linking Stored Red Blood Cell Metabolism to Transfusion Recipient Iron Homeostasis Pathophysiology in Critically-Ill Children

Background: Transfusion of packed red blood cells (pRBCs) is a common, life-saving therapy for many critically-ill children. However, routine blood bank storage results in the progressive accumulation of a series of RBC modifications, termed the "storage lesion," that reduce RBC recovery in vivo, may affect transfusion recipient physiology, and may be implicated in transfusion-related adverse events. In particular, circulating non-transferrin-bound iron (NTBI) in recipients can be increased after pRBC transfusion. NTBI promotes bacterial growth, induces oxidative injury, and inversely correlates with post-transfusion recovery, all of which may increase the risk of sepsis, multiorgan failure, and death. We aimed to determine whether the metabolic state of donor pRBCs at the time of transfusion correlates with recipient NTBI levels, which serves as a marker of hemolysis and iron homeostasis in critically-ill pediatric patients. Study Design/Methods: Critically-ill pediatric patients, aged < 21-years-old with a minimum weight of 5kg in the pediatric intensive care unit at a single-site tertiary care hospital, who required pRBC transfusion, were prospectively enrolled after parental consent (n = 49 patients). Patient blood samples were obtained pre-transfusion and 4 hours following the patient's first qualifying pRBC transfusion, and analyzed for markers of iron and heme homeostasis, hemolysis, liver function, and inflammation utilizing standard laboratory methods. pRBC transfusate (1 mL) was frozen at -80°C immediately at the end of transfusion for each transfused patient. Hydrophilic and lipophilic compounds were extracted separately using methanol:acetonitrile:water or methanol, respectively, before analysis using high-throughput LC-MS based metabolomics and lipidomics platforms to determine the relative abundance of small molecule compounds. Metabolite levels in the pRBC transfusates were correlated with the differences in pre- and post-transfusion clinical laboratory values and selected patient parameters (e.g. blood type, patient age, and storage age of the pRBC unit at the time of transfusion), using Pearson correlation coefficients and associated p-values. Results: The relative levels of 221 unique metabolites and lipids in the pRBC transfusates were correlated with the corresponding transfusion recipient's clinical laboratory values and selected patient parameters. Analysis of Pearson's correlations between the levels of these compounds in the donor pRBC transfusates and the pre- and post-transfusion differences in the recipients' NTBI levels (ΔNTBI) demonstrated statistically significant correlations for 24 metabolites (R = 0.28-0.43, p = 0.0022-0.0476, see Table). Analysis of recipient clinical laboratory biomarkers associated with recipient ΔNTBI revealed a positive correlation with increases in serum iron (R = 0.41, p = 0.0034) but not with other changes in recipient clinical laboratory biomarkers. The age of the stored pRBCs at the time of transfusion did not correlate with ΔNTBI levels (R = 0.24, p = 0.0912). Conclusions: Statistically significant correlations of donor pRBC transfusate metabolites with transfusion recipient ΔNTBI are primarily seen with hypoxanthine, lactate, metabolites of glutathione homeostasis, amino acids, unsaturated free fatty acids, acyl-carnitines, and metabolites of indole and tryptophan metabolism. These data suggest that higher levels of oxidative stress, proteolysis, membrane remodeling in stored donor pRBCs, and, possibly, microbiome metabolism in the pRBC donor, are associated with significant increases in recipient plasma NTBI and serum iron levels, likely from hemolysis of storage-damaged RBCs. The levels of these metabolic biomarkers of the RBC storage lesion correlate better with ΔNTBI than the storage age of the donor unit, supporting the notion that pRBC metabolic age is not equivalent to pRBC chronologic age, and that the former may be more relevant to transfusion recipient pathophysiology in critically-ill children. Table. Disclosures Nemkov: Omix Technologies, Inc.: Equity Ownership, Other: Founder. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. D'Alessandro:Hemanext, Inc.: Membership on an entity's Board of Directors or advisory committees; Omix Technologies, Inc.: Other: Founder.

Low-molecular-mass iron complexes in blood plasma of iron-deficient pigs do not originate directly from nutrient iron.

Nutrient iron entering the blood binds transferrin (TFN)d, which delivers iron to cells in the body. In healthy individuals, ∼30% of TFN is iron-bound while the remainder is unbound (apo-TFN). TFN saturates the plasma of individuals with iron-overload diseases such as hereditary hemochromatosis, prompting release of a poorly-defined low-molecular-mass (LMM) iron species called non-transferrin-bound iron (NTBI). An experiment was devised to directly detect NTBI in plasma of iron-deficient pigs and to assess the role of the liver which is known to bind NTBI. Catheters were surgically installed in the portal vein (PV) and either the caudal vena cava or the cranial vena cava. After the animals recovered, 57Fe II ascorbate was injected into the stomach via a feeding tube. Blood was removed through the catheters before and after injection; plasma became 57Fe-enriched after injection. 57Fe-enriched plasma was passed through a 10 kDa cutoff membrane and the flow-through solution (FTS) was subjected to size-exclusion liquid chromatography (LC). The eluent flowed into an ICP-MS where 56Fe and 57Fe were detected. Low-intensity iron peaks with masses of 400-1600 Da were observed, but none became enriched in 57Fe after injection. Rather, the injected 57Fe bound to apo-TFN. Viewed naively, this implies that nutrient-derived 57Fe in healthy mammals passes from the intestines to apo-TFN without first entering the blood as a LMM intermediate. In this case, nutrient iron exported from intestinal enterocytes of healthy individuals may quickly bind apo-TFN such that LMM iron species do not accumulate in blood plasma. Some 57Fe from the FTS may have adsorbed onto the column. In any event, the LMM iron species in plasma that eluted from the column must have originated from iron stored within the body, perhaps in macrophages - not directly from nutrient iron absorption. The liver absorbed and released LMM iron species, but the effect was modest, consistent with its role as a dynamic iron buffer. Passage through the liver also altered the distribution of different forms of TFN present in the PV.

Green tea extract modulates oxidative tissue injury in beta-thalassemic mice by chelation of redox iron and inhibition of lipid peroxidation

Iron overload in patients with β-thalassemia can cause oxidative organ dysfunction. Iron chelation along with antioxidant supplementation can ameliorate such complications and prolong lives. Green tea extract (GTE) rich in epigallocatechin-3-gallate (EGCG) exhibits anti-oxidation and iron chelation properties in β-knockout thalassemic (BKO) mice diagnosed with iron overload. We investigated the effects of GTE and deferiprone (DFP) alone in combination with one another, and upon the levels of redox-active iron, lipid-peroxidation product, insulin and hepcidin in BKO mice. A state of iron overload was induced in the mice via a trimethylhexanoyl-ferrocene supplemented (Fe) diet for 3 months, and the mice were treated daily with either: DFP (50 mg/kg), DFP (50 mg/kg) plus GTE (50 mg EGCG equivalent/kg), or GTE alone for 2 months. Plasma non-transferrin bound iron (NTBI), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepcidin and insulin; tissue iron and MDA were measured. DFP, GTE and GTE + DFP effectively decreased plasma MDA (p < 0.05), NTBI and ALT, and increased plasma hepcidin and insulin. All the treatments also reduced iron accumulation and MDA production in both the pancreas and liver in the mice. However, the combination therapy demonstrated no advantages over monotherapy. The findings suggest GTE improved liver and pancreatic β-cell functions in iron-overloaded β-thalassemia mice by diminishing redox iron and free radicals, while inhibiting lipid peroxidation. Consequently, there are indications that GTE holds significant potential for clinical use.

Iron Absorption in Iron-Deficient Women, Who Received 65 mg Fe with an Indonesian Breakfast, Is Much Better from NaFe(III)EDTA than from Fe(II)SO₄, with an Acceptable Increase of Plasma NTBI. A Randomized Clinical Trial.

Plasma non-transferrin-bound iron (NTBI) is potentially harmful due to the generation of free radicals that cause tissue damage in vascular and other diseases. Studies in iron-replete and iron-deficient subjects, receiving a single oral test dose of Fe(II)SO4 or NaFe(III)EDTA with water, revealed that FeSO4 was well absorbed when compared with NaFeEDTA, while only the Fe(II) compound showed a remarkable increase of NTBI. As NaFeEDTA is successfully used for food fortification, a double-blind randomized cross-over trial was conducted in 11 healthy women with uncomplicated iron deficiency. All subjects received a placebo, 6.5 mg FeSO4, 65 mg FeSO4, 6.5 mg NaFeEDTA, and 65 mg NaFeEDTA with a traditional Indonesian breakfast in one-week intervals. Blood tests were carried out every 60 min for five hours. NTBI detection was performed using the fluorescein-labeled apotransferrin method. Plasma iron values were highly increased after 65 mg NaFeEDTA, twice as high as after FeSO4. A similar pattern was seen for NTBI. After 6.5 mg of NaFeEDTA and FeSO4, NTBI was hardly detectable. NaFeEDTA was highly effective for the treatment of iron deficiency if given with a meal, inhibiting the formation of nonabsorbable Fe-complexes, while NTBI did not exceed the range of normal values for iron-replete subjects.

Non‐transferrin‐bound iron and oxidative stress during allogeneic hemopoietic stem cell transplantation in patients with or without iron overload

Non‐transferrin‐bound iron and oxidative stress during allogeneic hemopoietic stem cell transplantation in patients with or without iron overload

Effects of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac function and Ca2+ regulation in iron-overloaded thalassemic mice

Although disturbance of cardiac Ca2+ regulation is involved in the pathophysiology of iron-overload cardiomyopathy, the obvious mechanisms involved in the dysregulation of iron-induced cardiac Ca2+ are unclear. Moreover, the roles of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac intracellular Ca2+ transients and Ca2+ regulatory proteins in thalassemic mice are still unknown. We tested the hypothesis that treatment with either deferiprone or efonidipine attenuated cardiac Ca2+ dysregulation and led to improved left ventricular (LV) function in iron-overloaded thalassemic mice. Wild-type (WT) mice and β-thalassemic (HT) mice were fed with either a normal diet (ND) or a high iron-diet (FE) for 90 days. Then, the FE-fed mice were treated with either deferiprone (75 mg/kg/day) or efonidipine (4 mg/kg/day) for 30 days. ND-fed HT mice had an increase in T-type calcium channels (TTCC) and an increased level of sarcoplasmic reticulum Ca2+-ATPase (SERCA), compared with ND-fed WT mice. Chronic iron feeding led to an increase in TTCC and expression of SERCA proteins in FE-fed WT mice. Moreover, chronic iron overload led to increased plasma non-transferrin bound iron (NTBI) and cardiac iron deposition, impaired cardiac intracellular Ca2+ transients including decreased intracellular Ca2+ transient amplitude, rising rate and decay rate, as well as impaired LV function as indicated by a decreased %LV ejection fraction (%LVEF) in both WT and HT mice. Our findings showed that treatment with either deferiprone (DFP) or efonidipine (EFO) showed similar benefits in reducing plasma NTBI and cardiac iron deposition, and improving %LVEF from 84.3 (WT) to 89.3 (DFP) and 89.2 (EFO) treatment; and from 84.2 (HT) to 88.8 (DFP) and 89.5 (EFO) treatment, however there was no improvement in the regulation of cardiac Ca2+ in iron-overloaded thalassemic mice. These findings provide the understanding of the effects of these drugs on the iron-overloaded heart in thalassemic mice and suggest that an alternative intervention that could improve calcium regulation under this condition is needed to improve the therapeutic outcome. Moreover, whether the benefits of the TTCC blocker is via its inhibition of the TTCC alone or together with its ability to chelate iron are still unclear and need further investigation.

Iron accelerates hemoglobin oxidation increasing mortality in vascular diseased guinea pigs following transfusion of stored blood.

Non-transferrin-bound iron (NTBI) and free hemoglobin (Hb) accumulate in circulation following stored RBC transfusions. This study investigated transfusion, vascular disease, and mortality in guinea pigs after stored RBC transfusion alone and following cotransfusion with apo-transferrin (apo-Tf) and haptoglobin (Hp). The effects of RBC exchange transfusion dose (1, 3, and 9 units), storage period (14 days), and mortality were evaluated in guinea pigs with a vascular disease phenotype. Seven-day mortality and the interaction between iron and Hb as cocontributors to adverse outcome were studied. Concentrations of iron and free Hb were greatest after transfusion with 9 units of stored RBCs compared with fresh RBCs or stored RBCs at 1- and 3-unit volumes. Nine units of stored RBCs led to mortality in vascular diseased animals, but not normal animals. One and 3 units of stored RBCs did not cause a mortality effect, suggesting the concomitant relevance of NTBI and Hb on outcome. Cotransfusion with apo-Tf or Hp restored survival to 100% following 9-unit RBC transfusions in vascular diseased animals. Our data suggest that increases in plasma NTBI and Hb contribute to vascular disease-associated mortality through iron-enhanced Hb oxidation and enhanced tissue injury.

Hepcidin Protects Against Extracellular Infections By Eliminating Non-Transferrin-Bound Iron

The production of the iron-regulatory hormone hepcidin is strongly induced by infection and inflammation, causing hypoferremia. This is proposed to be a host defense response to limit microbial replication. Patients with hereditary hemochromatosis or β-thalassemia, who are hepcidin-deficient and iron-overloaded, are susceptible to infections with gram-negative bacteria. The specific mechanism by which hepcidin and hypoferremia mediate resistance to infection, and the spectrum of microbes affected, are poorly understood.We tested the role of hepcidin in infections with several groups of bacteria: 1) siderophilic gram-negative Yersinia enterocolitica and Vibrio vulnificus, known to have increased virulence in patients with iron overload; 2) clinically common gram-negative Klebsiella pneumoniae and gram-positive Staphylococcus aureus; and 3) primarily intracellular Mycobacterium tuberculosis.In vivo role of hepcidin and iron was assessed by comparing survival and tissue bacterial burden between wild type (WT) and hepcidin knockout (KO) mice. Hepcidin KO mice were tested both in iron-loaded and iron-depleted condition to assess for any iron-independent roles of hepcidin in infection.Hepcidin played the most prominent host defense role in infections with siderophilic pathogens Y. enterocolitica and V. vulnificus. At inocula where WT mice were resistant and had 0% mortality, iron-loaded hepcidin KO mice were highly susceptible to these infections and displayed 100% mortality (Figure). Compared to WT mice, iron-loaded hepcidin KO were also more susceptible to infection with K. pneumoniae, and displayed more rapid and higher mortality (Figure). In all of these models, treatment of infected iron-loaded hepcidin KO mice with hepcidin analogs prevented or decreased mortality. Short-term administration of hepcidin analogs altered serum iron concentrations but not iron stores, indicating that only extracellular iron availability plays a role in infection with these extracellular, gram-negative pathogens. Additional experiments indicated that the protective effect of hepcidin and minihepcidin was not caused by any direct antimicrobial activity but by their effect on extracellular iron.In contrast, in mouse models of infection with gram-positive S. aureus or with M. tuberculosis, there was no difference in susceptibility between hepcidin KO and WT mice. We further tested the effect of parenteral iron administration in WT mice infected with S. aureus and M. tuberculosisbut iron loading of these mice had no effect on bacterial burden. These results indicated that neither hepcidin nor iron availability affect the virulence of these bacteria.We next examined the mechanism by which hepcidin deficiency promotes the growth of gram- negative pathogens. Hepcidin causes a decrease in iron-transferrin concentrations, however iron-transferrin is accessible only to a few highly host-adapted pathogens and most bacteria cannot utilize this form of iron. On the other hand, non-transferrin-bound iron (NTBI), a form of iron that appears in the circulation when the iron-binding capacity of transferrin is exceeded, may be readily utilizable by microbes. Indeed, mice that had increased susceptibility to infection in our models also had detectable NTBI in plasma. We developed an in vitro model to test the dependence of bacterial growth on iron-transferrin or NTBI concentrations. Human plasma was supplemented with graded amounts of ferric ammonium citrate and agar plates generated with a range of increasing transferrin saturation and eventually increasing NTBI concentrations. V. vulnificus, Y. enterocolitica and K. pneumoniaeonly grew when sufficient iron was added to saturate transferrin and generate NTBI. Similarly, bacterial CFU counts in vivo correlated with the presence of NTBI in mouse models but not with transferrin saturation when NTBI was absent.Using mouse models of different bacterial infections, we demonstrated that hepcidin is essential for resistance to infection with certain gram-negative pathogens, but plays no role in the susceptibility to intracellular M. tuberculosis or gram-positive S. aureus. We further provide evidence that the primary host defense function of hepcidin is the elimination of NTBI, and this can be potentiated by the use of synthetic hepcidin analogues to prevent progression of infection and associated mortality. [Display omitted] DisclosuresGanz:Intrinsic LifeSciences: Equity Ownership; Merganser biotech: Consultancy, Equity Ownership; Silarus Therapeutics: Equity Ownership. Nemeth:Merganser Biotech: Consultancy, Equity Ownership; Intrinsic LifeSciences: Equity Ownership; Silarus Therapeutics: Equity Ownership, Research Funding.

SLC39A14 Is Required for the Development of Hepatocellular Iron Overload in Murine Models of Hereditary Hemochromatosis

Nearly all forms of hereditary hemochromatosis are characterized by pathological iron accumulation in the liver, pancreas, and heart. These tissues preferentially load iron because they take up non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. Yet, how tissues take up NTBI is largely unknown. We report that ablation of Slc39a14, the gene coding for solute carrier SLC39A14 (also called ZIP14), in mice markedly reduced the uptake of plasma NTBI by the liver and pancreas. To test the role of SLC39A14 in tissue iron loading, we crossed Slc39a14(-/-) mice with Hfe(-/-) and Hfe2(-/-) mice, animal models of type 1 and type 2 (juvenile) hemochromatosis, respectively. Slc39a14 deficiency in hemochromatotic mice greatly diminished iron loading of the liver and prevented iron deposition in hepatocytes and pancreatic acinar cells. The data suggest that inhibition of SLC39A14 may mitigate hepatic and pancreatic iron loading and associated pathologies in iron overload disorders.

Evaluation of a novel oral iron chelator 1-(N-acetyl-6-aminohexyl)-3-hydroxypyridin-4-one (CM1) for treatment of iron overload in mice

Desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) are promising effective iron chelators for the treatment of iron overload in b-thalassemia patients; nonetheless, their side effects have also been reported. 3-Hydroxypyridinone derivatives are being developed as a safer new chelator and in combined chelation therapy. We evaluated the iron-chelating activity of 1-(N-acetyl-6-aminohexyl)-3-hydroxypyridin-4-one (CM1) in iron-loaded C57BL6 mice. The feeding of a ferrocene-supplemented diet (Fe diet) to mice resulted in iron overload, detectable plasma nontransferrin-bound iron (NTBI) and labile plasma iron (LPI), and increases of red cell membrane iron, plasma malondialdehyde (MDA) and excessive tissue iron deposits. Like DFP, the CM1 lowered the levels of the membrane non-heme iron, the NTBI and LPI (p

Evaluating Toxic Free Iron and Options for Keeping It At Bay in Iron Overloaded Patients.

AbstractAbstract 2110An important goal in iron chelation is preventing tissue iron overload by maintaining low levels of plasma non-transferrin bound iron (NTBI). This is important in periods between chelator administration, when the plasma iron might surpass not only transferrin- but also chelator-iron binding capacities, thereby raising plasma NTBI and exposing cells to infiltrating forms of iron. We aimed to assess the ability of different chelation protocols to maintain patients free of the redox active and chelatable component of NTBI referred to as labile plasma iron (LPI) and the correlation between LPI and eLPI (equivalent to NTBI including mobilizable and chelated iron) with respect to compliance. Patients & Methods:49 TMps (23 M, 26 F, mean age 35.5±11 years were included. Patients received deferoxamine (DFO)=8–40 (mg/kg) and/or deferiprone (DFP)=74–100 and/or deferasirox (DFX)=7.6–25 for >3 years. Blood samples were drawn at trough levels (12 hr for DFP & 24 hr for DFO and/or DFX) and at peak plasma levels of chelator (2 hrs post DFP and/or DFX p/o). LPI and eLPI were determined with the FeROS™ assay (Aferrix, Israel). Stata 11.0 was used for statistical analysis. The numbers of patients receiving a particular regime were adequate for comparison in the two combination regimes that included deferiprone. Results:28/49 (57%) of TMps had normal basal LPI (<0.45μM/l) and 90% (19/21) normalized within 2 h of chelation. Baseline eLPI and LPI were negatively related to compliance (Spearman’s rho: −0.28, p=0.05 and Spearman’s rho: −0.38, p=0.007, respectively). A total of 34 TM patients (23 on DFP+DFO, 11 on DFX+DFO) were included in the comparison analysis based on measurements presented in Table 1.Table 1Summary statisticsOverall (34)DFP & DFO (23)DFP & DFX (11)Age†35.1 ± 11.635.4 ± 10.034.4 ± 15.1Gender, females, n (%)16/34 (47.1%)11/23 (47.8%)5/11 (45.5%)LPI_BF†0.71 ± 0.770.85 ± 0.830.40 ± 0.56LPI_AF†0.09 ± 0.230.04 ± 0.10.20 ± 0.36eLPI_BF†1.52 ± 1.311.66 ± 1.421.21 ± 1.03eLPI_AF†1.53 ± 1.151.64 ± 1.171.30 ± 1.13LPI change†−0.61 ± 0.74−0.80 ± 0.79−0.20 ± 0.42eLPI change†0.01 ± 0.98−0.02 ± 1.170.08 ± 0.36probability of reducing LPI, n (%)19/23 (82.6%)15/15 (100%)4/8 (50.0%)probability of increasing eLPI, n (%)15/34 (44.1%)11/23 (47.8%)4/11 (36.4%)probability of normalizing LPI, n (%)14/16 (87.5%)13/13 (100%)1/3 (33.3%)Compliance†78.1 ± 18.272.9 ± 17.988.7 ± 14.1None of the two LPI and two eLPI measurements differed between the two treatment groups (all p-values >0.05). Reduction of LPI was significantly higher in DFP+DFO compared to DFP+DFX (p=0.004). Linear regression estimated that DFP+DFO achieved 0.32 μM higher reduction compared to DFP+DFX (results adjusted for baseline LPI). Detailed regression results: DFP+DFO vs DFP+DFX: b-coefficient=0.32 (95% ci: 0.06–0.59, p=0/019), LPI-bf: b-coefficient=0.85 (95% ci: 0.67–1.03, p<0.001). The R2 of 0.896 shows 89.6% of the variation of LPI decrease can be explained by the model, indicating a good fit.†mean ± SD. [Display omitted] The same trends were confirmed by analysis of the probability of increasing eLPI, reducing LPI and normalizing LPI. No differences were observed in the proportion of patients who increased their baseline eLPI between the two regimes (p>0.05). In contrast, patients on DFP+DFO were 11.0 times more likely to reduce their baseline LPI levels compared to DFP+DFX (exact logistic regression, odds ratio (OR)=11.0, 95% ci: 1.05 - +∞, p=0.045). Similarly, patients treated with DFP+DFO were 14.7 times more likely to normalize their baseline LPI levels compared to DFP+DFX (exact logistic regression, OR =14.7, 95% ci: 1.0 - +∞, p=0.05). Discussion:The DFP+DFO combination seems to be superior to DFP+DFX in reducing LPI despite the lower compliance with the former. As the DFP is in both regimes our results indicate that the DFP is more efficient in combination with DFO than with DFX. The concept of having chelator present in the circulation at all times is logical as it would protect from the infiltrating forms of iron and is attainable with the present repertoire of chelators. This preliminary study suggests that the measurement of LPI provides a convenient and immediate index of chelation efficacy, adequacy of doses of chelator prescribed, and patient compliance. Repeating such measurements every 3 months should be useful in guiding patient management. Disclosures:Berdoukas:Apopharma: Consultancy. Cabantchik:Aferrix: Consultancy.