Iron overload in hematological disorders.

While most common symptom of impairment of iron homeostasis is iron deficiency anemia, some hematological disorders are associated with iron overload (IO). These disorders are related mainly to chronic severe hemolytic anemia, where red blood cells (RBC) or their precursors are destroyed prematurely (hemolyzed), leading to anemia that cannot be compensated by increased production of new RBC. In such cases, IO is mainly due to repeated RBC transfusions and/or increased uptake of iron in the gastrointestinal tract. Normally, iron is present in the plasma and in the cells bound to compounds that render it redox inactive. Iron overload leaves a fraction of the iron free (labile iron pool) and redox active, leading to the generation of excess free radicals such as the reactive oxygen species. This condition upsets the cellular redox balance between oxidants and antioxidants, leading to oxidative stress. The free radicals bind to various cellular components, thereby becoming toxic to vital organs. Oxidative stress may also affect blood cells, such as RBC, platelets and neutrophils, exacerbating the anemia, and causing recurrent infections and thrombotic events, respectively. The toxic effect of IO can be decreased by treating the patients with iron chelators that enter cells, bind free iron and remove it from the body through the urine and feces. Iron toxicity may be also ameliorated by treatment with anti-oxidants that scavenge free radicals and/or correct their damage. The use of iron chelators is widely accepted when started in young patients with severe chronic anemia, but is still debatable as a therapeutic modality for older patients suffering from IO due to myelodysplastic syndromes. It should be noted that in addition to preventing iron toxicity, some compounds with iron chelator activity may also benefit other aspects of hematological disorders. These aspects include stimulation of platelet production, inhibition of leukemic cell proliferation and induction of their differentiation. Compounds with such multiple activities may prove beneficial for at least some patients with leukemia and myelodysplastic syndromes.

New Insights on ß-Thalassemia in the Palestinian Population of Gaza: High Frequency and Milder Phenotype Among Homozygous IVS-I-1 (HBB: c.92+1G>A) Patients with High Levels of Hb F.

ß-Thalassemia (ß-thal) is a very common disease in the Palestinian population of the Gaza Strip. We studied their mutation frequency and clinical features. Thirteen different mutations were identified. The most common mutation was IVS-I-1 (G>A) (HBB: c.92+1G>A), which was prevalent in 31.5% of the thalassemia alleles studied. The IVS-I-110 (G>A) (HBB: c.93-21G>A) mutation was found in 25.0% of the alleles. Homozygotes for the IVS-I-1 mutation had higher mean hemoglobin (Hb) levels, required less blood transfusions, and lower transferrin saturation than the homozygotes for the IVS-I-110 mutation. This milder phenotype was, most likely, the result of the persistent production of Hb F; it was 9-fold higher in absolute terms (g/dL) and 7.7-fold higher in relative terms (percentage of total Hb). About half of our IVS-I-1 patients carried the XmnI polymorphism, which is known to be associated with elevated Hb F levels.

Toxicity of iron overload and iron overload reduction in the setting of hematopoietic stem cell transplantation for hematologic malignancies.

Iron is an essential element for key cellular metabolic processes. However, transfusional iron overload (IOL) may result in significant cellular toxicity. IOL occurs in transfusion dependent hematologic malignancies (HM), may lead to pathological clinical outcomes, and IOL reduction may improve outcomes. In hematopoietic stem cell transplantation (SCT) for HM, IOL may have clinical importance; endpoints examined regarding an impact of IOL and IOL reduction include transplant-related mortality, organ function, infection, relapse risk, and survival. Here we review the clinical consequences of IOL and effects of IOL reduction before, during and following SCT for HM. IOL pathophysiology is discussed as well as available tests for IOL quantification including transfusion history, serum ferritin level, transferrin saturation, hepcidin, labile plasma iron and other parameters of iron-catalyzed oxygen free radicals, and organ IOL by imaging. Data-based recommendations for IOL measurement, monitoring and reduction before, during and following SCT for HM are made.

Pathophysiology and treatment of patients with beta-thalassemia - an update.

Thalassemia (thal) is an autosomal recessive, hereditary, chronic hemolytic anemia due to a partial or complete deficiency in the synthesis of α-globin chains (α-thal) or ß-globin chains (ß-thal) that compose the major adult hemoglobin (α 2ß 2). It is caused by one or more mutations in the corresponding genes. The unpaired globin chains are unstable; they precipitate intracellularly, resulting in hemolysis, premature destruction of red blood cell [RBC] precursors in the bone marrow, and a short life-span of mature RBCs in the circulation. The state of anemia is treated by frequent RBC transfusions. This therapy results in the accumulation of iron (iron overload), a condition that is exacerbated by the breakdown products of hemoglobin (heme and iron) and the increased iron uptake for the chronic accelerated, but ineffective, RBC production. Iron catalyzes the generation of reactive oxygen species, which in excess are toxic, causing damage to vital organs such as the heart and liver and the endocrine system. Herein, we review recent findings regarding the pathophysiology underlying the major symptoms of ß-thal and potential therapeutic modalities for the amelioration of its complications, as well as new modalities that may provide a cure for the disease.

The frequency of silent cerebral ischemia in patients with transfusion-dependent ß-thalassemia major.

The aim of this study is to determine the frequency of silent cerebral ischemia (SCI) in a group of patients with ß-thalassemia major and correlate them with demographic parameters, blood and spleen status, volume and frequency of transfusions. In this cross-sectional study, 40 ß-thalassemic patients over 10 years old who had no neurologic deficit were studied. Brain MRI was performed to detect SCI. Silent cerebral ischemia was classified according to number and size. Silent cerebral ischemia was found in 15 patients (37.5 %). Mean number of SCI was 6.73 ± 10.33 (1-40), and mean size of the brain lesions was 3.07 ± 2.81 mm (1-11 mm). The patients with SCI were significantly older (31.1 ± 6.5 vs. 25 ± 6.8 years, P = 0.009), and most of them were splenectomized (80% vs. 36 %, P = 0.01). Interestingly, 10 out of 15 patients with SCI had platelet count less than 500,000/mm(3). Eight of these patients (80 %) were splenectomized. Other variables had no statistically significant association with the presence of SCI. Older age and splenectomized multitransfused ß-thalassemic patients even with normal platelet count have a higher incidence of SCI. The effect of splenectomy is more significant in statistical analysis. In splenectomized patients with a high platelet count and even with normal platelet count, aspirin therapy is indicated. Based on the results, it seems that regular blood transfusions are not going to have a significant effect on the number and size of SCI.

The Effect of Fermented Papaya Preparation on Radioactive Exposure.

Exposure to ionizing radiation causes cellular damage, which can lead to premature cell death or accumulation of somatic mutations, resulting in malignancy. The damage is mediated in part by free radicals, particularly reactive oxygen species. Fermented papaya preparation (FPP), a product of yeast fermentation of Carica papaya Linn, has been shown to act as an antioxidant. In this study, we investigated the potential of FPP to prevent radiation-induced damage. FPP (0-100 µg/ml) was added to cultured human foreskin fibroblasts and myeloid leukemia (HL-60) cells either before or after irradiation (0-18 Gy). After 1-3 days, the cells were assayed for: intracellular labile iron, measured by staining with calcein; reactive oxygen species generation, measured with dichlorofluorescein diacetate; apoptosis, determined by phosphatidylserine exposure; membrane damage, determined by propidium iodide uptake; and cell survival, determined by a cell proliferation assay. DNA damage was estimated by measuring 8-oxoguanine, a parameter of DNA oxidation, using a fluorescent-specific probe and by the comet assay. These parameters were also assayed in bone marrow cells of mice treated with FPP (by adding it to the drinking water) either before or after irradiation. Somatic mutation accumulation was determined in their peripheral red blood cells, and their survival was monitored. FPP significantly reduced the measured radiation-induced cytotoxic parameters. These findings suggest that FPP might serve as a radioprotector, and its effect on DNA damage and mutagenicity might reduce the long-term effects of radiation, such as primary and secondary malignancy.

A mouse model to study thrombotic complications of thalassemia.

Patients with ß-thalassemia major and mainly intermedia have an increased risk for developing venous and arterial thrombosis which may be related to circulating pathological red blood cells (RBC) and continuous platelet activation. In the present study we used a modified thalassemic mice model in conjunction with a "real-time" carotid thrombus formation procedure to investigate thrombotic complications of thalassemia. Heterozygous Th3/+ mice, which lack one copy of their ß-major and ß-minor globin genes, exhibit anomalies in RBC size and shape, chronic anemia and splenomegaly which recapitulate the phenotype of human ß-thalassemia intermedia. Flow cytometry measurements showed higher reactive oxygen species generation, indicating oxidative stress, in platelets and RBC of the thalassemic mice compared with wild type mice concomitant with an increase in reduced glutathione content which may represent a compensatory response to oxidative stress, and exposed phosphatidylserine which indicates platelet activation. To elucidate the effect of thalassemia on the development of arterial thrombosis, we studied photochemical-induced real-time thrombus formation in the carotid artery of these mice. The results indicated a significantly shorter "time to occlusion" in the thalassemic mice compared to wild type mice, which was prolonged following in vivo aspirin treatment. We suggest that this mouse model may contribute to our understanding of platelet activation and the hypercoagulable state in thalassemia and lay foundations to screening of anti-platelet drugs as well as anti-oxidants as possible therapeutics for prevention of thrombosis in thalassemia patients.

Iron dosing in kidney disease: inconsistency of evidence and clinical practice.

The management of anemia in patients with chronic kidney disease (CKD) is difficult. The availability of erythropoiesis-stimulating agents (ESAs) has increased treatment options for previously transfusion-requiring patients, but the recent evidence of ESA side effects has prompted the search for complementary or alternative approaches. Next to ESA, parenteral iron supplementation is the second main form of anemia treatment. However, as of now, no systematic approach has been proposed to balance the concurrent administration of both agents according to individual patient's needs. Furthermore, the potential risks of excessive iron dosing remain a topic of controversy. How, when and whether to monitor CKD patients for potential iron overload remain to be elucidated. This review addresses the question of risk and benefit of iron administration in CKD, highlights the evidence supporting current practice, provides an overview of standard and potential new markers of iron status and outlines a new pharmacometric approach to physiologically compatible individualized dosing of ESA and iron in CKD patients.

Impact of iron overload and potential benefit from iron chelation in low-risk myelodysplastic syndrome.

Myelodysplastic syndromes (MDSs) are a group of heterogeneous clonal bone marrow disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, and potential for malignant transformation. Lower/intermediate-risk MDSs are associated with longer survival and high red blood cell (RBC) transfusion requirements resulting in secondary iron overload. Recent data suggest that markers of iron overload portend a relatively poor prognosis, and retrospective analysis demonstrates that iron chelation therapy is associated with prolonged survival in transfusion-dependent MDS patients. New data provide concrete evidence of iron's adverse effects on erythroid precursors in vitro and in vivo. Renewed interest in the iron field was heralded by the discovery of hepcidin, the main serum peptide hormone negative regulator of body iron. Evidence from ß-thalassemia suggests that regulation of hepcidin by erythropoiesis dominates regulation by iron. Because iron overload develops in some MDS patients who do not require RBC transfusions, the suppressive effect of ineffective erythropoiesis on hepcidin may also play a role in iron overload. We anticipate that additional novel tools for measuring iron overload and a molecular-mechanism-driven description of MDS subtypes will provide a deeper understanding of how iron metabolism and erythropoiesis intersect in MDSs and improve clinical management of this patient population.

Does erythropoietin have a role in the treatment of ß-hemoglobinopathies?

This review presents the indications and contraindications (pros and cons) for the potential use of erythropoietin (Epo) as a treatment in ß-thalassemia and sickle cell anemia (SCA). Its high cost and route of administration (by injection) are obvious obstacles, especially in underdeveloped countries, where thalassemia is prevalent. We believe that from the data summarized in this review, the time has come to define, by studying in vitro and in vivo models, as well as by controlled clinical trials, the rationale for treating patients with various forms of thalassemia and SCA with Epo alone or in combination with other medications.

Non-transfusion-dependent thalassemias.

Non-transfusion-dependent thalassemias include a variety of phenotypes that, unlike patients with beta (ß)-thalassemia major, do not require regular transfusion therapy for survival. The most commonly investigated forms are ß-thalassemia intermedia, hemoglobin E/ß-thalassemia, and α-thalassemia intermedia (hemoglobin H disease). However, transfusion-independence in such patients is not without side effects. Ineffective erythropoiesis and peripheral hemolysis, the hallmarks of disease process, lead to a variety of subsequent pathophysiologies including iron overload and hypercoagulability that ultimately lead to a number of serious clinical morbidities. Thus, prompt and accurate diagnosis of non-transfusion-dependent thalassemia is essential to ensure early intervention. Although several management options are currently available, the need to develop more novel therapeutics is justified by recent advances in our understanding of the mechanisms of disease. Such efforts require wide international collaboration, especially since non-transfusion-dependent thalassemias are no longer bound to low- and middle-income countries but have spread to large multiethnic cities in Europe and the Americas due to continued migration.

Cerebral infarction in ß-thalassemia intermedia: breaking the silence.

Despite remarkable advances in understanding cerebrovascular disease attributed to sickle cell anemia, data from other hemoglobinopathies have only recently started to emerge. Several brain magnetic resonance imaging studies confirm a high prevalence of silent ischemic lesions in patients with ß-thalassemia intermedia, especially in splenectomized adults who are transfusion-independent and those with elevated platelet counts. Large-vessel disease is also common in this patient population but without apparent association with silent white matter infarcts, leaving smaller arteriolar involvement as a potential explanation. The hypothesized pathophysiology is multifactorial with hypercoagulability and toxicity from free iron species playing major roles. The long-term sequelae of such covert findings is unknown, although experience from patients with sickle cell anemia confirms their association with subsequent overt stroke and neurocognitive deficits. The roles of transfusion and antiplatelet therapy to prevent the occurrence and progression of silent ischemic lesions in patients with ß-thalassemia intermedia should be the focus of future trials.

Frequency and distribution of asymptomatic brain lesions in patients with ß-thalassemia intermedia.

We aimed to determine the frequency of asymptomatic brain lesions in a group of patients with ß-thalassemia intermedia (ß-TI) and to evaluate correlation of asymptomatic brain lesions with splenectomy, thrombocytosis, blood transfusions, and clinical parameters. Ninety five neurologically intact patients with ß-TI were randomly enrolled in this cross-sectional study. Diffusion-weighted imaging brain MRI was performed in every patient to detect cerebral white matter lesions (WML). We found an overall frequency of 15 (15.8 %) for WMLs, 14 (23.7 %) in splenectomized, and 1 (2.8 %) in nonsplenectomized patients. The presence of WML was significantly associated with splenectomy (P = 0.008) and thrombocytosis (P = 0.009). However, after adjustment for splenectomy, thrombocytosis was not significantly associated with the presence of WML (P > 0.05). The number of patients with regular blood transfusions and normal MRI was not significantly higher compared to those with abnormal findings (52.5 % vs. 26.7 %; P = 0.092). In untransfused patients, hydroxyurea (HU) administration was associated with a lower incidence of WML (P < 0.001). Although in univariate analysis either splenectomy or thrombocytosis showed significant correlation with the presence of single or multiple WMLs, thrombocytosis by itself did not significantly contribute in developing asymptomatic brain lesions. The lack of significant correlation between lesions and regular blood transfusions could be related to the treatment with HU in untransfused patients, which increased fetal hemoglobin levels and improved the morphology and the pathological indices of the red blood cells. Larger prospective studies are suggested for the accurate evaluation of the correlation of these factors with developing asymptomatic brain lesions.

ß-thalassemia intermedia: a clinical perspective.

Our understanding of the molecular and pathophysiological mechanisms underlying the disease process in patients with ß-thalassemia intermedia has substantially increased over the past decade. Earlier studies observed that patients with ß-thalassemia intermedia experience a clinical-complications profile that is different from that in patients with ß-thalassemia major. In this article, a variety of clinical morbidities are explored, and their associations with the underlying disease pathophysiology and risk factors are examined. These involve several organs and organ systems including the vasculature, heart, liver, endocrine glands, bone, and the extramedullary hematopoietic system. The effects of some therapeutic interventions on the development of clinical complications are also discussed.

Evidence for tissue iron overload in long-term hemodialysis patients and the impact of withdrawing parenteral iron.

BACKGROUND/AIMS: Erythropoiesis in long-term hemodialyzed (LTH) patients is supported by erythropoietin (rHuEpo) and intravenous (IV) iron. This treatment may end up in iron overload (IO) in major organs. We studied such patients for the parameters of IO in the serum and in major organs. METHODS: Patients were treated with rHuEpo (6-8 x 10(3) units × 1-3/wk) and IV 100 mg ferric saccharate. RESULTS: Of 115 patients, 21 had serum ferritin (SF) > 1000 ng/mL. This group was further analyzed. Their SF and transferrin saturation (TSAT) were 2688 ± 1489 ng/mL and 54.2 ± 32.7%, respectively (vs. 125-360 ng/mL and 20-50% in normal controls). Serum hepcidin was 60.1 ± 29.5 nm (vs. 10.61 ± 6.44 nm in controls) (P < 0.001). Nineteen patients had increased malonyldialdehyde, a product of lipid peroxidation, indicating oxidative stress. T2* MRI disclosed in 19 of 21 patients moderate to severe IO in the liver and spleen, in three of eight patients in the pancreas, but in no patient in the heart. After stopping IV iron for a mean of 12 months, while continuing rHuEpo, the mean SF decreased in 11 patients to 1682 ng/mL and the mean TSAT decreased to 28%, whereas hemoglobin did not change indicating that tissue iron was utilized. CONCLUSION: High SF correlates with IO in the liver and spleen, but not in the heart.

The role of endocytic pathways in cellular uptake of plasma non-transferrin iron.

BACKGROUND: In transfusional siderosis, the iron binding capacity of plasma transferrin is often surpassed, with concomitant generation of non-transferrin-bound iron. Although implicated in tissue siderosis, non-transferrin-bound iron modes of cell ingress remain undefined, largely because of its variable composition and association with macromolecules. Using fluorescent tracing of labile iron in endosomal vesicles and cytosol, we examined the hypothesis that non-transferrin-bound iron fractions detected in iron overloaded patients enter cells via bulk endocytosis. DESIGN AND METHODS: Fluorescence microscopy and flow cytometry served as analytical tools for tracing non-transferrin-bound iron entry into endosomes with the redox-reactive macromolecular probe Oxyburst-Green and into the cytosol with cell-laden calcein green and calcein blue. Non-transferrin-bound iron-containing media were from sera of polytransfused thalassemia major patients and model iron substances detected in thalassemia major sera; cell models were cultured macrophages, and cardiac myoblasts and myocytes. RESULTS: Exposure of cells to ferric citrate together with albumin, or to non-transferrin-bound iron-containing sera from thalassemia major patients caused an increase in labile iron content of endosomes and cytosol in macrophages and cardiac cells. This increase was more striking in macrophages, but in both cell types was largely reduced by co-exposure to non-transferrin-bound iron-containing media with non-penetrating iron chelators or apo-transferrin, or by treatment with inhibitors of endocytosis. Endosomal iron accumulation traced with calcein-green was proportional to input non-transferrin-bound iron levels (r(2) = 0.61) and also preventable by pre-chelation. CONCLUSIONS: Our studies indicate that macromolecule-associated non-transferrin-bound iron can initially gain access into various cells via endocytic pathways, followed by iron translocation to the cytosol. Endocytic uptake of plasma non-transferrin-bound iron is a possible mechanism that can contribute to iron loading of cell types engaged in bulk/adsorptive endocytosis, highlighting the importance of its prevention by iron chelation.

Non-transferrin bound iron in Thalassemia: differential detection of redox active forms in children and older patients.

Non-transferrin bound iron (NTBI) is commonly detected in patients with systemic iron overload whose serum iron-binding capacity has been surpassed. It has been perceived as an indicator of iron overload, impending organ damage and a chelation target in poly-transfused thalassemia patients. However, NTBI is a heterogeneous entity comprising various iron complexes, including a significant redox-active and readily chelatable fraction, which we have designated as "labile plasma iron" (LPI). We found that LPI levels can be affected by plasma components such as citrate, uric acid, and albumin. However, the inclusion of a mild metal mobilizing agent in the LPI assay (designated here as "eLPI"), at concentrations that do not affect transferrin-bound iron, largely overcomes such effects and provides a measure of the full NTBI content. We analyzed three distinct groups of poly-transfused, iron overloaded thalassemia patients: non-chelated children (3-13 yrs, Gaza, Palestine), chelated adolescents-young adults (13-28 yrs, Israel), and chelated adults (27-61 yrs, Israel) for LPI and eLPI. The eLPI levels in all three groups were roughly commensurate (r(2) = 0.61-0.75) with deferrioxamine-detectable NTBI, i.e., DCI. In older chelated patients, eLPI levels approximated those of LPI, but in poly-transfused unchelated children eLPI was notably higher than LPI, a difference attributed to plasma properties affected by labile iron due to lack of chelation, possibly reflecting age-dependent attrition of plasma components. We propose that the two formats of NTBI measurement presented here are complementary and used together could provide more comprehensive information on the forms of NTBI in patients and their response to chelation.