The Effect of The Oral Iron Chelator Deferiprone on Iron Overload and Oxidative Stress in Patients with Myelodysplastic Syndromes A Study by the Israeli MDS Working Group.

BACKGROUND: Most patients with lower risk myelodysplastic neoplasms (MDS) become RBC transfusion-dependent, resulting in iron overload, which is associated with an increased oxidative stress state. Iron-chelation therapy is applied to attenuate the toxic effects of this state. Deferiprone (DFP) is an oral iron chelator, which is not commonly used in this patient population, due to safety concerns, mainly agranulocytosis. The purpose of this study was to assess the effect of DFP, on oxidative stress parameters in iron overloaded RBC transfusion-dependent patients with lower risk MDS. METHODS: Adult lower-risk MDS patients with a cumulative transfusion burden of >20 red blood cells units and evidence of iron overload (serum ferritin >1,000 ng/mL) were included in this study. DFP was administered (100 mg/kg/day) for 4 months. Blood samples for oxidative stress parameters and iron overload parameters were done at baseline and monthly: reactive oxygen species (ROS), phosphatidylserine, reduced glutathione, membrane lipid peroxidation, serum ferritin and cellular labile iron pool. The primary efficacy variable was ROS. Tolerability and side-effects were recorded as well. A paired t-test was applied for statistical analyses. RESULTS: Eighteen patients were treated with DFP. ROS significantly decreased in all cell lineages: median decrease of 58.6% in RBC, 33.3% in PMN, and 39.8% in platelets (p<0.01 for all). Other oxidative stress markers improved: phosphatidylserine decreased by 57.95%, lipid peroxidase decreased by 141.3%, and reduced gluthathione increased by 72.8% (p<0.01 for all). The iron-overload marker, cellular labile iron pool, decreased by 35% in RBCs, 44.3% in PMN, and 46.3% in platelets (p<0.01 for all). No significant changes were observed in SF levels. There were no events of agranulocytosis. All AEs were grade 1-2. CONCLUSIONS: Herein we showed preliminary evidence that DFP decreases iron-induced oxidative stress in MDS patients with a good tolerability profile (albeit a short follow-up period). No cases of severe neutropenia or agranulocytosis were reported. The future challenge is to prove that reduction in iron toxicity will eventually be translated into a clinically meaningful improvement.

Vasculo-toxic and pro-inflammatory action of unbound haemoglobin, haem and iron in transfusion-dependent patients with haemolytic anaemias.

Increasing evidence suggests that free haem and iron exert vasculo-toxic and pro-inflammatory effects by activating endothelial and immune cells. In the present retrospective study, we compared serum samples from transfusion-dependent patients with ß-thalassaemia major and intermedia, hereditary spherocytosis and sickle cell disease (SCD). Haemolysis, transfusions and ineffective erythropoiesis contribute to haem and iron overload in haemolytic patients. In all cohorts we observed increased systemic haem and iron levels associated with scavenger depletion and toxic 'free' species formation. Endothelial dysfunction, oxidative stress and inflammation markers were significantly increased compared to healthy donors. In multivariable logistic regression analysis, oxidative stress markers remained significantly associated with both haem- and iron-related parameters, while soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble endothelial selectin (sE-selectin) and tumour necrosis factor α (TNFα) showed the strongest association with haem-related parameters and soluble intercellular adhesion molecule 1 (sICAM-1), sVCAM-1, interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) with iron-related parameters. While hereditary spherocytosis was associated with the highest IL-6 and TNFα levels, ß-thalassaemia major showed limited inflammation compared to SCD. The sVCAM1 increase was significantly lower in patients with SCD receiving exchange compared to simple transfusions. The present results support the involvement of free haem/iron species in the pathogenesis of vascular dysfunction and sterile inflammation in haemolytic diseases, irrespective of the underlying haemolytic mechanism, and highlight the potential therapeutic benefit of iron/haem scavenging therapies in these conditions.

Fetal Hemoglobin in the Maternal Circulation - Contribution of Fetal Red Blood Cells.

The major hemoglobin (Hb) during fetal life is fetal Hb (Hb F). It is mostly replaced by adult Hbs before birth and during the first year of life. In adults, where Hb F comprises <2.0% of the total Hb, it is not homogenously distributed among the red blood cells (RBCs) but is concentrated in a few RBCs, termed F-cells. Interestingly, for reasons that are unclear, Hb F increases in the maternal circulation during pregnancy. This increased Hb F could have two potential origins that are not mutually exclusive: A) maternal origin, due to inducing environment of Hb F in the maternal erythroid precursors; B) fetal origin, due to fetal cells crossing the placenta and entering the maternal circulation. The question we present herein is whether the observed increased Hb F in the maternal circulation during pregnancy is, at least partially, derived from the fetal origin. Peripheral blood was obtained from normal neonates (1-3 days old), adult men and pregnant and non pregnant women. The RBCs were stained for Hb F and carbonic anhydrase (CA) using a fetal cell count kit and analyzed by flow cytometry. Fetal and adult F-cells were distinguished by their expression of Hb F and CA. Fetal F-cells were Hb F++/CA-, while adult F-cells were Hb F+/CA+. Comparing pregnant and non pregnant women samples (n = 10), we found six samples of pregnant women with 0.2-1.7% fetal cells, but none in the non pregnant group. These results support the possibility that at least part of the increase in Hb F during pregnancy is due to fetal cells entering the maternal circulation.

The JAK2V617F mutation in normal individuals takes place in differentiating cells.

The JAK2V617F mutation that results in a hyper-activation of the JAK2 kinase in the erythropoietin pathway is a molecular marker for myeloproliferative neoplasms. Using allele-specific Real-Time PCR, we detected the mutation in the blood of 17.3% (17/98) of normal donors; the mutant allele burden was, however, very low (<0.01% compared to >1% in polycythemia vera). It was much higher in differentiated blood cells in the peripheral blood than in undifferentiated CD34+ cells. Erythropoietin-stimulated differentiation of normal CD34+ cells in liquid culture increased the mutation frequency by 3.34-fold. When progenitors from 9 normal donors were grown in erythropoietin-stimulated semi-solid cultures, the mutation was found in 8.69% of the colonies, but only in <3% of the JAK2 alleles in each positive colony, suggesting that the mutation occurred only in a few cells per colony. In mouse erythroleukemia cells carrying human JAK2 DNA, wild-type or JAK2V617F, the frequencies of mutations from JAK2 wild-type to JAK2V617F and vice versa increased following erythroid differentiation. These results suggest that the mutation occurs and accumulates during differentiation. We hypothesize that genetic stability, which relies on DNA repair, is efficient in normal hematopoietic stem cells but is downgraded in differentiating cells, rendering them susceptible to mutations, including JAK2V617F.

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.

Heterogeneity of F cells in ß-thalassemia.

BACKGROUND: Fetal hemoglobin (HbF), which is largely replaced after birth by the adult Hb, is concentrated in a few "F cells." Their number significantly increases in certain physiologic and clinical situations, including in ß-thalassemia (ß-thal). Their quantification is used to detect fetal-maternal hemorrhage (FMH), where fetal cells enter the maternal circulation. We were confronted with a pregnant woman with ß-thal who was suspected to have FMH. To establish the usefulness of a flow cytometric procedure to differentiate between fetal cells and the maternal F cells, we screened adult ß-thal patients. STUDY DESIGN AND METHODS: Blood samples were simultaneously stained with fluorescent antibodies to HbF and to carbonic anhydrase (CA) isotype II, which is specific to adult red blood cells (RBCs). RESULTS: A heterogeneous distribution of RBCs with respect to HbF and CA expression was observed: adult non-F cells (CA+HbF-) and F cells (CA+HbF+/HbF++) as well as F cells with characteristics of fetal cells (CA-HbF++). CONCLUSIONS: The presence of CA-HbF++ RBCs in nonpregnant women, and even men, with thal indicates that the CA/HbF method is inappropriate for detection of FMH. The coexistence of F cells carrying fetal or adult markers suggests that they originate from two types of stem cell, adult and fetal, lineages. Normally, the fetal lineage is insignificant, but in ß-thal, as HbF-containing RBCs have a selective advantage, the "fetal" lineage gains significance.

Iron chelation improves ineffective erythropoiesis and iron overload in myelodysplastic syndrome mice.

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Red cell transfusion therapy for anemia in MDS results in iron overload, correlating with reduced overall survival. Whether the treatment of iron overload benefits MDS patients remains controversial. We evaluate underlying iron-related pathophysiology and the effect of iron chelation using deferiprone on erythropoiesis in NUP98-HOXD13 transgenic mice, a highly penetrant well-established MDS mouse model. Our results characterize an iron overload phenotype with aberrant erythropoiesis in these mice which was reversed by deferiprone-treatment. Serum erythropoietin levels decreased while erythroblast erythropoietin receptor expression increased in deferiprone-treated MDS mice. We demonstrate, for the first time, normalized expression of the iron chaperones Pcbp1 and Ncoa4 and increased ferritin stores in late-stage erythroblasts from deferiprone-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts. Importantly, erythroblast ferritin is increased in response to deferiprone, correlating with decreased erythroblast ROS. Finally, we confirmed increased expression of genes involved in iron uptake, sensing, and trafficking in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS.

Distribution and shedding of the membrane phosphatidylserine during maturation and aging of erythroid cells.

Maturation and aging of erythroid cells are accompanied by extensive remodeling of the membrane and a marked decrease in cell size, processes that are mediated by externalization and shedding of phosphatidylserine (PS). In the present study, we investigated the redistribution of PS in the plasma membrane of erythroid precursors during their maturation and of mature RBCs during senescence, and the involvement of changes in calcium (Ca)-flux in these processes. Maturation was studied by analyzing normal human bone marrow cells as well as cultured human normal erythroid precursors induced by erythropoietin and murine erythroleukemia cells induced by hexamethylene-bisacetamide. Senescence was studied in normal human peripheral RBCs following density fractionation. PS and Ca were determined by flow cytometry using annexin-V and Flu-3, respectively. The outer, inner and shed PS were quantified by a novel two-step binding inhibitory assay. The results indicate a bi-phasic modulation of intracellular Ca and PS externalization/shedding; both of which decreased during maturation and increased during aging. The role of intracellular Ca in PS externalization/shedding was demonstrated by modulating intracellular Ca: Ca was decreased by incubating the cells with an ion chelator (EDTA) or with decreasing concentrations of Ca, whereas treatment with the ionophore A23187 elevated intracellular Ca. The results showed that low Ca resulted in decreased outer and shed PS, whereas high Ca had the opposite effect. The results suggest that PS externalization and shedding are mediated by increased cellular Ca-flux, and that they play an important role in erythroid maturation and RBC senescence.

Peroxiredoxin II is essential for preventing hemolytic anemia from oxidative stress through maintaining hemoglobin stability.

The pathophysiology of oxidative hemolytic anemia is closely associated with hemoglobin (Hb) stability; however, the mechanism of how Hb maintains its stability under oxidative stress conditions of red blood cells (RBCs) carrying high levels of oxygen is unknown. Here, we investigated the potential role of peroxiredoxin II (Prx II) in preventing Hb aggregation induced by reactive oxygen species (ROS) using Prx II knockout mice and RBCs of patients with hemolytic anemia. Upon oxidative stress, ROS and Heinz body formation were significantly increased in Prx II knockout RBCs compared to wild-type (WT), which ultimately accelerated the accumulation of hemosiderin and heme-oxygenase 1 in the Prx II knock-out livers. In addition, ROS-dependent Hb aggregation was significantly increased in Prx II knockout RBCs. Interestingly, Prx II interacted with Hb in mouse RBCs, and their interaction, in particular, was severely impaired in RBCs of patients with thalassemia (THAL) and sickle cell anemia (SCA). Hb was bound to the decameric structure of Prx II, by which Hb was protected from oxidative stress. These findings suggest that Prx II plays an important role in preventing hemolytic anemia from oxidative stress by binding to Hb as a decameric structure to stabilize it.

Shedding of phosphatidylserine from developing erythroid cells involves microtubule depolymerization and affects membrane lipid composition.

Phosphatidylserine (PS), which is normally localized in the cytoplasmic leaflet of the membrane, flip-flops to the external leaflet during aging of, or trauma to, cells. A fraction of this PS undergoes shedding into the extracellular milieu. PS externalization and shedding change during maturation of erythroid cells and affect the functioning, senescence and elimination of mature RBCs. Several lines of evidence suggest dependence of PS shedding on intracellular Ca concentration as well as on interaction between plasma membrane phospholipids and microtubules (MTs), the key components of the cytoskeleton. We investigated the effect of Ca flux and MT assembly on the distribution of PS across, and shedding from, the membranes of erythroid precursors. Cultured human and murine erythroid precursors were treated with the Ca ionophore A23187, the MT assembly enhancer paclitaxel (Taxol) or the inhibitor colchicine. PS externalization and shedding were measured by flow cytometry and the cholesterol/phospholipids in RBC membranes and supernatants, by ¹H-NMR. We found that treatment with Taxol or colchicine resulted in a marked increase in PS externalization, while shedding was increased by colchicine but inhibited by Taxol. These results indicate that PS externalization is mediated by Ca flux, and PS shedding by both Ca flux and MT assembly. The cholesterol/phospholipid ratio in the membrane is modified by PS shedding; we now show that it was increased by colchicine and A23187, while taxol had no effect. In summary, the results indicate that the Ca flux and MT depolymerization of erythroid precursors mediate their PS externalization and shedding, which in turn changes their membrane composition.

Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro.

BACKGROUND: The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes. DESIGN AND METHODS: To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro. RESULTS: The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes. CONCLUSIONS: The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.

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.

Increased serum hepcidin levels during treatment with deferasirox in iron-overloaded patients with myelodysplastic syndrome.

Hepcidin is a major regulator of iron metabolism. We evaluated changes in serum hepcidin during 3 months of therapy with the iron-chelator deferasirox in patients with low-risk myelodysplastic syndrome and iron overload. Serum hepcidin was found to be high in these patients, correlated with their iron and oxidative status, and further increased by treatment with deferasirox. These findings support the concept that the hepcidin level represents a balance between the stimulating effect of iron overload and the inhibitory effects of erythropoietic activity and oxidative stress. These preliminary findings favour the rationale for iron chelation therapy in such patients.

Oxidative stress-induced membrane shedding from RBCs is Ca flux-mediated and affects membrane lipid composition.

Phosphatidylserine (PS), which is normally localized in the cytoplasmic leaflet of the membrane, undergoes externalization during aging or trauma of red blood cells (RBCS: ). A fraction of this PS is shed into the extracellular milieu. Both PS externalization and shedding are modulated by the oxidative state of the cells. In the present study we investigated the effect of calcium (Ca) flux on oxidative stress-induced membrane distribution of PS and its shedding and on the membrane composition and functions. Normal human RBCs were treated with the oxidant t-butyl hydroperoxide, and thalassemic RBCs, which are under oxidative stress, were treated with the antioxidant vitamin C or N-acetylcystein. The intracellular Ca content was modulated by the Ca ionophore A23187 and by varying the Ca concentration in the medium. Ca flux was measured by Fluo-3, PS externalization and shedding were measured by quantitative flow cytometry and membrane composition was measured by (1)H-NMR analysis of the cholesterol and phospholipids. The results indicated that increasing the inward Ca flux induced PS externalization and shedding, which in turn increased the membrane cholesterol/phospholipid ratio and thereby increased the RBC osmotic resistance. In addition, these processes modulated the susceptibility of RBCs to undergo phagocytosis by macrophages; while PS externalization increased phagocytosis, the shed PS prevented it. These results indicate that PS redistribution and shedding from RBCs, which are mediated by increased calcium, have profound effects on the membrane composition and properties and, thus, may control the fate of RBCs under physiological and pathological conditions.

Oxidative stress contributes to hemolysis in patients with hereditary spherocytosis and can be ameliorated by fermented papaya preparation.

In the present study, we questioned the role of oxidative stress in hereditary spherocytosis (HS), where red blood cells (RBC) have a shortened survival due to primary deficiency in membrane proteins. Using flow cytometry techniques, we showed that RBC derived from 17 HS patients of seven families generate more reactive oxygen species, membrane lipid peroxides, and less reduced glutathione than normal RBC. Following in vitro incubation of HS-RBC from seven patients with a fermentation bioproduct of Carica papaya (fermented papaya preparation (FPP)) with known antioxidative properties, oxidative stress markers were significantly reduced. Similar results were obtained following treatment with FPP for 3 months of 10 adult HS patients, as well as decreased tendency to undergo hemolysis. The hemoglobin levels increased by >1 g/dl, mean corpuscular hemoglobin concentration decreased by >1 g/dl, and the reticulocyte count decreased by 0.93%. Concomitantly, lactic dehydrogenase decreased by 17% and indirect bilirubin by 50%. A significant decrease in malonyldialdehyde was also detected. These data indicate that oxidative stress plays an important role in the pathophysiology of HS which can be ameliorated by an antioxidant such as FPP. Additional clinical trials with FPP and other antioxidants are warranted.

Transferrin-iron routing to the cytosol and mitochondria as studied by live and real-time fluorescence.

In the present study we analysed the mechanism of intracellular routing of iron acquired by erythroid cells via receptor-mediated endocytosis of Tf-Fe [Tf (transferrin)-iron]. Using real-time fluorimetry and flow cytometry, in conjunction with targeted fluorescent metal sensors, we monitored concurrently the cytosolic and mitochondrial changes in labile iron evoked by endocytosed Tf-Fe. In K562 human erythroleukaemia cells, most of the Tf-Fe was found to be delivered to the cytosolic labile iron pool by a saturable mechanism [60-120 nM Km (app)] that was quantitatively dependent on: Tf receptor levels, endosomal acidification/reduction for dislodging iron from Tf and ensuing translocation of labile iron into the cytosolic compartment. The parallel ingress of iron to mitochondria was also saturable, but with a relatively lower Km (app) (26-42 nM) and a lower maximal ingress per cell than into the cytosol. The ingress of iron into the mitochondrial labile iron pool was blocked by cytosol-targeted iron chelators, implying that a substantial fraction of Tf-Fe delivered to these organelles passes through the cytosol in non-occluded forms that remain accessible to high-affinity ligands. The present paper is the first report describing intracellular iron routing measured in intact cells in real-time and in quantitative terms, opening the road for also exploring the process in mixed-cell populations of erythroid origin.

The Redox Balance and Membrane Shedding in RBC Production, Maturation, and Senescence.

Membrane shedding in the form of extracellular vesicles plays a key role in normal physiology and pathology. Partial disturbance of the membrane-cytoskeleton linkage and increased in the intracellular Ca content are considered to be mechanisms underlying the process, but it is questionable whether they constitute the primary initiating steps. Homeostasis of the redox system, which depends on the equilibrium between oxidants and antioxidants, is crucial for many cellular processes. Excess oxidative power results in oxidative stress, which affects many cellular components, including the membrane. Accumulating evidence suggests that oxidative stress indirectly affects membrane shedding most probably by affecting the membrane-cytoskeleton and the Ca content. In red blood cells (RBCs), changes in both the redox system and membrane shedding occur throughout their life-from birth-their production in the bone marrow, to death-aging in the peripheral blood and removal by macrophages in sites of the reticuloendothelial system. Both oxidative stress and membrane shedding are disturbed in diseases affecting the RBC, such as the hereditary and acquired hemolytic anemias (i.e., thalassemia, sickle cell anemia, and autoimmune hemolytic anemia). Herein, I review some data-based and hypothetical possibilities that await experimental confirmation regarding some aspects of the interaction between the redox system and membrane shedding and its role in the normal physiology and pathology of RBCs.

Effect of iron chelators on labile iron and oxidative status of thalassaemic erythroid cells.

BACKGROUND/AIMS: Iron accumulation in vital organs such as heart and liver is a major pathology in beta-thalassaemia. It may also affect mature RBCs and developing erythroid precursors. The cellular damage is mainly caused by the labile iron pool (LIP) and is mediated by reactive oxygen species (ROS). We have previously shown that thalassaemic RBCs and their precursors have more LIP and ROS than their normal counterparts. We now report the effect of clinically relevant iron chelators on these parameters. METHODS: RBCs, reticulocytes and cultured erythroid precursors derived from patients with beta-thalassaemia were studied for LIP and oxidative stress parameters by flow-cytometry. RESULTS: In vitro treatment with deferiprone, deferasirox and deferoxamine reduced the cytosolic LIP in RBCs and reticulocytes, and both the cytosolic and mitochondrial LIP in cultured erythroid precursors. This was associated with reduced oxidative stress (ROS and external phosphatidylserine). While the effect of deferiprone and deferasirox was fast (within 10 min), deferoxamine affected these parameters after 24 h, suggesting a slower rate of entry. CONCLUSION: The chelators studied reduce the LIP and the oxidative status of thalassaemic RBC and their precursors. Whether these effects directly improve ineffective erythropoiesis and RBC survival remains to be shown.

Amelioration of oxidative stress in red blood cells from patients with beta-thalassemia major and intermedia and E-beta-thalassemia following administration of a fermented papaya preparation.

In beta-hemoglobinopathies, such as beta-thalassemia (thal) and sickle cell anemia, the primary defects are mutations in the beta-globin gene. However, many aspects of the pathophysiology are mediated by oxidative stress. Fermented papaya preparation (FPP), a natural health food product obtained by biofermentation of carica papaya, has been shown to limit oxidative stress both in vitro and in vivo. We studied the effect of FPP on two groups of beta-thal patients: beta-thal, major and intermedia, (in Israel) and E-beta-thal (in Singapore). The results indicated that in both groups FPP treatment increased the content of reduced glutathione (GSH) in red blood cells (RBC), and decreased their reactive oxygen species (ROS) generation, membrane lipid peroxidation, and externalization of phosphatidylserine (PS), indicating amelioration of their oxidative status, without a significant change in the hematological parameters. Since the turnover of the erythron is relatively slow, it is possible that longer duration of treatment, probably with the addition of an iron chelator, is required in order to achieve the latter goals.

N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells.

Many aspects of the pathology in beta-hemoglobinopathies (beta-thalassemia and sickle cell anemia) are mediated by oxidative stress. In the present study we tested a novel thiol compound, N-acetylcysteine amide (AD4), the amide form of N-acetyl cysteine (NAC) for its antioxidant effects. Using flow-cytometry, we showed that in vitro treatment of blood cells from beta-thalassemic patients with AD4 elevated the reduced glutathione (GSH) content of red blood cells (RBC), platelets and polymorphonuclear (PMN) leukocytes, and reduced their ROS. These effects resulted in a significant reduced sensitivity of thalassemic RBC to hemolysis and phagocytosis by macrophages. Intra-peritoneal injection of AD4 to beta-thalassemic mice (150 mg/kg) reduced the parameters of oxidative stress (p<0.001). Our results show the superiority of AD4, compared to NAC, in reducing oxidative stress markers in thalassemic cells both in vitro and in vivo.