Oxidative Status of Platelets in Normal and Thalassemic Blood.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3765-3765
Author(s):  
Johnny Amer ◽  
Eitan Fibach
Keyword(s):  
Oxidative Stress ◽  
Platelet Activation ◽  
Specific Antigen ◽  
Calcium Ionophore ◽  
Iron Chelator ◽  
Oxidative Status ◽  
Beta Thalassemia ◽  
Ros Generation ◽  
Continuous Exposure ◽  
Light Scatter

Abstract Thromboembolic complications, possibly involving chronic platelet activation, are an important cause of morbidity and mortality in beta-thalassemia. Oxidative stress, with the generation of reactive oxygen species (ROS), has been suspected to play a role in the pathophysiology of thalassemia and cardiovascular disorders. Previous investigations demonstrated that ROS profoundly affect platelet function and promote platelet activation. Other studies have shown that platelets themselves produce ROS upon activation. In the present study, we adapted flow cytometric techniques to measure oxidative-state markers, ROS generation and reduced glutathione (GSH), using 2′-7′-dichlorofluorescin diacetate and mercury orange, respectively, in platelets. GSH is the major intracellular antioxidant - an important scavenger of ROS. To exclude non-platelets from analysis, a two-parameter (side light scatter and forward light scatter) gate was set. The identity of the gated cells was verified by immunofluorescence staining for CD41 - a platelet-specific antigen. Using these techniques, the average Mean Fluorescence Channel (MFC) values of platelets from 46 normal donors and 22 beta-thalassemic donors were 176 ± 99 vs. 314 ± 81, respectively, for ROS and 319 ± 87 vs. 113 ± 47, respectively, for GSH. These results show that thalassemic platelets contain higher ROS and lower GSH levels than do normal platelets, indicating a state of oxidative stress. The relationship between platelet activation and oxidative status was determined by treating platelets with thrombin (0.1 U/ml), calcium ionophore (5 μM) or phorbol myristate acetate (400 ng/ml). All these treatments caused platelet activation as well as ROS generation; thalassemic platelets were more responsive than platelets from normal controls. In the absence of any known inherent abnormality in thalassemic platelets, the increased oxidative status was attributable to continuous exposure to oxidative insults from extra-platelet sources. Indeed, further investigation indicated that the oxidative status of normal platelets was increased by thalassemic plasma and was inhibited by the iron-chelator Desferoxamin. Iron and hemin, whose levels are increased in thalassemic plasma, stimulated the platelets’ oxidative stress. This was also affected by RBC: it was higher in normal platelets incubated with thalassemic RBC than when incubated with normal RBC. Normal RBC stimulated with hydrogen peroxide, a treatment which results in an elevated oxidative status, increased platelet ROS to a greater extent (3.3-fold) than did unstimulated RBC. These results suggest that thalassemic RBC, having higher than normal ROS, mediate oxidative stress in platelets directly, probably by contact or close proximity. Platelet oxidative stress was ameliorated by antioxidants such as N-acetyl-L-cysteine and vitamin C. Treatment with these agents of oxidant-stimulated platelets reduced ROS and enhanced the GSH level. The present results indicate that in thalassemia, platelets are in a state of oxidative stress, causing their chronic activation and possibly thromboembolic consequences. This situation may also prevail in other RBC anomalies, such as sickle cell anemia, Polycythemia Vera and Paroxysmal Nocturnal Hemoglobinuria, which are also associated with thromboembolic phenomena. Our findings raise the possibility of using antioxidants in addition to antithrombotic drugs as prophylactic treatment in these diseases.

Oxidative status of platelets in normal and thalassemic blood

2004 ◽  
Vol 92 (11) ◽  
pp. 1052-1059 ◽  
Author(s):  
Johnny Amer ◽  
Eitan Fibach
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Reduced Glutathione ◽  
Calcium Ionophore ◽  
Iron Chelator ◽  
Oxidative Status ◽  
Ros Generation ◽  
Continuous Exposure ◽  
Flow Cytometric ◽  
Oxidative State

SummaryChronic platelet activation may be involved in thromboembolic complications, a leading cause of morbidity and mortality in β-thalassemia. Oxidative stress, with the generation of reactive oxygen species (ROS), is suspected to play a role in the pathophysiology of thalassemia and cardiovascular disorders. In the present study, we adapted flow cytometric techniques to measure oxidative state markers, ROS generation and reduced glutathione (GSH) content in platelets. Our results show that platelets obtained from β-thalassemic patients contain higher ROS and lower GSH levels than do platelets from normal donors, indicating a state of oxidative stress. In the absence of any known inherent abnormality in thalassemia platelets, this may be attributed to continuous exposure to oxidative insults from extra-platelet sources. We found that exposure of platelets to oxidants such as hydrogen peroxide and tertbutylhydroperoxide or to the platelet activators thrombin, calcium ionophore or phorbol myristate acetate stimulated the platelets’ oxidative stress.This was also increased by plasma of thalassemia patients, and decreased following treatment of the plasma with the iron-chelator Desferoxamin. Iron and hemin, the levels of which are augmented in plasma of thalassemia patients, stimulated the platelets’ oxidative stress.The oxidative status of the platelets was also affected by red blood cells (RBC); it was higher in normal platelets incubated with thalassemic RBC than with normal RBC. Normal RBC stimulated with hydrogen peroxide had a greater effect on platelets than did unstimulated RBC.The platelets’ oxidative stress was ameliorated by antioxidants such as N-acetyl-L-cysteine and vitamin C. Our findings indicate that in thalassemia, platelets undergo a state of oxidative stress, leading to their activation and potentially to thromboembolic consequences, and suggest that this hypercoagulable state might be treated with antioxidants.

Oxidative Stress in Red Blood Cells, Platelets and Polymorphonuclear Leukocytes from Patients with Myelodysplastic Syndrome.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2632-2632
Author(s):  
Eitan Fibach ◽  
Hussam Ghoti ◽  
Johnny Amer ◽  
Asher Winder ◽  
Eliezer Rachmilewitz
Keyword(s):  
Oxidative Stress ◽  
Myelodysplastic Syndrome ◽  
Blood Cells ◽  
Cell Damage ◽  
Iron Chelator ◽  
Oxidative Status ◽  
Iron Chelators ◽  
Ros Generation ◽  
Transfusion Requirements

Abstract Myelodysplastic syndrome (MDS) is characterized by refractory cytopenias due to ineffective hematopoiesis. Some patients with severe anemia require multiple blood transfusions and develop iron overload. Consequently, reactive oxygen species (ROS) are generated concomitant with a decrease in cellular antioxidants such as reduced gluthatione (GSH). The generated oxidative stress contributes to cell damage, apoptosis and ineffective hematopoiesis. Using flow cytometry, we measured the oxidative state of RBC, platelets and PMN in 14 low-risk MDS patients and 25 normal donors. The results indicate that the majority of the patients had higher ROS in RBC (2.79-fold) and platelets (2.91-fold) and lower GSH in their RBC (3.4-fold) and platelets (2.1-fold) than normal (p<0.005). As for PMN, there were no significant differences in ROS, although GSH was significantly (p<0.1) lower in MDS compared with normal donors. The oxidative stress in MDS cells could be ameliorated by a short in vitro treatment with the iron-chelators deferrioxamine and deferiprone, or with the anti-oxidant N-acetylcysteine. These results suggest that the decrease in transfusion requirements with increase in platelet and PMN counts in MDS patients treated with deferrioxamine may be due to indirect antioxidant effect of the iron chelator and suggest that treatment with a combination of iron-chelators and anti-oxidants might be more effective. ROS generation and GSH content in MDS blood cells ROS generation and GSH content in MDS blood cells Effect of iron chelations and an antioxidant on the oxidative status of MDS cells Effect of iron chelations and an antioxidant on the oxidative status of MDS cells

The Oxidative Status of Valinomycin-Resistant Normal and Thalassemic Red Cells (RBCs).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3191-3191
Author(s):  
Johnny Amer ◽  
Zipora Etzion ◽  
Robert M. Bookchin ◽  
Eitan Fibach
Keyword(s):  
Oxidative Stress ◽  
Oxidant Stress ◽  
Thalassemia Major ◽  
Oxidative Status ◽  
Normal Blood ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Before And After ◽  
Mercury Orange

Abstract Normal high-K+, low-Na+ RBCs, suspended in low-K+ media and permeabilized to K+ with valinomycin, become dehydrated from net loss of KCl and water. A very small fraction of light, normal RBC and larger fractions of light, sickle cell anemia (SCA) and beta-thalassemia RBC were found to be “valinomycin-resistant” (val-res) due to their Na+/K+ gradient dissipation (PNAS2000;97: 8050; BLOOD2000;96:24b). In thalassemia and SCA, although the primary lesions involve the globin genes, the major damage to the RBC membranes is mediated by oxidative stress. We previously showed (Cytometry2004;60:73) that thalassemic RBC have higher reactive oxygen species (ROS) and lower reduced glutathione (GSH) levels than normal RBC before or after in vitro oxidant stress (treatment with hydrogen peroxide). Here, we examined the oxidative status of val-res RBC from normal and beta-thalassemia major blood. RBC suspended in a plasma-like buffer containing 15 mM KCl and 10 mM valinomycin for 45 min were then layered on arabinogalactone (Larex) with density δ=1.091, and spun at 15,000 g for 30 min. Val-res cells were identified as the low density (δ<1.091 g/ml) RBCs recovered from the interphase layer. The percent val-res RBC in beta-thalassemic samples (n = 10), was 84-fold higher (4.2 ± 0.4% (mean ± SD), range 2.5 to 6.0%) than in normal samples (0.05 ± 0.06%, range 0.02 to 0.1%) (n =10). To determine the oxidative status of the RBC, the cells were washed with PBS and stained for intracellular contents of ROS and GSH, using 2′-7′-dichlorofluoresein and Mercury Orange, respectively. RBC were analyzed by flow cytometry, using gating based on size and granularity. The Mean Fluorescence Channel (MFC) for each fluorochrome was computed. The results showed that valinomycin treatment, per se, did not affect ROS and GSH contents: MFC of the stained un-fractionated RBC was similar before and after treatment with valinomycin, indicating that large changes in MCHC had little or no effect on these measurements. In addition, the unfractionated RBC had ROS and GSH values comparable to those of the high density (val-sensitive) RBC which were recovered from the pellet of valinomycin-treated RBC following Larex fractionation. Measurements on six normal and six beta-thalassemic blood samples indicated that in each case val-res RBC had higher ROS (3.5-10 fold) and lower GSH (2.5-8 fold) levels than the unfractionated RBC or the val-sensitive RBC of the same sample. Compared with val-res cells from normal blood, thalassemic val-res RBC had higher capacity to produce ROS (1.7-fold) and had a lower GSH level (1.5-fold) compared with normal val-res RBC. These results confirm that, as with SCA, beta-thalassemia blood contains a higher percent of val-res RBC than normal blood. They show, further, that (i) both normal and thalassemic val-res RBC have higher oxidative status than other cells (val-sensitive) in the same sample; and that (ii) thalassemic val-res RBC have higher oxidative status than val-res RBC in normal blood. The present results are consistent with the possibility that oxidative stress may contribute to the generation of val-res RBCs, but do not establish a cause-effect relationship. Further studies will be needed to elucidate the origin and significance of these cells.

In Vitro and In Vivo Antioxidant Effect of Fermented Papaya Preparation (FPP) on Blood Cells of Beta-Thalassaemia Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1766-1766
Author(s):  
Eitan Fibach ◽  
Johnny Amer ◽  
Ada Goldfarb ◽  
Eliezer Rachmilewitz
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Peripheral Blood ◽  
Thalassemia Major ◽  
Oxidative Status ◽  
Beta Thalassemia ◽  
Thalassemia Intermedia

Abstract In sickle cell anemia (SCD) and thalassemia, although the basic lesions are mutations in the globin genes, the pathophysiology involves oxidative stress-mediated cell damage in the bone marrow (ineffective erythropoiesis due to apoptosis of early erythroid precursors) and in the peripheral blood (chronic hemolysis of mature RBC). In addition, some patients develop thromboembolic complications and recurrent bacterial infections, the etiology of which is related at least in part, to documented oxidative stress in platelets and neutrophils (PMN), respectively. To study the presence and the role of oxidative stress in thalassemia and SCD, we adapted flow cytometry techniques for measuring the generation of Reactive Oxygen Species (ROS), the content of reduced glutathione (GSH), membrane lipid peroxidation and externalization of phosphatidylserine (PS) moieties in RBC, platelets and PMN. Cells derived from the peripheral blood of patients with beta-thalassemia major, intermedia or SCD showed increased oxidative status (increased ROS, lipid peroxidation and PS externalization, and decreased GSH) compared with their normal counterparts. Incubating fresh blood samples from patients with thalassemia major and thalassemia intermedia with 10 mg/ml FPP for 16 hours at 37oC reduced the oxidative status of RBC as well as platelets and PMN. Experiments carried out in normal and thalassemic mice (Th3/+, a mouse model of human beta-thalassemia intermedia demonstrated that mice treated for one week with 10 mg/ml FPP (dissolved in the drinking water) had reduced oxidative stress compared to control mice. The in-vivo effect of FPP was tested on 9 patients with beta-thalassemia (6 - major and 3 - intermedia) treated with 3 gr FPP per os three times a day for 12–15 weeks. Following the treatment, the ROS in RBC, platelets and PMN decreased and the GSH increased in all patients (see table). Six of these patients responded by a modest increase in RBC, reticulocytes and hemoglobin levels. These results suggest that FPP may have an important clinical efficacy as an antioxidant in thalassemia and sickle cell anemia. The in vivo effect of FPP treatment of beta-thalassemia patients Baseline After treatment n Mean ± SE Mean ± SE P-value* * Paired samples t-test RBC 9 324.07 ± 29.19 209.55 ± 23.65 0.001 ROS Platelets 9 223.73 ± 26.49 109.11 ± 8.71 0.001 PMN 9 222.72 ± 46.42 117.61 ± 8.98 0.045 RBC 9 55.37 ± 5.37 94.88 ± 3.71 0.001 GSH Platelets 9 59.41 ± 4.98 97.55 ± 5.26 <0.0001 PMN 9 58.29 ± 5.35 90.06 ± 5.87 0.005

Oxidative Stress of RBC in a Murine Model of Beta-Thalassemia Can Be Reversed by Treatment with Antioxidants.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3643-3643
Author(s):  
Eitan Fibach ◽  
Johnny Amer ◽  
Eliezer Rachmilewitz ◽  
Ella Guy ◽  
Stefano Rivella
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Mouse Model ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Beta Thalassemia ◽  
Light Scatter ◽  
Intracellular Ros ◽  
Mercury Orange ◽  
Dichlorofluorescin Diacetate

Abstract Oxidative stress is a prominent contributor to the premature destruction of RBC as well as anemia in thalassemia and sickle cell anemia. The oxidative status within RBC is maintained by the balance between oxidative systems, such as Reactive Oxygen Species (ROS), and antioxidative systems, such as reduced glutathione (GSH). Using flow cytometric methods, we previously showed that RBC obtained from patients with thalassemia (Amer et al. Eur J Haematol70:84,2003; Cytometry60:73,2004) or sickle cell anemia (Amer et al. Blood, 104:972a,2004) exhibit oxidative stress. In the present study, we assessed the extent of RBC oxidative stress and the effects of antioxidant administration, using the thalassemic mouse model Th3/+. This model closely mimics the phenotype observed in patients affected by beta-thalassemia intermedia, such as low hemoglobin levels (7 to 9 gr/dL), splenomegaly and iron overload. In addition, the mature RBC have a shorter life-span and are characterized by anisocytosis, poikilocytosis and hypochromatism. RBC were derived from normal and thalassemic mice before and 4 hours after i.p. injection of the antioxidants N-acetyl cysteine (NAC), vitamin C (Vit. C) or tocotrinol - a mixture of vitamin E derivatives, at a dose of 150 mg/kg. Intracellular ROS was determined in dichlorofluorescin diacetate-stained RBC following stimulation with 2 mM H2O2; GSH content was assessed in RBC stained with mercury orange. Cells were analyzed by flow cytometry: RBC were gated according to size (forward light scatter) and granularity (side light scatter), their fluorescence was measured and the Mean Fluorescence Channel (MFC) was calculated. Fig. 1 shows the average MFC of ROS and GSH of normal and thalassemic mice treated or not treated with anti-oxidants (N=6 in each group). The results show a significantly higher (2.6-fold) production of ROS and lower (three-fold) levels of GSH in RBC from the thalassemic mice versus those in RBC from normal mice. Administration of antioxidants decreased the ROS of normal and thalassemic RBC by 1.4-fold and 2.6-fold, respectively, whereas GSH levels were significantly increased both in the normal (2.7 fold) and in the thalassemic (9.4-fold) RBC. The results show that the RBC of thalassemic mice are under oxidative stress that could be ameliorated by a short antioxidant treatment. Hence, this mouse model recapitulates the oxidative stress found in thalassemic patients and can serve as a model for studying the effects of antioxidant therapy. The flow cytometry methodology used is helpful in following up the results of the treatment and in evaluating its efficacy in reducing oxidative stress. Figure Figure

Oxidative Stress Induces Changes and Shedding of Membrane Phospholipids from Thalassemic RBCs - A NMR Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1782-1782
Author(s):  
Eitan Fibach ◽  
Inna Freikman ◽  
Johnny Amer ◽  
Jack S. Cohen ◽  
Israel Ringel
Keyword(s):  
Oxidative Stress ◽  
31P Nmr ◽  
Oxidative Status ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Membrane Phospholipids ◽  
Flip Flop ◽  
Nmr Study

Abstract Changes in the membrane phospholipid (PL) asymmetry is one of the hallmarks of apoptosis in nucleated cells. Although mature anucleated RBCs, do not undergo the classical pattern of apoptosis, upon trauma or aging they present changes in the membrane asymmetry. These changes include a flip-flop of phosphatidylserine (PS) from the inner leaflet of the membrane to its outer leaflet. This externalization of PS stimulates RBC phagocytosis by macrophages of the reticulo-endothelial system and their removal from the circulation. Oxidative stress is among the causes of PS externalization on RBCs. In beta-thalassemia and sickle cell disease, although the primary defects are mutations in the globin genes, oxidative stress is thought to mediate part of the damage to the RBCs, and particularly to its membrane, including PS externalization. In the present study, we used Nuclear Magnetic Resonance (NMR) spectroscopy to analyze normal and beta-thalassemic RBCs in order to study the relationship between their oxidative status and the content and shedding of their PL. Using 1H-NMR, we demonstrated a higher lactate/pyruvate ratio in thalassemic RBCs, confirming their state of oxidative stress. Using 31P-NMR, we measured the content of various PLs, and found 30±3% more phosphatidylcholine (PC), and unexpectedly, less PS in thalassemic RBCs than in normal RBCs. The PS was increased in thalassemic RBC, but not in normal RBC, by treatment with anti-oxidants (vitamin C and N-acetyl cysteine) and decreased by oxidants (t-butylhydroxyperoxide and H2O2) in normal and thalassemic RBCs. PC showed the opposite behavior, indicating a correlation between PS and PC contents and the oxidative status. Since RBCs with exposed PS have been reported to be more frequent in thalassemic blood than in normal blood, we hypothesized that the decrease in PS is a result of shedding from the external membrane, either as free PS moieties or as part of membrane vesicles. NMR analysis of blood plasma obtained from normal and thalassemic donors indicated a 2.6-fold and 1.8-fold increase in PS and PC, respectively in the latter plasma. In vitro incubation of RBC produced much higher PS in supernatants derived from thalassemic RBCs compared with those of normal RBCs. Anti-oxidants reduced the PS shedding from thalassemic RBCs into their supernatants while oxidants increased the PS shedding by normal RBCs. RBCs are known to shed membranous particles (termed vesicles or microparticles) in vitro and in vivo during their physiologic and pathological senescence. We studied this point by purifying microparticles from plasma and RBC supernatants of normal and thalassemic donors, and measuring the PLs content in their lipophilic extracts by 31P-NMR. We found that the PS content and its proportion out of the total PLs were higher in microparticles purified from thalassemic plasma (0.25±0.04 mM, 19% of the plasma total PS) or RBC supernatants than in normal plasma microparticles (0.045±0.06 mM, 9.5% of the plasma total PS) or supernatants. The results also show that although microparticles are enriched in PS compared to their intact RBCs, the bulk of the shed PS is not associated with microparticles. These results suggest that oxidative stress in RBCs causes them to shed their PS and that the increase in PC levels maybe be a compensating mechanism. The pathological consequences of these phenomena on the survival of RBCs in thalassemia warrants further study.

Improvement of Oxidative Stress Parameters in MDS Patients with Iron Overload Treated with Deferasirox

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2675-2675 ◽  
Author(s):  
Eliezer Rachmilewitz ◽  
Drorit Merkel ◽  
Hussam Ghoti ◽  
Johnny Amer ◽  
Arnon Nagler ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Iron Overload ◽  
Serum Ferritin ◽  
Blood Cells ◽  
Iron Chelation ◽  
Iron Chelator ◽  
Ros Generation ◽  
Iron Species ◽  
Oral Iron Chelator

Abstract Introduction: Using flourescence markers, evidence for oxidative stress has been documented by flow cytometry in red cells (RBC) and other blood cells of patients with congenital and acquired hemolytic anemias (e.g. thalassemia, sickle cell anemia, hereditary spherocytosis and paroxysmal nocturnal hemoglobinuria) as well as in myelodysplatic syndrome (MDS) (Eur. J. Hematol. 79:463,2008). These data included increased generation of reactive oxygen species (ROS) concomittant with a decrease in the cellular content of the major antioxidant reduced gluthatione (GSH), and increased membrane lipid peroxidation. Oxidative stress in these anemias is thought to be mainly due to the accumulation of non-transferrin-bound iron (NTBI) which affects iron homeostasis and results in excess extra-cellular and intra-cellular labile pools: labile plasma iron (LPI) and cellular labile iron pool (LIP) (Cytometry 73:22,2008), respectively, catalyzing ROS generation. The presence of these free iron components is correlated with the severity of hemolysis and the subsequent repetitive blood transfusions, resulting in iron-overload. The purpose of the present study was to measure changes in parameters of iron-overload and oxidative stress in iron-overloaded patients with MDS following treatment with the oral iron-chelator Deferasirox. Patients and Methods: Fifteen patients–5 males and 10 females-(mean age 66 ± 13 years) with “low risk” MDS (IPSS &lt; 1.0) received Deferasirox 20 mg/kg/day (12 patients) or 4–6 mg/kg/day (3 patients) due to side effects mainly gastrointestinal, increased creatinine and rash, for an average period of 95 days (63–163 days). All of them had evidence of iron-overload. Their mean number of transfusions was 65 ± 62, and mean ferritin levels before treatment was 3008 ± 1797 ng/ml. ROS, GSH, lipid peroxidation, LIP and LPI were measured every 3–4 weeks, in RBC, platelets and polymorphonuclear leukocytes (PMN). Blood cell counts, hemoglobin levels and serum ferritin were assayed simulteneously. Results: There was a statistically significant decrease in ROS (28%, p=0.006), lipid peroxidation (138%, p=0.008) and the LIP (23%, p=0.004) of RBC, concomittant with an increase in GSH (123%, p=0.001). GSH was also increased in platelets (48%, p=0.008) and PMN (72%, p=0.001), and LIP decreased in platelets (23%, p=0.004). In 8 patients the mean initial LPI levels of 0.39 units decreased to 0.12 units (p=0.028). There were no significant changes in hemoglobin levels or in any of the other types of blood cells. Mean serum ferritin levels increased in 8 patients by 964 ± 927 ng/ml and decreased in the rest by 1432 ± 1267 ng/ml. Discussion: The results clearly demonstrate that administration of Deferasirox to iron-overloaded MDS patients for a mean period of 3 months resulted in a significant decrease in parameters of oxidative stress mainly in RBC and platelets and in intra-(LIP) and extra-cellular (LPI) species of free iron. Serum ferritin, a major parameter in the assessment of the severity of iron-overload, was increased in 8 patients in the present study, similar to previously reported findings of increased ferritin levels at 12 months following iron chelation with Deferasirox (Am. J. Hematol.83:611–3, 2008). However, unlike iron stored in ferritin, the major risk of iron-overload is the presence of excess extra and intracellular iron species (LPI and LIP) which are playing an important role in ROS generation and consequent membrane damage in blood cels and in other major organs.. These preliminary data suggest that treatment with Deferasirox,, a once-a-day oral iron chelator, reduced the toxic iron species and several parameters of oxidative stress in iron overloaded MDS patients. Furthermore, the novel methodologies applied in this study may be useful for evaluating the severity of the iron-overload and for monitoring the efficacy of iron chelation therapy. Additional studies assessing the correlation of these changes to the long-term morbidity, mortality, and quality of life are definitely warranted in this patient population.

Platelet Oxidative Stress and its Relationship with Cardiovascular Diseases in Type 2 Diabetes Mellitus Patients

2019 ◽  
Vol 26 (22) ◽  
pp. 4145-4165 ◽  
Author(s):  
Mohammed El Haouari
Keyword(s):  
Oxidative Stress ◽  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Cardiovascular Diseases ◽  
Platelet Activation ◽  
Current Knowledge ◽  
Natural Antioxidants ◽  
Ros Generation

Enhanced platelet activation and thrombosis are linked to various cardiovascular diseases (CVD). Among other mechanisms, oxidative stress seems to play a pivotal role in platelet hyperactivity. Indeed, upon stimulation by physiological agonists, human platelets generate and release several types of reactive oxygen species (ROS) such as O2 -, H2O2 or OH-, further amplifying the platelet activation response via various signalling pathways, including, formation of isoprostanes, Ca2+ mobilization and NO inactivation. Furthermore, excessive platelet ROS generation, incorporation of free radicals from environment and/or depletion of antioxidants induce pro-oxidant, pro-inflammatory and platelet hyperaggregability effects, leading to the incidence of cardiovascular events. Here, we review the current knowledge regarding the effect of oxidative stress on platelet signaling pathways and its implication in CVD such as type 2 diabetes mellitus. We also summarize the role of natural antioxidants included in vegetables, fruits and medicinal herbs in reducing platelet function via an oxidative stress-mediated mechanism.

Platelets and PMN as Well as RBC of Patients with Sickle Cell Anemia Exhibit Oxidative Stress Which Can Be Corrected by Antioxidants.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3575-3575
Author(s):  
Johnny Amer ◽  
Hussam Ghoti ◽  
Eliezer Rachmilewitz ◽  
Koren Ariel ◽  
Eitan Fibach
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Sickle Cell ◽  
Clinical Manifestations ◽  
Oxidative Status ◽  
Polymorphonuclear Neutrophils ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Beta Thalassemia ◽  
Mercury Orange

Abstract Sickle cell disease (SCD) is caused by an abnormal hemoglobin (HbS), which results mainly in sickling and hemolysis of RBC. However, the platelets and the polymorphonuclear neutrophils (PMN) are also involved in the pathophysiology of the disease. Similar to the findings in thalassemia, some patients develop thromboembolic phenomena with hypercoagulable state, which is due in part to platelet activation. In addition, a growing body of evidence suggests that WBC, particularly PMN, are abnormal; their number is elevated during painful crises and the severity of the disease increases with their number. Several aspects of the changes in the three blood lineages are thought to result from oxidative stress, which represents the imbalance between enhanced generation of reactive oxygen species (ROS) and a low cellular content of antioxidants such as reduced glutathione (GSH), the major intracellular scavenger of ROS. In SCD, oxidative stress results primarily from the premature precipitation of the unstable HbS and the accumulation of excess iron, a catalyst in the formation of ROS. Accumulation of ROS may result in hemolysis of RBC, activation of platelets and a respiratory burst of PMN. We developed flow cytometry techniques for measuring oxidative-state markers, ROS generation and GSH content, simultaneously in RBC, platelets and PMN. Peripheral blood samples obtained from 15 normal donors, 10 patients with SCD and 5 SCD/beta-thalassemia patients (two with the IVS2-1 mutation and 3 with the IVS1-6 mutation) were studied. Out of the 15 SCD patients, 5 were splenectomized, and all were treated with folic acid, except one who was treated with hydroxyurea. The blood was mixed with 3% gelatin, and following 30 min. incubation the upper layer, containing RBC, platelets and WBC, was collected. ROS and GSH were measured by FACS analysis following staining with 2′, 7′-dichlorofuoresceine (DCF) and mercury orange, respectively. Cells were gated on the basis of size and granularity to include platelets, RBC or PMN, exclusively. The cells in each gate were analyzed for green (DCF) or orange (mercury orange) fluorescence and the Mean Fluorescence Channel (MFC) was calculated. The results showed that ROS production increased by 10 to 30-fold in RBC, platelets and PMN from SCD patients compared with that of their normal counterparts. Concomitantly, the GSH content decreased by 20–50% in the SCD cells. It was possible to modulate the oxidative status of cells from both normal donors and SCD patients: Exposure of the cells to oxidants such as hydrogen peroxide (2 mM), hemin (0.1 mM) or iron (ferric ammonium citrate - 0.1 mM) increased the oxidative status in all cell types, while antioxidants such as N-acetyl cysteine, vitamin C (both at 1 mM) and vitamin E (0.2 mM) significantly decreased the oxidative stress. Our results indicate that similar to the findings in thalassemia, in SCD, RBC and platelets, as well as PMN, are in a state of oxidative stress, which could in part account for the clinical manifestations. Addition of antioxidants, which reduced the ROS and enhanced the GSH content of the cells, could protect against oxidative damage. The flow cytometry techniques we developed may prove useful for studying the effects of various antioxidants and for monitoring the patient’s oxidative status during therapy thereby providing an objective, quantitative evaluation of their efficacy.

Clinical-Reported Outcomes Of Deferoxamine Versus Deferasirox In The Treatment Of Homozygous BetaThalassemia

2018 ◽  
Vol 9 (SPL1) ◽  
Author(s):  
Zeina A Munim Al-Thanoon ◽  
Zeina A Munim Al-Thanoon ◽  
Mustafa Basil ◽  
Nasih A Al-Kazzaz
Keyword(s):  
Serum Ferritin ◽  
Iron Chelation ◽  
Iron Chelator ◽  
Beta Thalassemia ◽  
Control Group ◽  
Current Standard ◽  
Cross Sectional ◽  
Significant Difference ◽  
Multiple Blood ◽  
Group 2

Iron chelation therapy with deferoxamine (DFO),the current standard for the treatment of iron overload in patients with betathalassemia,requires regular subcutaneous or intravenous infusions. This can lead to reduced quality of life and poor adherence,resulting in increased morbidity and mortality in iron-overloaded patients with beta-thalassemia. Deferasirox (DFX) is an orally administered iron chelator that has been approved for use in many countries. The requirement of an effective,well tolerated iron chelator with a less demanding mode of administration has led to the development of deferasirox. The present study was aimed to compare the satisfaction and compliance with deferoxamine versus deferasirox (Exjade®),a novel oral iron chelator in patients with transfusion - dependent beta- thalassemia. A cross-sectional,single-center investigation study was carried out in the Thalassemia Center of Ibn-Atheer Teaching Hospital in Nineveh province,Iraq. One hundred and eight thalassemic patients aged between 2- 20 years old having received multiple blood transfusions and a serum ferritin greater than 1500 ng/ml. Patients were randomised into two groups. Group 1 received deferoxamine at a dose of 20-50mg/kg/day and group 2 received deferasirox at the dose of 10-30 mg/kg/day. Another 56 apparently healthy volunteers were used as a control group. The assessment of chelation was done during the period between November 2013 and February 2014 by measurement of serum ferritin. Satisfaction and compliance was assessed by using a special questionnaire prepared by the researcher. Out of the 108 thalassemic patients enrolled there was no discontinuation in treatment with the two drugs under study. The serum ferritin did not change significantly in any of the chelation groups. In comparison with the patients who were treated with DFO,those receiving DFX reported a significantly higher rate of compliance and satisfaction (P < 0.05). However,no significant difference was observed between the two groups regarding their satisfaction (P > 0.05).Compliance with deferasirox (50 %) was more than that with deferoxamine (20 %). Satisfaction with deferoxamine was significantly lower than deferasirox (p= 0.00).

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