Reactive Oxygen Species In Erythrocytes Research Articles

High-throughput synchronous erythrocyte cellular antioxidant activity and protection screening of phenolic-rich extracts: Protocol validation and applications

Oxidative/nitrosative damage takes part in chronic disease development, which generates an urgent need for intervention and better therapies to manage them. The scientific community has demanded easy-to-run, cheap, and reliable methods for cellular antioxidant activity assays. This work standardised and validated an erythrocyte cellular antioxidant activity and membrane protection/injury (HERYCA-P) protocol to study food-derive extracts. The method measures intracellular reactive oxygen species (ROS) generation, lipoperoxidation, and haemolysis induced by 2,2′-azobis(2-amidinopropane) dihydrochloride. Quercetin decreased ROS generation by 50.4% and haemolysis by 2.2%, while ascorbic acid inhibited lipid peroxidation by 40.1%. Total phenolic contents of teas were correlated with decreased ROS generation (r = −0.924), lipoperoxidation (r = −0.951), and haemolysis (r = −0.869). The erythrocyte ROS generation and lipoperoxidation were also associated with CUPRAC (r = −0.925; r = −0.951) and hydroxyl radical scavenging activity (r = −0.936; r = −0.949). The precision rates of antioxidant standards and tea samples were below 15%. HERYCA-P is feasible as a complementary antioxidant assay for food matrices.

Perilla Fruit Oil-Fortified Soybean Milk Intake Alters Levels of Serum Triglycerides and Antioxidant Status, and Influences Phagocytotic Activity among Healthy Subjects: A Randomized Placebo-Controlled Trial.

This study aimed to develop perilla fruit oil (PFO)-fortified soybean milk (PFO-SM), identify its sensory acceptability, and evaluate its health outcomes. Our PFO-SM product was pasteurized, analyzed for its nutritional value, and had its acceptability assessed by an experienced and trained descriptive panel (n = 100) based on a relevant set of sensory attributes. A randomized clinical trial was conducted involving healthy subjects who were assigned to consume deionized water (DI), SM, PFO-SM, or black sesame-soybean milk (BS-SM) (n = 48 each, 180 mL/serving) daily for 30 d. Accordingly, health indices and analyzed blood biomarkers were recorded. Consequently, 1% PFO-SM (1.26 mg ALA rich) was generally associated with very high scores for overall acceptance, color, flavor, odor, taste, texture, and sweetness. We observed that PFO-SM lowered levels of serum triglycerides and erythrocyte reactive oxygen species, but increased phagocytosis and serum antioxidant activity (p < 0.05) when compared to SM and BS-SM. These findings indicate that PFO supplementation in soybean milk could enhance radical-scavenging and phagocytotic abilities in the blood of healthy persons. In this regard, it was determined to be more efficient than black sesame supplementation. We are now better positioned to recommend the consumption of PFO-SM drink for the reduction of many chronic diseases. Randomized clinical trial registration (Reference number 41389) by IRSCTN Registry.

Generation of reactive oxygen species in erythrocytes in patients with polytrauma and alcohol withdrawal complicated by alcoholic delirium

Generation of reactive oxygen species in erythrocytes in patients with polytrauma and alcohol withdrawal complicated by alcoholic delirium

Effects of Flavonoid Quercetin on Thrombo-Inflammatory Processes in Patients with Sickle Cell Disease

Introduction:Altered erythrocyte physiology in Sickle Cell Disease (SCD) results in severe vascular complications, including vaso-occlusion (VOC) along with various other manifestations, such as thrombosis and end organ damage. Moreover, platelet activation endothelial dysfunction and fibrin formation, are critical processes implicated in thrombo-inflammatory vasculopathy in SCD. Since flavonoids intake is associated with lowered adverse cardiovascular disease outcomes and cancer associated thrombosis, we are evaluating the effects of the flavonoid Isoquercetin (IQ) on thrombo-inflammatory biomarkers in patients with SCD (NCT:04514510). Here we report the mechanistic effects of Quercetin (Que), an aglycone that differs from IQ by the absence of 3-linked glucoside moiety, on markers of erythrocyte and platelet activation in patients with SCD.Methods:We studied patients with SCD (Genotype SS, mean age: 39 yrs, N=8) and ethnic matched controls (Genotype AA, mean age: 42 yrs, N=8). Erythrocyte reactive oxygen species (ROS) was measured in washed red cells by flow cytometry using the fluorescent probe 2‘, 7‘-dichlorofluorescein diacetate (DCFDA) in the presence or absence of Quercetin (Que) (100 µM). Effects of in vitro erythrocyte ROS induction by hydrogen peroxide (H 2O 2) and ROS inhibition by N-acetyl-L-cysteine (NAC) were evaluated. Baseline and agonist-induced (ADP 20 µM) platelet P-selectin expression in vitro in the presence or absence of Que (100 µM) was assessed by flow cytometry.Results:At baseline, SS erythrocytes demonstrated elevated levels of ROS reflected by greater florescent intensity with DCFDA when compared with erythrocytes from ethnic matched controls (SS mean= 1884, SD: 508.8 vs. AA mean: 1604, SD: 554.5; p value= 0.3798). Stimulation of AA erythrocytes in vitro with H 2O 2 led to accumulation of ROS that approached levels similar to those exhibited by SS erythrocytes at baseline (AA mean: 1890, SD: 748.3). Pre-treatment of SS and AA erythrocytes in vitro with the antioxidant NAC exhibited minimal effects on ROS in SS erythrocytes (SS baseline+NAC mean: 1942, SD: 526.8, p value 0.4609 and SS H 2O 2+NAC mean: 1889, SD: 437.4, p value 0.5469) but lowered ROS levels in both baseline and H 2O 2 treated AA erythrocytes (AA baseline+NAC mean: 1442, SD: 453.9, p value 0.1563 and AA H 2O 2+NAC mean: 1581, SD: 518.7, p value 0.078). Pre-treatment of SS and AA erythrocytes in vitro with Que substantially lowered erythrocyte ROS in patients and controls at the baseline (SS baseline ROS with Que mean: 1582, SD: 542.0, p value 0.0078; AA baseline ROS with Que mean: 1341, SD: 490.9, p value 0.0313; Fig. 1A), and after H 2O 2 stimulation (SS H 2O 2 induced ROS+Que mean: 1677, SD: 572.5, p value 0.0156; AA H 2O 2 induced ROS+Que mean: 1417, SD: 512, p value 0.0078). Pre-treatment of erythrocytes with a combination of Que and NAC did not reveal synergistic anti-oxidant activity (SS NAC+Que mean: 1616, SD: 464.8; AA NAC+Que mean: 1333, SD: 524.1).We simultaneously investigated the effects of Que on platelet P-selectin expression in the same subjects. As expected, baseline platelet surface P-selectin expression was higher in SS patients compared with controls (SS N=5, mean: 1.48%, Max: 4.9%, Min: 0.03%; AA N=4, mean: 0.89%, Max: 2.3%, Min: 0%; p value 0.55). Stimulation of platelet rich plasma (PRP) with ADP increased surface P-selectin expression in comparison with baseline, in both patients and controls (SS N=5, mean: 18.04%, Max: 40.4%, Min: 5.8%, p value 0.0047; AA N=4, mean: 20.60%, Max: 43.8%, Min: 5.4%; p value 0.0140). Pre-treatment of PRP with Que prior to ADP stimulation revealed a trend toward decreased platelet surface P-selectin expression in both patients and controls (SS N=5, mean: 10.49%, Max: 24.8%, Min: 3.1%; AA N=4, mean: 12.09%, Max: 21.9%, Min: 3.4%; Fig 1B) although this did not return to baseline.Conclusion:The flavonoid Quercetin appears to have a beneficial effect in lowering erythrocyte ROS and platelet surface P-selectin and warrant further evaluation as a strategy to ameliorate thrombo-inflammatory pathophysiology in sickle hemoglobinopathies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

UV Light-Activated GdYVO4:Eu3+ Nanoparticles Induce Reactive Oxygen Species Generation in Leukocytes Without Affecting Erythrocytes In Vitro.

Nanoparticles (NPs) have been reported to be promising enhancement agents for radiation therapy. The aim of the study was to assess the cytotoxicity of UV non-treated and UV pretreated GdYVO4:Eu3+ nanoparticles against erythrocytes and leukocytes by detecting eryptosis and reactive oxygen species (ROS) generation. Levels of intracellular ROS in erythrocytes and leukocytes using a ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), as well as eryptosis rate utilizing annexin V staining, following direct exposure to UV-activated and nonactivated NPs were detected by flow cytometry. Blood cells were collected from 9 intact WAG rats. Neither the UV light-untreated GdYVO4:Eu3+ NPs nor the treated ones promoted eryptosis and ROS generation in erythrocytes. Low concentrations of UV light-untreated NPs did not induce oxidative stress in leukocytes, evidenced by unaffected intracellular ROS levels. UV light treatment grants prooxidant properties to NPs, confirmed by NP-induced ROS overproduction in leukocytes. High concentrations of both UV light-treated and untreated NPs altered the redox state of leukocytes. UV light treatment imparts prooxidant properties to GdYVO4:Eu3+ NPs, making them promising radiosensitizing agents in cancer radiation therapy.

Modifications of proteins of membrane-cytoskeleton complex and production of reactive oxygen species in erythrocytes cryopreserved with polyethylene glycol

Protein modifications in the membrane-cytoskeleton complex (MCC) of human erythrocytes, as well as changes in the intensity of reactive oxygen species (ROS) production upon cell cryopreservation with polyethylene glycol (PEG) were investigated. The protein profile of ghosts of erythrocytes frozen with PEG has common features with both the control and cells frozen without cryoprotectant. PEG makes it possible to restrict the structural rearrangements of the main MCC proteins under the effect of extreme factors and to restrain the amount of high molecular weight polypeptide complexes induced by the protein-cross-linking reagent diamide at the control level, in contrast to cells frozen without a cryoprotectant. However, changes related to the protein peroxiredoxin 2 in ghosts of erythrocytes cryopreserved with PEG are also attributed to cells frozen without a cryoprotectant that may be associated with the activation of oxidative processes. This is evidenced by a 10-fold increase in ROS formation in erythrocytes frozen under PEG protection. Thus, upon cryopreservation of erythrocytes with PEG, certain disorders in MCC proteins may be associated with increased formation of ROS, which may contribute to the disorganization of the structural components of MCC and disrupt the stability of cryopreserved cells under physiological conditions.

Quantitative determination of primaquine-5,6-ortho-quinone and carboxyprimaquine-5,6-ortho-quinone in human erythrocytes by UHPLC-MS/MS

The antimalarial drug primaquine (PQ) causes methemoglobinemia and hemolysis in individuals with a genetic deficiency of glucose 6-phosphate dehydrogenase. Reactive oxygen species (ROS) generated by redox cycling of the metabolite primaquine-5,6-orthoquinone (POQ) in erythrocytes has been attributed to be responsible for the toxicity of PQ. Carboxyprimaquine (CPQ), the major human plasma metabolite of PQ, can also form the analogous carboxyprimaquine-5,6-orthoquinone (CPOQ) metabolite, which can also generate ROS in erythrocytes by redox cycling, thus contributing to the hematotoxicity of this drug. In order to study these pathways and characterize such effects in vivo, methods are needed for characterization and quantification of POQ and CPOQ in human erythrocytes. The purpose of this work was to develop a validated method for the quantitative determination of CPOQ and POQ metabolites in human erythrocytes, suitable for clinical studies of PQ metabolism. Several liquid-liquid extraction methods using different organic solvents had been investigated. The solvent mixture of water-methanol-acetonitrile (9:9:5, v/v) was shown to yield the best results for the two analytes. Chromatographic analysis of POQ and CPOQ in human erythrocytes was achieved on a high strength silica (HSS) column and gradient elution (water and acetonitrile, both containing 0.1% formic acid) by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Quantitative estimation of POQ and CPOQ was executed by monitoring ion pairs of m/z 260.23 > 175.03 and m/z 275.19 > 175.04, respectively. The method, which was validated for precision, accuracy, selectivity, and linearity, was successfully applied for the quantitative determination of POQ and CPOQ, the key metabolites of PQ in human erythrocytes in PQ clinical study.

The Production of Reactive Oxygen Species in Human Erythrocytes during Cryopreservation with Glycerol and Polyethylene Glycol

Abstract—The production of reactive oxygen species in human erythrocytes during incubation in the presence of a glycerol–mannitol mixture and polyethylene glycol with a molecular weight of 1500 Da, as well as during hypothermic storage of cells cryopreserved under their protection, was studied. Analysis of the production of reactive oxygen species was carried out by flow cytometry using fluorescence indices of dichlorofluorescein. It was found that incubation in the glycerol–mannitol mixture, as well as cryopreservation and subsequent hypothermic storage, did not cause activation of the production of reactive oxygen species in erythrocytes. Polyethylene glycol caused the intensification of the production of reactive oxygen species both during incubation and storage of cryopreserved erythrocytes under hypothermia. Peculiarities of changes in the intensity of the production of reactive oxygen species in erythrocytes under the influence of cryoprotective agents and cryopreservation may have a significant impact on the stability of cells under stress and their ability to maintain functional integrity after returning to physiological conditions.

Potential Modulation of Vascular Function by Nitric Oxide and Reactive Oxygen Species Released From Erythrocytes.

The primary role for erythrocytes is oxygen transport that requires the reversible binding of oxygen to hemoglobin. There are, however, secondary reactions whereby the erythrocyte can generate reactive oxygen species (ROS) and nitric oxide (NO). ROS such as superoxide anion and hydrogen peroxide are generated by the autoxidation of hemoglobin. NO can be generated when nitrite reacts with hemoglobin forming an HbNO+ intermediate. Both of these reactions are dramatically enhanced under hypoxic conditions. Within the erythrocyte, interactions of NO with hemoglobin and enzymatic reactions that neutralize ROS are expected to prevent the release of any generated NO or ROS. We have, however, demonstrated that partially oxygenated hemoglobin has a distinct conformation that enhances hemoglobin-membrane interactions involving Band 3 protein. Autoxidation of the membrane bound partially oxygenated hemoglobin facilitates the release of ROS from the erythrocyte. NO release is made possible when HbNO+, the hemoglobin nitrite-reduced intermediate, which is not neutralized by hemoglobin, is bound to the membrane and releases NO. Some of the released ROS has been shown to be transferred to the vasculature suggesting that some of the released NO may also be transferred to the vasculature. NO is known to have a major effect on the vasculature regulating vascular dilatation. Erythrocyte generated NO may be important when NO production by the vasculature is impaired. Furthermore, the erythrocyte NO released, may play an important role in regulating vascular function under hypoxic conditions when endothelial eNOS is less active. ROS can react with NO and, can thereby modulate the vascular effects of NO. We have also demonstrated an inflammatory response due to erythrocyte ROS. This reflects the ability of ROS to react with various cellular components affecting cellular function.

Levels of oxidative damage and proinflammatory cytokines are enhanced in patients with active vitiligo

Vitiligo is an autoimmune depigmenting skin disease characterised by loss of melanocytes wherein oxidative stress is proposed to be the initial triggering factor with subsequent immune dysregulation. This study aimed to evaluate the relationship, if any, between the generation of reactive oxygen species (ROS), markers of oxidative damage and circulating cytokines in patients with active vitiligo. The generation of ROS in erythrocytes and neutrophils was significantly higher in patients with active vitiligo than healthy controls. Alongside, markers of oxidative stress-mediated damage namely lipid peroxidation, DNA damage and protein carbonylation were evaluated. Patients with active vitiligo demonstrated increased lipid and DNA damage but minimal protein damage. There was a significant decline in the free radical scavenging capacity of active vitiligo cases. A positive correlation existed between baseline levels of ROS and lipid peroxidation as also DNA damage. Patients with active vitiligo demonstrated an increase in several proinflammatory (IL-6, TNF-α, IL-1β, IFN-γ and IL-8) and some anti-inflammatory/immunoregulatory (IL-5 and IL-10) cytokines. Importantly, the levels of IFN-γ and IL-10 consistently correlated with the generation of ROS, markers of damage and their free radical scavenging capacity. Taken together, patients with active vitiligo demonstrated an enhanced generation of ROS in erythrocytes and neutrophils which mediated lipid peroxidation, DNA damage and coupled with a decline in their antioxidant capacity created a pro-oxidant milieu that favoured tissue damage and potential generation of neoantigens, accounting for disease progression.

A study of the free radical scavenging effects of Piper betle leaf extract in patients with vitiligo.

Vitiligo is an idiopathic skin disease manifested by depigmented macules. It is characterised by melanocyte destruction, and redox imbalance is proposed to play a contributory role. The aim of this study was to analyze the effects of an ethanolic extract of Piper betle leaves on the generation of reactive oxygen species in erythrocytes sourced from vitiligo patients. The effect of Piper betle on the generation of reactive oxygen species in erythrocytes was measured by flow cytometry in patients with active and stable vitiligo versus healthy controls, using 5-(and-6)-chloromethyl-2'-7'-dichlorodihydrofluorescein diacetate. The generation of reactive oxygen species in erythrocytes was higher in patients with vitiligo (n = 23) compared to healthy controls (n = 18). The geometrical mean fluorescence channel was 23.05 ± 2.11 in patients versus 17.77 ± 1.79 in controls, P = 0.039. The levels of reactive oxygen species were higher in patients with active vitiligo. Treatment of erythrocytes with Piper betle in concentrations of 0.5 and 1.0 μg/ml significantly decreased the baseline levels of reactive oxygen species by 31.7% in healthy controls, and 47.6% and 44.3% in patients with active vitiligo, respectively. Piper betle effectively scavenged hydrogen peroxide, which was evident by a decrease in the geometrical mean fluorescence channel by 52.4% and 62.9% in healthy controls, and 45.0% and 57.0% in patients with active vitiligo. The study had a small sample size. Future studies should focus on evaluation of the antioxidant role of Piper betle at the lesional site. This pilot study indicates that patients with active vitiligo demonstrate enhanced generation of reactive oxygen species in erythrocytes, which was significantly reduced following ex vivo treatment with Piper betle.

Cellular Hydration and Oxidation As Phenotype Modifiers in Sickle Cell Anemia

Although Sickle Cell Anemia (SCA) is caused by a single nucleotide mutation in the beta globin gene, there is broad phenotypic variability in affected individuals. It would be highly advantageous to be able to predict which SCA patients are most likely to suffer severe complications and which are likely to benefit from specific treatments. Since two of the major pathologic mechanisms in SCA are erythrocyte dehydration and chronic inflammation, alterations in the expression and/or function of proteins affecting these processes may be responsible for heterogeneity in SCA phenotype. We have developed a 23-gene Next-Generation sequencing panel to identify variants in genes involved in erythrocyte hydration and in reactive oxygen species (ROS) generation and reduction in patients with SCA. We have collected blood samples from an initial cohort of 18 SCA patients with severe phenotype, defined by history of stroke or abnormal transcranial Doppler velocities, and 13 SCA patients with mild phenotype for DNA preparation and for specialized testing to evaluate erythrocyte hydration status and ROS generation.Advia Automated Cell Counter results provided the clearest indications of erythrocyte dehydration in patient blood samples. The ability to examine reticulocyte parameters made it possible to evaluate hydration regardless of the frequency of transfusion. Reticulocyte CHCM, MCV, and percent hyperdense cells were significantly different between the two groups and indicative of a greater degree of dehydration in the severe phenotype group.Osmoscans (using a LoRRca ektacytometer) demonstrated a highly significant left shift indicative of erythrocyte dehydration in both mild and severe groups relative to controls (O min and O hyper values significantly decreased). Both phenotypic groups also demonstrated a significantly lower EI max in relation to controls, indicating decreased deformability of sickle erythrocytes. Deformability scans revealed significant increases in SS ½ and decreases in EI max in both groups in relation to the controls. There were significant differences between mild and severe phenotype groups in osmoscan O min and O hyper values and in deformability assay SS ½ and EI max values. The severe group had values closer to the normal values, most likely due to the contribution of transfused blood in the severe phenotype group. There were no significant differences between the mild and severe SCA phenotype groups in intracellular cation levels (K+ and Na+) measured by flame emission spectroscopy, although both groups had significantly higher intracellular Na+ compared to normal controls.Erythrocyte ROS detection was performed using a flow cytometry DCFDA assay. Both mild and severe phenotype groups demonstrated a highly significant increase in ROS compared to controls. However there was not a significant difference in erythrocyte ROS between the mild and severe phenotype groups.DNA from patient blood samples has been sequenced using the Haloplex target enrichment system and Illumina high-throughput sequencing. We analyzed 23 genes that are likely candidates to be involved in erythrocyte hydration (ABCB6, ABCG5, ABCG8, AQP1, AQP3, ATP2B4, KCNN4, PIEZO1, RHAG, SLC9A1, SLC12A4, SLC12A6, SLC12A7, SLC2A1, SLC4A1, STOM, TRPC6, XK) or in ROS production or reduction (G6PD, NOX1, CYBB, NOX4, NOX5). At least 19 variants classified as possibly damaging have been detected in 10 of the genes. Future study will be directed toward exploring the phenotypic implications of identified variants.Results of this study should further our understanding of the pathogenesis of SCD and help identify biomarkers of disease severity which may ultimately help guide clinical management for individual patients. They may also suggest novel targets for development of new therapies for SCD based on the modulation of erythrocyte hydration and inflammation. DisclosuresQuinn:Amgen: Research Funding; Eli Lilly: Research Funding; Silver Lake Research Corporation: Consultancy. Joiner:Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Enhanced eryptosis contributes to anemia in lung cancer patients.

ObjectivesAnemia is a common complication of malignancy, which could result from either compromised erythropoiesis or decreased lifespan of circulating erythrocytes. Premature suicidal erythrocyte death, characterized by cell shrinkage and phosphatidylserine (PS) externalization, decreases erythrocyte lifespan and could thus cause anemia. Here, we explored whether accelerated eryptosis participates in the pathophysiology of anemia associated with lung cancer (LC) and its treatment.MethodsErythrocytes were drawn from healthy volunteers and LC patients with and without cytostatic treatment. PS exposure (annexin V-binding), cell volume (forward scatter), cytosolic Ca2+ (Fluo3 fluorescence), reactive oxygen species (ROS) production (DCFDA fluorescence) and ceramide formation (anti-ceramide antibody) were determined by flow cytometry.ResultsHemoglobin concentration and hematocrit were significantly lower in LC patients as compared to healthy controls, even though reticulocyte number was higher in LC (3.0±0.6%) than in controls (1.4±0.2%). The percentage of PS-exposing erythrocytes was significantly higher in LC patients with (1.4±0.1%) and without (1.2±0.3%) cytostatic treatment as compared to healthy controls (0.6±0.1%). Erythrocyte ROS production and ceramide abundance, but not Fluo3 fluorescence, were significantly higher in freshly drawn erythrocytes from LC patients than in freshly drawn erythrocytes from healthy controls. PS exposure of erythrocytes drawn from healthy volunteers was significantly more pronounced following incubation in plasma from LC patients than following incubation in plasma from healthy controls.ConclusionAnemia in LC patients with and without cytostatic treatment is paralleled by increased eryptosis, which is triggered, at least in part, by increased oxidative stress and ceramide formation.

Eryptosis as an Underlying Mechanism in Systemic Lupus Erythematosus-Related Anemia

Background: The progression of systemic lupus erythematosus (SLE) leads to anemia in patients, adversely affecting prognosis. The diverse causes of anemia may include excessive eryptosis or premature suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine (PS) exposure on the cell surface. The present study explored if SLE enhances eryptosis and the underlying mechanisms. Materials and Methods: Eryptosis was assessed using flow cytometry in healthy volunteers (n = 20) and anemic patients hospitalized for SLE (n = 22), for parameters including PS exposure, cell volume, cytosolic calcium ion (Ca²⁺) levels and reactive oxygen species (ROS) and ceramide abundance. These indicators were measured in erythrocytes of experimental subjects and erythrocytes treated with plasma from healthy volunteers or SLE patients. Results: The hemoglobin and hematocrit levels were significantly lower in anemic SLE patients than in healthy volunteers (***p<0.001, p<0.001, respectively). The percentage of PS-exposing erythrocytes was significantly higher in SLE patients than in healthy volunteers (p<0.001), accompanied by an increase in cytosolic Ca²⁺ levels, oxidative stress. The measurements of PS and Ca²⁺ levels were significantly higher in the erythrocytes of healthy volunteers following incubation in plasma of SLE patients than in plasma of healthy volunteers for 24h (***p<0.001, *p<0.05 respectively). Conclusion: Eryptosis is enhanced in SLE and may contribute to anemia. The probable underlying mechanisms may be an excessive formation of ROS in erythrocytes. Also, some plasma components may trigger eryptosis by increasing the cytosolic Ca²⁺ concentration.

Hemoglobin – source of reactive oxygen species

Erythrocytes are especially vulnerable to reactive oxygen species because of their direct role in oxygen transport. Moreover, hemoglobin contains iron ions (Fe²⁺), which catalyze both the Fenton reaction and lipid peroxidation. Reactive oxygen species in erythrocytes are also generated through nonenzymatic and enzymatic processes of heme degradation. The nonenzymatic process of heme degradation is initiated by e.g. hydrogen peroxide, whereas the process of enzymatic degradation is under the influence of heme oxygenase. In both cases biliverdin, carbon monoxide (CO) and iron ions (Fe²⁺) are generated. These products of heme degradation can initialize the oxidative processes within erythrocytes, but at low concentrations exhibit cytoprotective properties.

Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease

AbstractChronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. In this study, we demonstrate that a significant part of ROS production in sickle cells is mediated enzymatically by NADPH oxidase, which is regulated by protein kinase C, Rac GTPase, and intracellular Ca2+ signaling within the sickle RBC. Moreover, plasma from patients with SCD and isolated cytokines, such as transforming growth factor β1 and endothelin-1, enhance RBC NADPH oxidase activity and increase ROS generation. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte rigidity and fragility in SCD. Erythrocyte ROS generation, hemolysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive-feedback loop to drive the pathophysiology of sickle cell disease. These findings suggest a novel pathogenic mechanism in SCD and may offer new therapeutic targets to counteract inflammation and RBC rigidity and fragility in SCD.

Red blood cell extrudes nucleus and mitochondria against oxidative stress

Mammal red blood cells (erythrocytes) contain neither nucleus nor mitochondria. Traditional theory suggests that the presence of a nucleus would prevent big nucleated erythrocytes to squeeze through these small capillaries. However, nucleus is too small to hinder erythrocyte deformation. And, there is no sound reason to abandon mitochondria for the living cells. Here, we found that mammal erythrocyte reactive oxygen species (ROS) levels kept stable under diabetes, ischemia reperfusion, and malaria conditions or in vitro sugar/heme treatments, whereas bird erythrocyte ROS levels increased dramatically in these circumstances. Nuclear and mitochondrial extrusion may help mammal erythrocytes to better adapt to high-sugar and high-heme conditions by limiting ROS generation.

Thyroid hormone—induced haemoglobin changes and antioxidant enzymes response in erythrocytes

Thyroid hormones modulate haemoglobin and reactive oxygen species (ROS) production, leading to antioxidant changes. This study evaluated the antioxidant response to ROS in erythrocytes in hypothyroid and hyperthyroid rats. Wistar rats were divided into four groups: control; hyperthyroid (T4-12 mg 1(-1) in drinking water); sham operated (simulation of thyroidectomy); and hypothyroid (thyroidectomized). Four weeks after, blood was collected and haemoglobin and T(4) levels, lipid peroxidation (LPO), protein oxidation, superoxide dismutase (SOD), catalase (CAT) , glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities, and total radical antioxidant potential (TRAP) were measured. SOD, CAT and GST immunocontent was evaluated. Haemoglobin levels were increased in hyperthyroid erythrocytes. LPO and carbonyls were augmented (65% and 55%, respectively) in hyperthyroid and reduced (31% and 56%, respectively) in hypothyroid group. SOD and CAT activities have not changed, as well as CAT immunocontent. TRAP was diminished in both hyperthyroid and hypothyroid groups (36% and 37%, respectively). GST activity and immunocontent, as well as GPx activity, were increased in hyper and hypothyroid rats. The data suggest that thyroid hormone changes determine ROS concentration changes and decrease of some antioxidant defences that would lead to a compensatory answer of the GST and GPx enzymes, which could be consider as credible biomarkers.

Elevated Reactive Oxygen Species Production In Sickle Erythrocytes Is Modulated by a Pathway Involving Endothelin-1, TGFβ1, PKC, and Rac GTPases

AbstractAbstract 1634Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease. One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle erythrocytes. Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. The potential role of enzymatic mechanisms in ROS production in RBCs has not been fully explored. One candidate for enzyme-mediated ROS production in SS RBCs is NADPH oxidase, which is activated by the small Rho GTPases Rac1 and Rac2 in a variety of cell types (Hordijk P.L. Circ. Res. 2006; 98:453-462).Using flow cytometry with 5-(and 6-)-chloromethyl-2‘,7‘-dichlorodihydrofluorescein diacetate, a peroxide-sensitive probe, we determined that ROS generation is elevated in HbSS RBCs by 150–250% relative to that in HbAA RBCs. The NADPH oxidase NOX subunit homologs NOX1 and NOX5 were expressed in erythrocytes, and treatment of SS RBCs with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) reduced ROS generation in a dose-dependent manner. Inhibition of PKC or Rac activity by the small molecule-inhibitors calphostin and NSC23766, respectively, also resulted in decreased ROS production in HbSS-RBCs in a dose-dependent fashion, while PKC activation by phorbol 12-myristate 13-acetate (PMA) increased ROS production. This effect of PMA could be inhibited by parallel treatment with NSC23766, implicating a PKC-Rac axis in erythrocyte ROS production. Enzymatic ROS production in sickle RBCs was dependent on the availability of free intracellular calcium, since it was inhibited by BAPTA-AM, a cell-permeable calcium chelator. Moreover, we found that ROS generation in SS RBCs was modulated by humoral factors. Incubation of AA RBCs in blood type-matched SS patient plasma resulted in increased ROS generation, while the incubation of SS RBCs in AA plasma decreased ROS production. Immunoblotting of AA and SS RBC membranes with specific antibodies revealed receptors for the inflammatory signaling molecules TGFβ1 and Endothelin-1 (ET-1), both of which are present in elevated levels in the plasma of patients with SCD. Incubation of AA RBCs with TGFβ1 or ET-1 resulted in increased Rac activation and increased ROS production in these treated cells.Our results suggest that ROS production in sickle RBCs is mediated by NADPH oxidase through Ca2+-regulated PKC and Rac signals, which in turn are modulated by plasma TGFβ1 and ET-1 via their receptors. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte deformation and fragility in sickle cell disease. Erythrocyte ROS generation, RBC lysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive feedback loop to drive the pathophysiology of sickle cell disease. These data may offer new therapeutic targets to counteract inflammation and RBC fragility and deformation in sickle cell disease. Disclosures:No relevant conflicts of interest to declare.

Sickle Erythrocytes Have Increased Adducin Phosphorylation and Increased ROS Production Mediated by Signaling Pathways Involving Protein Kinase C and Rac GTPases.

Abstract 901Although sickle hemoglobin (HbSS) polymerization in hypoxic conditions is the trigger for the sickling deformation of red blood cells (RBC) containing HbSS, subsequent changes in the cytoskeleton and the mechanisms by which reversibly sickled cells become irreversibly malformed have not been fully elucidated. Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells (Schwartz.M., J.Cell Science 2004). Deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin (an F-actin capping protein) at Ser-726, a domain-target of protein kinase C (PKC) (Kalfa et al, Blood 2006).We evaluated the cytoskeleton phosphorylation changes in RBCs from patients with HbSS and from control subjects with hemoglobin AA (HbAA) by immunoblotting with phospho-specific antibodies. We found that phosphorylation of adducin at Ser-726 and of band 4.1 at Ser-331 were significantly increased in the erythrocyte cytoskeleton of HbSS-RBCs. This was associated with elevated PKC activity in these cells, compared with normal RBCs. ROS concentration was also elevated in HbSS-RBCs by 150-250% compared to that in HbAA-RBC, as determined by flow cytometry using 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, a peroxide-sensitive probe. Inhibition of PKC or Rac activity in HbSS-RBCs by small molecule-inhibitors, calphostin and NSC23766 respectively, resulted in decreased ROS production in a dose-dependent fashion, while PKC activation by phorbol 12-myristate 13-acetate (PMA) increased ROS production, implicating a PKC-Rac axis in erythrocyte ROS production. Additionally, Rac inhibition resulted in dose-dependent increase of adducin phosphorylation while PKC inhibition had the opposite effect. Preincubation of HbSS RBCs with calphostin appeared to decrease the subsequent susceptibility of these cells to sickling under hypoxic conditions, suggesting a role for PKC activity in the transition from normal to sickle morphology, likely through a combination of effects on volume regulation and membrane stability of the cells. Our results imply that aberrant cytoskeleton protein phosphorylation and ROS production in HbSS-RBCs, mediated by signaling pathways involving PKC and Rac GTPases, may contribute to membrane changes induced by cell sickling. Elucidation of these pathways may identify new therapeutic targets in sickle cell disease. Disclosures:No relevant conflicts of interest to declare.