Oxidative Stress In Red Blood Cells Research Articles

Exposure to Waterpipe Smoke Disrupts Erythrocyte Homeostasis of BALB/c Mice

The prevalence of waterpipe tobacco smoking (WPS) is increasing worldwide and is relatively high among youth and young adults. It has been shown, both experimentally and clinically, that WPS exposure adversely affects the cardiovascular and hematological systems through the generation of oxidative stress and inflammation. Our study aimed to evaluate the impact of WPS exposure on erythrocytes, a major component of the hematological system, of BALB/c mice. Here, we assessed the effect of nose-only WPS exposure for four consecutive weeks on erythrocyte inflammation, oxidative stress, and eryptosis. The duration of the session was 30 min/day, 5 days/week. Control mice were exposed to air. Our results showed that the levels of C-reactive protein, lipid peroxidation (LPO), superoxide dismutase, and total nitric oxide (NO) were significantly increased in the plasma of WPS-exposed mice. The number of erythrocytes and the hematocrit were significantly decreased in WPS-exposed mice compared with the control group. Moreover, there was an increase in the erythrocyte fragility in mice exposed to WPS compared with those exposed to air. The levels of lactate dehydrogenase, LPO, reduced glutathione, catalase, and NO were significantly increased in the red blood cells (RBCs) of WPS-exposed mice. In addition, erythrocytes of the WPS-exposed group showed a significant increase in ATPase activity, Ca2+, annexin V binding, and calpain activity. Taken together, our findings suggest that WPS exposure elevated inflammation and oxidative stress in the plasma and induced hemolysis in vivo. It also caused alterations of RBCs oxidative stress and eryptosis in vitro. Our data confirm the detrimental impact of WPS on erythrocyte physiology.

Ginseng total saponin improves red blood cell oxidative stress injury by regulating tyrosine phosphorylation and glycolysis in red blood cells

BackgroundOxidative stress is the main cause of many diseases, but because of its complex pathogenic factors, there is no clear method for treating it. Ginseng total saponin (GTS) an important active ingredients in Panax ginseng C.A. Mey (PG) and has potential therapeutic ability for oxidative stress due to various causes. However, the molecular mechanism of GTS in the treating oxidative stress damage in red blood cells (RBCs) is still unclear. PurposeThis study aimed to examine the protective effect of GTS on RBCs under oxidative stress damage and to determine its potential mechanism. MethodsThe oxidative stress models of rat RBCs induced by hydrogen peroxide (H2O2) and exhaustive swimming in vivo and in vitro was used. We determined the cell morphology, oxygen carrying capacity, apoptosis, antioxidant capacity, and energy metabolism of RBCs. The effect of tyrosine phosphorylation (pTyr) of Band 3 protein on RBCs glycolysis was also examined. ResultsGTS reduced the hemolysis of RBCs induced by H2O2 at the lowest concentration. Moreover, GTS effectively improved the morphology, enhanced the oxygen carrying capacity, and increased antioxidant enzyme activity, adenosine triphosphate (ATP) levels, and adenosine triphosphatase (ATPase) activity in RBCs. GTS also promoted the expression of membrane proteins in RBCs, inhibited pTyr of Band 3 protein, and further improved glycolysis, restoring the morphological structure and physiological function of RBCs. ConclusionsThis study shows, that GTS can protect RBCs from oxidative stress damage by improving RBCs morphology and physiological function. Changes in pTyr expression and its related pTyr regulatory enzymes before and after GTS treatment suggest that Band 3 protein is the main target of GTS in the treating endogenous and exogenous oxidative stress. Moreover, GTS can enhance the glycolytic ability of RBCs by inhibiting pTyr of Band 3 protein, thereby restoring the function of RBCs.

Analysis of non-physiological shear stress-induced red blood cell trauma across different clinical support conditions of the blood pump.

Systematic research into device-induced red blood cell (RBC) damage beyond hemolysis, including correlations between hemolysis and RBC-derived extracellular vesicles, remains limited. This study investigated non-physiological shear stress-induced RBC damage and changes in related biochemical indicators under two blood pump clinical support conditions. Pressure heads of 100 and 350mmHg, numerical simulation methods, and two in vitro loops were utilized to analyze the shear stress and changes in RBC morphology, hemolysis, biochemistry, metabolism, and oxidative stress. The blood pump created higher shear stress in the 350-mmHg condition than in the 100-mmHg condition. With prolonged blood pump operation, plasma-free hemoglobin and cholesterol increased, whereas plasma glucose and nitric oxide decreased in both loops. Notably, plasma iron and triglyceride concentrations increased only in the 350-mmHg condition. The RBC count and morphology, plasma lactic dehydrogenase, and oxidative stress across loops did not differ significantly. Plasma extracellular vesicles, including RBC-derived microparticles, increased significantly at 600min in both loops. Hemolysis correlated with plasma triglyceride, cholesterol, glucose, and nitric oxide levels. Shear stress, but not oxidative stress, was the main cause of RBC damage. Hemolysis alone inadequately reflects overall blood pump-induced RBC damage, suggesting the need for additional biomarkers for comprehensive assessments.

Assessment of Red Blood Cell Aggregation in Preeclampsia by Microfluidic Image Flow Analysis-Impact of Oxidative Stress on Disease Severity.

Preeclampsia (PE) is a hypertensive disease characterized by proteinuria, endothelial dysfunction, and placental hypoxia. Reduced placental blood flow causes changes in red blood cell (RBC) rheological characteristics. Herein, we used microfluidics techniques and new image flow analysis to evaluate RBC aggregation in preeclamptic and normotensive pregnant women. The results demonstrate that RBC aggregation depends on the disease severity and was higher in patients with preterm birth and low birth weight. The RBC aggregation indices (EAI) at low shear rates were higher for non-severe (0.107 ± 0.01) and severe PE (0.149 ± 0.05) versus controls (0.085 ± 0.01; p < 0.05). The significantly more undispersed RBC aggregates were found at high shear rates for non-severe (18.1 ± 5.5) and severe PE (25.7 ± 5.8) versus controls (14.4 ± 4.1; p < 0.05). The model experiment with in-vitro-induced oxidative stress in RBCs demonstrated that the elevated aggregation in PE RBCs can be partially due to the effect of oxidation. The results revealed that RBCs from PE patients become significantly more adhesive, forming large, branched aggregates at a low shear rate. Significantly more undispersed RBC aggregates at high shear rates indicate the formation of stable RBC clusters, drastically more pronounced in patients with severe PE. Our findings demonstrate that altered RBC aggregation contributes to preeclampsia severity.

Green Synthesised TiO2 Nanoparticles-Mediated Terenna asiatica: Evaluation of Their Role in Reducing Oxidative Stress, Inflammation and Human Breast Cancer Proliferation.

Oxidative stress and chronic inflammation interplay with the pathogenesis of cancer. Breast cancer in women is the burning issue of this century, despite chemotherapy and magnetic therapy. The management of secondary complications triggered by post-chemotherapy poses a great challenge. Thus, identifying target-specific drugs with anticancer potential without secondary complications is a challenging task for the scientific community. It is possible that green technology has been employed in a greater way in order to fabricate nanoparticles by amalgamating plants with medicinal potential with metal oxide nanoparticles that impart high therapeutic properties with the least toxicity. Thus, the present study describes the synthesis of Titanium dioxide nanoparticles (TiO2 NPs) using aqueous Terenna asiatica fruit extract, with its antioxidant, anti-inflammatory and anticancer properties. The characterisation of TiO2 NPs was carried out using a powdered X-ray diffractometer (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray diffraction (EDX), high-resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), and zeta-potential. TiO2 NPs showed their antioxidant property by scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals in a dose-dependent manner with an IC50 value of 80.21 µg/µL. To ascertain the observed antioxidant potential of TiO2 NPs, red blood cells (RBC) were used as an in vitro model system. Interestingly, TiO2 NPs significantly ameliorated all the stress parameters, such as lipid peroxidation (LPO), protein carbonyl content (PCC), total thiol (TT), superoxide dismutase (SOD), and catalase (CAT) in sodium nitrite (NaNO2)-induced oxidative stress, in RBC. Furthermore, TiO2 NPs inhibited RBC membrane lysis and the denaturation of both egg and bovine serum albumin, significantly in a dose-dependent manner, suggesting its anti-inflammatory property. Interestingly, TiO2 NPs were found to kill the MCF-7 cells as a significant decrease in cell viability of the MCF-7 cell lines was observed. The percentage of growth inhibition of the MCF-7 cells was compared to that of untreated cells at various doses (12.5, 25, 50, 100, and 200 µg/mL). The IC50 value of TiO2 NPs was found to be (120 µg/mL). Furthermore, the Annexin V/PI staining test was carried out to confirm apoptosis. The assay indicated apoptosis in cancer cells after 24 h of exposure to TiO2 NPs (120 µg/mL). The untreated cells showed no significant apoptosis in comparison with the standard drug doxorubicin. In conclusion, TiO2 NPs potentially ameliorate NaNO2-induced oxidative stress in RBC, inflammation and MCF-7 cells proliferation.

Increased retention of functional mitochondria in mature sickle red blood cells is associated with increased sickling tendency, hemolysis and oxidative stress.

Abnormal retention of mitochondria into mature red blood cells (RBCs) has been recently reported in sickle cell anemia (SCA) but their functionality and their role in the pathophysiology of SCA remain unknown. The presence of mitochondria within RBCs was determined by flow cytometry in 61 SCA patients and 10 healthy donors. Patients were classified according to the percentage of mature RBCs with mitochondria contained in the whole RBC population: low (0-4%), moderate (>4 and 8%). RBC rheological, hematological, RBC senescence and oxidative stress markers were compared between the three groups. RBC senescence and oxidative stress markers were also compared between mature RBC containing mitochondria and those without. The functionality of residual mitochondria in sickle RBCs was measured by high-resolution respirometry assay and showed detectable mitochondrial oxygen consumption in sickle mature RBCs but not in healthy RBCs. Increased levels of mitochondrial reactive oxygen species (ROS) were observed in mature sickle RBCs when incubated with Antimycin A vs without. In addition, mature RBCs retaining mitochondria exhibited greater levels of ROS compared to RBCs without mitochondria, as well as greater Ca2+, lower CD47 and greater phosphatidylserine exposure. Hematocrit and RBC deformability were lower, and the propensity of RBC to sickle under deoxygenation was higher, in the SCA group with a high percentage of mitochondria retention in mature RBCs. This study showed the presence of functional mitochondria in mature sickle RBCs, which could favor RBC sickling and accelerate RBC senescence, leading to increased cellular fragility and hemolysis.

Abstract WMP113: Red Blood Cells From Stroke Patients With Admission Hyperglycemia Induces Brain Endothelial Dysfunction

Background: Beyond its traditional role as oxygen carriers, red blood cells (RBCs) are increasingly recognized to participate in regulating vascular function. Many RBC disorders, such as sickle cell disease, are associated with vascular complications resulting in stroke. Our recent study revealed that admission hyperglycemia in stroke patients intensifies oxidative stress in RBCs. We hypothesize that RBCs that are altered by hyperglycemia may perturb endothelial function. Method: Brain endothelial cells were incubated with RBCs isolated from stroke patients with admission hyperglycemia (hyper-RBC) and normoglycemia (normo-RBC) (n=3/group). Mass spectrometry was used to profile the changes in endothelial cells. Functional alterations were assessed by gene set enrichment analysis (GSEA). Significant changes were verified using qPCR and western blot. Result: Compared to normo-RBC, hyper-RBC activates cell adhesion process and toll like receptor signaling in brain endothelial cells (Fig A, B). Cell adhesion molecules (ICAM1, VCAM1) and toll like receptor TLR4 were upregulated at both mRNA and protein levels (Fig C, D), which may promote vaso-occlusion and inflammation. In contrast, vasoregulation was deactivated by hyper-RBC (Fig A, B). The expression of eNOS was downregulated and the bioavailability of vasodilator NO was decreased, consistent with impaired vasodilation (Fig C, D). Notably, the changes induced by hyper-RBC could be reversed by pretreating hyper-RBC with ROS scavenger TEMPOL. The upregulation of ICAM1, VCAM1 and TLR4 in hyper-RBC-treated cells was attenuated by TEMPOL pretreatment, and eNOS expression and NO bioavailability were also restored by TEMPOL (Fig C, D). Conclusion: Intensified oxidative stress in RBCs induced by admission hyperglycemia in stroke patients can cause endothelial dysfunction. Further studies are underway to validate these targets, and ask whether these mechanisms may exacerbate brain injury in stroke patients.

Abstract 38: Admission Hyperglycemia In Stroke Patients Increases Oxidative Stress In Red Blood Cells

Background: Our recent study has shown that admission hyperglycemia in stroke patients is associated with red blood cell (RBC) dysregulation, however, the underlying mechanism is still unclear. In addition to transporting oxygen via hemoglobin, RBCs also carry a robust antioxidant system to remove the reactive oxygen species (ROS) continuously produced from hemoglobin autoxidation (Fig A). It has been reported that in vitro hyperglycemia can increase oxidative stress in RBCs. We thus hypothesize that acute hyperglycemia in stroke patients may also affect the antioxidant system in RBCs. Method: Stroke patients with hyperglycemia and normoglycemia (n=12/group) were prospectively recruited in accordance with IRB protocol. Blood was collected at 2 hr, 24 hr, 72 hr and 3 mon post stroke. RBCs were precipitated from whole blood, and leucocytes were removed with depletion filter. The expression and glycosylation of RBC antioxidants, such as catalase (CAT), superoxide dismutase type 1 (SOD1), glutathione peroxidase 1 (GPX1) and peroxiredoxin 2 (PRX2), were quantified using mass spectrometry-based selected reaction monitoring (SRM) method. Result: CAT, SOD1 and GPX1 showed gradually decreased expression in hyperglycemic stroke patients, which may impair the antioxidant capacity of RBCs (Fig B). The glycosylation level of PRX2 was increased in stroke-related hyperglycemia (Fig C), and increased glycosylation was associated with decreased PRX2 activity (Fig D). Consistent with the expression and glycosylation changes in antioxidants, total RBC antioxidant capacity was reduced in hyperglycemic patients, and ROS was significantly accumulated in RBCs (Fig E). Conclusion: Acute hyperglycemia in stroke patients intensifies RBC oxidative stress by affecting the expression and post-translational modification of RBC antioxidants. Larger studies are underway to verify these findings, and explore how hyperglycemia and RBC oxidative stress influence stroke outcome.

Abstract 37: Acute Hyperglycemia At Stroke Onset Is Associated With Abnormal Red Blood Cells

Background: Acute hyperglycemia at stroke onset is a common clinical problem and is an independent predictor of mortality and long-term disability. Abnormal red blood cells (RBCs) have long been recognized as a stroke risk factor and are associated with poor stroke outcome. Recent evidence has shown that short term hyperglycemia exposure can intensify oxidative stress in RBCs and cause hemolysis. We hypothesize that acute hyperglycemia may induce RBC dysfunction in stroke patients, which may be related to the poor outcome in those patients. Method: 93 stroke patients were consecutively recruited in accordance with IRB protocol. Patients with diabetes, prediabetes or HbA1C ≥ 5.7% were excluded. None of the patients had preexisting anemia. Hyperglycemia was defined as blood glucose &gt; 110 mg/dL at admission. RBC indices were documented for all the patients. Patients with major hemorrhagic events and multiple blood draws were excluded from the study. Clinical outcome was determined by mRankin Scale at 3 month (good: ≤ 2; poor: &gt; 2). Result: 45 (48.4%) were hyperglycemic and 48 (51.6%) were normoglycemic. Hyperglycemic patients showed gradually decreased RBC counts and hemoglobin content, while RBC distribution width (RDW) was gradually elevated (Fig. A). Subgroup analyses showed that the association of hyperglycemia with RBC alterations was not affected by sex and age, two factors that are known to affect RBC function (Fig. B). More importantly, patients with abnormally low RBC count, low hemoglobin and high RDW were at increased risk of developing poor outcome (Fig. C). These results remained robust after adjusting for other risk factors (Fig. C), suggesting that abnormal RBC parameters are independent predictors of poor stroke outcome. Conclusion: Our results revealed that acute hyperglycemia at stroke onset is associated with abnormal RBC indices, which are predictive of poor stroke outcome. Studies in a larger patient cohort with extended time course are still ongoing.

Influence of 24 h Simulated Altitude on Red Blood Cell Deformability and Hematological Parameters in Patients with Fontan Circulation.

Patients with Fontan circulation are particularly dependent on low pulmonary vascular resistance because their lungs are passively perfused. Hypoxia drives pulmonary vasoconstriction; thus, red blood cell (RBC) deformability and stability of hematological parameters might be of particular importance, because alterations during hypoxia might further influence circulation. This study aimed to measure respective parameters in patients with Fontan circulation exposed to normobaric hypoxia. A total of 18 patients with Fontan circulation (16 to 38 years) were exposed to normobaric hypoxia (15.2% ambient oxygen). Blood samples were taken in normoxia, after 24 h in hypoxia, and 60 min after return to normoxia. Blood count, RBC age distribution, EPO, RBC deformability, marker of RBC nitric oxide, oxidative state, and RBC ATP were measured. Hypoxia increased oxidative stress in RBC, but without affecting RBC deformability. RBC age distribution remained unaffected, although EPO concentrations increased, followed by a rise in reticulocyte count at an already high hematocrit. NO metabolism was not affected by hypoxia. Modest normobaric hypoxia for 24 h did not impair RBC deformability in patients with Fontan circulation; however, the oxidative system seemed to be stressed. Given the high baseline Hct in these patients, hypoxia-induced erythropoiesis could adversely affect rheology with more prolonged hypoxia exposure.

Bile acids profile and redox status in healthy infants.

At birth, human neonates are more likely to develop cholestasis and oxidative stress due to immaturity or other causes. We aimed to search for a potential association between bile acids profile, redox status, and type of diet in healthy infants. A cross-sectional, exploratory study enrolled 2-month-old full-term infants (n = 32). We measured plasma bile acids (total and conjugated), and red blood cell (RBC) oxidative stress biomarkers. The type of diet (breastfeeding, mixed, formula) was used as an independent variable. Plasma total bile acids medium value was 14.80 µmol/L (IQR: 9.25-18.00). The plasma-conjugated chenodeoxycholic acid percentage (CDCA%) correlated significantly and negatively with RBCs membrane-bound hemoglobin percentage (MBH%) (r = -0.635, p < 0.01) and with RBC-oxidized glutathione (r = -0.403, p < 0.05) levels. RBC oxidative stress biomarkers (especially MBH%) were predictors of conjugated CDCA%, and this predictive ability was enhanced when adjusted for the type of diet (MBH, r = 0.452, p < 0.001). Our data suggest that the bile acid profile might play a role in the regulation of redox status (or vice versa) in early postnatal life. Eventually, the type of diet may have some impact on this process. The conjugated CDCA% in plasma is negatively correlated with biomarkers of RBC oxidative stress in healthy infants. Specific biomarkers of RBC oxidative stress (e.g. MBH, GSH, GSSG) may be promising predictors of conjugated CDCA% in plasma. The type of diet may influence the predictive ability of hit RBC oxidative stress biomarkers (e.g. MBH, GSH, GSSG). Our findings suggest a link between plasma bile acids profile and the RBC redox status in healthy infants, eventually modulated by the type of diet. The recognition of this link may contribute to the development of preventive and therapeutic strategies for neonatal cholestasis.

Antioxidant Activity of Quercetin in a H2O2-Induced Oxidative Stress Model in Red Blood Cells: Functional Role of Band 3 Protein.

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 μM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42− uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 μM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.

Ginseng polysaccharide attenuates red blood cells oxidative stress injury by regulating red blood cells glycolysis and liver gluconeogenesis

Ethnopharmacological relevancePanax ginseng C.A. Mey (PG) is famous for “Qi-tonifying” effect, which has a medicinal history of more than 2 millennia. Modern pharmacology has confirmed that the “Qi-tonifying” effect of PG may be closely related to its pharmacological properties such as anti-oxidation, antineoplastic and treatment of cardiovascular disease. As one of the earliest cells affected by oxidative stress, RBCs are widely used in the diagnosis of diseases. Ginseng polysaccharide (GPS), is one of the major active components of PG, which plays an important role in resisting oxidative stress, affecting energy metabolism and other effects. However, the molecular mechanism explaining the “Qi-tonifying” effect of GPS from the perspective of RBCs oxidative damage has not been reported. Aim of the studyThis study aimed to investigate the protective effect of GPS on oxidatively damaged RBCs using in vitro and in vivo models and explore the molecular mechanisms from the perspective of glycolysis and gluconeogenesis pathways. To provides a theoretical basis for the future research of antioxidant drugs. Materials and methodsEstablished three different in vitro and in vivo research models: an in vitro model of RBCs exposed to hydrogen peroxide (H2O2) (40 mM), an in vivo model of RBCs from rats subjected to exhaustive swimming, and an in vitro model of BRL-3A cells exposed to H2O2 (25 μM). All three models were also tested in the presence of different concentrations of GPS. ResultsThe findings showed that GPS was the most potent antagonist of H2O2-induced hemolysis and redox inbalance in RBCs. In exhaustive exercise rats, GPS ameliorated RBVs hemolysis, including reducing whole-blood viscosity (WBV), improving deformability, oxygen-carrying and -releasing capacities, which was related to the enhancing of antioxidant capacity. Moreover, GPS promoted RBCs glycolysis in rats with exhaustive exercise by recovering the activities of glycolysis-related enzymes and increasing band 3 protein expression, thereby regulating the imbalance of energy metabolism caused by oxidative stress. Furthermore, we demonstrated that GPS improved antioxidant defense system, enhanced energy metabolism, and regulated gluconeogenesis via activating PPAR gamma co-activator 1 alpha (PGC-1α) pathway in H2O2-exposed BRL-3A cells. Mechanistically, GPS promoted glycolysis and protected RBCs from oxidative injury was partly dependent on the regulation of gluconeogenesis, as inhibition of gluconeogenesis by metformin (Met) attenuates the regulation of antioxidant enzymes and key enzymes of glycolytic by GPS in exhaustive exercise rats. ConclusionThis study demonstrates that GPS protects RBCs from oxidative stress damage by promoting RBCs glycolysis and liver gluconeogenesis pathways. These results may contribute to the study of new RBCs treatments to boost antioxidant capacity and protect RBCs against oxidative stress.

Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

BackgroundHypercalcemia induced by multiple myeloma (MM) affects the biological functions of excitable and non-excitable cells. However, red blood cells (RBCs) regulatory effect on calcium in hypercalcemia is still not fully understood.MethodsA total of 113 patients with MM osteolytic lesions were studied retrospectively. Flow cytometry and atomic absorption spectroscopy were used to detect calcium content. Immunofluorescence and Western blotting were used to investigate protein expression. GEO and miRNA databases were used to screen miRNAs. Exosomal miR-4261 migration was investigated by Transwell assay. Dual-luciferase assays confirmed the targeting relationship between miR-4261 and ATP2B4. An RBC oxidative stress model was constructed, and Omega-Agatoxin IVA was used to study the role of plasma membrane Ca2+-ATPase 4 (PMCA4) in RBCs.ResultsThe results showed that MM RBCs had calcium overload, and serum calcium levels increased as the number of RBCs decreased. The expression of PMCA4 in MM RBCs was significantly lower than in normal RBCs. The exosomal miR-4261 produced by MM cells could be transferred to RBCs to downregulate the expression of ATP2B4.ConclusionsStudies have confirmed that RBCs experience calcium overload in MM with osteolytic lesions, which is related to the downregulation of ATP2B4 by MM exosomal miR-4261.

The relationship between glycosylated hemoglobin level and red blood cell storage lesion in blood donors.

Glycosylated hemoglobin (HbA1c), not routinely screened in blood donors, is associated with morphological, biochemical, and functional abnormalities of red blood cells (RBCs) and with enhanced oxidative stress. We aimed to explore HbA1c levels in blood donors and their effect on RBC storage. An analytical cross-sectional study was conducted on 875 eligible blood donors aged 18-60 years from May 1, 2021, to August 30, 2021. Two selected groups of donors (HbA1c <6.5%, n=10; HbA1c ≥ 6.5%, n=10) exhibiting as similar as possible baseline values (such as age, sex, and living habits, etc.) were recruited for blood donation in leukoreduced CPDA-1units. RBC morphological, biochemical, structural, and oxidative stress states were measured during 5-35 days of storage. Elevated HbA1c prevalence was 37%, including 31.7% (277/875) in the prediabetes range (HbA1c 5.7%-6.4%) and 5.4% (47/875) in the diabetes range (HbA1c ≥ 6.5%). Age, body mass index (BMI), smoking, and alcohol consumption were the main factors influencing the HbA1c levels. During storage, high-HbA1c group had abnormal RBC morphology, impaired membrane function, and ion imbalance (higher mean corpuscular volume, distribution width, hemolysis rate, potassium ion efflux, and phosphatidylserine exposure) as compared with low HbA1c group. Additionally, RBC oxidative stress was significantly increased in donors with high HbA1c levels during 21-35 days. Blood donors proportion with abnormal HbA1c levels was relatively high, and donor HbA1c levels may be associated with stored RBCs capacity. Our study provides new insights into the different effects of donor HbA1c levels on RBC storage lesions.

An Ex Vivo Model of Oxidatively Stressed Red Blood Cells Demonstrates a Role for Exogenous Amino Acids in Enhancing Red Blood Cell Function and Morphology

The capacity of mature red blood cells (RBCs) to respond to oxidative stress is limited due to lack of a full complement of organelles and as such, when faced with an oxidative environment, they rely on their endogenous antioxidant capacity (including superoxide dismutase, catalase, peroxiredoxin and glutathione) to protect against cellular damage. Low blood glutathione activity has been reported in several red cell disorders leading to increased oxidative stress. Targeting oxidative stress has thus been proposed as a secondary treatment in multiple anemia-causing diseases, such as sickle cell disease (SCD) and malaria, although its overall efficacy remains unclear. As glutathione itself is not permeable through the RBC membrane, treatment with cell-permeable amino acid precursors of glutathione (glutamine, cysteine and/or glycine) is a potential strategy to expand the RBC's antioxidant capacity and alleviate oxidative stress. Indeed, L-glutamine has recently been approved as a therapeutic for SCD, although the mechanistic basis for its effect is not clear. To fill this gap in our understanding, we performed detailed characterization of biophysical phenotype, morphology, and intracellular redox environment of oxidatively stressed RBCs in environments with varying amounts of available precursor amino acids.To assess the impact of exogenous amino acid precursors on the RBC's glutathione antioxidant capacity, we exposed mature RBCs from healthy adults to hydrogen peroxide (H 2O 2) and co-incubated with media that included glutamine, cysteine, and/or glycine. As catalase has the ability to scavenge high levels of exogenously fluxed H 2O 2, we performed these experiments using sodium azide to block catalase activity, enabling us to model oxidatively stressed RBCs. We performed osmotic gradient ektacytometry to quantify RBC deformability and hydration status, and assessed RBC morphology using osmotic-adjusted fixation techniques and scanning electron microscopy. As previously documented, H 2O 2 exposure in sodium azide-treated healthy RBCs was associated with decreased deformability, decreased hydration and increased numbers of echinocytes in a dose-dependent manner. We monitored red cell phenotypic changes following co-incubation with glutamine, cysteine, and/or glycine individually and in combination to test whether these amino acids extended the RBC's antioxidant abilities and contributed to improved function and morphology. We found that supplementation with all three amino acids in combination significantly improved both deformability and hydration of H 2O 2-stressed RBCs, as opposed to treatment with glutamine alone. To directly assess whether the exogenous amino acids were in fact contributing to less intracellular oxidative stress in RBCs, we quantified intracellular reactive oxygen species (ROS) using 2‘, 7‘ -dichlorofluorescein diacetate (DCFDA), a cell permeable dye used to measure ROS production. As expected, H 2O 2 exposure alone was associated with elevated intracellular ROS inside RBCs in both a time- and dose-dependent manner. Supplementation with the three amino acid cocktail during H 2O 2 stress resulted in a reduction in the level of intracellular ROS activity.In summary, we documented that exogenous added amino acids reduce oxidative damage in RBCs and we hypothesize that this protection occurs via the glutathione antioxidant pathways. In future studies, we plan to investigate the impact of exogenous amino acids on sickled and irreversibly sickled RBCs (ISCs) in the context of SCD, and on uninfected and infected RBCs in the context of malaria. DisclosuresKim-Shapiro: Beverage Operations LLC: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: co-inventor on a patent related to the use of nitrite under cardiovascular conditions, and a co-author on patents related to treatment of hemolysis.

The Role of RBC Oxidative Stress in Sickle Cell Disease: From the Molecular Basis to Pathologic Implications.

Sickle cell disease (SCD) is an inherited monogenic disorder and the most common severe hemoglobinopathy in the world. SCD is characterized by a point mutation in the β-globin gene, which results in hemoglobin (Hb) S production, leading to a variety of mechanistic and phenotypic changes within the sickle red blood cell (RBC). In SCD, the sickle RBCs are the root cause of the disease and they are a primary source of oxidative stress since sickle RBC redox state is compromised due to an imbalance between prooxidants and antioxidants. This imbalance in redox state is a result of a continuous production of reactive oxygen species (ROS) within the sickle RBC caused by the constant endogenous Hb autoxidation and NADPH oxidase activation, as well as by a deficiency in the antioxidant defense system. Accumulation of non-neutralized ROS within the sickle RBCs affects RBC membrane structure and function, leading to membrane integrity deficiency, low deformability, phosphatidylserine exposure, and release of micro-vesicles. These oxidative stress-associated RBC phenotypic modifications consequently evoke a myriad of physiological changes involved in multi-system manifestations. Thus, RBC oxidative stress in SCD can ultimately instigate major processes involved in organ damage. The critical role of the sickle RBC ROS production and its regulation in SCD pathophysiology are discussed here.

Using redox potential as a feasible marker for banked blood quality and the state of oxidative stress in stored red blood cells.

BackgroundStored red blood cells (RBCs) may undergo oxidative stress over time, with functional changes affecting oxygen delivery. Central to these changes are oxidation‐reduction (redox) reactions and redox potential (RP) that must be maintained for cell function. RP imbalance can lead to oxidative stress that may contribute to storage lesions. This study's purpose was to identify changes in RP over time in banked RBCs, and among RBCs of similar age.MethodsMultiple random RBC segments from RBC units were tested (n = 32), ranging in age from 5 to 40 days, at 5‐day intervals. RP was recorded by measuring open circuit potential of RBCs using nanoporous gold electrodes with Ag/AgCl reference. RP measures were also performed on peripheral venous blood from 10 healthy volunteers. RP measures were compared between RBC groups, and with volunteer blood.ResultsStored RBCs show time‐dependent RP increases. There were significant differences in Day 5 RP compared to all other groups (p ≤ 0.005), Day 10–15 vs. ages ≥ Day 20 (p ≤ 0.025), Day 20–25 vs. Day 40 (p = 0.039), and all groups compared to healthy volunteers. RP became more positive over time suggesting ongoing oxidation as RBCs age; however, storage time alone was not predictive of RP measured in a particular unit/segment.ConclusionsThere are significant differences in RP between freshly stored RBCs and all others, with RP becoming more positive over time. However, storage time alone does not predict RP, indicating RP screening may be an important measure of RBC oxidative stress and serve as an RBC quality marker.

An Antioxidant Nanoparticle Enhances Exercise Performance in Rat High-intensity Running Models.

Although the adverse effects of excessively generated reactive oxygen species (ROS) on the body during aerobic exercise have been debated, there are few reports on the remarkable effects of the application of conventional antioxidants on exercise performance. The conventional antioxidants could not enhance exercise performance due to their rapid excretion from the body and serious adverse effects on the cellular respiratory system. In this study, impact of the original antioxidant self-assembling nanoparticle, redox-active nanoparticle (RNP), is investigated on the exercise performance of rats during running experiments. With an increase in the dose of the administered RNP, the all-out time of the rat running extends in a dose-dependent manner. In contrast, with an increase in the dose of the low-molecular-weight (LMW) antioxidant, the all-out running time of the rats decreases. The control group and LMW antioxidant treated group decrease in the number of red blood cells (RBCs) and increase oxidative stress after running. However, the RNP group maintains a similar RBC level and oxidative stress as that of the sedentary group. The results suggest that RNP, which shows long-blood circulation without disturbance of mitohormesis, effectively removes ROS from the bloodstream to suppresses RBC oxidative stress and damage, thus improving exercise performance.

Oxidative stress as an underlying mechanism of anticancer drugs cytotoxicity on human red blood cells' membrane

The aim of this study is to investigate the direct in vitro effects of anticancer drugs on red blood cells (RBCs) and to explore the underlying mechanism, mainly by measuring RBCs oxidative stress (OS) status. After RBCs direct contact with fourteen (14) anticancer drugs, several parameters were assessed including: cellular turbidity, methemoglobin (metHb) generation, released Hb and Hb stability. Moreover, intracellular Hb, considered as new molecular target of anticancer drugs, was quantified inside RBCs. MDA level, the main biomarker of OS, was simultaneously measured. The cellular turbidity reveled severe (docetaxel “TXT”, 0.03 ± 0.002), moderate (methotrexate “MTX”, 0.49 ± 0.009), or none (5-fluorouracil “5-FU”, 0.76 ± 0.029) membrane cytotoxicity (MC). An inverse relationship between cell concentration, released Hb and metHb content was obtained. High metHb generation, revealing intense OS, was also mostly expressed in paclitaxel “TXL” and etoposide “VP16”. Further, epirubicin “EPI” and “TXT” induced important oxidation of membrane lipids with 0.32 ± 0.014 and 0.26 ± 0.004, respectively. Also, MTX (0.17 ± 0.006) and doxorubicin “DOX” (0.32 ± 0.034) affected significantly Hb stability by a direct contact with molecule. These findings demonstrated that anticancer drugs have the ability to induce membrane damages by the exacerbation of OS through membrane lipid peroxidation and Hb oxidation even inside RBCs.