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Chloroquine (CQ) is a 4-aminoquinoline derivative largely employed in the management of malaria. CQ treatment exploits the drug's ability to cross the erythrocyte membrane, inhibiting heme polymerase in malarial trophozoites. Accumulation of CQ prevents the conversion of heme to hemozoin, causing its toxic buildup, thus blocking the survival of Plasmodium parasites. Recently, it has been reported that CQ is able to exert antiviral properties, mainly against HIV and SARS-CoV-2. This renewed interest in CQ treatment has led to the development of new studies which aim to explore its side effects and long-term outcome. Our study focuses on the effects of CQ in non-parasitized red blood cells (RBCs), investigating hemoglobin (Hb) functionality, the anion exchanger 1 (AE1) or band 3 protein, caspase 3 and protein tyrosine phosphatase 1B (PTP-1B) activity, intra and extracellular ATP levels, and the oxidative state of RBCs. Interestingly, CQ influences the functionality of both Hb and AE1, the main RBC proteins, affecting the properties of Hb oxygen affinity by shifting the conformational structure of the molecule towards the R state. The influence of CQ on AE1 flux leads to a rate variation of anion exchange, which begins at a concentration of 2.5 µM and reaches its maximum effect at 20 µM. Moreover, a significant decrease in intra and extracellular ATP levels was observed in RBCs pre-treated with 10 µM CQ vs. erythrocytes under normal conditions. This effect is related to the PTP-1B activity which is reduced in RBCs incubated with CQ. Despite these metabolic alterations to RBCs caused by exposure to CQ, no signs of variations in oxidative state or caspase 3 activation were recorded. Our results highlight the antithetical effects of CQ on the functionality and metabolism of RBCs, and encourage the development of new research to better understand the multiple potentiality of the drug.

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BACKGROUND: Oxidative 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. PURPOSE: This study aimed to examine the protective effect of GTS on RBCs under oxidative stress damage and to determine its potential mechanism. METHODS: The 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. RESULTS: GTS 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. CONCLUSIONS: This 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.

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BACKGROUND: The triad "insulin resistance, prediabetes, diabetes" is three independent neologies with characteristic features and development. In addition, each are characterized by progression and the possibility of transition from one form to other. Due to the fact that diabetes is one of the common diseases associated with high rates of disability, it is necessary to improve diagnostic methods and educational regimens for successful prevention and treatment of the disease. OBJECTIVE: We investigated Band 3 protein (B3p) level, osmotic resistance of erythrocytes, the total antioxidant activity (TAA) of blood serum, level of HbA1 in group patients with insulin resistance (IR), prediabetes, and Type 2 Diabetes Mellitus (T2DM) and comparative with health control group. METHODS: We used original, accurate research methods that measure the essence of the studied quantities. RESULTS: Disruptions of glucose and insulin homeostasis ay lead to the initiation of oxidative stress (in our study demonstrated by a decrease of TAA of blood serum) increased redox-sensitive PTP activity and aberrant band 3 phosphorylation, potentially leading to reduced erythrocyte deformability. At the same time glycation of Hb during T2DM may affect its cross-link with membrane proteins, in particular with B3p, and although appears to limit its cross-linking and decrease its clusterization ability, induces alterations in the cytoskeletal matrix, and thereby decrease erythrocytes' osmotic resistance making them more susceptible to hemolysis. CONCLUSIONS: The osmotic resistance of the erythrocytes can be used as a sensitive marker for the detection of the early stages of hyperglycemia (prediabetes). This set of clinical trials will make it possible to identify diseases that make up the triad at an early stage. Early detection of disorders and continued research in this direction will help in the development of a diagnostic scheme for the prevention of such patients. Based on our data, research into anti-oxidation drugs is very important. With the help of the array of studies described in the article and antioxidant treatment, the likelihood of successful treatment will increase.

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Band 3 protein is an important channel protein in the erythrocyte membrane which mediates the anion transport process inside and outside the cell membrane,as well as contributes to the maintenance of erythrocyte morphology,and has important physiological functions.However,the distribution state of this protein in the primary cell membrane is not known.Cryo-scanning electron microscopy enables imaging of the surface morphology of biological samples in a near-physiological state.In order to investigate the distribution of band 3 protein on erythrocyte membranes under physiological conditions,the present study utilized 5-nm gold nanoparticles modified with the antibodies to specifically bind to the band 3 protein on human blood erythrocyte membranes and imaged them by cryo-scanning electron microscopy,to obtain distribution of band 3 protein on human blood erythrocyte membranes.The results showed that the membrane proteins on the erythrocyte membranes tended to be clustered and distributed to form ″protein islands″,and band 3 proteins were mainly distributed in these protein islands,which were tightly connected with each other to form several functional microregions to play their respective roles.

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Mercury is a toxic heavy metal widely dispersed in the natural environment. Mercury exposure induces an increase in oxidative stress in red blood cells (RBCs) through the production of reactive species and alteration of the endogenous antioxidant defense system. Recently, among various natural antioxidants, the polyphenols from extra-virgin olive oil (EVOO), an important element of the Mediterranean diet, have generated growing interest. Here, we examined the potential protective effects of hydroxytyrosol (HT) and/or homovanillyl alcohol (HVA) on an oxidative stress model represented by human RBCs treated with HgCl2 (10 µM, 4 h of incubation). Morphological changes as well as markers of oxidative stress, including thiobarbituric acid reactive substance (TBARS) levels, the oxidation of protein sulfhydryl (-SH) groups, methemoglobin formation (% MetHb), apoptotic cells, a reduced glutathione/oxidized glutathione ratio, Band 3 protein (B3p) content, and anion exchange capability through B3p were analyzed in RBCs treated with HgCl2 with or without 10 µM HT and/or HVA pre-treatment for 15 min. Our data show that 10 µM HT and/or HVA pre-incubation impaired both acanthocytes formation, due to 10 µM HgCl2, and mercury-induced oxidative stress injury and, moreover, restored the endogenous antioxidant system. Interestingly, HgCl2 treatment was associated with a decrease in the rate constant for SO42- uptake through B3p as well as MetHb formation. Both alterations were attenuated by pre-treatment with HT and/or HVA. These findings provide mechanistic insights into benefits deriving from the use of naturally occurring polyphenols against oxidative stress induced by HgCl2 on RBCs. Thus, dietary supplementation with polyphenols might be useful in populations exposed to HgCl2 poisoning.

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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.

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Aging is a process characterised by a general decline in physiological functions. The high bioavailability of reactive oxygen species (ROS) plays an important role in the aging rate. Due to the close relationship between aging and oxidative stress (OS), functional foods rich in flavonoids are excellent candidates to counteract age-related changes. This study aimed to verify the protective role of Açaì extract in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. Markers of OS, including ROS production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, as well as the anion exchange capability through Band 3 protein (B3p) and glycated haemoglobin (A1c) have been analysed in erythrocytes treated with d-Gal for 24 h, with or without pre-incubation for 1 h with 0.5-10 µg/mL Açaì extract. Our results show that the extract avoided the formation of acanthocytes and leptocytes observed after exposure to 50 and 100 mM d-Gal, respectively, prevented d-Gal-induced OS damage, and restored alterations in the distribution of B3p and CD47 proteins. Interestingly, d-Gal exposure was associated with an acceleration of the rate constant of SO42- uptake through B3p, as well as A1c formation. Both alterations have been attenuated by pre-treatment with the Açaì extract. These findings contribute to clarify the aging mechanisms in human erythrocytes and propose functional foods rich in flavonoids as natural antioxidants for the treatment and prevention of OS-related disease conditions.

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Aging is a multi-factorial process developing through a complex net of interactions between biological and cellular mechanisms and it involves oxidative stress (OS) as well as protein glycation. The aim of the present work was to verify the protective role of Quercetin (Q), a polyphenolic flavonoid compound, in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. The anion-exchange capability through the Band 3 protein (B3p) measured by the rate constant of the SO42- uptake, thiobarbituric acid reactive substances (TBARS) levels-a marker of lipid peroxidation-total sulfhydryl (-SH) groups, glycated hemoglobin (A1c), and a reduced glutathione/oxidized glutathione (GSH-GSSG) ratio were determined following the exposure of erythrocytes to 100 mM d-Gal for 24 h, with or without pre-incubation with 10 µM Q. The results confirmed that d-Gal activated OS pathways in human erythrocytes, affecting both membrane lipids and proteins, as denoted by increased TBARS levels and decreased total sulfhydryl groups, respectively. In addition, d-Gal led to an acceleration of the rate constant of the SO42- uptake through the B3p. Both the alteration of the B3p function and oxidative damage have been improved by pre-treatment with Q, which preferentially ameliorated lipid peroxidation rather than protein oxidation. Moreover, Q prevented glycated A1c formation, while no protective effect on the endogenous antioxidant system (GSH-GSSG) was observed. These findings suggest that the B3p could be a novel potential target of antioxidant treatments to counteract aging-related disturbances. Further studies are needed to confirm the possible role of Q in pharmacological strategies against aging.

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BACKGROUND: Erythrocyte aggregation is a phenomenon that is commonly found in several pathological disease states: stroke, myocardial infarction, thermal burn injury, and COVID-19. Erythrocyte aggregation is characterized by rouleaux, closely packed stacks of cells, forming three-dimensional structures. Healthy blood flow monodisperses the red blood cells (RBCs) throughout the vasculature; however, in select pathological conditions, involving hyperthermia and hypoxemia, rouleaux formation remains and results in occlusion of microvessels with decreased perfusion. OBJECTIVES: Our objective is to address the kinetics of rouleaux formation with sudden cessation of flow in variable temperature and oxygen conditions. METHODS: RBCs used in this in vitro system were obtained from healthy human donors. Using a vertical stop-flow system aligned with a microscope, images were acquired and analyzed for increased variation in grayscale to indicate increased aggregation. The onset of aggregation after sudden cessation of flow was determined at proscribed temperatures (37-49°C) and oxygen (0%, 10%), and in the presence and absence of 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Both autologous and homologous plasma were tested. RESULTS: RBCs in autologous plasma aggregate faster and with a higher magnitude with both hyperthermia and hypoxemia. Preventing deoxyhemoglobin from binding to band 3 with DIDS (dissociates the cytoskeleton from the membrane) fully blocks aggregation. Further, RBC aggregation magnitude is greater in autologous plasma. CONCLUSIONS: We show that the C-terminal domain of band 3 plays a pivotal role in RBC aggregation. Further, aggregation is enhanced by hyperthermia and hypoxemia.

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Selenium is a chalcogen element that is essential in animals, but is highly toxic when ingested above the nutritional requirement. Selenite is used as a supplement in patients receiving total parenteral nutrition. However, the therapeutic and toxic doses of selenite are separated by a narrow range. This ambivalent character of selenite implies the presence of cellular mechanisms that precisely control selenite homeostasis. Here, we investigated mechanisms that determine cellular susceptibility to selenite exposure. The resistance to selenite exposure was significantly different among cell lines. We determined the expression levels of TPMT (thiopurine S-methyltransferase) and SLC4A1 (solute carrier family 4 member 1), which encode selenium methyltransferase and selenite transporter, respectively. We also examined the effect of inhibition of Band 3 protein activity, which is encoded by SLC4A1, on the cellular sensitivity to selenite. The data suggest that the expression level of SLC4A1 is the determinant of cellular sensitivity to selenite.

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Objective: The impact of acute inflammation, revealed by C-reactive protein (CRP) plasma levels, has been studied on the erythrocytes anion exchanger Band 3 protein.Methods: Anion exchange capability through Band 3 protein, lipid peroxidation, -SH membrane groups and intracellular GSH levels have been measured on erythrocytes from patients with CRP >8 mg/L.Results: Under acute inflammation, a significant increase in anion exchange capability, increased lipid peroxidation, decreased-SH groups and GSH content were observed. Serum CRP levels recovery (after one week) was associated to -SH groups and GSH recovery, but not to anion exchange capability restoration. After 2 months, a total recovery of all parameters was observed.Conclusion: Band 3 protein anion exchange capability is affected by acute inflammation; the accelerated rate of anion exchange may be mainly due to lipid peroxidation, rather than to -SH groups oxidation; erythrocytes renewal could be needed to have a total recover of their function.

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【Objective】 To investigate the quality changes of suspended red blood cells (SRBCs) prepared from the blood of Tibetan high Hb population, and explore the availability and safety of blood collected from Tibetan high Hb population. 【Methods】 The voluntary blood donors were grouped according to the Hb concentration at the initial screening: female blood donors from Tibet Autonomous Region (>3 500 m) with Hb≥190 g/L and male blood donors with Hb≥210 g/L were classified as plateau high hemoglobin group. A total of 13 male blood donors from Tibet Autonomous Region were recruited. And the female blood donors (n=13) with Hb(115~165) g/L and male blood donors (n=12) with Hb(120~185) g/L from Chengdu were classified as control group. Whole blood of 200 mL specification was centrifuged to remove the plasma, and MAP additive solution was added to prepare SRBCs, then SRBCs were divided into four aliquots (50 mL/bag and stored at 4℃. Parameters as blood routine, free Hb and hemolysis rate were measured aseptically at day 1, 14, 21, 35 of storage. And 10 mL SRBCs was used to extract membrane proteins for tyrosine phosphorylation detection of band 3 protein. 【Results】 The RBCs counts(×1012/L), hematocrit(%) and hemoglobin(g/L) of Tibetan high Hb group and control group were 6.76±0.95 vs 4.65±0.52, 63.3±6.8 vs 43.1±4.4 and 214.4±19.8 vs 143.2±16.9 (P<0.01). The erythrocyte deformability test on the day 1, 14, 21, 35 of storage showed that the deformability of SRBCs prepared from Tibetan high Hb group was significantly lower than that of the control group under shear stress of 3, 5.33, 9.49, 16.87, and 30 Pa, while the hemolysis rate of SRBCs prepared from the Tibetan high Hb group and the control group on the day 1, 14, 21, 35 were 0.050 2±0.040 2 vs 0.022 2±0.011 1, 0.055 4±0.043 vs 0.032 1±0.028 7, 0.061 2±0.025 9 vs 0.034 3±0.031 7 and 0.069 6±0.032 0 vs 0.044 0±0.033 3 (P<0.05). Western blotting showed that the cytoplasmic N-terminal Y21 of band 3 protein of SRBCs prepared from Tibetan high Hb group was highly phosphorylated. 【Conclusion】 The deformability of SRBCs prepared from the Tibetan high Hb group was significantly lower while the hemolysis rate of SRBCs was higher than that of the control group. The hemolysis rate of the SRBCs at the end of storage prepared from the Tibetan high Hb group meets the requirements of the national standard GB18469-2012(<0.8%). The increase of hemolysis rate of SRBCs prepared from the Tibetan high Hb group was closely related to the phosphorylation of band 3 protein.

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Aging, a time-dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d-galactose ( d-Gal)-induced aging model. Anion exchange capability, measured by the rate constant of SO4²- uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d-Gal for 24 h. Our results show that: (i) in vitro application of d-Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d-Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age-related disturbances.

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The main trend of current research in neurodegenerative diseases (NDDs) is directed towards the discovery of novel biomarkers for disease diagnostics and progression. The pathological features of NDDs suggest that diagnostic markers can be found in peripheral fluids and cells. Herein, we investigated the thermodynamic behavior of the peripheral red blood cells (RBCs) derived from patients diagnosed with three common NDDs-Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) and compared it with that of healthy individuals, evaluating both fresh and aged RBCs. We established that NDDs can be differentiated from the normal healthy state on the basis of the variation in the thermodynamic parameters of the unfolding of major RBCs proteins-the cytoplasmic hemoglobin (Hb) and the membrane Band 3 (B3) protein. A common feature of NDDs is the higher thermal stability of both Hb and B3 proteins along the RBCs aging, while the calorimetric enthalpy can distinguish PD from ALS and AD. Our data provide insights into the RBCs thermodynamic behavior in two complex and tightly related phenomena-neurodegenerative pathologies and aging, and it suggests that the determined thermodynamic parameters are fingerprints of the altered conformation of Hb and B3 protein and modified RBCs' aging in the studied NDDs.

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BACKGROUND/AIMS: Hereditary Spherocytosis (HS) is the most common erythrocyte membrane disorder causing hemolytic anemia. The wide heterogeneity of both clinical and laboratory manifestations of HS contributes to difficulties associated with the diagnosis of this disorder. Although massive data previously reported worldwide, there is yet no data on HS among the Tunisian population. Here we aim to characterize HS in Tunisian patients at biochemical and cellular levels, identify the membrane protein deficiency, and compare the accuracy of the diagnostic tests to identify the most appropriate assay for HS diagnosis. METHODS: We investigated 81 patients with hemolytic anemia and 167 normal controls. The exploration of HS based on clinical and family history, physical examination, and the results of laboratory tests: blood smear, osmotic fragility test (OFT), cryohemolysis test (CT), pink test (PT), eosine-5'-maleimide (EMA) test, and erythrocyte membrane protein electrophoresis. RESULTS: We identified 21 patients with HS, classified as severe (6/21;28.5%), moderate (10/21;47.6%), and mild (5/21;23.8%). The most prevalent protein deficiency was the band 3 protein detected in ten Tunisian HS patients. The EMA test showed a high specificity (97.5%) and sensitivity (94.7%) for HS diagnosis compared to the other screening tests. Interestingly, fourteen among sixteen patients presenting with homozygous sickle cells HbSS showed an increase of EMA fluorescence intensity compared to other anemic patients. CONCLUSION: Our study highlights the efficiency of the EMA dye for the detection of HS whatever the nature of the involved protein deficiency. We report for the first time, the most prevalent protein deficiency among Tunisians with HS. Moreover, we found that the combination of the EMA-binding test with PT or incubated OFT improves the diagnosis sensitivity while maintaining a good specificity.

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Band 3 protein (B3p), anion transporter, allows the HCO3- /Cl- exchange across plasma membrane and plays an important role for erythrocytes homeostasis. In addition, B3p is linked to proteins cytoskeleton, thus contributing to cell shape and deformability, essential to erythrocytes adjustment within narrowest capillaries. Taking into account that erythrocytes are a suitable cell model to investigate the response of the oxidative stress effects, B3p functions, and specifically anion exchange capability, determining the rate constant for SO42- uptake, has been considered. As, in the latter years, rising attention has been addressed to membrane transport system, and particularly to this protein, the present mini-review has been conceived to report the most recent knowledge about B3p, with specific regard to its functions in oxidative stress conditions, including oxidative stress-related diseases.

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d-Galactose (d-Gal), when abnormally accumulated in the plasma, results in oxidative stress production, and may alter the homeostasis of erythrocytes, which are particularly exposed to oxidants driven by the blood stream. In the present investigation, the effect of d-Gal (0.1 and 10 mM, for 3 and 24 h incubation), known to induce oxidative stress, has been assayed on human erythrocytes by determining the rate constant of SO42- uptake through the anion exchanger Band 3 protein (B3p), essential to erythrocytes homeostasis. Moreover, lipid peroxidation, membrane sulfhydryl groups oxidation, glycated hemoglobin (% A1c), methemoglobin levels (% MetHb), and expression levels of B3p have been verified. Our results show that d-Gal reduces anion exchange capability of B3p, involving neither lipid peroxidation, nor oxidation of sulfhydryl membrane groups, nor MetHb formation, nor altered expression levels of B3p. d-Gal-induced %A1c, known to crosslink with B3p, could be responsible for rate of anion exchange alteration. The present findings confirm that erythrocytes are a suitable model to study the impact of high sugar concentrations on cell homeostasis; show the first in vitro effect of d-Gal on B3p, contributing to the understanding of mechanisms underlying an in vitro model of aging; demonstrate that the first impact of d-Gal on B3p is mediated by early Hb glycation, rather than by oxidative stress, which may be involved on a later stage, possibly adding more knowledge about the consequences of d-Gal accumulation.

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Mercury (Hg) is a global environmental pollutant that affects human and ecosystem health. With the aim of exploring the Hg-induced protein modifications, intact human erythrocytes were exposed to HgCl2 (1-60 µM) and cytosolic and membrane proteins were analyzed by SDS-PAGE and AU-PAGE. A spectrofluorimetric assay for quantification of Reactive Oxygen Species (ROS) generation was also performed. Hg2+ exposure induces alterations in the electrophoretic profile of cytosolic proteins with a significant decrease in the intensity of the hemoglobin monomer, associated with the appearance of a 64 kDa band, identified as a mercurized tetrameric form. This protein decreases with increasing HgCl2 concentrations and Hg-induced ROS formation. Moreover, it appears resistant to urea denaturation and it is only partially dissociated by exposure to dithiothreitol, likely due to additional protein-Hg interactions involved in aggregate formation. In addition, specific membrane proteins, including band 3 and cytoskeletal proteins 4.1 and 4.2, are affected by Hg2+-treatment. The findings reported provide new insights into the Hg-induced possible detrimental effects on erythrocyte physiology, mainly related to alterations in the oxygen binding capacity of hemoglobin as well as decreases in band 3-mediated anion exchange. Finally, modifications of cytoskeletal proteins 4.1 and 4.2 could contribute to the previously reported alteration in cell morphology.

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Hyperglycemia is considered a threat for cell homeostasis, as it is associated to oxidative stress (OS). As erythrocytes are continuously exposed to OS, this study was conceived to verify the impact of either diabetic conditions attested to by glycated hemoglobin (Hb) levels (>6.5% or higher) or treatment with high glucose (15-35 mM, for 24 h) on erythrocyte homeostasis. To this aim, anion exchange capability through the Band 3 protein (B3p) was monitored by the rate constant for SO42- uptake. Thiobarbituric acid reactive species (TBARS), membrane sulfhydryl groups mostly belonging to B3p, glutathione reduced (GSH) levels, and B3p expression levels were also evaluated. The rate constant for SO42- uptake (0.063 ± 0.001 min-1, 16 min in healthy volunteers) was accelerated in erythrocytes from diabetic volunteers (0.113 ± 0.001 min-1, 9 min) and after exposure to high glucose (0.129 ± 0.001in-1, 7 min), but only in diabetic volunteers was there an increase in TBARS levels and oxidation of membrane sulfhydryl groups, and a decrease in both GSH and B3p expression levels was observed. A combined effect due to the glycated Hb and OS may explain what was observed in diabetic erythrocytes, while in in vitro hyperglycemia, early OS could explain B3p anion exchange capability alterations as proven by the use of melatonin. Finally, measurement of B3p anion exchange capability is a suitable tool to monitor the impact of hyperglycemia on erythrocytes homeostasis, being the first line of high glucose impact before Hb glycation. Melatonin may be useful to counteract hyperglycemia-induced OS at the B3p level.