d-Galactose Decreases Anion Exchange Capability through Band 3 Protein in Human Erythrocytes

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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Melatonin Protects Band 3 Protein in Human Erythrocytes against H2O2-Induced Oxidative Stress

Molecules ◽

10.3390/molecules24152741 ◽

2019 ◽

Vol 24 (15) ◽

pp. 2741 ◽

Cited By ~ 9

Author(s):

Rossana Morabito ◽

Alessia Remigante ◽

Angela Marino

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Protein Expression ◽

Beneficial Effect ◽

Anion Exchange ◽

Rate Constant ◽

Cell Shape ◽

Band 3 ◽

Band 3 Protein ◽

Expression Levels

The beneficial effect of Melatonin (Mel), recognized as an anti-inflammatory and antioxidant compound, has been already proven to prevent oxidative stress-induced damage associated to lipid peroxidation. As previous studies modeled the impact of oxidative stress on Band 3 protein, an anion exchanger that is essential to erythrocytes homeostasis, by applying H2O2 at not hemolytic concentrations and not producing lipid peroxidation, the aim of the present work was to evaluate the possible antioxidant effect of pharmacological doses of Mel on Band 3 protein anion exchange capability. The experiments have been performed on human erythrocytes exposed to 300 μM H2O2-induced oxidative stress. To this end, oxidative damage has been verified by monitoring the rate constant for SO4= uptake through Band 3 protein. Expression levels of this protein Mel doses lower than 100 µM have also been excluded due to lipid peroxidation, Band 3 protein expression levels, and cell shape alterations, confirming a pro-oxidant action of Mel at certain doses. On the other hand, 100 µM Mel, not provoking lipid peroxidation, restored the rate constant for SO4= uptake, Band 3 protein expression levels, and H2O2-induced cell shape alterations. Such an effect was confirmed by abolishing the endogenous erythrocytes antioxidant system. Therefore, the present findings show the antioxidant power of Mel at pharmacological concentrations in an in vitro model of oxidative stress not associated to lipid peroxidation, thereby confirming Band 3 protein anion exchange capability measurement as a suitable model to prove the beneficial effect of Mel and support the use of this compound in oxidative stress-related diseases affecting Band 3 protein.

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Effect of cadmium on anion exchange capability through Band 3 protein in human erythrocytes

Journal of Biological Research - Bollettino della Società Italiana di Biologia Sperimentale ◽

10.4081/jbr.2018.7203 ◽

2018 ◽

Vol 91 (1) ◽

Cited By ~ 1

Author(s):

Rossana Morabito ◽

Alessia Remigante ◽

Benedetta Arcuri ◽

Angela Marino ◽

Marco Giammanco ◽

...

Keyword(s):

Protein Expression ◽

Anion Exchange ◽

Rate Constant ◽

Band 3 ◽

Human Erythrocytes ◽

Band 3 Protein ◽

Toxic Heavy Metal ◽

Expression Levels ◽

Sh Groups ◽

The Impact

The efficiency of Band 3 protein, mediating HCO3-/Cl- exchange across erythrocytes membrane, is reduced by oxidative stress. The aim of the present study was to verify whether Band 3 protein efficiency is compromised by treatment with Cadmium (Cd2+), an extremely toxic heavy metal known to interfere with antioxidant enzymes, energy metabolism, gene expression and cell membranes. To this end, the rate constant for SO4= uptake through Band 3 protein (accounting for velocity of anion exchange) was measured along with membrane –SH groups, Malonyldialdehyde (MDA) and Band 3 protein expression levels in Cd2+ -treated human erythrocytes (300 µM, 1 mM). Our results show that Cd2+ reduced the rate constant for SO4= uptake, with a significant increase in MDA levels at both concentrations and with a reduction in –SH groups observed after 1 mM Cd2+ treatment, whereas Band 3 protein expression levels were unchanged in both conditions. In conclusion: i) Cd2+ reduces Band 3 protein efficiency via different mechanisms depending on metal concentration and with unchanged expression levels; ii) the assessment of Band 3 protein anion exchange capability is a good tool to assay the impact of heavy metals on cell homeostasis and, possibly, useful for diagnosis and monitoring of devalopment of Cd2+ toxicity-related pathologies.

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High Glucose Concentrations Affect Band 3 Protein in Human Erythrocytes

Antioxidants ◽

10.3390/antiox9050365 ◽

2020 ◽

Vol 9 (5) ◽

pp. 365 ◽

Cited By ~ 1

Author(s):

Rossana Morabito ◽

Alessia Remigante ◽

Sara Spinelli ◽

Giulia Vitale ◽

Vincenzo Trichilo ◽

...

Keyword(s):

Anion Exchange ◽

Rate Constant ◽

High Glucose ◽

Thiobarbituric Acid ◽

Band 3 ◽

Sulfhydryl Groups ◽

Band 3 Protein ◽

Expression Levels ◽

The Impact

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.

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Impact of acute inflammation on Band 3 protein anion exchange capability in human erythrocytes

Archives of Physiology and Biochemistry ◽

10.1080/13813455.2020.1764048 ◽

2020 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Rossana Morabito ◽

Alessia Remigante ◽

Marika Cordaro ◽

Vincenzo Trichilo ◽

Saverio Loddo ◽

...

Keyword(s):

Anion Exchange ◽

Acute Inflammation ◽

Band 3 ◽

Human Erythrocytes ◽

Band 3 Protein

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Pyridoxine Decreases Oxidative Stress on Human Erythrocyte Membrane Protein in vitro

The Open Biochemistry Journal ◽

10.2174/1874091x01913010037 ◽

2019 ◽

Vol 13 (1) ◽

pp. 37-44 ◽

Cited By ~ 1

Author(s):

Margarita Velásquez ◽

Darío Méndez ◽

Carlos Moneriz

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Membrane Protein ◽

Oxidative Damage ◽

Erythrocyte Membrane ◽

Cumene Hydroperoxide ◽

Protein Carbonylation ◽

Human Erythrocytes ◽

Red Cell Membrane

Background: Pyridoxine has reduction and prevention against the levels of reactive oxygen species in in vitro studies. However, the biochemical mechanism that explains this behavior has not yet been fully clarified. Objective: To evaluate the effect of pyridoxine against oxidative damage on the membrane of human erythrocytes. Methods: Cumene hydroperoxide was used to induce oxidative stress in protein and lipid. Human erythrocytes were incubated with pyridoxine and cumene hydroperoxide, either alone or together for 8 h. Oxidative damage was determined by measuring lipid peroxidation and membrane protein carbonylation. Results: The results indicate that the malondialdehyde concentration decreased with increasing concentration of pyridoxine. The membrane protein content also decreased with increasing concentration of vitamin B6, which was confirmed by the decreased signal intensity in the western blot when compared to control without pyridoxine. Results demonstrate that pyridoxine can significantly decrease lipid peroxidation and protein carbonylation in red cell membrane exposed to high concentrations of oxidant agent. Conclusion: Pyridoxine showed a protective effect against the oxidative stress in human erythrocytes in vitro, inhibiting the carbonylation and the oxidative damage of erythrocyte membrane proteins. To date, such an effect has not yet been reported in terms of protein oxidation.

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Natural Antioxidants Beneficial Effects on Anion Exchange through Band 3 Protein in Human Erythrocytes

Antioxidants ◽

10.3390/antiox9010025 ◽

2019 ◽

Vol 9 (1) ◽

pp. 25 ◽

Cited By ~ 6

Author(s):

Alessia Remigante ◽

Rossana Morabito ◽

Angela Marino

Keyword(s):

Anion Exchange ◽

Natural Antioxidants ◽

Band 3 ◽

Erythrocyte Membranes ◽

Band 3 Protein ◽

Ion Distribution ◽

Beneficial Effects ◽

Cross Links

Band 3 protein (B3p) exchanging Cl− and HCO3− through erythrocyte membranes is responsible for acid balance, ion distribution and gas exchange, thus accounting for homeostasis of both erythrocytes and entire organisms. Moreover, since B3p cross links with the cytoskeleton and the proteins underlying the erythrocyte membrane, its function also impacts cell shape and deformability, essential to adaptation of erythrocyte size to capillaries for pulmonary circulation. As growing attention has been directed toward this protein in recent years, the present review was conceived to report the most recent knowledge regarding B3p, with specific regard to its anion exchange capability under in vitro oxidative conditions. Most importantly, the role of natural antioxidants, i.e., curcumin, melatonin and Mg2+, in preventing detrimental oxidant effects on B3p is considered.

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Effect of toluene on erythrocyte membrane stability under in vivo and in vitro conditions with assessment of oxidant/antioxidant status

Toxicology and Industrial Health ◽

10.1177/0748233709346758 ◽

2009 ◽

Vol 25 (8) ◽

pp. 545-550 ◽

Cited By ~ 15

Author(s):

Ismail Karabulut ◽

Z. Dicle Balkanci ◽

Bilge Pehlivanoglu ◽

Aysen Erdem ◽

Ersin Fadillioglu

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Unsaturated Fatty Acids ◽

Osmotic Fragility ◽

Human Erythrocytes ◽

Antioxidant Enzyme Activities ◽

Oxidative Stress Parameters ◽

Stress Parameters

Toluene, an organic solvent used widely in the industry, is highly lipophilic and accumulates in the cell membrane impeding transport through it. Its metabolites cause oxygen radical formation that react with unsaturated fatty acids and proteins in erythrocytes leading to lipid peroxidation and protein breakdown. In this study, we aimed to investigate the membrane stabilizing and the oxidative stress—inducing effects of toluene in human erythrocytes. Measurements of osmotic fragility, mean corpuscular volume (MCV), oxidative stress parameters and antioxidant enzyme activities were performed simultaneously both in individuals exposed to toluene professionally (in vivo) and human erythrocytes treated with toluene (in vitro). To measure osmotic fragility, erythrocytes were placed in NaCl solutions at various concentrations (0.1% [blank], 0.38%, 0.40%, 0.42%, 0.44%, 0.46%, 0.48% and 1% [stock]). Percentage of haemolysis in each solution was calculated with respect to the 100% haemolysis in the blank solution. The erythrocyte packs prepared at the day of the above-mentioned measurements were kept at —80°C until the time for determination of malonyldialdehyde and protein carbonyl levels, and catalase (CAT) and glutathione peroxidase activities as indicators of oxidative stress. Toluene increased oxidative stress parameters significantly both in vivo and in vitro; it also caused a significant decrease in the activities of antioxidant enzymes. Osmotic fragility was altered only in the case of in vitro exposure. In conclusion, toluene exposure resulted in increased lipid peroxidation and protein damage both in vivo and in vitro. Although, it is natural to expect increased osmotic fragility due to oxidative properties of toluene, its membrane-stabilizing effect overcame the oxidative properties leading to decreased osmotic fragility or preventing its deterioration in vitro and in vivo toluene exposures, respectively, in the present study.

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