Vitamin E Analogue Protects Red Blood Cells against Storage-Induced Oxidative Damage.

BACKGROUND: To investigate i) the effects of Trolox® or mannitol, which represent two different classes of antioxidants, on oxidative changes generated in manually isolated red blood cells (RBCs) from citrate-phosphate-dextrose (CPD) preserved whole blood, followed by up to 20 days refrigerated storage, and ii) whether Trolox supplemented to the blood bank-manufactured saline-adenine-glucose-mannitol (SAGM) preserved RBC units would offer better storage conditions compared with SAGM alone. METHODS: The percentage of hemolysis and extracellular activity of lactate dehydrogenase (LDH) was measured to assess RBC membrane integrity. Lipid peroxidation, reduced glutathione (GSH) levels and total antioxidant capacity (TAC) were quantified by thiobarbituric acid-reactive substances (TBARS), Ellman's reagent and 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS.+) based assay, respectively. RESULTS: Trolox was little more effective than mannitol in protecting against progressive RBC hemolysis. Trolox (0.125-3.125 mmol/l) inhibited storage-induced leakage of LDH, lipid peroxidation, and to a lesser extent GSH depletion. Mannitol at these concentrations neither inhibited TBARS formation nor prevented GSH depletion. RBC units stored in SAGM-Trolox had significantly lower hemolysis, LDH leakage, and lipid peroxidation level compared to RBCs stored in SAGM. CONCLUSION: There is evidence of the beneficial effects of supplementing RBC-additive solutions with membrane-interacting antioxidants such as vitamin E analogues.

Influence of Pre-Storage Irradiation on the Oxidative Stress Markers, Membrane Integrity, Size and Shape of the Cold Stored Red Blood Cells.

BACKGROUND: To investigate the extent of oxidative damage and changes in morphology of manually isolated red blood cells (RBCs) from whole blood, cold stored (up to 20 days) in polystyrene tubes and subjected to pre-storage irradiation (50 Gy) and to compare the properties of SAGM-preserved RBCs stored under experimental conditions (polystyrene tubes) with RBCs from standard blood bag storage. METHODS: The percentage of hemolysis as well as the extracellular activity of LDH, thiobarbituric acid-reactive substances, reduced glutathione (GSH), and total antioxidant capacity (TAC) were measured. Changes in the topology of RBC membrane, shape, and size were evaluated by flow cytometry and judged against microscopy images. RESULTS: Irradiation caused significant LDH release as well as increased hemolysis and lipid peroxidation, GSH depletion, and reduction of TAC. Prolonged storage of irradiated RBCs resulted in phosphatidylserine exposure on the cell surface. By day 20, approximately 60% of RBCs displayed non-discoid shape. We did not notice significant differences in percentage of altered cells and cell volume between RBCs exposed to irradiation and those not exposed. CONCLUSION: Irradiation of RBC transfusion units with a dose of 50 Gy should be avoided. For research purposes such as studying the role of antioxidants, storage of small volumes of RBCs derived from the same donor would be more useful, cheaper, and blood-saving.

Vitamin C and Trolox decrease oxidative stress and hemolysis in cold-stored human red blood cells.

OBJECTIVES: To investigate the effects of sodium ascorbate (SA) (5-3125 µM) and a combination of SA and Trolox (25 and 125 µM) on oxidative changes generated in red blood cells (RBCs) followed by up to 20 days refrigerated storage. METHODS: RBCs were isolated from CPD-preserved human blood. Percentage of hemolysis and extracellular activity of lactate dehydrogenase (LDH) were measured to assess the RBC membrane integrity. Lipid peroxidation (LPO), glutathione (GSH) and total antioxidant capacity (TAC) were quantified by thiobarbituric acid-reactive substances, Ellman's reagent and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) [Formula: see text]-based assay, respectively. RESULTS: SA failed to reduce the storage-induced hemolysis and RBC membrane permeability. Addition of SA resulted in a concentration-independent LPO inhibition and increased TAC. A combination of SA/Trolox supplemented to the RBC medium significantly inhibited hemolysis, LDH leakage, LPO, GSH depletion and enhanced TAC. DISCUSSION: The effects of vitamin C action are closely concentration-dependent and may be modulated by a variety of compounds (e.g. Hb degradation products) released from RBCs during the prolonged storage, changing its properties from anti- to pro-oxidative. The two different class antioxidants (SA/Trolox) could possibly cooperate to be good potential RBC storage additives ensuring both antiradical and membrane stabilizing protection.

A moderate protective effect of quercetin against γ-irradiation- and storage-induced oxidative damage in red blood cells for transfusion.

PURPOSE: To investigate the extent of γ-irradiation-induced oxidative membrane damage and antioxidant activity of quercetin in long-term, cold stored (4°C) acid-citrate-dextrose- preserved human red blood cells (RBC). MATERIALS AND METHODS: The extracellular activity of lactate dehydrogenase (LDH) was measured to assess RBC membrane integrity. Lipid peroxidation and reduced glutathione (GSH) levels were quantified by thiobarbituric acid-reactive substances (TBARS) and Ellman's reagent, respectively. RESULTS: During storage of non-irradiated RBC (up 21 days) the LDH activity in the supernatant increased with time. In contrast to a low dose of ionizing radiation (30 Gy), irradiation at higher, but still clinically relevant doses, of 40-50 Gy resulted in elevation of the post-storage extracellular LDH activity. Quercetin (2-50 µM) dissolved in dimethyl sulfoxide (DMSO) significantly increased the LDH release in the irradiated and non-irradiated RBC, reflecting an increase of RBC membrane permeability. In the presence of ethanol as a solvent quercetin protected RBC against storage-induced oxidative damage - it inhibited the LDH release, GSH depletion, and lipid peroxidation. CONCLUSION: The level of protection offered by quercetin against the radiation- and storage-induced oxidative damage to RBC does not seem to be sufficient to warrant its application as an additive for conservation purposes. The findings indicate that the solvent can modulate a response of RBC to water-insoluble antioxidants changing their properties from anti-oxidative to pro-oxidative.

Does quercetin protect human red blood cell membranes against γ-irradiation?

OBJECTIVES: Radioprotective potential of quercetin, a powerful free radical scavenger, was investigated in human red blood cells (RBCs) and in isolated RBC membranes exposed to γ-irradiation-induced oxidative stress. METHODS: RBCs and RBC membrane suspensions were irradiated (50 Gy) in the presence of quercetin (2-50 µM). Oxidative damage of the membranes was analysed by protein carbonyl measurement (enzyme-linked immunosorbent assay). In RBCs, the concentration of glutathione (GSH) was determined. Lipid peroxidation in RBCs, and for comparison in plasma and peripheral lymphocytes, was quantified by the amount of thiobarbituric acid-reactive substances (TBARS). Radiation-induced damage of the RBC membrane integrity was evaluated by the degree of haemolysis. RESULTS: Quercetin (50 µM) brought back the level of carbonyls to normal in γ-irradiated RBC membrane proteins and inhibited radiation-induced lipid peroxidation in plasma and lymphocytes, by 75 and 96%, respectively. However, it moderately decreased reduced/oxidized glutathione (GSH/GSSG) ratio and significantly increased TBARS concentrations, by 60 and 28% in irradiated and non-irradiated RBCs, respectively. Haemolysis rate was much higher in RBCs irradiated in the presence of quercetin vs. non antioxidant. DISCUSSION: In non-cellular systems (RBC membranes or plasma) and in lymphocytes, quercetin shows antioxidative/radioprotective activity but in whole RBCs it acts as a pro-oxidant and a cytotoxic substance. The possible mechanisms of such action are discussed.

Aronia melanocarpa as a protector against nitration of fibrinogen.

Fibrinogen (Fg) also known as coagulation factor I represents about 4% of the total human plasma proteins. The main function of Fg is its involvement in last phase of blood coagulation cascade, when thrombin-induced conversion of dissolved plasma fibrinogen into an insoluble fibrin clot occurs. The reaction of fibrinogen with peroxynitrite causes both structural modifications and changes of the biological properties of this plasma glycoprotein. Recently, there is an increased interest in the screening of natural products present in fruits, vegetables and herbs for their possible antioxidative activities. Therefore, the aim of our study was to estimate the effect of extract from berries of Aronia melanocarpa against nitrative and oxidative damage induced by peroxynitrite. The extract from A. melanocarpa (0.5-50 µg/ml) added to Fg 10 min before peroxynitrite (100 µM) significantly inhibited both the formation of the high molecular weight protein aggregates and nitration of Fg molecule. The extract also abolished peroxynitrite-induced inhibition of fibrinogen polymerization (by 95% at 50 µg/ml). The obtained results indicate that natural extract from berries of A. melanocarpa has protective effects against peroxynitrite-induced nitrative damage of plasma fibrinogen, and therefore may contribute in the prevention of peroxynitrite-related cardiovascular or inflammatory diseases.

Irradiation dose-dependent oxidative changes in red blood cells for transfusion.

PURPOSE: To investigate the extent of γ-irradiation-induced oxidative protein and lipid damage in long-term (up to 21 days) cold stored (4°C) erythrocytes (RBC) and in plasma from whole blood anticoagulated with acid-citrate-dextrose (ACD-A). MATERIALS AND METHODS: Lipid peroxidation, protein carbonyl group (CO) and thiol levels were quantified by the amount of thiobarbituric acid-reactive substances (TBARS), enzyme-linked immunosorbent assay (ELISA) and with Ellman reagent, respectively. RESULTS: Irradiation (40-50 Gy) enhanced lipid peroxidation in the RBC membrane (at day 1 and after 21 storage days); the increase was storage time-dependent. In pre-irradiated (30-50 Gy) and long-term stored RBC membrane protein CO level was higher vs. non-irradiated. Irradiation resulted in RBC membrane protein thiol level elevation, most likely being a result of conformational changes and/or the polypeptide chain fragmentation. Similar to RBC, irradiation of plasma resulted in the increased TBARS generation. In plasma, significant protein CO elevation (at dose of 50 Gy) and protein thiol reduction (30-50 Gy) was observed. CONCLUSION: These findings clearly indicate that irradiation at clinically relevant doses enhances the degree of lipid peroxidation and oxidative protein damage in the membranes of stored RBC. The oxidative stress markers may be considered as additional parameters for RBC quality assessment in the blood banks.