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.

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) -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.

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.