Halina Malgorzata Zbikowska

Generation of reactive oxygen species in blood platelets.

The generation of superoxide anion radicals (O . - 2 ) and the other reactive oxygen species (ROS) was estimated by means of cytochrome c reduction and chemiluminescence, as well in resting blood platelets and in platelets stimulated by thrombin in the presence or absence of some inhibitors of pathways involved in platelet activation. We used allopurinol (xanthine oxidase inhibitor), wortmannin (PI 3-kinase inhibitor) and staurosporine (protein kinase C inhibitor). To determine the involvement of the glutathione in ROS generation, we used L-buthionine sulfoximine (BSO) which blocks GSH synthesis. Our results confirmed that thrombin stimulates the production of ROS concomitant with metabolism of arachidonate and production of malonyldialdehyde (MDA) in blood platelets ( P < 0.05) and showed that, in the presence of inhibitors, the generation of ROS in platelets (resting and stimulated) was reduced. This indicates that xanthine oxidase, PI 3-kinase or protein kinase C take part in the formation of ROS in blood platelets. Moreover, adhesion of platelets to fibrinogen and secretion of adenine nucleotides from platelets after wortmannin and staurosporine action was also inhibited. BSO not only decreased GSH level, but also reduced the amount of ROS; a correlation between the depletion of GSH and the decrease of ROS was observed ( R = -0.987; P < 0.02). It is concluded that in blood platelets, ROS are produced in the receptor-mediated signaling pathways and platelet activation (arachidonic acid metabolism, the glutathione cycle, metabolism of phosphoinositoides and due to xanthine oxidase). Our results support the importance of ROS in platelet function.

Radical scavenging and antioxidant effects of Matricaria chamomilla polyphenolic-polysaccharide conjugates.

Matricaria chamomilla L. (MC), a member of the Asteraceae family, is one of the oldest medicinal plants, widely used worldwide for a variety of healing applications. Its recommendations, derived from both traditional and modern medicine, include numerous disorders such as inflammation, ulcers, wounds, gastrointestinal disorders, stomach ache, pharyngitis, rheumatic pain, as well as the other ailments. This work is focused on another aspect of the biological activity of chamomile polyphenolic-polysaccharide conjugates--their antioxidant properties in the protection of blood plasma components against in vitro oxidative stress. Measurements of DPPH and ABTS radical scavenging indicated considerable anti-free radical action of MC. Pre-incubation of blood plasma with MC considerably diminished the extent of ONOO(-)-induced oxidative modifications such as protein carbonyl groups, SH groups, 3-nitrotyrosine, as well as the formation of lipid hydroperoxides. The analysis of the FRAP assay result shows a considerable increase of ferric reducing ability of blood plasma in the presence of MC. The results obtained in this study indicate that polyphenolic-polysaccharide conjugates isolated from M. chamomilla substances possess antioxidant properties. The M. chamomilla macromolecular glycoconjugates may be useful in the creation of new natural-based medications or dietary supplements, helpful in the prevention and treatment of oxidative stress-mediated disorders.

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

Abstract 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.

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

Abstract 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.

Red cabbage anthocyanins as inhibitors of lipopolysaccharide-induced oxidative stress in blood platelets

LPS is a Gram-negative bacteria endotoxin, which is an important pro-inflammatory agent. Blood platelets take part both in inflammatory processes and in pathogenesis of septic shock following accumulation of LPS. As a platelet agonist LPS causes the intraplatelet overproduction of ROS/RNS that are responsible for adverse modifications in the structure of platelet compounds being associated with a development of platelet-dependent diseases. Existing evidence suggests that anthocyanins (ATH) are able to protect the circulatory system. The antioxidative properties of ATH are believed to be mainly responsible for their positive health effects. The main goal of the present in vitro study was to investigate the potential protective properties of red cabbage ATH against oxidative damage induced by LPS in blood platelets. Exposure of platelets to LPS resulted in carbonyl group increase, 3-nitrotyrosine formation, lipid peroxidation and O2(•-) generation. We have shown that ATH extract effectively decreased oxidative stress induced by LPSs. The in silico analysis demonstrated that both cyanin and LPS were located at the same region of human TLR4-MD-2 complex. Our findings suggest that there could be two-way ATH platelet protection mechanism, by their antioxidant properties and directly by binding with TLRs.

Haemostatic properties of human plasma subjected to a sterilizing dose of gamma irradiation in the presence of ascorbate.

The objective was to study the effects of gamma irradiation, in the presence of sodium ascorbate, on coagulation/fibrinolytic activity of fresh frozen plasma to be applied to inactivate the transfusion-transmitted viruses in plasma-derived products. Plasma was irradiated (50 kGy total dose, on dry ice) using a 60Co source. The plasma proteins were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blot and the following parameters estimated: prothrombin time, functional fibrinogen concentration, thrombin-induced fibrinogen polymerization, plasminogen activity, and tissue-type plasminogen activator-induced conversion of plasminogen to plasmin. In irradiated plasma a moderate fragmentation of the most labile plasma proteins was found. The prothrombin time was prolonged (1.5-fold), functional fibrinogen was significantly reduced (60%), fibrinogen polymerization was impaired, plasminogen was predominantly maintained (90%) and tissue-type plasminogen activator-induced conversion of plasminogen to plasmin was unchanged. Ascorbate (25 mmol/l) raised the level of functional fibrinogen in irradiated plasma (to 50%; P = 0.0245) and slightly accelerated its polymerization. The small protective effect of ascorbate might be due to inhibition of the radiation-induced fibrinogen oxidation and/or fragmentation but addition of other antioxidants/stabilizers would be crucial when a high irradiation dose, an effective treatment for inactivation of the most resistant viruses, is applied.

Polyphenolic-polysaccharide conjugates from plants of Rosaceae/Asteraceae family as potential radioprotectors.

Polyphenolic-polysaccharide macromolecular, water-soluble glycoconjugates, isolated from the selected medicinal plants of Rosaceae/Asteraceae family: from leaves of Fragaria vesca L., Rubus plicatus Whe. et N. E., and from flowering parts of Sanguisorba officinalis L., and Erigeron canadensis L., were investigated for their ability to protect proteins and lipids of human plasma against γ-radiation-induced oxidative damage. Treatment of plasma with plant conjugates (6, 30, 150 μg/ml) prior exposure to 100 Gy radiation resulted in a significant inhibition of lipid peroxidation, evaluated by TBARS levels; conjugates isolated from E. canadensis and R. plicatus and a reference flavonoid quercetin showed similar high potential (approx. 70% inhibition, at 6 μg/ml). The conjugates prevented radiation-induced oxidation of protein thiols and significantly improved plasma total antioxidant capacity, estimated with Ellmans reagent and ABTS(.+) assay, respectively. The results demonstrate by the first time a significant radioprotective capability of the polyphenolic-polysaccharide conjugates isolated from E. canadensis, R. plicatus, S. officinalis and to the less extent from F. vesca. The abilities of these substances to inhibit radiation-induced lipid peroxidation and thiol oxidation in plasma seems to be mediated, but not limited to ROS scavenging activity.

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.

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

Abstract 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 Ellmans 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.

The role of ascorbate and histidine in fibrinogen protection against changes following exposure to a sterilizing dose of γ-irradiation

Sodium ascorbate and histidine were employed to protect fibrinogen against modifications followed by a γ-irradiation process that could potentially inactivate the blood-borne viruses in plasma-derived products. Fibrinogen was irradiated (50 kGy total dose, on dry ice) using a 60Co source. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. Carbonyl groups were measured by the 2,4-dinitrophenylhydrazine-coupled method, and the fibrinogen clotting activity was assessed by different functional assays. In irradiated fibrinogen, the carbonyl group concentration was elevated three-fold versus control; and moderate fragmentation of largely Aα and Bβ chains was revealed. The rate of thrombin-catalyzed fibrinogen polymerization was inhibited (average 50%) with normal fibrinopeptide release and with a minor decrease of total clottable fibrinogen and α-polymer formation. Ascorbate reduced the incorporation of carbonyls to the fibrinogen molecule (by > 50% at 50 mmol/l; P < 0.001). Contrary to ascorbate, which alone delayed the fibrinogen polymerization rate, histidine abolished irradiation-induced inhibition of fibrinogen polymerization (by 80% at 50 mmol/l; P < 0.001). In conclusion, even though ascorbate effectively protects fibrinogen from oxidation due to its adverse effects on fibrinogen function, it may not serve as a suitable radioprotective. On the contrary, the first definite evidence is provided that radiation-sterilized fibrinogen in the presence of histidine greatly retains its clotting capability.