t-BOOH-induced oxidative damage in sickle red blood cells and the role of flavonoids

Sickle cell anemia is a genetic disease characterized by an increase in generation of reactive oxygen species, abnormal iron release and low antioxidant activity which can lead to cell injury. Several therapies have been used to decrease the oxidative damage in these patients. In this study, we investigated the effect of flavonoids (quercetin and rutin) on the oxidation of red blood cells (RBC) from sickle cell anemia patients following exposure of the cells to tert-butyl hydroperoxide (t-BOOH). Quercetin provided greater protection against Hb oxidation, the binding of Hb to membrane and lipid peroxidation than did rutin. Quercetin (150 muM) reduced Hb oxidation by 30% and increased the level of oxyHb from 17.5 to 29 muM. Rutin prevented Hb oxidation only at concentrations higher than 200 muM and did not prevent the binding of Hb to RBC membrane. These distinct effects of the flavonoids probably reflect their structural characteristics. Thus, quercetin, which possesses a suitable structure for free-radical scavenging and ion quelation, was a more effective antioxidant than rutin. The presence of rutinose at position C-3 in rutin may impair its antioxidant effect. The presence of ascorbic acid enhanced the protective effect of quercetin and rutin against oxidative stress in sickle Hb and lipid peroxidation. This synergistic action helped to maintain a constant supply of flavonoids and thus, rescue the cells from the injury caused by free radicals and iron ions. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.574173212412

Effect of thiol reagents on functional properties and heme oxidation in the hemoglobin of Geochelone carbonaria

The reaction of thiol reagents with G. carbonaria hemoglobin was studied, and the oxygen equilibrium and kinetic of oxidation of derivatives determined. The oxygen affinity and kinetic of oxidation of hemoglobin derivatives were modified to various extents depending on the nature of thiol reagents used. Diamide yielded approximately 80% polymeric hemoglobin, although the oxidation kinetic, and the functional properties, were practically invariant (T-1/2 =10.0 min.; P50 = 5.0 mm Hg at pH 7.4; alkaline Bohr effect = -0.64). Iodoacetamide did not modify the electrophoretic pattern significantly, although all the free SH groups of hemoglobin were alkylated. A P50 of 2.5 mmHg at pH 7.4 and the Bohr effect of -0.15 were obtained; the T-1/2 Of about 6.4 min. was shorter than that for un-modified Hb. Similar T-1/2 were obtained for Hb treated with oxidized glutathione, which produced polymeric Hb and glutathionyl-Hb. The oxygen binding characteristics showed that both of Hb derivatives, glutathionyl-Hb and polymeric Hb, maintain the capacity to transport the gas.40235536

T-booh-induced Oxidative Damage In Sickle Red Blood Cells And The Role Of Flavonoids.

Sickle cell anemia is a genetic disease characterized byan increase in generation of reactive oxygen species, abnormal iron release and low antioxidant activity which can lead to cell injury. Several therapies have been used to decrease the oxidative damage in these patients. In this study, we investigated the effect of flavonoids (quercetin and rutin) on the oxidation of red blood cells (RBC) from sickle cell anemia patients following exposure of the cells to tert-butyl hydroperoxide (t-BOOH). Quercetin provided greater protection against Hb oxidation, the binding of Hb to membrane and lipid peroxidation than did rutin. Quercetin (150 microM) reduced Hb oxidation by 30% and increased the level of oxyHb from 17.5 to 29 microM. Rutin prevented Hb oxidation only at concentrations higher than 200 microM and did not prevent the binding of Hb to RBC membrane. These distinct effects of the flavonoids probably reflect their structural characteristics. Thus, quercetin, which possesses a suitable structure for free-radical scavenging and ion quelation, was a more effective antioxidant than rutin. The presence of rutinose at position C(3) in rutin may impair its antioxidant effect. The presence of ascorbic acid enhanced the protective effect of quercetin and rutin against oxidative stress in sickle Hb and lipid peroxidation. This synergistic action helped to maintain a constant supply of flavonoids and thus, rescue the cells from the injury caused by free radicals and iron ions.57124-

Quercetin diminishes the binding of hemoglobin to the red blood cell membrane

Hemoglobin (Hb) oxidation leads to the formation of hemichrome, which binds to the membrane and causes red blood cell removal by the reticuloendothelial system. In the present investigation, the effect of flavonoids on Hb oxidation and their binding to red blood cell (RBC) membranes were studied using tert-butyl hydroperoxide (tert-BOOH) to promote oxidative stress. The intrinsic antioxidant activity of RBC was able to prevent the binding of Hb to the membrane at tert-BOOH concentrations up to 0.4 mM. At higher concentrations, a brown pellet was observed and represented the appearance of membrane-bound oxidized Hb. Oxidations performed in membrane-free Hb solutions with an identical oxidative system showed less Hb oxidation. These observations suggest that erythrocyte membrane lipid peroxidation enhances the oxidative damage of Hb, increasing its binding to membranes. Quercetin partially protected Hb against oxidation by tert-BOOH and reduced the levels of the membrane bound hemichrome. Lipid peroxidation was also significantly suppressed by quercetin. Rutin and morin had little effect in preventing Hb binding to RBC membranes, indicating the importance of structure in the antioxidant properties of flavonoids. In the absence of oxidant, the peroxidation of erythrocyte membrane and isotonic hemolysis were protected by quercetin. These results suggest that quercetin displays a beneficial role on aging of RBC.41556

Effect Of Thiol Reagents On Functional Properties And Heme Oxidation In The Hemoglobin Of Geochelone Carbonaria.

The reaction of thiol reagents with G. carbonaria hemoglobin was studied, and the oxygen equilibrium and kinetic of oxidation of derivatives determined. The oxygen affinity and kinetic of oxidation of hemoglobin derivatives were modified to various extents depending on the nature of thiol reagents used. Diamide yielded approximately 80% polymeric hemoglobin, although the oxidation kinetic, and the functional properties, were practically invariant (T1/2 = 10.0 min.; P50 = 5.0 mm Hg at pH 7.4; alkaline Bohr effect = -0.64). Iodoacetamide did not modify the electrophoretic pattern significantly, although all the free SH groups of hemoglobin were alkylated. A P50 of 2.5 mmHg at pH 7.4 and the Bohr effect of -0.15 were obtained; the T1/2 of about 6.4 min. was shorter than that for un-modified Hb. Similar T1/2 were obtained for Hb treated with oxidized glutathione, which produced polymeric Hb and glutathionyl-Hb. The oxygen binding characteristics showed that both of Hb derivatives, glutathionyl-Hb and polymeric Hb, maintain the capacity to transport the gas.40355-6

Quercetin Diminishes The Binding Of Hemoglobin To The Red Blood Cell Membrane

Hemoglobin (Hb) oxidation leads to the formation of hemichrome, which binds to the membrane and causes red blood cell removal by the reticuloendothelial system. In the present investigation, the effect of flavonoids on Hb oxidation and their binding to red blood cell (RBC) membranes were studied using tert-butyl hydroperoxide (tert-BOOH) to promote oxidative stress. The intrinsic antioxidant activity of RBC was able to prevent the binding of Hb to the membrane at tert-BOOH concentrations up to 0.4 mM. At higher concentrations, a brown pellet was observed and represented the appearance of membrane-bound oxidized Hb. Oxidations performed in membrane-free Hb solutions with an identical oxidative system showed less Hb oxidation. These observations suggest that erythrocyte membrane lipid peroxidation enhances the oxidative damage of Hb, increasing its binding to membranes. Quercetin partially protected Hb against oxidation by tert-BOOH and reduced the levels of the membrane bound hemichrome. Lipid peroxidation was also significantly suppressed by quercetin. Rutin and morin had little effect in preventing Hb binding to RBC membranes, indicating the importance of structure in the antioxidant properties of flavonoids. In the absence of oxidant, the peroxidation of erythrocyte membrane and isotonic hemolysis were protected by quercetin. These results suggest that quercetin displays a beneficial role on aging of RBC.415563Luna, E.J., Kidd, G.H., Branton, D., Identification by peptide analysis of the spectrin-binding protein in human erythrocytes (1979) J Biol Chem, 254, pp. 2526-2532Hargreaves, W., Giedd, K.N., Verkleij, A., Reassociation of ankyrin with band 3 in erythrocyte membranes and in lipid vesicles (1980) J Biol Chem, 255, pp. 11965-11972Bennett, V., Stenbuck, P.J., The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membrane (1979) Nature, 280, pp. 468-473Bennett, V., Stenbuck, P.J., Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane (1980) J Biol Chem, 255, pp. 6424-6432Shaklai, N., Yguerabide, J., Ranney, H.M., Classification and localization of hemoglobin binding sites on the red blood cell membrane (1977) Biochemistry, 16, pp. 5585-5592Salhany, J.M., Cordes, K.A., Gaines, E.D., Light-scattering measurements of hemoglobin binding to the erythrocyte membrane. Evidence for transmembrane effects related to a disulfonic stilbene binding to band 3 (1980) Biochemistry, 19, pp. 1447-1454Waugh, S.M., Low, P.S., Hemichrome binding to band 3: Nucleation of Heinz bodies on the erythrocyte membrane (1985) Biochemistry, 24, pp. 34-39Kiefer, C.R., Trainor, J.F., McKenney, J.B., Hemoglobin-spectrin complexes: Interference with spectrin tetramer assembly as a mechanism for compartmentalization of band 1 and band 2 complexes (1995) Blood, 86, pp. 366-371Bates, D., Winterbourn, C.C., Haemoglobin denaturation, lipid peroxidation and haemolysis in phenylhydrazine-induced anaemia (1984) Biochim Biophys Acta, 798, pp. 84-87Snyder, L.M., Fortier, N.L., Trainor, J., Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking (1985) J Clin Invest, 76, pp. 1971-1977Fortier, N., Snyder, L.M., Garver, F., The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity (1988) Blood, 71, pp. 1427-1431Itano, H.A., Hirota, K., Vedvick, T.S., Ligands and oxidants in ferrihemichrome formation and oxidative hemolysis (1977) Proc Natl Acad Sci USA, 74, pp. 2556-2560French, J.K., Winterboum, C.C., Carrell, R.W., Mechanism of oxyhaemoglobin breakdown on reaction with acetylphenylhydrazine (1978) Biochem J, 173, pp. 19-26Trotta, R.J., Sullivan, S.G., Stern, A., Lipid peroxidation and hemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Dependence on glucose metabolism and hemoglobin status (1981) Biochim Biophys Acta, 678, pp. 230-237Van Der Zee, J., Van Steveninck, J., Koster, J.F., Inhibition of enzymes and oxidative damage of red blood cells induced by t-butylhydroxperoxide-derived radicals (1989) Biochim Biophys Acta, 980, pp. 175-180Slater, T., Free radical mechanisms in tissue injury (1984) Biochem J, 222, pp. 1-15Pryor, W., Oxy-radicals and related species: Their formation, lifetimes, and reactions (1986) Annu Rev Physiol, 48, pp. 657-667Yagi, K., Lipid peroxidation and human diseases (1987) Chem Phys Lipids, 45, pp. 337-351Hollman, P.C.H., Katan, M.B., Absorption, metabolism and health effects of dietary flavonoids in man (1997) Biomed Pharmacother, 51, pp. 305-310Van Aker, F.A.A., Schouten, O., Haenen, G.R.M.M., Flavonoids can replace α-tocopherol as an antioxidant (2000) FEBS Lett, 473, pp. 145-148Bors, W., Michel, C., Saran, M., Flavonoid antioxidants: Rate constants for reactions with oxygen radicals (1994) Methods Enzymol, 234, pp. 420-429Jovanovic, S.V., Steenken, S., Tosic, M., Flavonoids as antioxidants (1994) J Am Chem Soc, 116, pp. 4846-4851Ratty, A.K., Das, N.P., Effects of flavonoids on nonenzymatic lipid peroxidation: Structure-activity relationship (1988) Biochem Med Metab Biol, 39, pp. 69-79Laughton, M.J., Evans, P.J., Moroney, M.A., Inhibition of mammalian 5-lipooxygenase and cyclooxygenase by flavonoids and phenolic dietary additives. Relationship to antioxidant activity and to iron ion-reducing ability (1991) Biochem Pharmacol, 42, pp. 1673-1681Decharneux, T., Dubois, F., Beauloye, C., Effect of various flavonoids on lysosomes subjected to an oxidative or an osmotic stress (1992) Biochem Pharmacol, 44, pp. 1243-1248Korkina, L.G., Afanas'ev, I.B., Antioxidant and chelating properties of flavonoids (1997) Adv Pharmacol, 38, pp. 151-163Afanas'ev, I.B., Dorozhko, A.I., Brodskii, A.V., Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation (1989) Biochem Pharmacol, 38, pp. 1763-1769Saija, A., Scalese, M., Lanza, M., Flavonoids as antioxidant agents: Importance of their interaction with biomembranes (1995) Free Radic Biol Med, 19, pp. 481-486Ferrali, M., Signorini, C., Caciotti, B., Protection against oxidative damage of erythrocyte membrane by the flavonoid quercetin and its relation to iron chelating activity (1997) FEBS Lett, 416, pp. 123-129Winterbourn, C.C., Oxidative reactions of hemoglobin (1990) Methods Enzymol, 186, pp. 265-272Stocks, J., Normandy, T.L., The autoxidation of human red cell lipids induced by hydrogen peroxide (1971) Br J Haematol, 20, pp. 95-111Stern, A., Red cell oxidative damage (1985) Oxidative Stress, pp. 75-90. , Sies H, ed. 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Citrate diminishes hypothalamic acetyl-CoA carboxylase phosphorylation and modulates satiety signals and hepatic mechanisms involved in glucose homeostasis in rats

The hypothalamic AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway is known to play an important role in the control of food intake and energy expenditure. Here, we hypothesize that citrate, an intermediate metabolite, activates hypothalamic ACC and is involved in the control of energy mobilization. Initially, we showed that ICV citrate injection decreased food intake and diminished weight gain significantly when compared to control and pair-fed group results. In addition, we showed that intracerebroventricular (ICV) injection of citrate diminished (80% of control) the phosphorylation of ACC, an important AMPK substrate. Furthermore, citrate treatment inhibited (75% of control) hypothalamic AMPK phosphorylation during fasting. In addition to its central effect, ICV citrate injection led to low blood glucose levels during glucose tolerance test (GTT) and high glucose uptake during hyperglycemic-euglycemic clamp. Accordingly, liver glycogen content was higher in animals given citrate (ICV) than in the control group (23.3 +/- 2.5 vs. 2.7 +/- 0.5 mu g mL(-1) mg(-1), respectively). Interestingly, liver AMPK phosphorylation was reduced (80%) by the citrate treatment. The pharmacological blockade Of beta(3)-adrenergic receptor (SR 59230A) blocked the effect of ICV citrate and citrate plus insulin on liver AMPK phosphorylation. Consistently with these results, rats treated with citrate (ICV) presented improved insulin signal transduction in liver, skeletal muscle, and epididymal fat pad. Similar results were obtained by hypothalamic administration of ARA-A, a competitive inhibitor of AMPK. Our results suggest that the citrate produced by mitochondria may modulate ACC phosphorylation in the hypothalamus, controlling food intake and coordinating a multiorgan network that controls glucose homeostasis and energy uptake through the adrenergic system. (c) 2008 Elsevier Inc. All rights reserved.8225-261262127

TNF-alpha acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient - Effects on leptin and insulin signaling pathways

Acting in the hypothalamus, tumor necrosis factor-alpha (TNF-alpha) produces a potent anorexigenic effect. However, the molecular mechanisms involved in this phenomenon are poorly characterized. In this study, we investigate the capacity of TNF-alpha to activate signal transduction in the hypothalamus through elements of the pathways employed by the anorexigenic hormones insulin and leptin. High dose TNF-a promotes a reduction of 25% in 12 h food intake, which is an inhibitory effect that is marginally inferior to that produced by insulin and leptin. In addition, high dose TNF-a increases body temperature and respiratory quotient, effects not reproduced by insulin or leptin. TNF-alpha, predominantly at the high dose, is also capable of activating canonical pro-inflammatory signal transduction in the hypothalamus, inducing JNK, p38, and NF kappa B, which results in the transcription of early responsive genes and expression of proteins of the SOCS family. Also, TNF-a activates signal transduction through JAK-2 and STAT-3, but does not activate signal transduction. through early and intermediary elements of the insulin/leptin signaling pathways such as IRS-2, Akt, ERK and FOXO1. When co-injected with insulin or leptin, TNF-a, at both high and low doses, partially impairs signal transduction through IRS-2, Akt, ERK and FOXO1 but not through JAK-2 and STAT-3. This effect is accompanied by the partial inhibition of the anorexigenic effects of insulin and leptin, when the low, but not the high dose of TNF-alpha is employed. In conclusion, TNF-alpha, on a dose-dependent way, modulates insulin and leptin signaling and action in the hypothalamus. (c) Published by Elsevier Inc.2851050105

A novel beta-globin variant: Hb Pocos de Caldas [beta 61(E5)Lys -> Gln]

A novel hemoglobin (Hb) variant was found during a screening program for hemoglobinopathies in blood donors at the Hematology Center of the State University of Campinas, Campinas, Brazil. The carrier was a 30-year-old Caucasian woman of Native Indian and Italian origin, born in Pocos de Caldas, a city in the State of Minas Gerais, Southeastern Brazil. The new Hb was distinguished as an abnormal band, faster than Hb A, at alkaline pH (Fig. I a), that moved like Hb A at acid pH.([1]) Isoelectrofocusing (IEF) with an REP Hemoglobin IEF Kit (Helena Laboratories, Beaumont, TX, USA), at pH ranges varying from 6.0 to 8.0, showed a fast band moving to the anode (Fig. 1b). Urea Triton X-100 polyacrylamide gel electrophoresis([2]) showed the presence of a beta chain variant that was barely discernible from the normal beta chain (Fig. 1c). Stability tests([1]) were normal. The Hb oxygen equilibria were performed by the method of Rossi-Fanelli and Antonini,([3]) in stripped Hb and in the presence of organic polyphosphate at a pH between 7.0 and 8.0. Corroborating the clinical manifestation, Hb Pocos de Caldas showed functional properties similar to that of normal Hb. [GRAPHICS] Molecular analyses involved the amplification of a 661 bp fragment of the beta-globin gene, using primers P1 (5'-GCCAAGGACAGGTACGGCTGTCATC-3') DNA opposite strand and familial analysis, which revealed that the carrier's mother, brother, and sister also have the same beta-globin alteration (see Table 1). This is the fourth description of a mutation at beta61(E5)Lys: Hb N-Seattle (-->Glu) was found in a Black American blood donor,([5]) Hb Hikari (-->Asn) detected in a Japanese family([6]) and Hb Bologna (-->Met) encountered in a North Italian family.([7]) The mutations found at this position (external contacts of the Hb molecule) do not cause clinical manifestations, since all the carriers described so far have been asymptomatic.26438538