Action of pelargonidin on hyperglycemia and oxidative damage in diabetic rats: implication for glycation-induced hemoglobin modification

Glycation-modified hemoglobin in diabetes mellitus has been suggested to be a source of enhanced catalytic iron and free radicals causing pathological complications. The present study aims to verify this idea in experimental diabetes. Pelargonidin, an anthocyanidin, has been tested for its antidiabetic potential with emphasis on its role against pathological oxidative stress including hemoglobin-mediated free radical reactions. Male wistar rats were grouped as normal control, streptozotocin-induced diabetic control, normal treated with pelargonidin and diabetic treated with pelargonidin. Pelargonidin-treated rats received one time i.p injection of the flavonoid (3 mg/kg bodyweight). Biochemical parameters were assayed in blood samples of different groups of rats. Liver was used for histological examinations. Pelargonidin treatment normalized elevated blood glucose levels and improved serum insulin levels in diabetic rats. Glucose tolerance test appeared normal after treatment. Decreased serum levels of SOD and catalase, and increased levels of malondialdehyde and fructosamine in diabetic rats were reverted to their respective normal values after pelargonidin administration. Extents of hemoglobin glycation, hemoglobin-mediated iron release, iron-mediated free radical reactions and carbonyl formation in hemoglobin were pronounced in diabetic rats, indicating association between hemoglobin glycation and oxidative stress in diabetes. Pelargonidin counteracts hemoglobin glycation, iron release from the heme protein and iron-mediated oxidative damages, confirming glycated hemoglobin-associated oxidative stress in diabetes.     

A simple method to overcome the interference of hemoglobin in the detection of reporter genes in vivo

The determination of chemiluminescent intensity of reporter gene expression in vivo is generally disturbed by the presence of hemoglobin. Current methods consist in using perfusion to eliminate blood from investigated tumors or organs. In this work we propose a simple method to overcome this difficulty. The method consists in establishing an absorbance-dependence plot of the ratio R% = phi/phi(0) between the chemiluminescent intensities measured when hemoglobin is present or absent. For every measurement of the luminescent intensity phi on sample containing blood, if the absorbance A of the hemoglobin is determined, it allows one to have the intensity ratio R% which in turn gives the corrected intensity phi(0) when the absorption by hemoglobin is eliminated. The method is particularly adapted for comparative measurements of transfection levels in tumors where perfusion cannot be easily performed.     

Energy storage in the primary step of the photocycle of bacteriorhodopsin.

A pulsed-dye laser low temperature photocalorimeter is used to study the enthalpy differences between light-adapted bacteriorhodopsin (bR568) and its primary photoproduct (K) at 77 K. A key feature of our experimental method is the use of the laser-induced photostationary state as an internal reference. Analyses of the forward (bR leads to K), reverse (K leads to bR), and mixed (bR in equilibrium K) photoreactions were carried out to measure delta H12 = EK - EbR. All three experiments yielded identical values of delta H12 within experimental error (delta Have12 = 15.8 +/- 2.5 kcal mol-1). Accordingly, the primary event in the photocycle of light-adapted bacteriorhodopsin stores approximately 30% of the absorbed photon energy at the 568-nm absorption maximum. We observe that the quantum yields phi f1(bR leads to K) and phi r2(K leads to bR) add up to unity within experimental error: phi f1 + phi r2 = 1.02 +/- 0.19 for phi f1 in the range 0.28-0.33. A theoretical analysis of energy storage in K suggests that at least one-half of the enthalpy difference between K and bR is associated with charge separation accompanying chromophore isomerization.     

Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography

Since glutathionyl-hemoglobin has been suggested to be a clinical marker of oxidative stress in human blood and given the growing biological relevance of oxidative stress as a pathogenic factor in several diseases, we describe a method to measure glutathionyl-hemoglobin concentration in erythrocytes, by using cation-exchange high-pressure liquid chromatography with UV detection. The glutathionyl-hemoglobin peak has been identified on the basis of the following findings: (a) the peak increased when the sample was incubated with oxidized glutathione; (b) the peak disappeared when the sample was reduced with dithiothreitol, with the simultaneous increase of that corresponding to hemoglobin A(0); (c) the peak could be detected by incubating hemoglobin A(0) with reduced glutathione; (e) deconvoluted mass spectrum of the glutathionyl-hemoglobin peak showed a 16172.0-Da molecular mass, corresponding to hemoglobin beta bound to glutathione. Glutathionyl-hemoglobin concentration has been determined in erythrocytes of 40 healthy subjects, with a mean value of 2.58+/-0.7%, calculated as the percentage of its peak area ratio to that of total hemoglobin (HbA(0)+HbA(2)+HbA(1C)+glutathionyl-hemoglobin). The availability of a simple and reproducible method to detect glutathionyl-hemoglobin concentration in blood could be useful in monitoring oxidative stress, and for investigating the efficacy of antioxidant therapies in clinical trials.     

Oxidative stress during ex vivo hemodialysis of blood is decreased by a novel hemolipodialysis procedure utilizing antioxidants

The high cardiovascular mortality in patients receiving hemodialysis (HD) has been attributed, in part, to oxidative stress. Here we examined the effectiveness of antioxidants introduced by means of a novel hemolipodialysis (HLD) procedure in terms of reducing oxidative stress during ex vivo blood circulation. Oxidative stress was studied in a model HD system resembling the extracorporeal circulation of blood during clinical HD. Blood circulation produced an increase of up to 280% in free hemoglobin levels and an increase of 320% in electronegative LDL (LDL(-)) subfraction. A significant correlation between LDL(-) and free hemoglobin levels confirmed previous findings that LDL(-) formation during ex vivo circulation of blood can be mediated by the oxidative activity of free hemoglobin. These effects were significantly attenuated during HLD using a dialysis circuit containing vitamin E with or without vitamin C. By contrast, HLD with vitamin C alone had a marked pro-oxidant effect. TBARS, lipid hydroperoxides, vitamin E and beta-carotene content in LDL were not significantly altered by the HD procedure. These findings demonstrate the occurrence of oxidative stress in human plasma where lipoproteins are a target and indicate antioxidant-HLD treatment as a specific new approach to decreasing the adverse oxidative stress frequently associated with cardiovascular complications in high-risk populations of uremic patients.     

Posttransition state desolvation of the hydrophobic core of the src-SH3 protein domain

The folding thermodynamics of the src-SH3 protein domain were characterized under refolding conditions through biased fully atomic molecular dynamics simulations with explicit solvent. The calculated free energy surfaces along several reaction coordinates revealed two barriers. The first, larger barrier was identified as the transition state barrier for folding, associated with the formation of the first hydrophobic sheet of the protein. phi values calculated from structures residing at the transition state barrier agree well with experimental phi values. The microscopic information obtained from our simulations allowed us to unambiguously assign intermediate phi values as the result of multiple folding pathways. The second, smaller barrier occurs later in the folding process and is associated with the cooperative expulsion of water molecules between the hydrophobic sheets of the protein. This posttransition state desolvation barrier cannot be observed through traditional folding experiments, but is found to be critical to the correct packing of the hydrophobic core in the final stages of folding. Hydrogen exchange and NMR experiments are suggested to probe this barrier.     

Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells

Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The defective Hb-membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.     

Hemoglobin S-thiolation during peroxide-induced oxidative stress in chicken blood

Protein S-thiolation or protein-glutathione mixed disulfide (PSSG) occurs when cells are exposed to oxidative stress, and has been implicated in several cellular functions. The S-thiolation of hemoglobin as well as other abundant proteins is proposed to participate as a redox buffer, being part of the antioxidant protection system of the cell during the oxidative challenge. We studied the oxidative stress caused by peroxides (H(2)O(2), cumene and tert-butyl hydroperoxide) on chicken blood by measuring the thiol/disulfide status. Chicken blood under peroxide treatment showed a time- and concentration-dependent increase in glutathione disulfide (GSSG) and PSSG. GSSG peaked immediately after treatment (1 min), while PSSG increased progressively over time, showing a maximum after about 30 min. The system recovered after 140 min of incubation, with GSSG and PSSG then barely reaching control values. The S-thiolation of hemoglobin was monitored under nondenaturing PAGE, and the fraction of S-thiolated hemoglobin, or Hb A1, rose in a dose-dependent fashion and was proportional to total S-thiolation, measured as PSSG. This significant correlation indicates that hemoglobin is the major S-thiolated protein in chicken erythrocytes treated with peroxides. The present work shows the behavior of chicken blood under peroxide treatment; it anticipated that chicken hemoglobin thiol groups can actively participate in the redox processes of erythrocytes exposed to oxidative stress, and that hemoglobin is the major S-thiolated protein. This further corroborates the hypothesis that abundant proteins, such as hemoglobin, may take part in the cellular antioxidant defense system.     

Optical tweezers as a new biomedical tool to measure zeta potential of stored red blood cells

During storage, red blood cells (RBCs) for transfusion purposes suffer progressive deterioration. Sialylated glycoproteins of the RBC membrane are responsible for a negatively charged surface which creates a repulsive electrical zeta potential. These charges help prevent the interaction between RBCs and other cells, and especially among each RBCs. Reports in the literature have stated that RBCs sialylated glycoproteins can be sensitive to enzymes released by leukocyte degranulation. Thus, the aim of this study was, by using an optical tweezers as a biomedical tool, to measure the zeta potential in standard RBCs units and in leukocyte reduced RBC units (collected in CPD-SAGM) during storage. Optical tweezers is a sensitive tool that uses light for measuring cell biophysical properties which are important for clinical and research purposes. This is the first study to analyze RBCs membrane charges during storage. In addition, we herein also measured the elasticity of RBCs also collected in CPD-SAGM. In conclusion, the zeta potential decreased 42% and cells were 134% less deformable at the end of storage. The zeta potential from leukodepleted units had a similar profile when compared to units stored without leukoreduction, indicating that leukocyte lyses were not responsible for the zeta potential decay. Flow cytometry measurements of reactive oxygen species suggested that this decay is due to membrane oxidative damages. These results show that measurements of zeta potentials provide new insights about RBCs storage lesion for transfusion purposes.     

Mitochondria in exercise-induced oxidative stress

In recent years it has been suggested that reactive oxygen species (ROS) are involved in the damage to muscle and other tissues induced by acute exercise. Despite the small availability of direct evidence for ROS production during exercise, there is an abundance of literature providing indirect support that oxidative stress occurs during exercise. The electron transport associated with the mitochondrial respiratory chain is considered the major process leading to ROS production at rest and during exercise. It is widely assumed that during exercise the increased electron flow through the mitochondrial electron transport chain leads to an increased rate of ROS production. On the other hand, results obtained by in vitro experiments indicate that mitochondrial ROS production is lower in state 3 (ADP-stimulated) than in state 4 (basal) respiration. It is possible, however, that factors, such as temperature, that are modified in vivo during intense physical activity induce changes (uncoupling associated with loss of cytochrome oxidase activity) leading to increased ROS production. The mitochondrial respiratory chain could also be a potential source of ROS in tissues, such as liver, kidney and nonworking muscles, that during exercise undergo partial ischemia because of reduced blood supply. Sufficient oxygen is available to interact with the increasingly reduced respiratory chain and enhance the ROS generation. At the cessation of exercise, blood flow to hypoxic tissues resumes leading to their reoxygenation. This mimics the ischemia-reperfusion phenomenon, which is known to cause excessive production of free radicals. Apart from a theoretical rise in ROS, there is little evidence that exercise-induced oxidative stress is due to its increased mitochondrial generation. On the other hand, if mitochondrial production of ROS supplies a remarkable contribution to exercise-induced oxidative stress, mitochondria should be a primary target of oxidative damage. Unfortunately, there are controversial reports concerning the exercise effects on structural and functional characteristics of mitochondria. However, the isolation of mitochondrial fractions by differential centrifugation has shown that the amount of damaged mitochondria, recovered in the lightest fraction, is remarkably increased by long-lasting exercise.     

Model-Based Analysis of Mechanisms Responsible for Sleep-Induced Carbon Dioxide Differences

This work describes a comprehensive mathematical model of the human respiratory control system which incorporates the central mechanisms for predicting sleep-induced changes in chemical regulation of ventilation. The model integrates four individual compartments for gas storage and exchange, namely alveolar air, pulmonary blood, tissue capillary blood, body tissues, and gas transport between them. An essential mechanism in the carbon dioxide transport is its dissociation into bicarbonate and acid, where a buffering mechanism through hemoglobin is used to prevent harmfully low pH levels. In the current model, we assume high oxygen levels and consider intracellular hydrogen ion concentration as the principal respiratory control variable. The resulting system of delayed differential equations is solved numerically. With an appropriate choice of key parameters, such as velocity of blood flow and gain of a non-linear controller function, the model provides steady-state results consistent with our experimental observations measured in subjects across sleep onset. Dynamic predictions from the model give new insights into the behaviour of the system in subjects with different buffering capacities and suggest novel hypotheses for future experimental and clinical studies.     

A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo

Free radical formation in heme proteins is recognised as a factor in mediating the toxicity of peroxides in oxidative stress. As well as initiating free radical damage, heme proteins damage themselves. Under extreme conditions, where oxidative stress and low pH coincide (e.g., myoglobin in the kidney following rhabdomyolysis and hemoglobin in the CSF subsequent to subarachnoid hemorrhage), peroxide can induce covalent heme to protein cross-linking. In this paper we show that, even at neutral pH, the heme in hemoglobin is covalently modified by oxidation. The product, which we term OxHm, is a "green heme" iron chlorin with a distinct optical spectrum. OxHm formation can be quantitatively prevented by reductants of ferryl iron, e.g., ascorbate. We have developed a simple, robust, and reproducible HPLC assay to study the extent of OxHm formation in the red cell in vivo. We show that hemoglobin is oxidatively damaged even in normal blood; approximately 1 in 2,000 heme groups exist as OxHm in the steady state. We used a simple model (physical exercise) to demonstrate that OxHm increases significantly during acute oxidative stress. The exercise-induced increase is short-lived, suggesting the existence of an active mechanism for repairing or removing the damaged heme proteins.     

Disruption of iron homeostasis and lung disease

As a result of a direct exchange with the external environment, the lungs are exposed to both iron and agents with a capacity to disrupt the homeostasis of this metal (e.g. particles). An increased availability of catalytically reactive iron can result from these exposures and, by generating an oxidative stress, this metal can contribute to tissue injury. By importing this Fe³⁺ into cells for storage in a chemically less reactive form, the lower respiratory tract demonstrates an ability to mitigate both the oxidative stress presented by iron and its potential for tissue injury. This means that detoxification is accomplished by chemical reduction to Fe²⁺ (e.g. by duodenal cytochrome b and other ferrireductases), iron import (e.g. by divalent metal transporter 1 and other transporters), and storage in ferritin. The metal can subsequently be exported from the cell (e.g. by ferroportin 1) in a less reactive state relative to that initially imported. Iron is then transported out of the lung via the mucociliary pathway or blood and lymphatic pathways to the reticuloendothelial system for long term storage. This coordinated handling of iron in the lung appears to be disrupted in several acute diseases on the lung including infections, acute respiratory distress syndrome, transfusion-related acute lung injury, and ischemia–reperfusion. Exposures to bleomycin, dusts and fibers, and paraquat similarly alter iron homeostasis in the lung to affect an oxidative stress. Finally, iron homeostasis is disrupted in numerous chronic lung diseases including pulmonary alveolar proteinosis, transplantation, cigarette smoking, and cystic fibrosis.     

A novel device for the non-invasive measurement of free hemoglobin in blood bags.

The production of red blood cell concentrates from human donors is a very expensive procedure and human resources are in short supply. Under perfect storage conditions at a temperature of 2-6 degrees C, a blood bag must be used within 35-49 days (in Germany). Visual inspection of the bag for apparent hemolysis by a blood bank physician is a crucial but subjective quality control assessment. Since an interruption of the cold chain cannot be definitely ruled out, bags are often disposed of prematurely for safety reasons. There is currently no method of testing a closed blood bag with respect to hemolysis for its suitability to be used in a transfusion. The proposed optical measuring device is a hemoglobin sensor which determines the free hemoglobin in standard erythrocyte concentrates without opening the bag. The optical measurements are done on the flexible tube connected to the main bag. The optical measurements were evaluated using standard hemoglobin solutions with an accuracy of 0.005 g/dL. These investigations show that in the future each blood bag can be tested non-invasively for its content of free hemoglobin. This will contribute to decreasing the wastage rate of red blood cell concentrates.     

Oxidative modification and change of intramolecular hydrophobicity of hemoglobin A during incubation of human erythrocytes in Fenton's medium

It has been established by method of fluorescentive spectroscopy with using of zond FNA (N-phenilnaftilamine) that under incubation of human erythrocytes in Fenton's system (under intensive generation of oxygen active forms) the hydrophobicity of major hemoglobin fraction is lowered. The changes in the state of hemoglobin A intramolecular structure are correlated with formation of oxidative modification products. It has been shown that aldehydes and ketones of neutral character, which may be oxidative modification products of hydrophobic aminoacid residues, have the highest coefficient of correlation (r =-0.95).     

A monoclonal antibody detects macrophage maturation antigen which appears independently of class II antigen expression. Reactivity of monoclonal EBM11 with bovine macrophages

A mAb EBM11, raised against human macrophages [M phi] was found to detect a bovine M phi diameter-subpopulation. The Ag was strictly intracellular and was expressed in M phi only at a certain state of maturation. Its expression was regulated independently of the activation state of the cells, as revealed by treating M phi in vitro with bovine rIFN-alpha I1, IFN-gamma, or TNF-alpha, all potent M luminal diameter activators. Such treatment had no apparent effect on Ag expression. The Ag was present in 1 to 5% of peripheral blood leukocytes, i.e., up to 20% of circulating blood monocytes, in both normal noninfected cattle and in cattle persistently infected with bovine viral diarrhoea virus. Blood monocytes of the latter group were activated in vivo, but apparently did not reach a more mature state than found in noninfected animals. After an acute infection with bovine herpes virus type 1, the frequency and total number of EBM11+ cells decreased dramatically in inverse relationship to an equally significant increase in frequency and total number of circulating monocytes. In cryostat sections of normal tissues, the EBM11 mAb reacted with sinus M phi and M phi in germinal centers of lymphoid tissues, with alveolar M phi and liver Kupffer cells. In skin it reacted with few scattered M phi in the dermis, but not with epidermal Langerhans cells. This latter feature distinguishes the bovine system from the human. In virus-induced inflammatory processes in skin and keratinized epithelia EBM11+ cells constituted a subpopulation of the infiltrating M phi. The data obtained suggest that EBM11 mAb could be useful both for the elucidation of differentiation/maturation pathways of cells of the monocyte-macrophage lineage as well as for studies of M phi-virus interactions in virus infections. In either aspect cattle could provide a useful comparative model.     

Tyrosine residues as redox cofactors in human hemoglobin: implications for engineering nontoxic blood substitutes

Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and protein-based free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves "through-protein" electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the alpha and beta chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the alpha chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the beta chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.     

Mechanisms of haptoglobin protection against hemoglobin peroxidation triggered endothelial damage

Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria, and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute-phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused by free Hb are found within the cardiovascular system and Hb-triggered oxidative toxicity toward the endothelium has been promoted as a potential mechanism. The molecular mechanisms of this toxicity as well as of the protective activities of Hp are not yet clear. Within this study, we systematically investigated the structural, biochemical, and cell biologic nature of Hb toxicity in an endothelial cell system under peroxidative stress. We identified two principal mechanisms of oxidative Hb toxicity that are mediated by globin degradation products and by modified lipoprotein species, respectively. The two damage pathways trigger diverse and discriminative inflammatory and cytotoxic responses. Hp provides structural stabilization of Hb and shields Hb''s oxidative reactions with lipoproteins, providing dramatic protection against both pathways of toxicity. By these mechanisms, Hp shifts Hb''s destructive pseudo-peroxidative reaction to a potential anti-oxidative function during peroxidative stress.     

Electrophoretic patterns of hemoglobin and oxygen binding properties of blood of anostomidae fishes from Parana-Pardo-Grande hydrographic basin (S?o Paulo State, Brazil)

The electrophoretic patterns of hemoglobin (Hb) and the functional properties of blood of seven migratory fish species, in two different kinds of habitat during both the rainy and dry seasons, were studied. All species showed multiple hemoglobins and very similar electrophoretic patterns except for Schizodon nasutus, which showed two cathodal components. The functional properties of blood reflect those electrophoretic patterns, since all species showed very little or no Bohr effect during each situation studied. Data on Leporinus obtusidens blood (during the rainy season), from both lotic and lentic environments, showed a normal Bohr effect (phi = -0.073). Similarly, Schizodon nasutus and Leporinus friderici from a lotic environment during the dry season were compared and showed a reverse Bohr effect (phi = 0.042). In the other 14 situations, only Leporinus elongatus from a lentic environment during the rainy season showed a statistically significant difference in log P50 values from the other fishes.     

Evidence against a role of ketone bodies in the generation of oxidative stress in human erythrocytes by the application of reliable methods for thiol redox form detection

Aim of this study was to reconsider the previously suggested contribution of ketone bodies in causing oxidative damage in human red blood cells (RBCs) in the light of our recent findings demonstrating some methodological pitfalls that can occur during detection of hematic thiols. RBCs were incubated at 37 °C with 20 mM ketone bodies and analyzed with time for their content of glutathione, glutathione disulfide and S-glutathionylated proteins (in both the hemoglobin and membrane skeletal protein fraction). No changes in the concentrations of glutathione and its related forms were evidenced. Differently from previous reports, our results suggest that ketone bodies do not mediate generation of oxidative stress in human RBCs.