Properties Of Red Blood Cell Membrane Research Articles

Probing Red Blood Cell Membrane Microviscosity Using Fluorescence Anisotropy Decay Curves of the Lipophilic Dye PKH26.

Red blood cell (RBC) aggregation and deformation are governed by the molecular processes occurring on the membrane. Since several social important diseases are accompanied by alterations in RBC aggregation and deformability, it is important to develop a diagnostic parameter of RBC membrane structural integrity and stability. In this work, we propose membrane microviscosity assessed by time-resolved fluorescence anisotropy of the lipophilic PKH26 fluorescent probe as a diagnostic parameter. We measured the fluorescence decay curves of the PKH26 probe in the RBC membrane to establish the optimal parameters of the developed fluorescence assay. We observed a complex biphasic profile of the fluorescence anisotropy decay characterized by two correlation times corresponding to the rotational diffusion of free PKH26, and membrane-bounded molecules of the probe. The developed assay allowed us to estimate membrane microviscosity ηm in the range of 100-500 cP depending on the temperature, which paves the way for assessing RBC membrane properties in clinical applications as predictors of blood microrheological abnormalities.

Changes in Red Blood Cell Membrane Properties: The Role of Metabolic Syndrome Components

Metabolic syndrome (MetS) is a cluster of metabolic, hormonal and hemodynamic disorders that contribute to a change in the structural and functional status of erythrocytes and contribute to dysregulation of their cation transport function, where Ca2+ -dependent potassium channels (KCa channels) play an important role. A MetS model was performed using male Wistar rats, which were divided into control and experimental groups. Rats in the control group were fed standard rat chow. Rats in the experimental group were exposed to a high-fat and high-carbohydrate (HFHC) diet for 12 weeks. The data obtained indicate that the HFHC diet led to obesity, high blood pressure, hyperglycemia, impaired glucose tolerance, and dyslipidemia. The level of glutathione (GSH) decreased in the erythrocytes of rats suffering from MetS, but the level of malondialdehyde (MDA) increased. It was shown that the amplitude of the membrane potential of erythrocytes of rats with MetS changed depending on the acting agent: when stimulated with calcium ionophore A23187 it decreased, when the redox system ascorbat –phenazine methosulfate was used, it increased compared to the control group. The data obtained indicate that a HFHC diet leads to changes in the physical and chemical properties of the erythrocyte membrane.

Mechanisms of Hemolysis During Sepsis.

Cell-free hemoglobin is increasingly playing a more central role in the pathogenesis of sepsis being proved to be a potent predictor of patient's outcome. It is crucial, hence, to further investigate the mechanisms of sepsis-induced hemolysis with the aim of deriving possible therapeutic principles. Herein, we collected the most important previously known triggers of hemolysis during sepsis, which are (1) transfusion reactions and complement activation, (2) disseminated intravascular coagulation, (3) capillary stopped-flow, (4) restriction of glucose to red blood cells, (5) changes in red blood cell membrane properties, (6) hemolytic pathogens, and (7) red blood cell apoptosis. Graphical Abstract ᅟ.

The changes of red blood cell viscoelasticity and sports anemia in male 24-hr ultra-marathoners

BackgroundIn endurance sports, stress, dehydration and release of chemical factors have been associated with red blood cell (RBC) alterations of structure and function, which may contribute to sports anemia, a well-observed phenomenon during long-distance running. Until now, the investigation of the changes of viscoelastic properties of RBC membrane, a decisive factor of RBC deformability to avoid hemolysis, is lacking, especially in an Oriental population. Methodsnineteen runners were prospectively recruited into our study. Hematological parameters were analyzed before and immediately after the 2015 Taipei 24H Ultra-Marathon Festival, Taiwan. Video particle tracking microrheology was used to determine viscoelastic properties of each RBC sample by calculating the dynamic elastic modulus G′(f) and the viscous modulus G″(f) at frequency f = 20 Hz. ResultsHaptoglobin, RBC count, hemoglobin, hematocrit, mean cell hemoglobin, plasma free hemoglobin and unsaturated iron-binding capacity values of the recruited runners showed a statistically significant drop in the post-race values. Blood concentration of reticulocyte and ferritin were significantly higher at post-race compared with pre-race. 15 out of the 19 runners had a concurrent change in the elastic and the viscous moduli of their RBCs. Changes in the elastic and the viscous moduli were correlated with changes in the RBC count, hemoglobin and hematocrit. ConclusionViscoelasticity properties, the elastic modulus G′(f) and the viscous modulus G″(f) of RBCs are associated with endurance exercise-induced anemia.

Influence of Standard Laboratory Procedures on Measures of Erythrocyte Damage

The ability to characterize the mechanical properties of erythrocytes is important in clinical and research contexts: to diagnose and monitor hematologic disorders, as well as to optimize the design of cardiovascular implants and blood circulating devices with respect to blood damage. However, investigation of red blood cell (RBC) properties generally involves preparatory and processing steps. Even though these impose mechanical stresses on cells, little is known about their impact on the final measurement results. In this study, we investigated the effect of centrifuging, vortexing, pipetting, and high pressures on several markers of mechanical blood damage and RBC membrane properties. Using human venous blood, we analyzed erythrocyte damage by measuring free hemoglobin, phosphatidylserine exposure by flow cytometry, RBC deformability by ektacytometry and the parameters of a complete blood count. We observed increased levels of free hemoglobin for all tested procedures. The release of hemoglobin into plasma depended significantly on the level of stress. Elevated pressures and centrifuging also altered mean cell volume (MCV) and mean corpuscular hemoglobin (MCH), suggesting changes in erythrocyte population, and membrane properties. Our results show that the effects of blood handling can significantly influence erythrocyte damage metrics. Careful quantification of this influence as well as other unwanted secondary effects should thus be included in experimental protocols and accounted for in clinical laboratories.

Vascular dysfunction-associated with Alzheimer's disease.

Our attention is focused on the study of a new model based on the red blood cell (RBC) and on its interaction with amyloid beta peptide 1-42 (Aβ). RBC are highly deformable to assist blood flow in the microcirculation. For this reasons RBC abnormalities could contribute to Alzheimer's disease (AD) by obstructing oxygen delivery to brain, causing hypoxia. In our work, considering that RBC membrane contains, among blood elements, higher acetylcholinesterase (AChE) levels, we can assume that in blood occurs a mechanism similar to the one which occurs at the neuronal level leading to an increase of Aβ toxicity mediated by its binding with AChE, located on the RBC external face. Furthermore, since mechanical properties of RBC membrane are regulated by a number of molecular components of signalling and/or regulatory pathways, of these, particular interest has been addressed toward Nitric Oxide (NO) metabolism, due to its dependence to AChE.

Numerical computation of the elastic and mechanical properties of red blood cell membrane using the higher-order Cauchy–Born rule

This paper employs the higher-order gradient theory to study the elastic and mechanical properties of red blood cell (RBC) membrane using the higher-order Cauchy–Born rule as an atomistic-continuum constitutive model that directly incorporates the microstructure of the spectrin network. The triangulated structure of the spectrin network is used to identify a representative cell or microstructure for the model as a symmetrical hexagon, which was then used together with the coarse-grained Helmholtz free energy density to construct a strain energy density function. Effects of the area and volume constraint coefficients on elastic and mechanical properties of RBC membrane were studied by conducting numerical experiments. The dependence of the membrane properties on various microstructure parameters and temperature was also studied. Finally, we investigated the mechanical response of the RBC membrane when subjected to tensile, shear and area dilation loading conditions using a representative microstructure. The results obtained shows that the elastic and mechanical properties of the membrane vary with increase in area and volume constraint coefficients; it also shows that these elastic and mechanical properties are affected by temperature and membrane microstructure parameters, which also influence the response of the membrane under various loading conditions.

Large Deformation Properties of Red Blood Cell Membrane Based on a Higher Order Gradient Quasi-continuum Model.

Based on the proposed higher order gradient quasi-continuum model, the numerical investigations of the basic mechanical properties and deformation behaviors of human red blood cell (RBC) membrane under large deformation at room temperature (i.e., 300 K) are carried out in the present paper. The results show that RBC membrane is a nonlinear hyperelastic material. The mechanical properties of RBC membrane is dominated by isotropic nature at the stage of initial deformation, however, its anisotropic material properties emerge clearly with the loading increasing. The out-of-plane wrinkling of RBC membrane upon shear loading can be reproduced numerically. With the use of the so-called higher order Cauchy-Born rule as the kinematic description, the bending stiffness of RBC membrane can be considered conveniently.

Lipid peroxidation affects red blood cells membrane properties in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune, chronic inflammatory, non-organ specific disease with an important morbimortality affecting several organs and systems. Oxidative stress is a well documented mechanism of red blood cells (RBC) mechanical impairment. Free radicals could produced, through lipid peroxidation, physical and chemical alterations in the cellular membrane properties modifying its composition, packing and lipid distribution on the membrane erythrocyte. The aim of the present work is to study the lipid peroxidation in the RBC membrane in SLE patients (n = 42) affecting so far the lipid membrane fluidity and erythrocyte deformability in comparison with healthy controls (n = 52). Malonildialdehyde (MDA) is a subrogate assessing lipidic peroxidation, rigidity index estimating erythrocyte deformability and the anisotropy coefficient estimating lipid membrane fluidity were used. Our results show that MDA values are increased, while erythrocyte deformability and membrane fluidity are significantly decreased in erythrocyte membrane from SLE patients in comparison with normal controls. The association of thiobarbituric acid reactive substances (TBARS) with membrane lipid fluidity and erythrocyte deformability confirms that the damage of membrane properties is produced by lipid peroxidation.

Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels

Dehydrated hereditary stomatocytosis (DHS) is a genetic condition with defective red blood cell (RBC) membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations inthe mechanically activated PIEZO1(FAM38A) ion channel were associated with DHS. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated DHS cases, we identifythree novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for DHS. All the DHS-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in RBCs of DHS patients. Our findings also suggest a new role for mechanotransduction in RBC biology and pathophysiology.

Red Blood Cell Membrane Fluctuations and their Mechanisms: Passive Versus Active

Red blood cells (RBCs) are soft and flexible biconcave discs, which are able to pass through capillaries with diameters several times smaller than the RBC size. The RBC deformability also results in noticeable membrane fluctuations, which must be correlated with RBC membrane properties. However, it is still under debate whether RBC membrane fluctuations are simply passive thermal undulations or whether a red cell also experiences active fluctuations which are driven by a metabolic activity or other cell processes. We will present direct evidence that the RBC undulations are not solely passive thermal fluctuations, which has been obtained from a set of different experiments and simulations using a high spatiotemporal resolution: from 10 microseconds to several seconds in time and up to 20 nanometers in space. Experimental results show a violation of the fluctuation-dissipation theorem (FDT) for freshly prepared RBCs indicating the existence of active processes. However, the FDT is satisfied for starved cells demonstrating that the membrane fluctuations are passive when the energy supply is absent. Experiments also show a considerable change in the fluctuation amplitudes for fresh and starved cells. Subsequently, we perform simulations which fully mimic and quantify the experiments. We are able to quantitatively extract RBC membrane properties including shear elasticity, bending rigidity, and membrane viscosity. Furthermore, we test several models for active fluctuations, which mimic different possible mechanisms including spectrin network remodeling, ion pumps, and change in the spontaneous membrane curvature. Simulation results agree well with experimental data and suggest that several processes mentioned above may contribute to active RBC fluctuations. We will discuss which processes are more likely to take place.

Mechanical characteristics of human red blood cell membrane change due to C60 nanoparticle infiltration

The mechanical characteristics of human red blood cell (RBC) membrane change due to C(60) nanoparticle (NP) infiltration have been investigated in the present work. Using experimental approaches, including optical tweezer (OT) stretching and atomic force microscopy (AFM) indentation, we found that RBCs in the presence of C(60) NPs are softer than normal RBCs. The strain-stress relations of both normal and C(60) infiltrated RBC membranes are extracted from the data of AFM indentation, from which we proved that C(60) NP infiltration can affect the mechanical properties of RBC membrane and tend to weaken the tensile resistance of lipids bilayers. In order to explain this experimental phenomenon, a mechanical model has been developed. Based on this model, the strain-stress relations of both normal and C(60) infiltrated lipid bilayers are calculated with consideration of intermolecular interactions. The theoretical results are in great agreement with the experimental results. The influence of C(60) NP concentration on the mechanical properties of RBC membrane is successfully predicted. Higher concentrations of C(60) NPs in the lipid bilayers will lead to increased damage to the cell membrane, implying that the dosage of C(60) NPs should be controlled in medical applications.

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol⁻¹. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.

Combined Simulation and Experimental Study of Large Deformation of Red Blood Cells in Microfluidic Systems

We investigate the biophysical characteristics of healthy human red blood cells (RBCs) traversing microfluidic channels with cross-sectional areas as small as 2.7×3μm. We combine single RBC optical tweezers and flow experiments with corresponding simulations based on dissipative particle dynamics (DPD), and upon validation of the DPD model, predictive simulations and companion experiments are performed in order to quantify cell deformation and pressure-velocity relationships for different channel sizes and physiologically relevant temperatures. We discuss conditions associated with the shape transitions of RBCs along with the relative effects of membrane and cytosol viscosity, plasma environments, and geometry on flow through microfluidic systems at physiological temperatures. In particular, we identify a cross-sectional area threshold below which the RBC membrane properties begin to dominate its flow behavior at room temperature; at physiological temperatures this effect is less profound.

Liposomes alter thermal phase behavior and composition of red blood cell membranes

Unilamellar liposomes composed of natural phospholipids provide a new promising class of protective agents for hypothermic storage, cryopreservation, or freeze-drying of red blood cells (RBCs). In this study, FTIR spectroscopy, MALDI-TOF MS, and colorimetric assays were used to investigate the effects of liposomes composed of a homologous series of linear saturated phosphatidylcholine phospholipids (18:0; 16:0; 14:0; 12:0) on RBC membranes. RBCs were incubated with liposomes at 37°C and both the liposomal and the RBC fraction were analyzed after incubation. FTIR studies showed that liposomes composed of short acyl chain length lipids cause an increase in RBC membrane conformational disorder at suprazero temperatures, whereas long acyl chain length lipids were found to have little effects. The increased lipid conformational disorder in the RBC membranes coincided with a decrease in the cholesterol-to-phospholipid ratio. The opposite effects were found in the liposomes after incubation with RBCs. MALDI-TOF MS analysis showed the presence of short acyl chain length lipids (14:0 and 12:0) in RBC membranes after incubation, which was not observed after incubation with liposomes containing long acyl chain length lipids (18:0 and 16:0). Liposomes alter RBC membrane properties by cholesterol depletion and lipid addition.

Extracellular haemoglobin, oxidative stress and quality of red blood cells relative to perioperative blood salvage

The quality of blood-pack units in perioperative blood salvage was studied with respect to the presence of extracellular haemoglobin (Hb) and in terms of oxidative stress and the mechanical properties of red blood cells (RBC). The study was performed on blood-pack units and patients' blood after retransfusion. Results were compared to those obtained in patients prior to autotransfusion. Free Hb, non-protein thiols (as antioxidant marker) and markers of oxidative stress (conjugated dienes, thiobarbituric reactive substances and protein carbonyl content) were measured. The mechanical properties of RBC were evaluated by their osmotic fragility, membrane fluidity by measuring the fluorescence anisotropy of an extrinsic probe and permeability by measurement of extracellular K(+) and lactate dehydrogenase. Despite washing, extracellular Hb concentrations remained high (up to 0.7 g/L in blood-pack units) and was associated with a decrease of haptoglobin in patients, despite a concomitant inflammatory syndrome. In blood-pack units, we observed a decrease in antioxidant markers along with an increase in oxidative stress markers in association with an alteration of RBC membrane properties. Haemolysis must be limited during perioperative blood salvage in order to prevent exposure to oxidative stress and improve the efficiency of autotransfusion.

Plasma Dependent Reduction in Red Blood Cell Aggregation After Dextran Sulfate Low‐density Lipoprotein Apheresis—Implications for Rheological Studies

Red blood cell (RBC) aggregation is increased in familial hypercholesterolemia, and is reduced significantly after low density lipoprotein (LDL) apheresis. The purpose of the present study was to clarify whether this reduction depends on changes in plasma composition, RBC membrane properties, or both. RBC aggregation was determined in a computerized cell flow-properties analyzer, before and after LDL apheresis. We compared RBC aggregation in autologous plasma with aggregation in a plasma-free standard solution (0.5% of dextran 500 kDa) to define the separate contributions of plasma and cellular properties to the observed RBC aggregation. RBC aggregation in autologous plasma was reduced by 35.5% after LDL apheresis (P=0.01) but was not significantly affected when measured in dextran 500. This suggests that LDL apheresis attenuated RBC aggregation by altering plasma composition rather than RBC membrane properties. These results are relevant to the understanding of hemorheological changes which follow therapeutic apheresis in hypercholesterolemic patients.

Comparison of Hematologic Data in World Elite Junior Speed Skaters and in Non-Athletic Juniors

During the world junior speed skating championship 2002 all athletes (60 males, 56 females) were subjected to hematologic blood testing one day before the competition as requested by International Skating Union--ISU. This study aimed to obtain hematological reference values for junior athletes, whilst the influence of endurance training on hematologic variables of young athletes was studied. Hematologic results of athletes were compared to results of non-athletes matched by age and gender (14 males, 17 females). The blood analysis was done on an ADVIA 120. To compare measurement of ferritin, erythropoietin, and soluble transferrin receptor in serum as well as in EDTA-plasma, serum and EDTA-blood was obtained from the control group. In hemoglobin and hematocrit we found no significant difference between the two groups, whereas the number of erythrocytes was lower in athletes. The mean corpuscular volume was higher in athletes, whilst the corpuscular hemoglobin content was only marginally higher in athletes than in non-athletes. Consequently corpuscular hemoglobin concentration mean was lower in athletes than in non-athletes. There was no difference of erythropoietin and soluble transferrin receptor, whilst in ferritin we found a difference between the groups. Endurance training does not change the values of hemoglobin and hematocrit. Increased mean corpuscular volume and decreased corpuscular hemoglobin concentration mean could be a result of changed properties of red blood cell-membrane caused by acidosis and higher osmolality during the training. In junior athletes we did not find an iron overload as described in some adult athletes.

Blood Viscosity of the Neonate

After completing this article, readers should be able to: 1. List the determinants of blood viscosity in the neonate. 2. List the causes of polycythemia and hyperviscosity in the neonate. 3. List the signs, symptoms, and diagnostic criteria for polycythemia and hyperviscosity. 4. Describe steps for prevention and treatment of polycythemia and hyperviscosity in the neonate. Blood viscosity is defined as resistance to the movement of blood. In a circular vessel or tube, the resistance (R) increases with increasing viscosity (V) of the moving fluid and with the resistance resulting from the vessel geometry (Z): R=Z · V. Thus, blood viscosity describes the contribution of blood rheologic factors to blood flow resistance. However, blood viscosity depends on several factors, and the importance of these factors differs among various vessels. The major determinants of blood viscosity are the hematocrit, plasma viscosity, red blood cell (RBC) aggregation, RBC deformability, leukocyte properties, vessel diameter, and the shear forces acting on the RBC. (1) The hematocrit generally is assumed to be the most important determinant of blood viscosity. Blood viscosity rises exponentially with increasing hematocrit (Fig. 1). A marked rise in blood viscosity is seen when the hematocrit exceeds 0.65 L/L, which typically is defined as the threshold for hyperviscosity of the blood. However, in narrow arteries and arterioles and in capillaries, the plasma viscosity becomes the dominant determinant of blood viscosity. The fetal hematocrit increases from 0.33 L/L in the 12th week of gestation to 0.45 L/L in the 30th week and 0.50 L/L in the 40th week at term. At birth, the hematocrit may rise markedly as a result of “blood transfusion” from the placenta to the neonate. (2) A postnatal hematocrit of 0.45 to 0.65 L/L is considered normal in the healthy term neonate. Figure 1. Blood viscosity in tubes that have a diameter of 50 …

Alterations in human red blood cell membrane properties induced by the lipopolysaccharide from Proteus mirabilis S1959

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant ( P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly ( P<0.05) decreased the relative rotational correlation time of ISL and significantly ( P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h w/ h s ratio. These results indicated increased membrane protein mobility, mainly in the spectrin–actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.