Red blood cell membrane defects

We present an overview of the currently known molecular basis of red cell membrane disorders. A detailed discussion of the structure of the red cell membrane and the pathophysiology and clinical aspects of its disorders is reported. Generally speaking, hereditary spherocytosis (HS) results from a loss of erythrocyte surface area. The mutations of most cases of HS are located in the following genes: ANK1, SPTB, SLC4A1, EPB42 and SPTA1, which encode for ankyrin, spectrin beta-chain, the anion exchanger 1 (band 3), protein 4.2 and spectrin alpha-chain, respectively. Hereditary elliptocytosis (HE) reflects a diminished elasticity of the skeleton. Its aggravated form, hereditary pyropoikilocytosis (HPP), implies that the skeleton undergoes further destabilization. The mutations responsible for HE and HPP, lie in the SPTA1 and SPTB gene, and in the EPB41 gene encoding protein 4.1. Allele alpha LELY is a common polymorphic allele, which plays the role of an aggravating factor when it occurs in trans of an elliptocytogenic allele of the SPTA1 gene. Southeast Asian ovalocytosis derives from a change in band 3. The genetic disorders of membrane permeability to monovalent cations required a positional cloning approach. In this respect, channelopathies represent a new frontier in the field. Dehydrated hereditary stomatocytosis (DHS) was shown to belong to a pleiotropic syndrome: DHS + fetal edema + pseudohyperkalemia, which maps 16q23-24. Splenectomy is strictly contraindicated in DHS and another disease of the same class, overhydrated hereditary stomatocytosis, because it increases the risk of thromboembolic accidents.     

Red blood cell magnetophoresis

The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.     

Red blood cell membrane fluctuations and shape controlled by ATP-induced cytoskeletal defects

We show theoretically how adenosine 5'-triphosphate (ATP)-induced dynamic dissociations of spectrin filaments (from each other and from the membrane) in the cytoskeleton network of red blood cells (RBC) can explain in a unified manner both the measured fluctuation amplitude as well as the observed shape transformations as a function of intracellular ATP concentration. Static defects can be induced by external stresses such as those present when RBCs pass through small capillaries. We suggest that the partially freed actin at these defect sites may explain the activation of the CFTR membrane-bound protein and the subsequent release of ATP by RBCs subjected to deformations. Our theoretical predictions can be tested by experiments that measure the correlation between variations in the binding of actin to spectrin, the activity of CFTR, and the amount of ATP released.     

Red cell membrane elasticity as determined by flow channel technique.

The elasticity of red cell membrane was determined in a rectangular flow channel under controlled shear flow. The relation between shear stress and cell extension ratio (lambda) has been analyzed with the use of Evans' two-dimensional model. The deformed cell shapes observed experimentally agreed well with the model with lambda up to 1.4. The best correlation was found at lambda = 1.2. The analysis suggests a nonlinear extensional membrane modulus in the low stress range encountered in the flow channel. In terms of an appropriate strain parameter, the elastic modulus is shown to rise toward the level encountered in micropipette aspiration experiments. The implications of the present findings in modeling of cell mechanics and in cell hemolysis are discussed.     

Red cell membrane lipids in hemoglobinopathies

The complex mixture of lipids and proteins of the red blood cell membrane is well maintained during the life of the cell. Lipid analysis of the red cell reveals hundreds of phospholipid molecular species and cholesterol that differ with respect to their (polar) head group, and (apolar) side chains. These molecules move rapidly in the plane, as well as across the lipid bilayer. This dynamic movement is highly organized. In the plane of the bilayer, areas enriched in certain lipids accommodate protein structure and modulate function. While lipids move across the bilayer, the organization is highly asymmetric. Amino phospholipids are mainly found on the inside and choline containing phospholipids on the outside. Both the composition and organization of the red cell membrane is maintained throughout the life of the red cell by an intricate mechanism that involves enzymes, transporters and cytosolic factors. Key proteins that maintain red blood cell lipid organization have recently been identified. Alterations in these mechanisms, as the result of the globin mutations in sickle cell disease or thalassemia will lead to loss of membrane viability, apoptosis during erythropoiesis, early demise of the cell in the circulation, and when these cells are not removed appropriately their presence has pathologic consequences.     

Red blood cell fatty acids are associated with depression in a case-control study of adolescents

IntroductionEpidemiological studies suggest that reduced intakes and/or blood levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are associated with increased risk for depression in adults, but data on adolescents are scarce. The objective of this study was to determine whether red blood cell (RBC) levels of EPA+DHA (the omega-3 index) and/or the overall RBC fatty acid profile differ between depressed adolescents (cases) and non-depressed adolescents (controls).Patients and MethodsWe measured the RBC fatty acid composition of cases admitted to the hospital for depression (n=150) and compared it to that of controls (n=161).ResultsCases and controls had similar ages, gender proportions, and body mass index (BMI) distributions, but there was a significant difference in racial/ethnic composition due to differences in recruitment sites. The unadjusted odds ratio for case status was 0.72 (95% CI; 0.55–0.95) for a 1% absolute increase in the omega-3 index. A multivariable logistic regression model was used to determine which fatty acids were useful in classifying cases and controls; BMI, age, gender, and race/ethnicity were forced into the model. Seven fatty acids were selected (DHA, myristic, stearic, oleic, trans linoleic, trans palmitoleic, and alpha-linolenic acids) to optimize the model fit to the data. In the adjusted model, the odds ratio was 0.67 (95% CI; 0.49–0.93) for a 1 SD increase in DHA. Adding the seven fatty acid profile to the basic model increased the area under the ROC curve by 12.6% (7.5%–17.6%).Discussion and ConclusionThese findings support the hypothesis that adolescent depression is associated with a perturbed RBC fatty acid pattern which includes a reduced omega-3 index. Intervention studies with EPA and DHA should be conducted in this vulnerable population for which few, safe therapeutic options currently exist.     

Red blood cell membrane water permeability increases with length of ex vivo storage

Water transport across the red blood cell (RBC) membrane is an essential cell function that needs to be preserved during ex vivo storage. Progressive biochemical depletion during storage can result in significant conformational and compositional changes to the membrane. Characterizing the changes to RBC water permeability can help in evaluating the quality of stored blood products and aid in the development of improved methods for the cryopreservation of red blood cells. This study aimed to characterize the water permeability (L_p), osmotically inactive fraction (b), and Arrhenius activation energy (E_a) at defined storage time-points throughout storage and to correlate the observed results with other in vitro RBC quality parameters. RBCs were collected from age- and sex-matched blood donors. A stopped flow spectrophotometer was used to determine L_p and b by monitoring changes in hemoglobin autofluorescence when RBCs were exposed to anisotonic solutions. Experimental values of L_p were characterized at three different temperatures (4, 20 and 37 °C) to determine the E_a. Results showed that L_p, b, and E_a of stored RBCs significantly increase by day 21 of storage. Degradation of the RBC membrane with length of storage was seen as an increase in hemolysis and supernatant potassium, and a decrease in deformability, mean corpuscular hemoglobin concentration and supernatant sodium. RBC osmotic characteristics were shown to change with storage and correlate with changes in RBC membrane quality metrics. Monitoring water parameters is a predictor of membrane damage and loss of membrane integrity in ex vivo stored RBCs.     

Red blood cell mechanics and capillary blood rheology

Blood contains a high vol fraction of erythrocytes (red blood cells), which strongly influence its flow properties. Much is known about the mechanical properties of red cells, providing a basis for understanding and predicting the rheological behavior of blood in terms of the behavior of individual red cells. This review describes quantitative theoretical models that relate red cell mechanics to flow properties of blood in capillaries. Red cells often flow in single file in capillaries, and rheological parameters can then be estimated by analyzing the motion and deformation of an individual red cell and the surrounding plasma in a capillary. The analysis may be simplified by using lubrication theory to approximate the plasma flow in the narrow gaps between the cells and the vessel walls. If red cell shapes are assumed to be axisymmetric, apparent viscosities are predicted that agree with determinations in glass capillaries. Red cells flowing in microvessels typically assume nonaxisymmetric shapes, with cyclic “tank-treading” motion of the membrane around the interior. Several analyses have been carried out that take these effects into account. These analyses indicate that nonaxisymmetry and tank-treading do not significantly influence the flow resistance in single-file or two-file flow.     

Net proton secretion as a parameter for nitrate uptake

Summary Nitrate uptake and its link to net proton secretion in wheat (Triticum aestivum L. cv. Tassilo, Caribo, and Astron) were investigated using a pH-stat system. Since nitrate is taken up in symport with protons, nitrate and proton fluxes should be correlated. Nitrate concentration in the medium, measured by HPLC, decreased in a linear manner. The addition of nitrate caused a drop in net proton secretion rate to negative values (net proton influx). Once nitrate concentration had been lowered to a well defined level, net proton secretion rate started to recover. This critical nitrate concentration depended on the initial nitrate concentration in the medium. A technique to derive nitrate uptake rates from time courses of net proton secretion was developed and is described. Briefly, this method requires the initial nitrate concentration and the time until the minimal net proton secretion rate is achieved. Results determined with this technique were found in excellent agreement to simultaneous direct measurements of nitrate uptake by HPLC. Measurement of net proton secretion therefore can be used as a parameter for nitrate uptake and as a screening method for uptake efficiency. This method was used to compare three varieties of a high nitrogen efficiency breeding line of wheat. The originally less nitrogen efficient variety outperformed the actually sold cultivar in nitrate uptake rate.     

Red blood cell shapes as explained on the basis of curvature elasticity.

Assuming that the shape of red blood cells is controlled by the curvature elasticity of the surrounding membrane, we fit theoretical shapes to the contours Evans and co-workers determined by interference microscopy. Very good agreement is obtained for disc shapes. The fit is not so good for less common shapes, which may result from Evans' parametric representation and from the interference of shear elasticity.     

Red blood cell mechanics and capillary blood rheology.

Blood contains a high vol fraction of erythrocytes (red blood cells), which strongly influence its flow properties. Much is known about the mechanical properties of red cells, providing a basis for understanding and predicting the rheological behavior of blood in terms of the behavior of individual red cells. This review describes quantitative theoretical models that relate red cell mechanics to flow properties of blood in capillaries. Red cells often flow in single file in capillaries, and rheological parameters can then be estimated by analyzing the motion and deformation of an individual red cell and the surrounding plasma in a capillary. The analysis may be simplified by using lubrication theory to approximate the plasma flow in the narrow gaps between the cells and the vessels walls. If red cell shapes are assumed to be axisymmetric, apparent viscosities are predicted that agree with determinations in glass capillaries. Red cells flowing in microvessels typically assume nonaxisymmetric shapes, with cyclic "tank-treading" motion of the membrane around the interior. Several analyses have been carried out that take these effects into account. These analyses indicate that nonaxisymmetry and tank-treading do not significantly influence the flow resistance in single-file or two-file flow.     

Red blood cell phagocytosis following hexokinase inactivation

Hexokinase inactivating antibodies were loaded into human erythrocytes using an encapsulation procedure based on hypotonic haemolysis, isotonic resealing and reannealing. Red blood cells loaded with anti-hexokinase IgG showed 20 percent residual hexokinase activity and reduced glycolytic activity. ⁹Incubation of these cells in the presence of an oxidizing agent such as terbutyl hydroperoxide (TBH) and then in autologous plasma, promoted opsonization by autologous IgG and complement deposition, but not haemolysis. Furthermore, the antihexokinase IgG loaded cells treated with TBH were actively recognized and phagocytosed by macrophages. Thus, metabolic impairment of human erthrocytes promotes autologous IgG binding, C3 deposition and phagocytosis, a mechanism already reported for the removal of senescent erythrocytes.     

Red blood cell dielectrophoresis in axisymmetric fields

Dielectrophoretic velocities of human red blood cells in an axisymmetric field were measured as a function of the applied voltage and the distance from the axis of symmetry. The voltage of the alternating electric field (frequency 2 MHz), applied between two concentric cylindrical metal electrodes (outer and inner radii 0.24 and 1 mm, respectively), was varied up to 19 V. Two kinds of mediums were used: (a) 90% of 2.1% glycine solution and 10% of 5.5% glucose solution and (b) 5.4% sorbitol solution. The results have shown that in both mediums the cell velocities are proportional to the square of the applied voltage and inversely proportional to the cube of the distance from the axis of symmetry, as predicted by the theory. The coefficient of proportionality (dielectrophoretic coefficient) is on the order of 10(-25) A2S4kg-1. It depends on the donor of red blood cells and might be used for diagnostic purposes. These results will be used in future investigations of membrane adhesion, stability and fusion.     

Red blood cell dielectrophoresis in axisymmetric fields

Dielectrophoretic velocities of human red blood cells in an axisymmetric field were measured as a function of the applied voltage and the distance from the axis of symmetry. The voltage of the alternating electric field (frequency 2 MHz), applied between two concentric cylindrical metal electrodes (outer and inner radii 0.24 and 1 mm, respectively), was varied up to 19 V. Two kinds of mediums were used: (a) 90% of 2.1% glycine solution and 10% of 5.5% glucose solution and (b) 5.4% sorbitol solution. The results have shown that in both mediums the cell velocities are proportional to the square of the applied voltage and inversely proportional to the cube of the distance from the axis of symmetry, as predicted by the theory. The coefficient of proportionality (dielectrophoretic coefficient) is on the order of 10⁻²⁵ A²s⁴kg⁻¹. It depends on the donor of red blood cells and might be used for diagnostic purposes. These results will be used in future investigations of membrane adhesion, stability and fusion.