Effects of rHuEPO treatment on red blood cell osmotic resistance

Red blood cell osmotic resistance (RBCOR) is defined as resistance to osmotic changes in cell integrity after their exposure to hypotonic saline solution. The investigation examined the effect of rHuEPO on RBCOR in hemodialysed patients. The study included 58 patients aged 49 +/- 14 years, treated by hemodialysis for 59 +/- 43 months on average. Half of the patients received rHuEPO for anemia correction. RBCOR was determined in all patients as 3 values: hemolysis start point (HSP), hemolysis end point (HEP) and middle osmotic resistance (MOR). The patients underwent laboratory checkup for parameters characteristically changed in the uremic syndrome. In the control group of healthy subjects (n = 16) RBCOR was only determined. No differences were found in the average values of HSP, HEP and MOR between the rHuEPO treated group of patinets and the untreated group. Compared to healthy individuals, the hemodialysed patients displayed significantly higher values of HSP, HEP and MOR. The only one significant correlation of RBCOR and routine laboratory features was found between MOR and predialytic serum concentrations of calcium (r = 0.28, p < 0.05) and hydrogen ions (r = 0.37, p < 0.05). Our results suggest that the administration of rHuEPO does not affect RBCOR in hemodialysed patients, that RBCOR is not always reduced in this population and that it correlates with a small number of laboratory parameters characteristic for the uremic syndrome.     

Effects of nitric oxide on red blood cell deformability

In addition to its known action on vascular smooth muscle, nitric oxide (NO) has been suggested to have cardiovascular effects via regulation of red blood cell (RBC) deformability. The present study was designed to further explore this possibility. Human RBCs in autologous plasma were incubated for 1 h with NO synthase (NOS) inhibitors [N(omega)-nitro-l-arginine methyl ester (l-NAME) and S-methylisothiourea], NO donors [sodium nitroprusside (SNP) and diethylenetriamine (DETA)-NONOate], an NO precursor (l-arginine), soluble guanylate cyclase inhibitors (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one and methylene blue), and a potassium channel blocker [triethylammonium (TEA)]. After incubation, RBC deformability at various shear stresses was determined by ektacytometry. Both NOS inhibitors significantly reduced RBC deformability above a threshold concentration, whereas the NO donors increased deformability at optimal concentrations. NO donors, as well as the NO precursor l-arginine and the potassium blocker TEA, were able to reverse the effects of NOS inhibitors. Guanylate cyclase inhibition reduced RBC deformation, with both SNP and DETA-NONOate able to reverse this effect. These results thus indicate the importance of NO as a determinant of RBC mechanical behavior and suggest its regulatory role for normal RBC deformability.     

Effects of buffers and pH on rabbit red blood cell hexokinase

The kinetic properties of rabbit red blood cell hexokinase in different buffer systems have been studied. At pH 8.0 the reaction velocity (v) is about 30% higher in glycylglycine compared to Tris, Tea, Hepes or ammonium acetate buffers. The enzyme stability, heat-dependence and spectral properties of the enzyme are also affected by the buffer utilized. None of the following kinetic properties of red blood cell hexokinase varies with pH in the range 6.8-8.5: Km of glucose; Km of ATP and Ki of glucose 6-phosphate.     

Effects of dextran molecular weight on red blood cell aggregation

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biologic and biophysical interest, yet the mechanistic details governing the process are still being explored. Although a depletion model with osmotic attractive forces due to polymer depletion near the RBC surface has been proposed for aggregation by the neutral polyglucose dextran, its applicability at high molecular mass has not been established. In this study, RBC aggregation was measured over a wide range of dextran molecular mass (70 kDa to 28 MDa) at concentrations ≤2 g/dL. Our results indicate that aggregation does not monotonically increase with polymer size; instead, it demonstrates an optimum dextran molecular mass around 200-500 kDa. We used a model for depletion-mediated RBC aggregation to calculate the expected depletion energies. This model was found to be consistent with the experimental results and thus provides new insight into polymer-RBC interactions.     

Elastic energy of curvature-driven bump formation on red blood cell membrane.

Model calculations were performed to explore quantitative aspects of the discocyte-echinocyte shape transformation in red blood cells. The shape transformation was assumed to be driven by changes in the preferred curvature of the membrane bilayer and opposed by the elastic shear rigidity of the membrane skeleton. The energy required for echinocyte bump formation was calculated for a range of bump shapes for different preferred curvatures. Energy minima corresponding to nonzero bump heights were found when the stress-free area difference between the membrane leaflets or the spontaneous curvature of the membrane became sufficiently large, but the calculations predict that the membrane can tolerate significant differences in the resting areas of the inner and outer leaflets or significant spontaneous curvature without visible changes in shape. Thus, if the cell is near the threshold for bump formation, the calculations predict that small changes in membrane properties would produce large changes in cellular geometry. These results provide a rational framework for interpreting observations of shape transformations in red cells and for understanding the mechanism by which small changes in membrane elastic properties might lead to significant changes in geometry.     

Effects of cell lysis on the rheological behavior of red blood cell suspensions

Rheological studies of lysed cell suspensions are performed with a magneto acoustic ball microrheometer. Two methods for lysing the cells are developed in order to provide cell volume concentrations identical to control intact cell suspensions. The first uses a freeze-thaw technique and the second uses sonication. It is found that cell suspensions disrupted by sonication have a lower viscosity than intact suspensions, whereas cell suspensions lysed by the freeze-thaw method exhibit a higher viscosity. Sonication is discovered to have a detrimental impact on the cell membrane, and to cause complete destruction of the cell membrane structure. Measurements of the steady state viscosity show that indeed the presence of the membrane is not detected, and that what is measured is mainly the viscosity of the hemoglobin solution. On the other hand, freeze-thaw results indicate that at least two phenomena occur. The first phenomenon, occurring during the first freeze-thaw cycle, produces an increase in viscosity and in viscoelasticity. The second one, taking place after subsequent freeze-thaw cycles, induces a decrease in the bulk rheological properties. Several possible mechanisms are presented to explain the observed phenomena.     

Effects of pressure on red blood cell geometry during micropipette aspiration

Micropipette aspiration is a potentially useful and accurate technique to measure red blood cell (RBC) geometry. Individual RBCs are partially aspirated and from the resulting sphere diameter, total cell length, and pipette diameter, membrane area and cell volume can be calculated. In this study we have focused on possible shape artifacts associated with the aspirated portion of RBC. We observed that the apparent RBC geometry (calculated area and volume) changed markedly (P < 0.001) with the applied aspiration pressure; for normal human RBC the area increased by 5.6 +/- 0.6% and volume decreased by 4.7 +/- 0.6% when the aspiration pressure was increased from 20 to 100 mm water. The calculated membrane area dilation modulus was 7.4 dyn/ cm, which is far below the expected value, and microscopic observations revealed a membrane folding artifact as a possible artifact. These assumptions were strengthened by using a short-duration (3 s) pressure peak of 20-100-20 mm water. The folding then disappeared permanently, but a small (0.31 +/- 0.09%; P < 0.001) area decrease was detected which yields a realistic dilation modulus of 215 dyn/cm. We conclude that membrane folding can critically affect RBC micropipette measurements and that a transient pressure peak can unfold the RBC membrane, thus allowing accurate measurements of RBC geometry.     

大鼠力竭运动诱导的氧化应激损伤对红细胞变形性的影响

目的:探讨一次性力竭运动诱导的氧化应激反应对大鼠红细胞的抗氧化能力和细胞变形性的影响。方法:大鼠分为3组(n=10):对照组(Control)、适度运动组(MRE)和力竭运动组(ERE)。力竭运动组大鼠运动的前20 min保持5%的坡度和20 m/min的速度,20 min后调整为15%的坡度和25 m/min的速度,直至运动力竭。适度运动组大鼠在5%的坡度和20 m/min的速度下跑40 min。检测各组大鼠红细胞的抗氧化能力,并对氧化应激反应诱导的红细胞膜蛋白巯基水平、膜脂质过氧化水平和膜蛋白SDS-Page电泳条带变化进行了分析。通过激光衍射法对不同运动组大鼠红细胞变形性进行了检测。结果:力竭运动条件下大鼠红细胞受到严重的氧化应激损伤,红细胞内抗氧化能力下降。导致膜脂质过氧化损伤和膜蛋白巯基交联为主的蛋白聚簇化,形成高分子聚合物(HMW)。力竭组大鼠红细胞变形性(0.314±0.013 at 3 Pa and 0.534±0.009 at 30 Pa)显著低于对照组(0.41±0.01 at 3 Pa and 0.571±0.008 at 30 Pa;P0.05 and P0.01,respectively)和适度运动组。结论:力竭运动诱导的氧化损伤导致了红细胞变形能力(EI)的显著下降,使红细胞在微循环的转运受到限制,导致组织缺血缺氧进而引起休克、死亡等运动性疾病。     

Effects of chitooligosaccharides on human red blood cell morphology and membrane protein structure

Recent studies of chitosan have increased the interest in its conversion to chitooligosaccharides (COSs) because these compounds are water-soluble and have potential use in several biomedical applications. Furthermore, such oligomers may be more advantageous than chitosans because of their much higher absorption profiles at the intestinal level, which permit their facilitated access to systemic circulation and potential distribution throughout the entire human body. In that perspective, it is important to clarify their effect on blood further, namely, on human red blood cells (RBCs). The aim of this work was thus to study the effect of two COS mixtures with different molecular weight (MW) ranges, <3 and <5 kDa, at various concentrations (5.0-0.005 mg/mL) on human RBCs. The interactions of these two mixtures with RBC membrane proteins and with hemoglobin were assessed, and the RBC morphology and surface structure were analyzed by optical microscopy (OM) and atomic force microscopy (AFM). In the presence of either COS mixture, no significant hemolysis was observed; however, at COS concentrations >0.1 mg/mL, changes in membrane binding hemoglobin were observed. Membrane protein changes were also observed with increasing COS concentration, including a reduction in both alpha- and beta-spectrin and in band 3 protein, and the development of three new protein bands: peroxiredoxin 2, calmodulin, and hemoglobin chains. Morphologic evaluation by OM showed that at high concentrations COSs interact with RBCs, leading to RBC adhesion, aggregation, or both. An increase in the roughness of the RBC surface with increasing COS concentration was observed by AFM. Overall, these findings suggest that COS damage to RBCs was dependent on the COS MW and concentration, and significant damage resulted from either a higher MW or a greater concentration (>0.1 mg/mL).     

Effects of semicarbazide on oxidative processes in human red blood cell membranes

Semicarbazide can interfere with oxidative processes in the red blood cell membrane via different modes of action.Treatment of human red blood cell membranes with O₃ results, among other effects, in cross-linking of membrane proteins and inhibition of glyceraldehyde-3-phosphate dehydrogenase activity. Semicarbazide inhibits these effects by acting as an O₃ scavenger. The effect of semicarbazide as an O₃ scavenger is complicated by the fact that ozonolysis of semicarbazide yields a product that causes inhibition of glyceraldehyde-3-phosphate dehydrogenase.Glyceraldehyde-3-phosphate dehydrogenase inhibition can also be provoked by incubation of membrane suspensions with O₃-treated phospholipids. Semicarbazide prevented this effect by interaction with an inhibitory O₃-phospholipid reaction product.Protoporphyrin-induced photodynamic cross-linking of membrane proteins is chemically distinct from O₃-induced cross-linking. Photodynamic cross-linking is also inhibited by semicarbazide, in this case via reaction with a histidine photooxidation product.     

Effects of sphingolipids overload on red blood cell properties in Gaucher disease

Gaucher disease (GD) is a genetic disease with mutations in the GBA gene that encodes glucocerebrosidase causing complications such as anaemia and bone disease. GD is characterized by accumulation of the sphingolipids (SL) glucosylceramide (GL1), glucosylsphingosine (Lyso‐GL1), sphingosine (Sph) and sphingosine‐1‐phosphate (S1P). These SL are increased in the plasma of GD patients and the associated complications have been attributed to the accumulation of lipids in macrophages. Our recent findings indicated that red blood cells (RBCs) and erythroid progenitors may play an important role in GD pathophysiology. RBCs abnormalities and dyserythropoiesis have been observed in GD patients. Moreover, we showed higher SL levels in the plasma and in RBCs from untreated GD patients compared with controls. In this study, we quantified SL in 16 untreated GD patients and 15 patients treated with enzyme replacement therapy. Our results showed that the treatment significantly decreases SL levels in the plasma and RBCs. The increased SL content in RBCs correlates with abnormal RBC properties and with markers of disease activity. Because RBCs lack glucocerebrosidase activity, we investigated how lipid overload could occur in these cells. Our results suggested that SL overload in RBCs occurs both during erythropoiesis and during its circulation in the plasma.     

Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell

Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much research has therefore focused on RBC and cardiovascular disorders of mouse and humans. RBCs also host malaria parasites. Recently we presented an in-depth proteome for the human RBC. Here we present directly comparable data for the mouse RBC as membrane-only, soluble-only, and combined membrane-bound/soluble proteomes (comprising, respectively, 247, 232, and 165 proteins). All proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function, and in comparison with the human RBC, were classified as orthologs, family-related, or unique. Splice isoforms were identified, and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinated or partially degraded complexes. Overall there was close concordance between mouse and human proteomes, confirming the unexpected RBC complexity. Several novel findings in the human proteome have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function.