Enhanced sensitivity to calcium in Duchenne muscular dystrophy.

The ionophore A23187 causes an increase in the Ca content of human erythrocytes and a Ca-dependent increase in K efflux (Gardos effect). These changes are associated with a reduction in osmotic fragility and cell size. Treatment of erythrocytes from patients with Duchenne muscular dystrophy with A23187 results in ⁴⁵Ca uptake comparable to that of erythrocytes from control subjects. However, the reduction in osmotic fragility and K content observed in dystrophic erythrocytes is twofold greater than in control erythrocytes. These results indicate that an alteration in the regulation of erythrocyte membrane function by Ca occurs in Duchenne muscular dystrophy. This alteration may be responsible for other changes in erythrocyte membrane properties observed in Duchenne muscular dystrophy.     

Mechanical lysis of human erythrocytes. Membrane stabilization by plasma proteins]

Mechanical properties of erythrocyte membranes play an important role in red cell functions. Stability of human erythrocytes under deforming mechanical tensions which occur in the rapidly moving fluid is studied. The activation energy of the mechanical hemolysis determined by the temperature dependence of the hemolysis rate is 55 + 7 kJ/mol. The fragility of erythrocytes rises sharply as the salt concentrations increase. Glutaric dialdehyde forms a certain number of interprotein bonds which increase the fragility of erythrocytes. The mechanical stability of the erythrocyte membrane falls at high (0.5 M) ethanol concentrations. Blood plasma proteins, particularly human serum albumin, have a pronounced stabilizing effect. The hemolysis occurring during the rapid mixing is not probably associated with an osmotic mechanism since high sucrose concentrations do not prevent this process. The mechanical hemolysis depends both on the deforming tension arising in the membrane and on the state of the erythrocyte membrane.     

Effects of temperature, oxygen and carbon dioxide on osmotic fragility of carp, Cyprinus carpio L., erythrocytes

The in vitro effects of gases and temperature on the osmotic fragility of carp erythrocytes were studied. At the three different temperatures analyzed (5, 11 and 20°C) there was no noticeable modification in erythrocyte membrane osmotic resistance. Osmotic fragility of red blood cells was altered by CO₂ and air treatment, as compared to the standard procedure. This suggests the need to take into account a possible moderate hypoxia that develops in the routine procedure of nucleated erythrocyte osmotic fragility tests.     

Comparison of erythrocyte osmotic fragility among ectotherms and endotherms at three temperatures

1. Comparison of erythrocyte osmotic fragility (EOF) between various ectotherms and endotherms was investigated at 5, 25, and 38 degrees C. 2. We hypothesized that ectotherms might possess erythrocytes whose osmotic fragility would be less affected by temperature than those of endotherms. 3. Ectotherm erythrocytes were much more osmotically resistant than those of endotherms. 4. The EOF of ectotherms and endotherms showed similar responses to temperature. 5. It does not appear that the osmotic fragility of erythrocytes from ectotherms in this study are adapted to be less affected by temperature than those of endotherms. The highly osmotic resistant erythrocytes of ectotherms may alleviate the need for further adaptation for osmotic resistance.     

Some effects of the trypanocidal drug isometamidium on encapsulation in bovine carrier erythrocytes

Bovine erythrocyte exposure to isometamidium chloride causes increased osmotic fragility. Control cells tolerated up to 1 mg/ml drug with no effects. Carrier erythrocytes were highly susceptible to drug, with increased osmotic fragility and decreased encapsulation potential of sucrose and inulin. Scanning electron micrographs of control and carrier erythrocytes exposed to drug revealed the formation of enkephalocytes with carrier erythrocytes. Control erythrocytes showed greater tolerance to the drug. Apparently, access of the drug to the interior of the erythrocyte membrane allows the drug to be more interactive with the membrane.     

Osmotic properties of bovine erythrocytes aged in vivo

The osmotic properties of bovine erythrocytes aged in vivo were studied by the modified microhematocrit method. The osmotic fragility of older red cells decreases due to their larger relative osmotically non-active volume. Relative critical cell volume of bovine erythrocytes does not alter significantly with cell age. The age dependent change in the osmotic fragility of human red blood cells, the reverse of that found for bovine erythrocytes, is due to a different alteration of the critical cell volume during intravascular erythrocyte aging.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Vanadate increases oxygen affinity and affects enzyme activities and membrane properties of erythrocytes

Incubation of blood with vanadate markedly increases the affinity of hemoglobin for oxygen, decreases the deformability of erythrocytes, reduces their osmotic fragility and alters their morphology, determining the appearance of equinocytic forms. Since vanadate is easily taken up by the erythrocytes and binds hemoglobin, these effects might result from interactions of vanadate with hemoglobin and with membrane proteins at the glycerate-2, 3-P₂ and/or ATP binding site. In addition, vanadate inhibits phosphoglycerate mutase, phosphoglucomutase and adenylate-kinase activities from hemolysates, suggesting a possible inhibitory effect on erythrocyte metabolism     

Effects of Proteus mirabilis lipopolysaccharides with different O-polysaccharide structures on the plasma membrane of human erythrocytes

The effects of O33 and O49 P. mirabilis lipopolysaccharides (LPSs) on human erythrocyte membrane properties were examined. Physical parameters of the plasma membrane, such as membrane lipid fluidity, physical state of membrane proteins, and osmotic fragility, were determined. The fluidity of the lipids was estimated using three spin-labeled stearic acids of doxyl derivatives: 5-doxylstearic acid, 12-doxylstearic acid, and 16-doxylstearic acid. All the applied labels locate to different depths of the lipid layer and provide information on the ordering of phospholipid fatty acyl chain mobility. LPSs O49 increased the membrane lipid fluidity in the polar region of the lipid bilayer as indicated by spin-labeled 5-doxylstearic acid. An increase in fluidity was also observed in the deeper region using 12-doxylstearic acid only for O33 LPSs. The highest concentration of O33 LPSs (1 mg/ml) increased the motion of membrane proteins detected by the spin-label residue of iodoacetamide. These results showed different actions of O33 and O49 LPSs on the plasma membrane due to the different chemical structures of O-polysaccharides. P. mirabilis O33 and O49 LPSs did not induce changes in the membrane cytoskeleton, osmotic fragility and lipid peroxidation of erythrocytes. On the other hand a rise in the content of carbonyl compounds was observed for the highest concentrations of O33 LPS. This result indicated protein oxidation in the erythrocyte membrane. Lipid A, the hydrophobic part of LPS, did not change the membrane lipid fluidity and osmotic fragility of erythrocytes. Smooth and rough forms of P. mirabilis LPSs were tested for their abilities for complement-mediated immunohemolysis of erythrocytes. Only one out of seven LPSs used was a potent agent of complement-mediated hemolysis. It was rough, Ra-type of P. mirabilis R110 LPS. The O-polysaccharide-dependent scheme of reaction is presented.     

Glutaraldehyde effect on the osmotic fragility and rate of haemolysis of human erythrocytes: a kinetic study

The effect of glutaraldehyde treatment on the osmotic fragility and rate of haemolysis of human erythrocytes was studied in the temperature domain 22-42 degrees C at different aldehyde concentrations from 0.010 to 0.040% (w/v). The osmotic fragility linearly decreases at increasing temperatures for all glutaraldehyde concentrations with approximately the same slope as for the untreated erythrocytes. Rather similar effects are produced on the rate of haemolysis. It was not possible to define unique activation energies. The maximal haemolysis becomes smaller after the treatment with increasing concentrations of glutaraldehyde but more erythrocytes are no longer able to tolerate normally non-haemolysing osmotic stretching, this suggesting an artificial "ageing" of erythrocytes. The formation of a membrane protein skeleton as a result of cross-linking is inferred, while the changes in the cytoskeletal spectrin network seem to be less important.     

Protective effect of capsaicin on the osmotic fragility of human erythrocytes

Capsaicin exerts a stabilizing effect on erythrocytes making them more resistant to lysis under hypotonic stress. The protective action of capsaicin on osmotic fragility (OF) was not receptor mediated since no dose responsive effect was observed. The results suggest that this protective effect of capsaicin on OF is due to a direct interaction of capsaicin with the erythrocyte membrane rather than due to any alteration in the intracellular metabolism of erythrocytes.     

低强度激光对人红细胞生物物理特性的影响

研究不同功率的低强度He-Ne激光对正常人体红细胞流变学特性影响。以正常人体红细胞为研究对象,测量了低强度He-Ne激光在不同照射时间、不同功率条件下红细胞的变形、取向、膜流动性、膜的微粘度和渗透脆性的变化情况。结果表明:照射后红细胞的变形性和膜流动性增强、渗透脆性下降。照射对红细胞流变学特性影响显著,其中激光能量为0.24 J、照射血样为2 mL时取得的照射效果最佳。     

Beneficial influence of dietary curcumin, capsaicin and garlic on erythrocyte integrity in high-fat fed rats

In rats rendered hyperlipidemic by maintaining them on a high-fat diet (30%) for 8 weeks, inclusion of spice principles [curcumin (0.2%) or capsaicin (0.015%)] or garlic (2.0%) in the diet produced significant hypotriglyceridemic effect. Plasma cholesterol remained unaffected in high-fat treatment. Hepatic triglyceride content was significantly higher in high-fat fed rats, and this increase was effectively countered by inclusion of the hypolipidemic spice agents -- curcumin, capsaicin or garlic in the diet. The lipid profile of erythrocyte membranes of hyperlipidemic rats was similar to basal controls. An examination of the osmotic fragility of erythrocytes in various groups indicated that the red blood cells of hyperlipidemic rats display a slight resistance to osmotic lysis. Inclusion of spice principles [curcumin (0.2%) or capsaicin (0.015%)] or garlic (2.0%) in the diet, which produced the hypotriglyceridemic effect, appeared to beneficially correct this altered osmotic fragility of erythrocytes. Activities of ouabain-sensitive Na(+),K(+)-ATPase as well as acetylcholinesterase of erythrocyte membranes in high-fat fed rats remained unaltered. Activity of Ca(2+),Mg(2+)-ATPase in erythrocyte membrane was significantly decreased in high-fat fed animals, whereas dietary spice principles and garlic countered this reduction in enzyme activity. In the absence of any change in the cholesterol/phospholipid molar ratio in the erythrocyte membrane, a decreased activity of membrane-bound Ca(2+),Mg(2+)-ATPase could have probably contributed to the accumulation of intracellular calcium leading to the diminished deformability of the erythrocytes in high-fat fed rats.     

Alterations of hemorheological parameters and tubulin content in erythrocytes from diabetic subjects

Treatment of human erythrocytes with high glucose concentrations altered the content and distributions of three tubulin isotypes, with consequent reduction of erythrocyte deformability and osmotic resistance. In erythrocytes from diabetic subjects (D erythrocytes), (i) tubulin in the membrane-associated fraction (Mem-Tub) was increased and tubulin in the sedimentable fraction (Sed-Tub) was decreased, (ii) deformability was lower than in erythrocytes from normal subjects (N erythrocytes), and (iii) detyrosinated/acetylated tubulin content was higher in the Mem-Tub fraction and tyrosinated/acetylated tubulin content was higher in the Sed-Tub fraction, in comparison with N erythrocytes. Similar properties were observed for human N erythrocytes treated with high glucose concentrations, and for erythrocytes from rats with streptozotocin-induced diabetes. In N erythrocytes, high-glucose treatment caused translocation of tubulin from the Sed-Tub to Mem-Tub fraction, thereby reducing deformability and inducing acetylation/tyrosination in the Sed-Tub fraction. The increased tubulin acetylation in these cells resulted from inhibition of deacetylase enzymes. Increased tubulin acetylation and translocation of this acetylated tubulin to the Mem-Tub fraction were both correlated with reduced osmotic resistance. Our findings suggest that (i) high glucose concentrations promote tubulin acetylation and translocation of this tubulin to the membrane, and (ii) this tubulin is involved in regulation of erythrocyte deformability and osmotic fragility.     

Binding of ovarian steroids to erythrocytes in patients with sickle cell disease; effects on cell sickling and osmotic fragility

Ovarian steroids appear to influence the manifestations of sickle cell disease (SCD); oestrogens can adversely affect erythrocyte function, whereas progestogens may inhibit sickling and decrease the osmotic fragility of erythrocytes. The aims of the present studies were: (i) to characterise the binding of oestradiol and progesterone to erythrocytes from women with HbSS, HbSC and HbAA genotypes; (ii) to investigate whether steroids modulate susceptibility to sickling or osmotic fragility of HbSS and HbAA erythrocytes. Erythrocytes were incubated for 1h with [3H]-steroids at 4 and 37 degrees C. Binding of both oestradiol and progesterone was independent of temperature and steroid concentration, but was decreased by sequential "washing" of erythrocytes in fresh incubation buffer. Binding capacity was 80 +/- 6% greater for oestradiol (versus progesterone) in all three genotypes, and binding of both steroids was decreased by > or = 70% in HbSS erythrocytes compared to HbSC or HbAA erythrocytes. Pre-incubation of erythrocytes with 35 microM oestradiol or 30 microM progesterone had no significant effect on susceptibility of HbSS and HbAA erythrocytes to sickling, or on osmotic fragility. We conclude that both oestradiol and progesterone bind in a low affinity, non-saturable manner to erythrocytes with decreased binding in cells from women with HbSS. However, steroid binding does not affect susceptibility to sickling or osmotic fragility irrespective of haemoglobin genotype.     

Comparison of membrane structure,osmotic fragility,and morphology of multiple sclerosis and normal erythrocytes

Erythrocyte membranes from multiple sclerosis (MS) patients and normal individuals were studied by electron spin resonance spectroscopy, osmotic fragility tests, scanning electron microscopy (SEM) and fatty acid analysis of membrane lipids. There was no significant difference in the membrane fluidity between MS and normal erythrocytes using fatty acid spin labels with the nitroxide moiety on carbons 5, 12, or 16 from the carboxyl group. Linoleic acid, which has been reported to decrease the absolute electrophoretic mobility of only MS erythrocytes, increased the fluidity of MS and normal erythrocyte membranes to a similar extent. The osmotic fragility of MS erythrocytes obtained from outpatients was similar to normal control cells but the osmotic fragility of erythrocytes obtained from hospitalized MS patients was greater than normal. Scanning electron microscopy of MS erythrocytes revealed no gross abnormalities. Cells incubated with linoleic acid had transformed from discocytes into sphero-echinocytes with prominent membrane surface indentations but MS and normal erythrocytes appeared identical. Of the fatty acid content of the total lipid extract, erythrocytes from most, but not all, MS hospitalized patients and some patients with other demyelinating diseases had relatively less (P<.001) 18:2 than the normal cells. These results indicate that at least some of the abnormalities reported in MS erythrocytes may only be found in hospitalized patients and may be due to other complications of the disease. They also indicate that the reported abnormal effects of linoleic acid on the electrophoretic mobility of MS erythrocytes may be caused by some other mechanism than an effect on the fluidity of the bilayer.     

Estimation of cell membrane properties and erythrocyte red-ox balance in patients with metabolic syndrome

Metabolic syndrome (MS) is associated with occurrence of the many cardiovascular risk factors such as atherogenic dyslipidemia, visceral fat distribution, arterial hypertension and pro-thrombotic and pro-inflammatory status. In our study the effect of disorders that appear in MS on red-ox balance and erythrocyte cell membrane properties were estimated. The study comprised 50 patients with diagnosed MS and in 25 healthy subjects. Content of thiobarbituric acid reactive substances (TBARS) and catalase, superoxide dismutase and glutathione peroxidase activity were estimated in red blood cells. Moreover, conformation status of membrane proteins, membrane fluidity and osmotic fragility were evaluated. MS was found to manifest: (1) the increase of the concentration of TBARS in erythrocytes with no statistically significant differences in antioxidant enzymes activity, (2) disorders in the structure of erythrocyte cytoskeleton proteins, (3) the increase in membrane lipids fluidity at the depth of 5th and 12th carbon atom of fatty acid hydrocarbon chain and significantly decreased fluidity at the depth of 16th carbon atom, (4) increased erythrocyte osmotic fragility.     

Electron spin resonance, hematological, and deformability studies of erythrocytes from patients with Huntington's disease.

Electron spin resonance, hematologic, and deformability studies of erythrocytes from patients with Huntington's disease have been performed A decreased deformability of Huntington's disease erythrocytes compared to normal controls was demonstrated. No difference in erythrocyte hematologic indices, osmotic fragility, reticulocyte counts, or intracellular Na+ concentration was found. Huntington's disease serum had no demonstrable effect on electron spin resonance parameters of a protein-specific spin label attached to membrane proteins in control erythrocytes compared to the effect of control serum. This finding suggests that under the conditions employed no serum component or circulating factor is responsible for the changes in the physical state of membrane proteins in Huntington's disease erythrocytes (Butterfield, D.A., Oeswein, J.Q. and Markesbery, W.R. (1977) Nature 267, 453--455). No alteration in lipid fluidity of Huntington's disease erythrocyte membranes could be discerned suggesting that the underlying molecular defect in Huntington's disease involves a membrane protein. The results of the present studies on erythrocytes strongly support the concept that Huntington's disease is associated with a generalized membrane abnormality.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

The influence of metmyoglobin and ferrylmyoglobin on the human erythrocyte membrane

Preliminary experiments revealed that ferrylmyoglobin decayed more slowly in the absence than in the presence of intact erythrocytes and erythrocyte membranes. This suggested the existence of interactions between FerrylMb and the erythrocyte membrane. Subsequent studies examined the influence of FerrylMb on the membrane of intact erythrocytes and on isolated erythrocyte membranes. The incubation of intact erythrocytes with FerrylMb did not influence their osmotic fragility or the fluidity of their membranes; the level of peroxidation of the membrane lipids increased only slightly (there was only a slight increase in the level of membrane lipid peroxidation). The activity of acetylcholinesterase significantly increased after 15 minutes of incubation, whereas longer incubation did not lead to any changes in the activity of this enzyme. The incubation of isolated erythrocyte membranes with FerrylMb resulted in an increase in their fluidity and a significant rise in the level of lipid peroxidation.