Hypoosmotic hemolysis of erythrocytes by active carbonyl forms

Low-molecular-weight dicarbonyls formed during free radical peroxidation of polyene lipids (malondialdehyde) and autooxidation (glyoxal) or other oxidative transformations of glucose (methylglyoxal) are able to modify the structure of lipid-protein supramolecular complexes of cells. We investigated changes in the erythrocyte membrane structure after an 18-h exposure of human red blood cells in the presence of various natural dicarbonyls. The changes in the mechanical properties of the membrane after membrane modification by carbonyls were evaluated by the susceptibility of erythrocytes to hypoosmotic hemolysis. It has been shown that treatment of red blood cells with malondialdehyde increases the resistance of these cells to hypoosmotic hemolysis, whereas the malondialdehyde isomer, methylglyoxal, in contrast, makes red blood cells more sensitive to the action of hypoosmotic solutions. Paradoxically, a homologue of malondialdehyde, glyoxal, has no effect on hemolysis of red blood cells in hypoosmotic solutions. The findings point to the possibility of the multidirectional effect of low-molecular-weight dicarbonyls with similar structures on the structure and function of biological membranes.     

The kinetics of colloid osmotic hemolysis. I. Nystatin-induced lysis

A kinetic model of colloid osmotic hemolysis for cation-permeable cells has been developed. The model consists of three essential components. The first is a set of flux equations, under the assumption that the membrane potential is equal to the chloride equilibrium potential and that cation fluxes are described by the Goldman flux equation. The second is the osmotic equilibrium model of Freedman and Hoffman that takes into account the non-ideal osmotic behavior of erythrocytes. The third is an empirical relation between hemolysis and cell volume, developed from the lysis behavior in hypoosmotic media. Model simulations are compared with lysis experiments using the antibiotic nystatin to raise cation permeability. The form of the kinetics and inhibition of lysis by sucrose are described well by the model. In additional lysis experiments at different external pH the small pH dependence is accounted for by the model.     

Does calmodulin participate in the regulation of the Ca-pump of erythrocytes in vivo?

Using a highly effective chelator of Ca2+ and 45Ca, the concentration of Cai2+ in human and rat erythrocytes was measured both at normal and accelerated Ca2+ influx into the cells. No effect of the calmodulin-dependent reaction inhibitor R24571 was observed. The Ca-ATPase from saponin-treated erythrocytes was characterized by a high affinity for Ca2+ (K 0.5-0.7 microM). This value is 2-3 times as low as that for Ca2+ concentration causing a 50% increase of the Ca-ATPase activity in erythrocyte ghosts obtained during hypoosmotic hemolysis. The Ca-ATPase activity in saponin-treated erythrocytes did not change either under the effect of calmodulin or by R24571. It was assumed that calmodulin did not participate in the regulation of the Ca2+-pump operation in erythrocytes in vivo.     

Requirements for maximal enrichment of viable intraerythrocytic Plasmodium falciparum rings by saponin hemolysis

The purpose of the present study was to confirm the effectiveness of saponin hemolysis for concentrating ring-infected erythrocytes in Plasmodium falciparum cultures and to determine the actual numbers of the enriched parasites, not just percentage parasitemia. This is important because various molecular biology and vaccine development against malaria require useable quantities of pure culture with minimal number of uninfected erythrocytes at all stages. Synchronized cultures of three P. falciparum strains were exposed to 0.015% isotonic saponin solution for 30 minutes on ice. They were centrifuged and the pellets were treated again with saponin solution for 3-7 minutes. Initially, most of the cultures contained approximately 10(10) erythrocytes and 1-7% parasitemia, but at the end of the enrichment up to 10(8) of erythrocytes containing 90-99.8% parasitemia were recovered (maximal enrichment). From microscopic examination of the cells it was calculated that the hemolysis rate of uninfected and infected erythrocytes was circa 27 to 1, which could account for the enrichment. Studies by other investigators have suggested that P. falciparum merozoite invasion decreases erythrocyte membrane lipids, and it has been reported that reduction of membrane cholesterol could make erythrocytes saponin-resistant. The possibility that merozoite invasion made erythrocytes partially resistant to saponin hemolysis was strengthened by the observation that the proportions of multiple infections increased significantly in the enriched cultures. However, mature asexual parasites could not be concentrated by this method, suggesting possible differences between the membranes of erythrocytes containing ring forms and those of trophozoites and schizonts. Ring-infected erythrocytes freshly from malaria patients could also not be concentrated by the method described here, suggesting that the ability to induce saponin resistance in erythrocytes was acquired by the parasites in vitro.     

Lidocaine: An inhibitor in the free‐radical‐induced hemolysis of erythrocytes

Lidocaine was reported to protect erythrocytes from hemolysis induced by 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH). Since AAPH‐induced hemolysis was a convenient in vitro experimental system to mimic erythrocytes undergoing peroxyl radicals attack, the aim of this work was to investigate the antioxidant effect of lidocaine on AAPH‐induced hemolysis by chemical kinetics. As a result, one molecule of lidocaine can only trap 0.37 radical, much lower than melatonin. Meanwhile, lidocaine cannot protect erythrocytes from hemolysis induced by hemin, which the mechanism of hemolysis was due to the erythrocyte membrane destroyed by hemin. Accordingly, lidocaine protected erythrocytes by scavenging radicals preferentially rather than by stabilizing membrane. Moreover, the interactions of lidocaine with two radical species, including 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate) radical cation (ABTS^+?) and 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH), indicated that lidocaine can reduce ABTS^+? with 260 µM as the 50% inhibition concentration (IC₅₀) and cannot react with DPPH. Thus, lidocaine served as a reductant rather than a hydrogen donor to interact with radicals. Finally, the quantum calculation proved that, compared with the melatonin radical, the stabilization of N‐centered radical of lidocaine was higher than the amide‐type N‐centered radical but lower than the indole‐type N‐centered radical in melatonin. These results provided basic information for lidocaine to be an antiradical drug. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:81–86, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20267     

丹参酮lI-A磺酸钠及莨菪等药物对红细胞膜稳定性及钙调节蛋白(CaM)功能的影响

 为了进一步证实某些药物对红细胞膜稳定(即抗溶血)作用及抑制CaM功能与药理作用之间的相关性,本文选用丹参酮Ⅱ-A磺酸钠,戊脉安,奎尼丁和莨菪类药物,测定它们对大鼠红细胞溶血及CaM功能的影响。结果表明丹参酮Ⅱ-A磺酸钠,戊脉安,奎尼丁对红细胞溶血抑制率分别为100％,71％,32％,对CaM功能的抑制分别为71.8％,63.4％,46.4％。莨菪类药物有促溶血作用,对CaM功能的抑制也极弱。我们还发现以上药物对膜上Ca~(2+)+Mg~(2+)-ATP酶的直接作用亦有类似的规律。这种相关性可能与膜表面CaM与钙离子,靶酶及药物结合区的疏水性及药物本身的相对疏水性有关。     

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.     

Fusion of erythrolysosomes in epithelial cells of the sheep placenta

Summary In trophoblastic epithelial cells of the sheep placenta the breakdown of erythrocytes within complex erythrolysosomes was studied at the ultrastructural level.It was found that the formation of complex erythrolysosomes containing from two to several erythrocytes as a result of fusion of erythrolysosomes within the epithelial cells was a common occurrence when the epithelial cells engulfed a large number of erythrocytes. The erythrocytes enclosed in complex erythrolysosomes appear to be either in the same or in different stages of hemolysis.In the process of breakdown of erythrocytes within complex erythrolysosomes five successive stages of hemolysis could be distinguished. Acid phosphatase activity was demonstrated in the complex erythrolysosomes and appeared to be located in the angular interspaces between the erythrocytes and the lysosomal membrane. The fragmentation of complex erythrolysosomes with formation of small hemoglobin-containing lysosomes also occurred.The fusion of erythrolysosomes with formation of complex erythrolysosomes can be considered as an additional mechanism in the process of erythrocyte breakdown in the epithelial cells of the sheep placenta.     

Glycerol permeability of human fetal and adult erythrocytes and of a model membrane

When erythrocytes from different mammalian species are compared, the hemolysis rate in 0.3 m glycerol is seen to be directly related to the percentage of lecithin in the erythrocyte phospholipid. Since this percentage is higher in erythrocytes from human adults than in those from infants, the hemolysis times in 0.3 m glycerol were compared. As expected, hemolysis was more rapid in the adult cell, which is therefore more permeable to glycerol under these conditions. The permeability to glycerol of a film of erythrocyte lipids in vitro was next examined in a model system containing the two phases water and butanol. Lipid introduced into the bulk butanol appears as a film at the interface. When equal amounts of total lipid extracted from adult and fetal erythrocytes were introduced into the butanol phase of two such chambers, the initial flux of glycerol-(14)C across the lipid boundary was greater in the cell containing lipid from adult erythrocytes than in the cell containing fetal erythrocyte lipid. This difference corresponds qualitatively to the difference in hemolysis time measured in the intact erythrocytes.     

Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent

Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.     

Interaction of chemical carcinogens with plasma membranes: the effect of dimethylaminoazobenzene on erythrocyte osmotic fragility

The capacity of the hepatocarcinogen dimethylaminoazobenzene and two of its analogs to protect erythrocytes against hemolysis is demonstrated. The aminoazo dye compounds exhibit stabilization at relatively low final concentrations, suggestive of an efficient partitioning of the dye molecules into the erythrocyte membrane. Methyl groups at either the 2 or 3′ position of dimethylaminoazobenzene confer even greater stabilization potential to the basic dye structure. The application of the erythrocyte osmotic hemolysis assay in analysis of carcinogen-membrane interactions is discussed.     

Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane

Amphipathic peptides gramicidin S and melittin caused a characteristic colloid-osmotic hemolysis on human erythrocytes; that is, the peptides produced initially a small membrane lesion in erythrocyte membrane, followed by the release of hemoglobin. The size of membrane lesion increased with an increase in the concentration of peptide. Under the conditions causing membrane lesion, we observed the release of membrane fragments containing phospholipids. The present results show that both the peptides have the ability to stimulate the release of membrane fragments out of the cells and this brings about the perforation of molecules of small size, leading to a colloid-osmotic hemolysis.     

Effect of hypervitaminosis A on hemolysis and lipid peroxidation in the rat

Erythrocytes from rats fed large doses of Vitamin A alone, or large doses of vitamin A and vitamin E or diphenyl-p-phenylene diamine (DPPD) were studied for H2O2-induced hemolysis. The vitamin A-dosed rats were more susceptible than normal rats to H2O2-induced hemolysis. Hemolysis was not accompanied by lipid peroxidation. Nevertheless, the antioxidants vitamin E and DPPD inhibited hemolysis in erythrocytes from vitamin A-dosed rats. These antioxidants had the same inhibitory effect when they were included in the diet or added to erythrocyte suspensions in vitro. Erythrocytes from vitamin A-dosed rats with or without added vitamin E or DPPD were less susceptible than the erythrocytes from normal rats to osmotic challenge, showing that vitamin A was present in levels sufficient to alter the structure of the erythrocyte membrane. These studies show that oxidative hemolysis occurs when the erythrocyte membrane is modified. Furthermore, this oxidative hemolysis is unrelated to lipid peroxidation.     

Cholesterol sulfate inhibits the fusion of Sendai virus to biological and model membranes

Cholesterol sulfate is a component of several biological membranes. In erythrocytes, cholesterol sulfate inhibits hypotonic hemolysis, while in sperm, it can decrease fertilization efficiency. We have found cholesterol sulfate to be a potent inhibitor of Sendai virus fusion to both human erythrocyte and liposomal membranes. Cholesterol sulfate also raises the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine as demonstrated by differential scanning calorimetry and 31P nuclear magnetic resonance spectrometry. Although hexagonal phase structures are not readily found in biological membranes, there is a correlation between the effects of membrane additives on bilayer/non-bilayer equilibria and membrane stabilization. It is proposed that the ability of cholesterol sulfate to alter the physical properties of membranes contributes to its stabilization of biological membranes and the inhibition of membrane fusion.     

Genistein abrogates pre-hemolytic and oxidative stress damage induced by 2,2'-Azobis (Amidinopropane)

The pre-hemolytic mechanism induced by free radicals initiated from water-soluble 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) and its reversal by genistein was investigated in human erythrocytes. The time course of K+ efflux compared to the occurrence of hemolysis suggests that AAPH-induced hemolysis occurs indirectly via pore formation and band 3 oxidation as expected. However, genistein inhibited hemolysis, LDH release and membrane protein oxidation but not K+ efflux. This indicated that erythrocyte protein oxidation possibly in the hydrophobic core plays a significant role in the membrane pre-hemolytic damage. Chemiluminescence (CL) analysis carried out in non-lysed erythrocytes treated with AAPH showed a dramatic increase in CL indicating both reduced levels of antioxidants and increased membrane lipid peroxide. The V0 value was also increased up to 6 times, denoting a high degree of membrane peroxidation very early in erythrocyte membrane damage. The whole process was inhibited by genistein in a dose-dependent manner. These results indicate that the genistein inhibited both hemolysis and pre-hemolytic damage and also hindered membrane lipid peroxide formation and protein oxidation. In addition, it is suggested that pre-hemolytic damage is mediated mainly by the oxidation of both phospholipid and protein located in the deeper hydrophobic region of the membrane.     

Inhibition of monosodium urate monohydrate-mediated hemolysis by vitamin E

Microcrystals of monosodium urate monohydrate(MSUM)induce cytolysis and hemolysis inerythrocytes.In this report,we studied the effect of vitamin E on MSUM-mediated hemolysis in humanerythrocytes.Vitamin E significantly inhibited hemolysis induced by MSUM.The hydroxyl group in thechromanol ring of vitamin E is dispensable for protecting erythrocytes against hemolysis induced by MSUM,indicating that the inhibitory effect of vitamin E is not due to its antioxidant properties.However,both thechromanol ring and the isoprenoid side chain are important for vitamin E to suppress MSUM-induced hemolysis.Our current study suggests that vitamin E inhibits hemolysis induced by MSUM as a membrane stabilizer.     

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of shear stress and free radicals induced by ultrasound on erythrocytes

The present study was undertaken to elucidate the mechanism of hemolysis induced by ultrasound. Ar or N2O gas was used to distinguish between cavitation with or without free radical formation (hydroxyl radicals and hydrogen atoms). Free radical formation was examined by the method of spin trapping combined with ESR. After sonication of erythrocyte suspensions, several structural and functional parameters of the erythrocyte membrane--hemolysis, membrane fluidity, membrane permeability, and membrane deformability--were examined. Although free radical formation was observed in the erythrocyte suspensions sonicated in the presence of Ar, no free radical formation was observed in the presence of N2O. However, the hemolysis behavior induced by ultrasound was similar in the presence of Ar or N2O. The membrane fluidity, permeability, and deformability of the remaining unlysed erythrocytes after sonication in the presence of Ar or N2O were unchanged and identical to those of the control cells. On the other hand, after gamma irradiation (700 Gy), the hemolysis behavior was quite different from that after sonication, and the membrane properties were significantly changed. These results suggest that hemolysis induced by sonication was due to mechanical shearing stress arising from cavitation, and that the membrane integrity of the remaining erythrocytes after sonication was the same as that of control cells without sonication. The triatomic gas, N2O, may be useful for ultrasonically disrupting cells without accompanying free radical formation.     

The relationship of membrane lipids to species specific hemolysis by hemolytic factors from Stomoxys calcitrans (L.) (Diptera: Muscidae)

Posterior-midgut homogenate from female stable flies prepared at 12 h after feeding hemolyzed erythrocytes from 6 different mammalian species more readily than homogenate prepared at 22 h. A significant correlation was obtained between the per cent sphingomyelin content of the erythrocyte membrane and the time required for lysis by the 12 h homogenate. Erythrocytes with low sphingomyelin content were more sensitive to lysis than cells with high sphingomyelin. No such correlation exists for hemolysis by 22 h homogenate. Mean corpuscular volume and osmotic fragilities of erythrocytes were not related to hemolysis either by 12 or 22 h homogenate. Determination of phospholipase C and sphingomyelinase activities showed that the hydrolysis rate of phospholipase C in homogenates prepared at 12–14 h was almost twice as much as sphingomyelinase activity. Whereas hydrolysis rates in 22–24 h homogenate were not different and markedly reduced compared to the 12–14 h homogenate. The times required for erythrocyte hemolysis related to the phospholipase C and sphingomyelinase activity profiles suggests that these enzyme activities participate in the in vitro hemolysis of red blood cells. Bovine and human erythrocytes change their biconcave contour into a spiculated spherical shape when they are exposed to midgut homogenate. This shape change is interpreted as a detergent induced modification of the red cell membrane which renders the erythrocytes more vulnerable to hemolysis.