Colloid Osmotic Hemolysis Research Articles

Prehemolytic impact of phenothiazine drugs on the attachment of spectrin network in red blood cells.

Chlorpromazine, thioridazine, and trifluoperazine are phenothiazine drugs that cause colloid-osmotic hemolysis of human erythrocytes by unknown mechanism. To clarify this mechanism, the impact of these drugs on the βsp (1.4 MHz) and γ1sp (9 MHz) dielectric relaxations was investigated. Each relaxation was shown to reduce its strength on the severing specific bridge that connects the spectrin network with the lipid membrane. For βsp relaxation, this is the spectrin-actin-glycophorin C bridge while for γ1sp relaxation this is the spectrin-ankyrin-band 3 bridge.

Continuous and Delayed Photohemolysis Sensitized With Methylene Blue and Iron Oxide Nanoparticles (Fe3O4)

This research present the sensitization of methylene blue (MB), as a potential photodynamic therapy photo sensitizer which showed phototoxicity for many tumor cells in vitro incorporated with iron oxide nanoparticles (Fe3O4, IO-NP), which offer magnificent interaction both inside and outside the surface of biomolecules together with red blood cells (RBC's) with significant change in hemolysis process. The study investigated the sensitization of continuous photohemolysis (CPH) for MB and MB with IO-NP, delayed photohemolysis (DPH) at different irradiation temperature (Tirr). The photohemolysis rate for CPH at room temperature has a power dependence of 0.39 ± 0.05 with relative of steepness of 1.25 ± 0.02 and for different concentration of MB and power dependent of 0.15 ± 0.03 with relative steepness of 1.34 ± 0.01 for different MB and IO-NP. Logistic and Gompertz functions were applied as appropriate mathematical models to fit the collected experimental data for CPH and DPH respectively, and to calculate fractional photohemolysis rate with minimum errors. The Logistic function parameter; α, the hemolysis rate, increases with increasing concentrations of MB and decreases with increasing IO-NP concentrations in the presence of 6 μg/ml of MB. The parameter β; the time required to reduce the maximum number of RBCs to one half of its value, decreases with increasing MB concentration and increases with increasing IO-NP concentrations in the presence of 6 pg/ml of MB. In DPH at different Tirr, the Gompertz parameter; a, fractional hemolysis ratio, is independent of temperature in both case MB and MB plus IO-NP, while the parameter; b, rate of fractional hemolysis change, increases with increasing Tirr, in both case MB and MB plus IO-NP. The apparent activation energy of colloid-osmotic hemolysis is 9.47±0.01 Kcal/mol with relative steepness of 1.31 ± 0.05 for different MB and 6.06±0.03 Kcal/mol with relative steepness of 1.41 ± 0.09 for MB with iron oxide. Our results suggest that Logistic equation is the best fit for the CPH and Gompertz function for the DPH. Both models predict also that the relative steepness is independent of the light dose, sensitizer and IO-NP concentrations.

Functional significance of the intermediate conductance Ca2+-activated K+ channel for the short-term survival of injured erythrocytes

Increased cytosolic Ca(2+) concentrations activate Gardos K(+) channels in human erythrocytes with membrane hyperpolarization, efflux of K(+), Cl⁻, and osmotically obliged H₂O resulting in cell shrinkage, a phenomenon referred to as Gardos effect. We tested whether the Gardos effect delays colloid osmotic hemolysis of injured erythrocytes from mice lacking the Ca(2+)-activated K(+) channel K(Ca)3.1. To this end, we applied patch clamp and flow cytometry and determined in vitro as well as in vivo hemolysis. As a result, erythrocytes from K(Ca)3.1-deficient (K(Ca)3.1(-/-)) mice lacked Gardos channel activity and the Gardos effect. Blood parameters, reticulocyte count, or osmotic erythrocyte resistance, however, did not differ between K(Ca)3.1(-/-) mice and their wild-type littermates, suggesting low or absent Gardos channel activity in unstressed erythrocytes. Oxidative stress-induced Ca(2+) entry and phospholipid scrambling were significantly less pronounced in K(Ca)3.1(-/-) than in wild-type erythrocytes. Moreover, in vitro treatment with α-toxin from Staphylococcus aureus, which forms pores in the cellular membrane, resulted in significantly stronger hemolysis of K(Ca)3.1(-/-) than of wild-type erythrocytes. Intravenous injection of α-toxin induced more profound hemolysis in K(Ca)3.1(-/-) than in wild-type mice. Similarly, intra-peritoneal application of the redox-active substance phenylhydrazine, an agent for the induction of hemolytic anemia, was followed by a significantly stronger decrease of hematocrit in K(Ca)3.1(-/-) than in wild-type mice. Finally, malaria infection triggered the activation of K(Ca)3.1 and transient shrinkage of the infected erythrocytes. In conclusion, K(Ca)3.1 channel activity and Gardos effect counteract hemolysis of injured erythrocytes, thus decreasing hemoglobin release into circulating blood.

A novel non-lens betagamma-crystallin and trefoil factor complex from amphibian skin and its functional implications.

BackgroundIn vertebrates, non-lens βγ-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.Principal FindingsA naturally existing 72-kDa complex of non-lens βγ-crystallin (α-subunit) and trefoil factor (β-subunit), named βγ-CAT, was identified from frog Bombina maxima skin secretions. Its α-subunit and β-subunit (containing three trefoil factor domains), with a non-covalently linked form of αβ2, show significant sequence homology to ep37 proteins, a group of non-lens βγ-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. βγ-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. βγ-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K+ efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the α-subunit with Western blotting. Furthermore, βγ-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of βγ-CAT (25–50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC50 10 nM) and apoptosis. βγ-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited βγ-CAT induced vacuole formation, significantly inhibited βγ-CAT induced cell detachment, suggesting that βγ-CAT endocytosis is important for its activities.Conclusions/SignificanceThese findings illustrate novel cellular functions of non-lens βγ-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.

Pleurotolysin, a Novel Sphingomyelin-specific Two-component Cytolysin from the Edible Mushroom Pleurotus ostreatus, Assembles into a Transmembrane Pore Complex

Self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly and membrane insertion of transmembrane pores. Here we purified pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied the pore-forming properties of the cytolysin. Pleurotolysin consisted of non-associated A (17 kDa) and B (59 kDa) components, which cooperatively caused leakage of potassium ions from human erythrocytes and swelling of the cells at nanomolar concentrations, leading to colloid-osmotic hemolysis. Hemolytic assays in the presence of poly(ethylene glycol)s with different hydrodynamic diameters suggested that pleurotolysin formed membrane pores with a functional diameter of 3.8-5 nm. Pleurotolysin-induced lysis of human erythrocytes was specifically inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Experiments including solubilization of pleurotolysin-treated erythrocytes with 2% (w/v) SDS at 25 degrees C and SDS-polyacrylamide gel electrophoresis/Western immunoblotting showed that pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes by a sucrose gradient centrifugation. Pleurotolysin A and B formed an SDS-stable, ring-shaped complex of the same dimensions on sphingomyelin-cholesterol liposomes as well.

Enhancement of heme-induced membrane damage by the anti-malarial clotrimazole: the role of colloid-osmotic forces.

Two recent studies have demonstrated that clotrimazole, a well-known potential antifungal agent, inhibits the in vitro growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum. In a previous study, we suggested that clotrimazole acts as an anti-malarial agent by inhibiting heme catabolism in the malaria parasite and by enhancing heme-induced membrane damage. In this paper, we examined the mechanism of action by measuring hemolysis as an indicator of membrane damage. Our results showed that clotrimazole does not promote the binding of heme to membranes, and that the enhancement of heme-induced hemolysis by clotrimazole is not caused by lipid peroxidation or by oxidation of thiol groups in membrane proteins. Instead, clotrimazole inhibits glutathione-dependent heme degradation, resulting in an enhancement of heme-induced hemolysis. We also found that clotrimazole increases the susceptibility of erythrocytes to hypotonic lysis in the presence of heme and that sucrose could inhibit hemolysis induced by heme-clotrimazole complexes. Thus, it appears that the enhancement of heme-induced hemolysis by clotrimazole in our experiments is due to a colloid osmotic hemolysis mechanism. The hydrophobicity and the large molecular size of the heme-clotrimazole complex might be key factors for induction of hemolysis.

Relation between K+ leakage and damage to band 3 in photodynamically treated red cells.

Potassium leakage is one of the first events that appear after photosensitization of red blood cells. This event may subsequently lead to colloid osmotic hemolysis. The aim of our study was to determine which photodynamically induced damage is responsible for increased membrane cation permeability. This was done by studying the effect of dimethylmethylene blue (DMMB)-mediated photodynamic treatment (PDT) on different membrane transport systems. Inhibition of band 3 activity (anion transport) showed a comparable light dose dependency as PDT-induced potassium leakage, whereas glycerol transport activity was inhibited only at higher light doses. Dipyridamole (DIP), an inhibitor of anion transport, protects band 3 against DMMB-induced damage, and prevents the increase in cation permeability of the membrane. Damage to glycerol transport was partially reduced when PDT was performed in the presence of DIP. Because DIP has no affinity for the glycerol transporter, this protection might result from the reduced photodamage to band 3. These results support the hypothesis that band 3 might be involved in glycerol transport. Glucose transport was not affected by DMMB-mediated PDT. The present results are the first to show a causal relationship between DMMB-mediated photodamage to band 3 and increased cation permeability of red blood cells.

Effect of Stobadine on Carbon Tetrachloride-Induced Erythrocyte Membrane Changes in Rats

Our previous study showed that stobadine is effective against ischemia/reperfusion-induced gastric mucosal injury. The present study examined the ability of stobadine to protect erythrocyte membrane against free radical injury after long-term carbon tetrachloride (CCl 4) application. The erythrocyte membrane changes were established using colloid-osmotic hemolysis. The significant increase of colloid-osmotic hemolysis was found in animals treated with CCl 4. CCl 4 also increased formation of thiobarbituric acid-reactive substances (TBARs) and decreased thiol group content. Stobadine in both doses (10.0 and 20.0 mg · kg −1) protected erythrocyte membrane against CCl 4-induced injury. The membrane lipid bilayer is the most affected part of the erythrocyte membrane. In presence of stobadine, CCl 4-induced lipid peroxidation was partially or totally prevented whereas the level of total membrane thiols was increased. Based on these results, it can be concluded that protective effect of stobadine on CCl 4-induced erythrocyte membrane changes should be related to its antioxidant properties.

Changes of erythrocyte microrheology during normal pregnancy and after delivery

Objectives: The changes of blood hemorheology during pregnancy are still the subject of many studies. Erythrocyte membrane deformability (ED) as one of the main components forming blood hemorheology was studied in the present paper. Study design: The changes in ED were measured in healthy non-pregnant women and in three groups of pregnant women in the first, second and third trimestre. The last group consisted of women after delivery. The colloid-osmotic hemolysis method in relation to the change ionic strength was used for ED measurements. The significance of differences was evaluated by the unpaired Student's t-test. Results: The results revealed that ED during pregnancy progressively decreases but after delivery significantly increases. Statistical differences between means in all three trimesters were significant in comparison with non-pregnant women. Conclusion: We conclude that the changes of ED are important for the normal course of gravidity and are probably connected to hormonal changes during pregnancy and after delivery.

Mechanism of hemolysis of human erythrocytes exposed to monosodium urate monohydrate crystals. Preliminary characterization of membrane pores

Microcrystals of monosodium urate monohydrate (MSUM) have the ability to cause rapid hemolysis of erythrocytes. The nature of the initial MSUM crystal-erythrocyte membrane binding interaction was investigated over a range of different ionic strength media. There was negligible binding of MSUM to erythrocyte ghost membranes in low ionic strength media such as isotonic mannitol but binding was dramatically increased in isotonic NaCl/mannitol solutions or isotonic mannitol containing 1 mM Ca 2+. Hemolysis induced by MSUM crystals was preceded by the leakage of K + from the cells suggesting a colloid-osmotic mechanism of hemolysis. The inclusion of large (oligosaccharide) molecules in the extracellular media or the modulation of the extracellular solution tonicity inhibited both the rate and extent of hemolysis supporting the concept of MSUM-induced pores followed by colloid osmotic hemolysis.

Experimental studies of some microrheological factors in serious brain ischemia after stobadine pretreatment.

Serious brain ischemia was induced by occlusion of cerebral arteries in dogs. The occlusion time was 7 min. The blood was collected at various intervals of reperfusion (5, 60, 180, 240 min and 24 h). Thirty minutes before ischemization, stobadine was given (1, 2, or 5 mg/kg). The changes of erythrocyte membrane fluidity were evaluated using colloid-osmotic hemolysis induced by brilliant cresyl blue. In the control group (without stobadine) the colloid-osmotic hemolysis was significantly increased immediately after ischemization and after 5 and 60 min. However, after 240 min of reperfusion, a significant decrease of hemolysis was observed. The increase of colloid-osmotic hemolysis after ischemization in the control group was prevented after stobadine pretreatment. The thrombotization of microcirculation that was observed in the control group was not present after stobadine pretreatment.

Membrane electrofusion yields in membrane fractions obtained with a colloid-osmotic hemolysis and electrohemolysis procedure

Electrofusion yields in two subpopulations of erythrocyte ghosts, as obtained by fractionation based on either electroporation sensitivity or osmotic fragility, are not significantly different. This implies that whatever membrane property responds to electroporation or osmotic fragility, that property does not also modulate electrofusion yield. Other experiments showed that, unlike osmotic-based hemolysis, electrohemolysis of erythrocytes is accompanied by a cell volume increase, due to swelling, which does not exceed 120%–125% of the control volume while pure osmotic swelling can easily increase the volume up to 150%–160% of the control volume. Also, control experiments involving osmotic hemolysis of erythrocytes can be used to develop a simple and convenient method to estimate the relative volume fraction occupied by hemoglobin in erythrocytes.

Gramicidin, VDAC, porin and perforin channels

This review covers the miscellaneous channels that do not fall w=ithin the domain of the other reviews in this section. Three different channel systems will be reviewed: the gramicidin A channel; the large weakly selective channels that are present in the outer membrane of mitochondria (VDACs) and bacteria (porin); and the large channels whose function is to kill cells by colloid osmotic hemolysis (perform).

Effects of chlordecone and chlordecone alcohol on isolated ovine erythrocytes

Chlordecone (CHLO, 14-30 microM) and chlordecone alcohol (CHLO ALC, 10-23 microM) altered the permeability of isolated ovine erythrocytes as evidenced by a concentration- and time-dependent induction of K+ efflux and hemolysis. Hemolysis, but not K+ efflux, was markedly delayed when the erythrocytes were suspended in isotonic sucrose. CHLO-induced and CHLO ALC-induced hemolysis and K+ efflux were dependent on the pH of the external media. Raising the pH from 6.5 to 9.4 inhibited CHLO-induced K+ efflux and hemolysis, whereas CHLO ALC-induced K+ efflux and hemolysis were increased. Low concentrations of both compounds (1-4 microM) protected erythrocytes against hypotonic hemolysis. Neither CHLO (30 microM) nor CHLO ALC (23 microM) induced the release of trapped K+ from KSCN-loaded unilamellar liposomes made from erythrocyte lipids. It is postulated that CHLO and CHLO ALC interact with membrane proteins and increase the permeability of the membrane to cations. This interaction leads to colloid-osmotic hemolysis.

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.

Perspectives on hydrogen peroxide and drug-induced hemolytic anemia in glucose-6-phosphate dehydrogenase deficiency

G-6-Pd-deficiency is a genetic disorder of erythrocytes in which the inability of affected cells to maintain NAD(P)H levels sufficient for the reduction of oxidized glutathione results in inadequate detoxification of hydrogen peroxide through glutathione peroxidase. Although a variety of free-radical species may be produced during the interaction of xenobiotic agents with erythctrocytes and hemoglobin, the inability to destroy peroxides seems to be the hallmark of the disease. Colloid osmotic hemolysis is seldom observed in this disorder and it is possible that hydroxyl radicals derived from peroxide damage both lipid and protein constituents of the plasma membrane so that its intrinsic mechanical properties are altered. Erythrocytes with damaged membranes become less deformable and may be subjected to mechanical entrapment in the microcircultion. Ultimate recognition of damaged cell and sequestration by phagocytes leads to anemia.

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

A kinetic model of colloid osmotic hemolysis for cation-permeable cell 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.

Erythropoietic protoporphyria: lipid peroxidation and red cell membrane damage associated with photohemolysis.

The mechanism by which long wavelength ultraviolet light hemolyzes red cells obtained from patients with erythropoietic protoporphyria (EPP) was investigated. Previous studies had suggested that irradiation of these red cells with wavelengths of light capable of eliciting dermatological manifestations led to oxygen-dependent colloid osmotic hemolysis through the formation of peroxides. In the present report, lipid peroxidation during in vitro irradiation of EPP red cells with long ultraviolet light was demonstrated by: (a) the formation of 2-thiobarbituric acid reactants; (b) the presence of conjugated diene bonds in red cell lipid; and (c) the selective loss of unsaturated fatty acids proportional to the number of carbon-carbon double bonds in each. Irradiation of EPP red cells was also shown to result in the formation of hydrogen peroxide.Before photohemolysis there was a decline in cell membrane sulfhydryl groups and a loss in activity of the cell membrane enzyme acetylcholinesterase. These parameters provide further evidence suggesting that the cell membrane is a primary site of the photohemolytic effect of long ultraviolet light in EPP red cells. Further evaluation of the radiation-induced inactivation of EPP red cell acetylcholinesterase was performed by radiating mixtures containing bovine erythrocyte acetylcholinesterase and protoporphyrin IX. These studies revealed that the rate of decline in enzyme activity is accelerated by the addition of linoleic acid, an unsaturated fatty acid, but not by palmitic acid, a saturated fatty acid. Partial protection against both photohemolysis and acetylcholinesterase decline is provided by alpha-to-copherol. This lipid antioxidant loses its activity during the irradiation of EPP red cells suggesting that it is utilized in this process.

Metabolic aspects of the photodynamic effect of protoporphyrin in protoporphyria and in normal red blood cells

The photodynamic action of protoporphyrin on erythrocytes of patients with erythropoietic protoporphyria and on sensitized normal red blood cells leads to K + loss and Na + gain, resulting finally in colloid osmotic hemolysis. To elucidate the pathochemical background of this process, the ATP and glutathione-producing metabolic systems and the activity of other cellular enzymes was studied during irradiation, in the prelytic period. The glucose consumption, intracellular ATP level and catalase activity were not influenced by the photochemical process, whereas the cellular glutathione concentration decreased during irradiation. The activity of cellular acetylcholinesterase and of membrane ATPase decreased in the prelytic period, the (Na + + K +)-dependent ATPase being more sensitive than the Mg 2+-stimulated ATPase. Measurements of 86Rb + uptake demonstrated a decreasing active Rb + transport with a concomitant increase of passive Rb + permeability during irradiation. The results indicated that the cellular membrane is presumably the primary target of the protoporphyrin-induced photodynamic process.

Photoreactions associated with in vitro hemolysis in erythropoietic protoporphyria.

Abstract— –Photohemolysis of erythrocytes. from patients with erythropoietic protoporphyria results from damage to the cell membrane following photoexcitation of the protoporphyrin molecule by 400 nm radiation. Photochemical reactions were assessed for their roles in initiating cell destruction in an in vitro model in which erythrocytes from patients with erythropoietic protoporphyria were irradiated. Electron transfer properties were demonstrated using 2,3,6‐trichloroindophenol as an electron acceptor. Photohemolysis of these abnormal cells is due to cell membrane damage initiating colloid osmotic hemolysis. Additional data are offered demonstrating that this type of photohemolysis is oxygen‐dependent, associated with peroxide formation, and preceded by increased osmotic fragility. An hypothesis explaining the interrelationship of the photochemistry of protoporphyrin with the photobiologic observation of hemolysis is offered.