PH-dependence of detergent-induced hemolysis and vesiculation of erythrocytes

The influence of pH of the medium on the parameters of detergent-induced fast hemolysis and vesiculation of human erythrocytes was studied. In the range of pH 6.3-7.2 neither the extent nor the rate of the vesiculation induced by 25 microM sodium dodecyl sulfate (SDS) changed. However, a decrease of pH from 8.0 to 5.8 strongly modified both the extent and the rate of the hemolysis induced by SDS. Within the range of pH 8.0-6.4, the effect can be ascribed to the increase of the positive charge of the membrane. This could lead to the accumulation of the membrane-bound anion detergent and, hence, to the change of the hemolysis parameters. Non-charged detergent Triton X-100 did not display any pH-dependence. At pH between 6.4 and 5.8 the extent and rate of hemolysis changed in a complicated manner. The kinetic curves of hemolysis could be approximated by a single exponential within the pH range between 8.0 and 7.2. Upon further reduction of pH, a second exponential component, with a larger time constant, appeared in the kinetic curves. At 5.8 < pH < 7.2, the contribution of the "fast" hemolysis dropped virtually to zero, with pK about 6.0. This points to a structural transition of the membrane, possibly involving histidine. We suggest that the parameters of the detergent-induced hemolysis are sensitive to the changes of the charge and structural state of erythrocyte membrane.     

Parameters of hemolysis by sodium dodecyl sulfate as an indicator of the structural states of erythrocyte membranes]

The relationship between the parameters (percentage and rate) of hemolysis by sodium dodecyl sulfate and the time of incubation of human erythrocyte suspensions in glucose-free medium at 37 degrees C was studied. The polyphasic changes in the parameters were found, which depend on the mode of pretreatment: ATP depletion by iodoacetate, heat denaturation of spectrin, and treatment of cells by valinomycin. It was found that the percentage and rate of detergent hemolysis do not always change in parallel.     

Preservation of resuspended red cell concentrates. Rate of vesiculation and of spontaneous hemolysis

In resuspended red cell concentrates addition of sucrose, mannitol and sorbitol (30 mM final concentration each) to the SAG medium (150 mM NaCl, 50 mM glucose, 1.25 mM adenine) results in a significant reduction of the spontaneous hemolysis of the cells to about 25% after 3 weeks and to about 40% after 6 weeks preservation. Furthermore, in comparison to the SAG medium the vesiculation rate is reduced to about 40% after 3 weeks preservation. Clear cut differences in the effects between the three additives could not be found. The addition of guanosine (1.25 mM final concentration) to the SAG-sucrose or SAG-sorbitol medium has no significant effects on hemolysis and vesiculation.     

pH-dependent hemolysis and cell fusion of rhabdoviruses

Human erythrocytes pretreated with fungal semialkali protease or trypsin became susceptible to hemagglutination by vesicular stomatitis virus (VSV) and rabies virus. Both viruses exhibited extensive hemolytic and fusion activities against erythrocytes pretreated with these enzymes. The hemolysis and fusion were pH dependent and the activities were most apparent at pH 5.0 and decreased with increase in pH. However, VSV still exhibited slight hemolytic activity at neutral pH. Hemolysis was also dependent on the dose of virus and was inhibited by treatment of the viruses with antiviral antibody. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis of erythrocyte membranes suggested that most of the carbohydrates were removed from the membrane proteins by the treatment with proteolytic enzymes.     

The mechanism of bile salt-induced hemolysis.

The hemolytic activities of sodium deoxycholate (DChol) and its tauro-conjugate (TDChol) and glyco-conjugate (GDChol) were analysed. 50 % hemolysis occurred in 30 min at pH 7.3, at the concentrations of these detergents equal to 0.044, 0.042 and 0.040 % respectively. These values are below their critical micellar concentrations. Based on its kinetics, this hemolysis is classified as being of permeability type. The detergents increase the permeability of erythrocyte membranes to KCl, and colloid osmotic hemolysis occurs. The minimum of hemolytic activity of the three cholates is at about pH 7.5. A very high increase in hemolytic activity occurs at pHs below 6.8, 6.5 and 6.2 for DChol, TDChol, and GDChol, respectively. These values are close to the pK(a) for DChol (6.2), but much higher than the pK(a) for TDChol (1.9) and GDChol (4.8). It is therefore suggested that the increase in hemolytic activity is not a result of the protonation of the anionic groups of the cholates. At acidification below pH 6, the kinetics of DChol induced hemolysis change to the damage type characterised by nonselective membrane permeability. Such a transition is not observed in TDChol and GDChol induced hemolysis. It is therefore suggested that the change in the type of hemolysis depends on protonation of the anionic group of cholates.     

Suppression of high-pressure-induced hemolysis of human erythrocytes by preincubation at 49 degrees C.

When human erythrocytes were preincubated at 37-52 degrees C under atmospheric pressure before exposure to a pressure of 200 MPa at 37 degrees C, the value of hemolysis was constant (about 43%) up to 45 degrees C but became minimal at 49 degrees C. The results from anti-spectrin antibody-entrapped red ghosts, spectrin-free vesicles, and N-(1-pyrenyl)iodoacetamide-labeled ghosts suggest that the denaturation of spectrin is associated with such behavior of hemolysis at 49 degrees C. The vesicles released at 200 MPa by 49 degrees C-preincubated erythrocytes were smaller than those released by the treatment at 49 degrees C or 200 MPa alone. The size of vesicles released at 200 MPa was independent of preincubation temperature up to 45 degrees C, and the vesicles released from 49 degrees C-preincubated erythrocytes became smaller with increasing pressure up to 200 MPa. Thus, hemolysis and vesiculation under high pressure are greatly affected by the conformation of spectrin before compression. Since spectrin remains intact up to 45 degrees C, the compression of erythrocytes at 200 MPa induces structural changes of spectrin followed by the release of large vesicles and hemolysis. On the other hand, in erythrocytes that are undergoing vesiculation due to spectrin denaturation at 49 degrees C, compression produces smaller vesicles, so that the hemolysis is suppressed.     

Peroxidation of liposomes in the presence of human erythrocytes and induction of membrane damage of erythrocytes by peroxidized liposomes

Hemolysis (Kobayashi, T., Takahashi, K., Yamada, A., Nojima, S. and Inoue, K. (1983) J. Biochem. 93, 675-680) and shedding of acetylcholinesterase-enriched membrane vesicles (diameter 150-200 nm) were observed when human erythrocytes were incubated with liposomes of phosphatidylcholine which contained polyunsaturated fatty acyl chains. These events occurring on erythrocyte membrane were inhibited by radical scavengers or incorporation of alpha-tocopherol into liposomes, suggesting that lipid peroxidation is involved in the process leading to membrane vesiculation and hemolysis. The idea was supported by findings that generation of chemiluminescence, formation of thiobarbituric acid reactive substance, accumulation of conjugated diene compounds in liposomes and decrease of polyunsaturated fatty acids in liposomes occurred concomitantly during incubation. Hemolysis was also suppressed by the addition of extra liposomes, insensitive to peroxidation, or of serum albumin even after the completion of peroxidation of liposomes. These results suggest that peroxidized lipids, responsible for vesiculation and hemolysis, may be formed first in liposomes and then gradually transferred to erythrocyte membranes. The accumulation of these lipids peroxides may eventually cause membrane vesiculation followed by hemolysis.     

The prevention of asbestos-induced hemolysis.

The hemolytic potency of different types of asbestos dusts and silica (min-u-sil) is described. Dust-induced hemolysis can be prevented to varying degrees by protection with erythrocyte membranes, tissue culture medium (containing 20% serum) and by pulmonary surfactant. It is suggested that the binding of relatively non-soluble serum or surfactant materials to asbestos dusts (particularly chrysotile) is important to prevent hemolysis. The relevance of the present study to the initiation of cellular damage by inhaled asbestos is discussed.     

Cold-induced hemolysis in a hypertonic milieu

Summary Suspension of human erythrocytes at 37° C in an environment made hypertonic by increasing concentrations of sodium chloride and sucrose was followed by hemolysis when the temperature was lowered to 0° C. Two distinct stages were involved in this hemolytic phenomenon, the first being incubation with hypertonic solute at some temperature above 20° C with an increasing effect up to 45° C, and the second stage consisting of lowering the temperature below 15° C with increasing hemolysis down to 0° C. The rate of cooling was not an important factor, but the presence of ions reduced the extent of cold-induced hemolysis in hypertonic sucrose. No significant release of membrane phospholipid and cholesterol accompanied this hemolysis. The solubilization of membrane protein components was investigated, with some differences appearing on sodium dodecyl sulfate polyacrylamide gel electrophoresis between hypertonic and isotonic supernatants. Spectrin could not be identified in solubilized form. Correlation of the temperatures of note in these studies with results from the literature on other biological effects of temperature-induced phase transitions in membrane lipids strongly points to the conclusion that such transitions are involved in the mechanism of cold-induced hypertonic hemolysis. It is postulated that the hypertonic milieu has resulted in membrane-protein alteration damage which prevents normal adaption to the new physical state of the membrane lipids during cooling.     

Mechanism of free radical-induced hemolysis of human erythrocytes: comparison of calculated rate constants for hemolysis with experimental rate constants.

We previously developed a simple competitive reaction model between lipid peroxidation and protein oxidation in erythrocyte membranes that accounts for radical-induced hemolysis of human erythrocytes. In this study, we compared the rate constants calculated from the hemolysis curves of erythrocytes in the presence of radical initiators with those obtained from experiments using erythrocyte ghosts treated with radicals. 2,2'-Azobis(amidinopropane) dihydrochloride and 2,2'-azobis(2,4-dimethylvaleronitrile) were used as radical initiators. Plots of the logarithm of concentration of the radical initiator against the logarithm of the rate constant gave straight lines. The slope of the lines for the calculated lipid peroxidation was nearly equal with the experimental value. Similar results were obtained for oxidation of membrane proteins, except for band 3 oxidation. The values for the rate constants calculated from hemolysis curves seem to be accurate. The slope of the lines for the calculated rate constants for proteins was larger than the experimental value for band 3 oxidation, because band 3 oxidation is accompanied by aggregation or redistribution of band 3 proteins to form hemolytic holes. These results indicate that the competitive reaction model may be useful for analyzing radical-induced hemolysis.     

A kinetics study of pig erythrocyte hemolysis induced by polyene antibiotics

The kinetics of the hemolysis induced by filipin is of the damage type, indicating the formation of large nonselective perforations of erythrocyte membranes. The process is relatively independent of the ionic composition of the incubation medium, and the differences between the hemolysis induced by filipin in pig and human erythrocytes are not significant. In a sucrose medium, filipin-induced hemolysis is inhibited in humans, whereas it is stimulated in pig erythrocytes. It is suggested that low ionic strength is the reason for the different modifications of complexation of filipin in pig and human erythrocyte membranes in a sucrose medium. The kinetics of the hemolysis induced in pig erythrocytes by amphotericin B and nystatin is of the permeability type, indicating the formation of selective channels in erythrocyte membranes and colloid osmotic hemolysis. The rate of the hemolysis, which is high in a KCl medium, is decreased in all the other media tested (CaCl2, MgCl2, potassium phosphate buffer, K2SO4, sucrose), although there are no changes in the kinetics of hemolysis. The results are interpreted as the formation of highly selective channels at a low concentration of the antibiotics. At increasing concentrations, channels of decreasing selectivity occur. The resistances of pig erythrocytes to amphotericin B and nystatin are lower than those of human erythrocytes.     

Dextran protection of erythrocytes from low-pH-induced hemolysis

Low-pH-induced hemolysis of erythrocytes is inhibited by dextrans. The protective effect was observed with dextrans larger than 40 kDa. Electron microscopy showed dextrans of 150 kDa in a tight association with the erythrocyte membrane. These results indicate that dextrans stop the low-pH-induced hemolysis by interacting with the acid-induced defects in the erythrocyte membrane [(1989) Biochim. Biophys. Acta, in press.     

Ceramide-enriched membrane domains in red blood cells and the mechanism of sphingomyelinase-induced hot-cold hemolysis

Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 degrees C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 degrees C. The mechanism of this phenomenon is not understood. PlcHR 2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, that is the prototype of a new phosphatase superfamily, induces hot-cold hemolysis. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot-cold hemolysis because the phenomenon occurs not only in human erythrocytes that contain both phosphatidylcholine (PC) and sphingomyelin (SM) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot-cold hemolysis induced by PlcHR 2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR 2 activity. After cooling down to 4 degrees C, the erythrocyte ghost membranes arising from hemolysis contain large, ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to hemolysis. We also interpret the slow hemolysis observed at 37 degrees C as a phenomenon of gradual release of aqueous contents, induced by the sphingomyelinase activity, as described by Ruiz-Arguello et al. [(1996) J. Biol. Chem. 271, 26616]. These hypotheses are supported by the fact that ceramidase, which is known to facilitate slow hemolysis at 37 degrees C, actually hinders hot-cold hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR 2 demonstrates the presence of ceramide-rich domains that are rigid at 4 degrees C but fluid at 37 degrees C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally, in liposomes composed of SM, PC, and cholesterol, which exhibit slow release of aqueous contents at 37 degrees C, addition of 10 mol % ceramide and transfer to 4 degrees C cause a large increase in the rate of solute efflux.     

Effects of cooling rate on thermal shock hemolysis

The increase in thermal shock hemolysis in hypertonic sodium chloride with increasing cooling rate was confirmed. Thermal shock damage was also induced by hypertonic solutions of sucrose but it decreased with increasing cooling rate. The effect of cooling rate on thermal shock hemolysis appears to be due to the time that the cells are in the hypertonic solutions. The extent of the stress of the temperature reduction was independent of the cooling rate. In hypertonic sodium chloride susceptibility to thermal shock damage increased with increasing time of exposure at +25 °C (0–5 min) before decreasing with time (5–50 min). In contrast, with hypertonic sucrose, thermal shock damage increased gradually with time of exposure. The protective effects of sucrose on thermal shock hemolysis at a given osmolality can be explained by the different solution properties (e.g., ionic strength) of hypertonic sodium chloride and sucrose. These results suggest that the role of thermal shock damage during slow freezing should be reexamined.     

High-pressure-induced hemolysis in papain-digested human erythrocytes is suppressed by cross-linking of band 3 via anti-band 3 antibodies

Upon exposure of human erythrocytes to a high pressure of 200 mPa, both hemolysis and vesiculation occur. The hemolysis of erythrocytes at 200 mPa was enhanced by removal of sialic acids from the membrane surface with papain. However, such enhancement was suppressed by cross-linking of band 3 via an anti-band 3 antibody (AB3A), which recognizes the exofacial domain of band 3, or by clustering of band 3 via Zn2+. On the other hand, the size of high-pressure-induced vesicles increased from 423 to 525 nm in diameter upon exposure to papain of erythrocytes, but decreased to 444 nm with following treatment with AB3A. In these vesicles, the content of spectrin relative to band 3 was almost the same. Furthermore, the band 3-cytoskeleton interactions in erythrocyte membranes remained unaltered upon treatment with papain and AB3A. Flow cytometric analysis demonstrated that papain-pretreated erythrocytes mainly produce open ghosts at 200 mPa and that the production of such open ghosts is suppressed by AB3A. Thus, upon removal of negative charges from the membrane surface, open ghosts are readily produced due to the release of larger vesicles under pressure. Upon cross-linking of band 3 via AB3A, however, the release of smaller vesicles at 200 mPa is facilitated so that high-pressure-induced hemolysis is suppressed.     

Erythrocyte hemolysis by detergents--a mathematical model and analysis of the concentration and kinetic curves

A mathematical model of erythrocyte lysis by detergents is developed which takes into consideration the kinetics of detergent binding to plasma membrane. Experimentally obtained sigmoidal kinetic and concentration curves of hemolysis are well described by the model. A comparative study is carried out in terms of the model of hemolytic action for five detergents: Triton X-100, sodium dodecylsulfate, sodium deoxycholate, cetyltrimethylammonium bromide, and cetylpyridinium chloride. The amount of detergent which should be bound to an erythrocyte membrane to induce lysis was found to be roughly the same for all detergents studied. However, detergents vary in their affinity to the membrane. Cetylpyridinium displays the highest affinity (and consequently the highest hemolytic activity), whereas deoxycholate has the least one.     

Lead-induced changes of cation-osmotic hemolysis in rats.

Erythrocyte microrheology changes were measured using cation-osmotic hemolysis (COH) in healthy rats and rats after 6 and 12 months of lead ingestion. Using COH, properties of two membrane constituents, spectrine membrane skeleton and membrane bilayer were studied. COH in rats after 12 months of lead ingestion was significantly lower only in the area with lower ionic strength (15.4-61.6 mmol x l(-1) of NaCl) (p < 0.01 resp. p < 0.05). No changes in COH were found after 6 months of continuous lead ingestion. The relation between cation-osmotic hemolysis and erythrocyte deformability is being discussed.     

Stoichiometry of hemolysis by the polyene antibiotic lucensomycin

The stoichiometry of hemolysis by the polyene antibiotic lucensomycin was investigated. It appears that hemolysis occurs only when a relatively high fraction (probably between 15 and 40%) of the cholesterol sites in the erythrocyte membrane have combined with the polyene. Also in phospholipid-cholesterol vesicles the increase of permeability requires occupancy of 40–50% of the existing cholesterol sites.As for the possible cooperative effect in the hemolytic process, it is probable that several (at least 9–10) lucensomycin-cholesterol adducts must interact on each side of the membrane to form an aqueous channel; the distribution of these adducts in the erythrocyte membrane occurs, however, apparently at random.     

Disulfiram may mediate erythrocyte hemolysis induced by diethyldithiocarbamate and 1,4-naphthoquinone-2-sulfonate.

The increase in 1,4-naphthoquinone-2-sulfonate (NQS)-induced hemolysis by the superoxide dismutase inhibitor diethyldithiocarbamate (DEDC) was formerly attributed to increased superoxide anion levels in the erythrocyte. Our results show that removal of DEDC after preincubation and prior to the addition of NQS did not produce a significant increase in hemolysis, which suggests that hemolysis is primarily caused by the reaction products of DEDC with NQS and not to the inactivation of superoxide dismutase. Disulfiram, the oxidized product of DEDC, was found to be the main product formed when excess DEDC was reacted with NQS. Oxygen uptake also occurred and hydrogen peroxide was formed. The latter caused the oxidation of DEDC to disulfiram as catalase prevented disulfiram formation. Disulfiram was found to readily hemolyze erythrocytes at low concentrations as well as to crosslink the proteins in the erythrocyte membrane. Furthermore, disulfiram-induced hemolysis was markedly enhanced in glutathione-depleted erythrocytes. Disulfiram was subsequently found to readily oxidize glutathione in red blood cells. When equimolar concentrations of DEDC and NQS were reacted, the major product formed was the diethyldithiocarbamate:1,4-naphthoquinone (DEDC:NQS) conjugate. However, the principal mediator of erythrocyte hemolysis when excess DEDC is reacted with 1,4-naphthoquinone-2-sulfonate is disulfiram, whose mode of action may be to modify membrane protein sulfhydryls.     

Exercise-induced hemolysis is caused by protein modification and most evident during the early phase of an ultraendurance race.

Whether structural changes of the erythrocyte membrane increase the susceptibility to hemolysis particularly of the relatively older cell population during the early phase of a 216-km ultrarace was tested in six male runners (age 53.6 +/- 10.4 yr, height 175.8 +/- 11.1 cm, body mass 75.9 +/- 8.4 kg). Erythrocyte membrane spectrins were lowest (P < 0.001) after 42 km (75.59 +/- 5.25% of prerace) and increased (P < 0.001) toward 216 km (88.27 +/- 3.37%). Susceptibility to osmotic hemolysis was highest (P < 0.01) after 42 km (107.34 +/- 3.02 mOsm sodium phosphate buffer) with almost identical (P > 0.05) values prerace (97.98 +/- 3.41 mOsm) and postrace (98.61 +/- 3.26 mOsm). Haptoglobin indicated intravascular hemolysis of 9.27 x 10(9) cells/l (P < 0.05) during the initial 84 km. Changes in hematocrit and plasma proteins indicated an estimated total net erythrocyte loss of 3.47 x 10(11) cells/l (P < 0.05) after 21 km. This was compensated by a gain in erythrocytes (P < 0.05) of 3.31 x 10(11) cells/l during the final 132 km. A main effect (P < 0.05) on erythropoietin suggests increased erythropoiesis throughout the race. Exercise-induced hemolysis reflects alterations in erythrocyte membrane spectrins and occurs particularly in the early phase of an ultraendurance race because of a relative older cell population.