A simple method for determination of red blood cell mechanical fragility in the rat

A method for measuring the mechanical fragility of red blood cells suitable for use in small laboratory animals, such as rats, is reported because of lack of such data in the literature. Whole blood is mixed with phosphate buffered saline in a tube containing glass beads. The tubes are rocked for 90 minutes, centrifuged and the percent hemolysis determined. Varying the osmolality of the saline suspending medium had little effect on the mechanical fragility of rat red cells prior to the NaCl concentrations at which a significant change in osmotic hemolysis occurred. The duration of rocking increased the mechanical fragility. Varying the pH (6.4-8.0) had no effect. The size of the glass beads changed the mechanical fragility as did varying temperature. The mean mechanical fragility of rat red blood cells was 46% hemolysis (80 adult male animals). Because of the small volume of blood required with this method, mechanical fragility of red cells of other small laboratory animals also may be determined.     

HEMOLYSIS OF CHICKEN BLOOD

1. The time-dilution curves are given for the hemolytic action of saponin, sodium taurocholate, and sodium oleate on nucleated chicken erythrocytes. 2. Saponin and sodium taurocholate cause hemolysis but leave the nuclei and ghosts in suspension, thereby making the end-point of hemolysis more arbitrary than the clear end-point for non-nucleated cell hemolysis. 3. The curves of hemolysis by saponin and taurocholate are shown to be of the same nature as are found in the hemolysis of non-nucleated cells. 4. Sodium oleate causes first hemolysis and then, in the stronger solutions, causes karyolysis. Two pairs of values for κ and c = ∞ are thus obtainable for the same reaction, one pair for the destruction of corpuscular membrane, the other pair for the destruction of the nucleus. 5. Viscosity changes are found in the lysin-cell system with strong concentrations of sodium taurocholate and sodium oleate. Time-viscosity curves are given for these changes. 6. Microscopically, the action of these lysins on the nucleated chicken red cell appears to be similar to their action on the non-nucleated erythrocytes.     

The use of ELISA to monitor amplified hemolysis by the combined action of osmotic stress and radiation: potential applications

A new assay has been developed to study the osmotic fragility of red blood cells (RBCs) and the involvement of oxygen-derived free radicals and other oxidant species in causing human red blood cell hemolysis. The amount of hemoglobin released into the supernatant, which is a measure of human red blood cell hemolysis, is monitored using an ELISA reader. This ELISA-based osmotic fragility test compared well with the established osmotic fragility test, with the added advantage of significantly reduced time and the requirement of only 60 mul of blood. This small amount of blood was collected fresh by finger puncture and was immediately diluted 50 times with PBS, thus eliminating the use of anticoagulants and the subsequent washings. Since exposure of RBCs to 400 Gy gamma radiation caused less than 5% hemolysis 24 h after irradiation, the RBC hemolysis induced by gamma radiation was amplified by irradiating the cell in hypotonic saline. The method was validated by examining the protective effect of Trolox, an analog of vitamin E and reduced glutathione (GSH), a well-known radioprotector, against human RBC hemolysis caused by the combined action of radiation and osmotic stress. Trolox, a known membrane stabilizer and an antioxidant, and GSH offered significant protection. This new method, which is simple and requires significantly less time and fewer RBCs, may offer the ability to study the effects of antioxidants and membrane stabilizers on human red blood cell hemolysis induced by radiation and oxidative stress and assess the osmotic fragility of erythrocytes.     

THE KINETICS OF IN VIVO HEMOLYTIC SYSTEMS

This paper is concerned with a variety of questions which bear on the occurrence of hemolysis in vivo, and with the possibility of regarding the contents of the blood stream as a hemolytic system in which a steady state is maintained by the production of new red cells to replace those which are destroyed. The material which is dealt with includes the following. 1. Mixtures of Lysins, Accelerators, and Inhibitors.—The effects of individual accelerators and inhibitors in mixtures, like the effects of individual lysins, are roughly additive in simple systems, the acceleration or inhibition produced by the individual substances being most conveniently measured in terms of R-values. 2. Normal Intravascular Lysins.—These probably play only a small part in red cell destruction unless their concentration rises to unusual levels, or unless their effects are enhanced by accelerators, or by the reduction of the concentration of normal inhibitors. The three normal in vivo hemolytic processes for which there is substantial evidence involve (a) the action of the bile salts and of the soaps derived from chyle, (b) the action of the spleen, and (c) the action of hemolytic substances derived from tissues. The recent observations of Maegraith, Findlay, and Martin on the presence of widely distributed tissue lysins are confirmed except for their conclusion that these lysins are species-specific. Species-specific tissue lysins, if present, are not the only lysins derivable from tissues by simple immersion in saline, for non-species-specific lytic substances can also be obtained, and seem to be similar to the "lysolecithin" which some regard as responsible for the action of the spleen on red cell fragility and shape. 3. Plasma Inhibitors.—About 30 per cent of the total inhibitory effect of plasma for saponin hemolysis is due to the contained cholesterol, while 25 per cent at most is due to the plasma proteins, particularly globulins. The remaining 45 per cent is probably accounted for by enhancing effects among the inhibitors; e.g., the enhancing effect of lecithin on the cholesterol inhibition. The mechanism of the inhibition is still incompletely understood; probably reactions between inhibitor and lysin and reactions between inhibitor and components of the red cell surface are both involved, and it is important to observe that the inhibitory effect of plasma or of a plasma constituent may be greater in systems containing one lysin than in systems containing another. No evidence for diffusible inhibitory substances in plasma has been found, and the variations observed in the inhibitory power of human plasma seem to be related to the combined concentrations of cholesterol, protein, and probably lecithin, rather than to the cholesterol content alone. For this reason the inhibitory power tends to be low under conditions of poor nutrition. 4. The Steady State and the Kinetics of Hemolysis In Vivo.—On the assumption that the steady state is the result of a balance between a process which produces red cells and a process which destroys them, equations have been developed for the way in which cells of different resistances are affected when the rate of destruction changes. A method for analyzing experimental curves is described and illustrated. In general, this part of the paper relates the level of the red cell count in the animal to the intensity of the hemolytic processes taking place in vivo, and does not lend itself to detailed abstraction.     

Hyperglycemia can cause membrane lipid peroxidation and osmotic fragility in human red blood cells

The present study has examined the effect of elevated glucose levels on membrane lipid peroxidation and osmotic fragility in human red blood cells (RBC). Defibrinated whole blood or RBC were incubated with varying concentrations of glucose at 37 degrees C for 24 h. RBC incubated with elevated levels of glucose showed a significantly increased membrane lipid peroxidation when compared with control RBC. A significant positive correlation was observed between the extent of glucose-induced membrane lipid peroxidation and the osmotic fragility of treated RBC. Glucose-induced membrane lipid peroxidation and osmotic fragility were blocked when RBC were pretreated with fluoride, an inhibitor of glucose metabolism; with vitamin E, an antioxidant; with para-chloromercurobenzoate and metyrapone, inhibitors of the cytochrome P-450 system; or with dimethylfurane, diphenylamine, and thiourea, scavengers of oxygen radicals. RBC treated with elevated glucose concentrations also showed an increase in NADPH levels. Exogenous addition of NADPH to normal RBC lysate induced membrane lipid peroxidation similar to that observed in the glucose-treated RBC. These data suggest that elevated glucose levels can cause the peroxidation of membrane lipids in human RBC.     

Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3 h in a 37 °C water bath, red blood cells were frozen in liquid nitrogen for 24 h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4 °C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04 ± 21.68% and significantly more than that of the unfrozen cells (0.95 ± 0.28%, P < 0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400 mM, the percentage of damaged cells was 1.97 ± 0.73% and similar to that of the unfrozen cells (P > 0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400 mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides (P < 0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4 °C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.     

Flow cytometric analysis of peripheral blood erythrocyte chimerism in alpha-thalassemic mice.

A rapid and reliable method for longitudinal studies on the degree of red cell chimerism following bone marrow transplantation of alpha-thalassemic recipient mice is presented. Blood obtained by tail clipping from transplanted mice was analyzed by measuring forward light scatter (FLS) distribution of red cells using a flow cytometer. Amplification and threshold of FLS were specifically adjusted. For flow cytometric analysis, the red cells needed to be suspended in hypotonic saline (103 mmol/l NaCl). Osmotic fragility testing showed that lysis of erythrocytes did not significantly influence the measurements. Flow cytometric measurement allowed for a rapid determination of the degree of red cell chimerism.     

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.     

Effect of inositol hexaphosphate on the transient behavior of red cells following a DMSO-induced osmotic pulse

An osmotic pulse can be used to incorporate inositol hexaphosphate (IHP) into red cells. The pulse is induced by equilibrating a red cell suspension with DMSO and then rapidly diluting with an isotonic IHP solution. Since IHP binds to hemoglobin and lowers the affinity for oxygen, this method may find application in the preparation of low-affinity cells for experimental and clinical use. The experiments reported here examined the dynamic changes of several red cell variables immediately following the osmotic pulse. The effect of IHP, which has been shown to dissociate red cell cytoskeletons, was evaluated by comparison with a matched phosphate-buffered saline (PBS) diluent. Red cell morphology, volume, and hemoglobin permeability were studied by fixing the cells at times ranging from 0.06 to 300 sec after dilution. Mechanical fragility was measured by subjecting the cells to a short period of shear stress at the same times after dilution. With both diluents, the cells underwent a rapid increase in volume followed by a return towards normal volume with a maximum at less than 250 msec. With IHP diluent, the period of hemoglobin permeability immediately followed the size peak and was completed by about 1 sec after dilution. PBS also induced a second leakage at longer times (10-120 sec), which resulted in a morphological dichotomy with ghosts and intact cells. The choice of diluent also affected sensitivity to shear stress. The IHP-treated cells had a mechanical fragility maximum at about 1 sec. The PBS-treated cells exhibited no enhanced mechanical fragility. An unexpected result was the inhibition of the second phase of lysis in PBS-treated cells by a properly timed shear stress.     

Lipopolysaccharide from Proteus mirabilis O29 induces changes in red blood cell membrane lipids and proteins

Alterations in red blood cell (RBC) plasma membranes, i.e. in lipids and proteins, and osmotic fragility of these cells after treatment with Proteus mirabilis O29 endotoxin (lipolysaccharide (LPS)) were examined using a spin labelling method. At the highest concentration of LPS, insignificantly decreased fluidity of membrane lipids was observed. Changes in conformation of membrane proteins were determined by two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (MSL) and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The analysis of spectra of MSL and ISL showed modifications in membrane proteins in red blood cells treated with the highest concentration of lipopolysaccharide. On the other hand, in the case of isolated membranes, disturbances in membrane were observed for all concentrations of LPS. The alterations in membrane lipids and proteins are paralleled in a significant rise in osmotic fragility of RBCs upon endotoxin treatment. These results provide experimental evidence that P. mirabilis O29 LPS causes deleterious changes in membranes of human red blood cells. They show that action of lipopolysaccharide mainly concerns the membrane cytoskeleton.     

The combination between hemolysins and red cells or ghosts,as studied with a radioactive lysin and with new color reactions

The quantity of a radioactive hemolysin, sodium dodecyl sulfonate-S³⁵, taken up by red cells from concentrations too small to produce hemolysis varies with the lysin concentration, and does so in a way which can be described by an adsorption isotherm. Attempts to use color reactions or surface tension measurements to determine the quantity of digitonin, saponin, and the bile salts taken up by red cells from hypolytic concentrations have failed, principally because chromogenic, and also surface-active, substances are liberated from the cells when the lysin is added. Color reactions with the anthrone reagent show that digitonin and saponin are both taken up by or fixed to red cell ghosts; the extent of the uptake, however, is uncertain, again because of the liberation of chromogenic substances. Comparison of the results of the various methods which measure the apparent amount of lysin fixed, or utilized in reactions between lysins and red cells or ghosts show discrepancies between results given by direct methods (measurement of radioactivity or of color) and indirect methods (addition of a second population after lysis of a first, and dependence of the position of the asymptote of the time-dilution curve on the number of red cells). The discrepancies are traceable to the inhibitory effects of substances liberated from the red cells or ghosts. The ease with which a lysin, once taken up by red cells, can be detached by diluting the system determines the extent to which the hemolytic reaction is "progressive," but has no observed connection with the quantity taken up in the first place. There is now ample evidence that lysis in systems containing simple hemolysins is a process involving two stages in time and two phases, and that it is usually complicated by reactions between the hemolysin and liberated inhibitory material.     

Structure-based design of a novel synthetic spiroketal pyran as a pharmacophore for the marine natural product spongistatin 1

SPIKET-P, a novel synthetic spiroketal pyran, was rationally designed as a pharmocophore for the tubulin depolymerizing marine natural product Spongistatin 1. SPIKET-P was prepared from the commercially available benzyl (R)-(-)-glycidyl ether using a versatile 11-step synthetic scheme in a stereocontrolled fashion. At nanomolar concentrations, SPIKET-P caused tubulin depolymerization in cell-free turbidity assays and exhibited potent cytotoxic activity against cancer cells as evidenced by destruction of microtubule organization, and prevention of mitotic spindle formation in human breast cancer cells.     

Danazol therapy renders red cells resistant to osmotic lysis

Danazol, an attenuated androgen, is useful in endometriosis, idiopathic thrombocytopenic purpura (ITP), and autoimmune hemolytic anemia (AIHA). However, its mechanism of action is unknown. We investigated the possibility that danazol affects cell membranes directly. Red cell osmotic fragility was studied in patients receiving danazol. A significant decrease in osmotic fragility was observed. Accompanying the change, peripheral blood smears showed many target cells and electron microscopy revealed extra folds in erythrocyte membranes. Twenty-two patients were studied prospectively before and after danazol. Osmotic fragility decreased significantly (P less than 0.001) in 1 month of therapy and progressed with further treatment. A rebound increase (P less than 0.01) was observed in 1 month after discontinuation of danazol among 16 patients. Incubation experiments showed that danazol-induced changes are not reversed with normal sera. Patient sera did not induce the changes in normal red cells. Danazol in vitro protected red cells from osmotic lysis at low concentrations but enhanced lysis at high concentrations. We suggest that danazol alters red cell membranes directly to increase their surface area, inducing target cell formation and increasing their resistance to osmotic lysis.     

t-Butyl hydroperoxide-induced changes in the physicochemical properties of human erythrocytes

Treatment of human erythrocytes with micromolar concentrations of $\text{t-}\text{butyl}$ hydroperoxide causes a variety of changes in the physical properties of the cells. Red cells exposed to concentrations of $\text{t-}\text{butyl}$ hydroperoxide of less than 750 μM for 15 min exhibited significant decreases in cellular and membrane deformability, increases in membrane-associated protein crosslinking, osmotic fragility and the viscosity of the intracellular hemoglobin solution. No changes in the volume or density of the cells were observed. Changes in cellular deformability are probably attributable solely to changes in the mechanical properties of the cell membrane. Conversely, when red cells are exposed to $\text{t-}\text{butyl}$ hydroperoxide concentrations in excess of 750 μM for 15 min they exhibited decreases in cellular deformability which may be related to increases in cell volume as well as membrane rigidity.     

THE ESCAPE OF HEMOGLOBIN FROM THE RED CELL DURING HEMOLYSIS

By means of measurements from cinematograph films of the time taken for human red cells to lose hemoglobin while hemolyzing, it is shown that small concentrations of saponin bring about a relatively small permeability of the cell membrane to the pigment, whereas large concentrations so destroy the membrane that the theoretical time for loss of pigment through a completely permeable membrane (0.16 second) is very nearly attained. These results are in agreement with those obtained from electrical measurements, and the dependence of permeability on lysin concentration can be explained on the basis of what is known about the rate of transformation of lysin as it reacts with the cell envelope. When cells are hemolyzed by hypotonic solutions, on the other hand, the permeability of the membrane to pigment is nearly constant, irrespective of the tonicity used to bring about lysis.     

Trypanosoma evansi: Effect of experimental infection on the osmotic fragility, lipid peroxidation and calcium-ATPase activity of rat red blood cells

Trypanosoma evansi is the causative agent of equine trypanosomoses. The disease is characterized by fever, anemia, and cachexia. Peroxidative damage of the red blood cells caused by the parasite, may contribute to the pathogenesis of the anemia seen in trypanosomoses. Consequently, we evaluated the hematocrit, the osmotic fragility of the red blood cells, the level of lipid peroxidation and the activity of the Ca-ATPase of red blood cell ghosts from rats experimentally infected with T. evansi. After 72 h inoculation, the hematocrit decreased from 49.5% to 33%; the osmotic fragility of the red blood cells was approximately 40% higher as compared to the healthy animals; and the red blood cell ghosts showed a higher level of lipid peroxidation and a lower Ca-ATPase activity than the red cell ghosts from the healthy animals. In vitro incubations of red blood cells from healthy animals with T. evansi, produced also a significant increase of the osmotic fragility of the red blood cells.     

太白银莲花皂苷B对胶质瘤细胞生长抑制的研究

目的:太白银莲花皂苷B(Anemone taipaiensis saponin B)是第一次从太白银莲花中经过系统化学分析和分离鉴定的皂苷之一,所以它的生物学效应目前仍然不清楚。在本研究中,我们首次体外研究太白银莲花皂苷B对胶质瘤细胞系的生物学效应,观察它对胶质瘤细胞增殖的的抑制作用。方法:采用四甲基偶氮唑蓝(MTT)法测定太白银莲花皂苷B对胶质瘤细胞生长曲线的影响,Hoechst 33342细胞核染色后荧光显微镜观察,采用光学显微镜观察细胞的形态学变化。结果:MTT实验结果显示太白银莲花皂苷B对胶质瘤细胞U87MG和U251MG有强烈的生长抑制作用,且具有剂量依赖性,应用SPSS18.0统计软件得出太白银莲花皂苷B对U87MG细胞72 h的抑制浓度为IC25=5.2μmol/L,IC50=6.7μmol/L and IC75=8.7μmol/L,U251细胞的抑制浓度为IC25=6.2μmol/L,IC50=7.9μmol/L and IC75=10.5μmol/L。Hoechst 33342细胞核染色荧光显微镜观察以及光学显微镜下细胞形态观察显示出典型的凋亡细胞形态学特征,经过皂苷B处理后,细胞皱缩成圆球形,细胞核碎裂或者致密浓染,向核膜边缘聚集,染色质浓缩为半月状、车轮状或者马蹄状,凋亡小体出现。这些特征在24 h时更明显。结论:体外实验初步显示,太白银莲花皂苷B对U87MG和U251MG细胞具有明显的增殖抑制作用,并具有促凋亡作用。     

Red blood cell permeabilization by hypotonic treatments, saponin, and anticancer avicins

Plasma membrane permeabilization by saponin and anticancer avicins was studied using light dispersion measurements, since high correlation between light dispersion changes and hemolysis has been demonstrated. Nevertheless, we observed that rat red blood cell swelling in moderately hypotonic media was accompanied by up to 20% decrease of light dispersion, when hemolysis was not yet detectable. Avicin G and avicin D were significantly more efficient than saponin in inducing cytotoxicity in PC3 human prostate cancer cells. We found that the preincubation of avicins with the plasma membrane, but not with the cytosolic fraction of previously lysed red blood cells, completely protected fresh cells against permeabilization. The data suggest that the plasma membrane can tightly bind the avicins, but not the saponin. Using the “osmotic protection” method with 100 mOsm PEGs of increasing molecular weight in isotonic media, the size of the pores generated by avicin G and avicin D in the plasma membrane was estimated to be higher than the hydrodynamic radius of PEG-8000. The obtained results indicate that the anticancer activity of avicin G and avicin D could be related, at least partially, to their high ability to permeabilize biological membranes. These data might represent interest for possible applications of these anticancer drugs in vivo.     

An Improved Diluent for Rubella Hemagglutination and Hemagglutination-Inhibition Tests

Rubella hemagglutinating (HA) antigen was prepared in BHK-21 tissue as 5% cell suspensions and from unconcentrated and 20× concentrated infected supernatant fluids. In some instances, unconcentrated fluids were treated with Tween 80 and ether; cell suspensions were treated with ether alone. Preparations were tested for HA activity in dextrose-gelatin-Veronal (DGV) buffer solutions; 0.85% NaCl; Sorenson's phosphate-buffered saline, pH 7.2; and a diluent of 0.9% NaCl, 0.1% CaCl₂ (anhydrous), and 0.1% MgSO₄·7H₂O. HA titers were consistently two- to fourfold higher in the saline with added Ca⁺⁺ and Mg⁺⁺ than in DGV. Hemagglutination-inhibition titers of paired human sera were the same in either diluent. It is suggested that the interaction between rubella HA antigen and the red cells of young (less than 1-day-old) chicks may be at least partially ion dependent and that titers are enhanced by increased quantities of divalent cations.     

Physiological (osmotic fragility) and morphological effects on red blood cells: action of phytic acid and stannous fluoride

Phytic acid occurs in foods derived from plants. We have investigated the possibility that phytic acid and stannous fluoride are capable of altering the physiological properties (osmotic fragility) and morphological properties of red blood cells (RBC). Osmotic fragility was unchanged by the presence of phytic acid and stannous fluoride in the studied concentrations, but RBC morphology was modified in the presence of the studied substances. In conclusion, the alterations to RBC morphology were not sufficient to promote modifications in osmotic fragility. Our results suggest that the chelating properties of phytic acid could be responsible for the observed effects.