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.     

Microturbidimetric determination of erythrocyte osmotic fragility in newborn,weanling and mature rats

A rapid, microturbidimetric method for recording red cell osmotic fragility using a Platelet Aggregometer is described. This method requires only 0.2 ml of whole blood and a fragility curve of 20 points can be determined in less than 1 hr. Measurement of the degree of hemolysis is based on the increasing transparency of the erythrocyte suspension when hemolysis takes place. Erythrocytes of immature animals are osmotically more resistant than those of adults and the change in osmotic resistance is not directly related to the percentage of reticulocytes.     

Experimental variations in sonic fragility of red cells

A magnetostriction oscillator has been used for determining the relative fragility of human red cells to 9000 cycle vibrations under some different environmental conditions. The destruction of the cells is a logarithmic function of time according to the equation See PDF for Equation. Hypertonic saline solution, saponin, preheating, alcohol, and ether in subhemolytic concentrations decrease the fragility of human red cells subjected to sonic oscillation. Hypotonic saline, preheating to 50°C. for 3 minutes, and hemolytic concentrations of ether increase the fragility of red cells to 9 kc. vibrations. Following preheating of the cells, and in the presence of saponin the destruction deviates slightly from a true logarithmic rate of hemolysis.     

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

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

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.     

Potentiating effect of chlorpromazine on glyceryl guaiacolate ether induced hemolysis of rat,dog and human blood in vitro

The hemolytic effect of glyceryl guiacolate ether (GGF) with and without chloromazine (CPZ) was studied in vitro on rat, dog and human blood. The lowest concentration of GGE which could produce hemolysis of rat red cells was 0.15 M. The time fpr 50% hemolysis (TH50) of blood depended upon the concentration of drug and dilution of blood. A higher concentration of GGE hemolyzed blood much faster than the lower. There was a progressive increase in the TH50 when 0.15 M GGF was tested on blood samples containing increasing numbers of red cells. CPZ in all cases had its own hemolytic effect at higher concentrations. In this regard rat blood was 10 times more sensitive than dog, and human. A striking potentiating effect of CPZ was observed on the hemolytic effect of GGE. The magnitude of potentiation in all cases was directly related to the concentrations of CPZ. Dog blood was found relatively more sensitive to the hemolytic effect of the combination of CPZ and GGE as compared to the rat and human, which acted alike.     

Effects of Silicate Polymers on Erythrocytes in Presence and Absence of Complement

The effects of silicates upon erythrocytes depend upon the degree of polymerization. Monomeric silicate does not appear to be taken up by red cells. Polymerized silicates are taken up and bound tightly. In the presence of small polymeric forms erythrocytes are lysed by complement. Larger polymers are bound to erythrocytes but do not sensitize to complement hemolysis. Larger polymers, however, are directly toxic and cause hemolysis in the absence of complement. Red cells exposed to complement-active polymers show characteristic alteration in morphology with the assumption of irregular bell shapes. Larger polymers cause the cells to become spherical before spontaneous rupture occurs. Large polymers cause erythrocyte agglutination but this is minimal or absent with small complement-active polymers. Complement-active polymers cause little or no change in osmotic fragility. Increase in mechanical fragility is a sensitive indication of the presence of larger, agglutinating polymers. The conversion of pneumococci from Gram positivity to negativity appears to be caused principally by complement-active polymers. Possible implications of polymer size and complement activity are discussed in relation to production of silicotic lesions by silica-containing ores.     

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.     

Antihemolytic effect of Rooibos tea (Aspalathus linearis) on red blood cells of Japanese quails.

The antihemolytic activity of Rooibos and black tea on Japanese quail erythrocytes was studied. Peroxide and hypotonic hemolysis of the red blood cells of quails, either fed with Rooibos tea supplemented food or fed without tea, was performed. Long-term consumption of Rooibos tea did not change the erythrocyte fragility to either peroxide or hypotonia induced hemolysis. However, Rooibos and black teas decreased peroxide induced hemolysis of erythrocytes incubated with each of them, but not hemolysis induced by hypotonic NaCl solution. Stronger inhibition of hemolysis has been obtained when a boiled water extract of Rooibos tea was used for the inhibition. The degree of inhibition was comparable with the effect of ascorbic acid.     

Freeze-drying of red blood cells at ultra-Low temperatures

The hemolysis of human red blood cells (RBCs) after freeze-drying and resuspension depends on the vacuum-drying temperature. In an experimental study, RBCs were first solidified based on a modified high-yield cryopreservation protocol in the presence of hydroxyethyl starch and maltose. Afterward, they were vacuum-dried in a special low-temperature freeze-drying device at selected shelf temperatures between -5 and -65 degrees C. Subsequently, the dried samples were resuspended in an isotonic, phosphate-buffered saline solution. The hemolysis was determined according to a modified saline stability test. It decreases with a decreasing shelf temperature until a minimum is reached at -35 degrees C. A further decrease of the shelf temperature has no beneficial effect; the hemolysis even increases. To interpret these results, we assume that the hemolysis depends on two contrary damaging effects: (1) the higher the shelf temperature, the higher the probability of structural damages occurring during drying; (2) the lower the shelf temperature, the lower the driving force for water transport; this may lead to an incomplete intracellular dehydration which means that the cells are not in a glassy state at ambient temperature.     

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.     

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.     

Comparison of rat blood preparation methods for acetaldehyde assay

A comparison is made of four previously described methods for the preparation of blood for acetaldehyde (AcH) assay in the rat. The spontaneous formation of AcH which occurs during the treatment of blood containing ethanol and the recovery of known amounts of AcH added to the blood were studied. The methods using sodium nitrite-sulfosalicylic acid or perchloric acid (PCA) in saline gave low levels of spontaneous formation (1 to 2 microM AcH for 48 mM ethanol). In the recovery studies it was seen that semicarbazide does not allow displacement of all the AcH; treatment of the blood with the reactant sodium nitrite-sulfosalycilic acid and use of the hemolysis method gave levels of recovery lower than 50%. Only treatment of the blood with perchloric acid in NaCl allowed all the AcH added to the blood to be recovered. In vivo, PCA in saline releases the AcH which was seen to remain bound in the red blood cells with the semicarbazide method. So the recommended procedure for accurate assay of blood AcH in the rat is cold deproteinization in PCA/saline before head-space gas chromatography. The levels of in vivo blood AcH (4.1 +/- 0.33 microM) obtained in the rat using this method for a blood alcohol concentration of 52 mM are lower than those previously described in the literature.     

Impact of blood manufacturing and donor characteristics on membrane water permeability and in vitro quality parameters during hypothermic storage of red blood cells

Several factors have been proposed to influence the red blood cell storage lesion including storage duration, blood component manufacturing methodology, and donor characteristics [1,18]. The objectives of this study were to determine the impact of manufacturing method and donor characteristics on water permeability and membrane quality parameters.Red blood cell units were obtained from volunteer blood donors and grouped according to the manufacturing method and donor characteristics of sex and age. Membrane water permeability and membrane quality parameters, including deformability, hemolysis, osmotic fragility, hematologic indices, supernatant potassium, and supernatant sodium, were determined on day 5 ± 2, day 21, and day 42. Regression analysis was applied to evaluate the contribution of storage duration, manufacturing method, and donor characteristics on storage lesion.This study found that units processed using a whole blood filtration manufacturing method exhibited significantly higher membrane water permeability throughout storage compared to units manufactured using red cell filtration. Additionally, significant differences in hemolysis, supernatant potassium, and supernatant sodium were seen between manufacturing methods, however there were no significance differences between donor age and sex groups.Findings of this study suggest that the membrane-related storage lesion is initiated prior to the first day of storage with contributions by both blood manufacturing process and donor variability. The findings of this work highlight the importance of characterizing membrane water permeability during storage as it can be a predictor of the biophysical and chemical changes that affect the quality of stored red blood cells during hypothermic storage.     

The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride

Human red blood cells, suspended in solutions of sodium chloride, have been frozen to temperatures between -2 and -14 degrees C and thawed, and the extent of hemolysis was measured. In parallel experiments, red cells were exposed to similar cycles of change in the composition of the suspending solution, but by dialysis at 21 degrees C. The tonicity of the saline in which the cells were initially suspended was varied between 0.6x isotonic and 4x isotonic; some samples from each experimental treatment were returned to isotonic saline before hemolysis was measured. It was found that the tonicity of the saline used to suspend the cells for the main body of the experiment affected the amount of hemolysis measured: raising the tonicity from 0.6x to 1x to 2x reduced hemolysis, both in the freezing and in the dialysis experiments, whereas raising the tonicity further to 4x reversed that trend. There was little difference between the freeze/thaw and the dialysis treatments for the cells suspended in 1x or 2x saline, whether or not the cells were returned to isotonic conditions. However, the cells suspended in 0.6x saline showed greater damage from freezing and thawing than from the comparable change in the composition of the solution, whether or not they were returned to isotonic conditions. Cells that were suspended in 4x saline and exposed to changes in salt concentration by dialysis showed less hemolysis when they were assayed in the 4x solution than cells that had received the comparable freezing/thaw treatment, but when the experiment included a return to isotonicity, the two treatments gave similar results. Returning the cells to isotonic saline had a negligible affect on the cells in 0.6x and 1x saline, but caused considerable hemolysis in the 2x and 4x samples, more so after dialysis than after freezing and thawing. We conclude that cells suspended in 0.6x and 4x saline behave differently from cells suspended in 1x and 2x saline and hence that cells suspended in a range of solutions of differing initial tonicity should not be treated as a homogeneous population. We argue that an effect of the unfrozen fraction of water (U) cannot be distinguished, within the framework of these freeze/thaw experiments alone, from an effect of initial tonicity, and that the biphasic nature of the correlation between haemolysis and U makes a causal connection improbable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of the functional and mechanical properties of platelet and red blood cells by nitric oxide donors

The effect of NO donors (sodium nitroprusside, S-nitrosoglutathione, dinitrosyl-iron complexes) on the functional and mechanical properties of human platelets and red blood cells has been investigated. It has been established by atomic force microscopy that NO donor-induced platelet disaggregation is accompanied by changes in the elastic properties of cells. It has been shown that, in the presence of NO donors, the detergent-induced hemolysis of red blood cells is delayed, and the elasticity modulus of these cells decreases. The results obtained indicate that NO donors regulate the structural and functional properties of platelets and red blood cells.     

Hemolysis and ATP Release from Human and Rat Erythrocytes under Conditions of Hypoxia: A Comparative Study

Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat erythrocytes. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5- and 2.5-fold, respectively. In rat erythrocytes a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL 67156. In these conditions we observed a positive correlation (R² = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia-induced ATP release from rat erythrocytes. The obtained results indicate a key role of hemolysis in the ATP release from red blood cells.     

Anemia and potassium permeability of red blood cells in analbuminemic rats

A mutant strain, Nagase analbuminemia rats (NAR), was established from Sprague-Dawley rats. Hematological evaluations were made on NAR of 4 to 52 weeks of age. NAR had an abnormally low number of red blood cells (RBC), a low hematocrit, a reduced hemoglobin concentration and an increased number of reticulocytes. Their plasma electrolyte level was normal. Osmotic fragility of RBC was slightly increased in the rats. Thus NAR shows a slight anemia. The in vitro experiments on RBC were performed. The incubation of blood showed a hemolytic tendency and elevated potassium efflux in the blood of NAR. In addition, an increased efflux of potassium was found in the RBC of NAR, when the RBC was washed with phosphate buffered saline and then was incubated with the saline containing CaCl2. This potassium efflux was prevented in the presence of rat albumin. These findings suggest that the deficiency of serum albumin may increase the permeability of potassium in erythrocyte membrane in NAR.     

Osmotic hemolysis and fragility A new model based on membrane disruption,and a potential clinical test

Red cell osmotic hemolysis has traditionally been defined by the loss of hemoglobin, in response to reduced osmotic pressure, as measured spectroscopically. Previous work from this laboratory using resistive pulse spectroscopy (RPS) has shown that in a mixed population of hemolyzing cell, ghosts can be detected as being more deformable, and hence appearing distinctly smaller, than the remaining intact cells. Other researchers using similar methods have reported detection of ghosts as apparently smaller objects, resulting from their greater sensitivity to dielectric breakdown. We now confirm both of these results, and demonstrate by kinetic studies that changes which occur in the rheological and electrical properties of ghosts are independent phenomena. We include in our analysis the explicit calculation of ghost and intact spherocyte resistivity after dielectric breakdown. The two different characterizations for ghosts are integrated into a proposed model of osmotic hemolysis based on known red blood cell membrane and cytoplasmic properties. This work provides both a theoretical and a practical foundation for RPS-based measures of osmotic fragility, including a potential new clinical test, measures which provide very early detection of the ultimate fate of osmotically stressed red cells.     

Influence of magnesium deficiency on the bioavailability and tissue distribution of iron in the rat

We investigated the effect of dietary magnesium (Mg) deficiency on the nutritive utilization and tissue distribution of iron (Fe). Wistar rats were fed an Mg-deficient diet (56 mg/kg) for 70 days. Absorbed Fe, Fe balance, number of the erythrocytes [red blood cells (RBC)] and leukocytes white blood cells (WBC)], hemoglobin (Hb), and Fe content were determined in samples of plasma, whole blood, skeletal muscle, heart, kidney, liver, spleen, femoral bone, and sternum obtained on experimental days 21, 35, and 70. The Mg-deficient diet significantly increased Fe absorption and Fe balance from week 5 until the end of the experimental period. This effect was accompanied by a significant decrease in the concentration of RBC and Hb from day 35, which caused the decrease in whole blood Fe seen on day 70. However, WBC were significantly increased from day 21 until the end of the experimental period. Mg deficiency significantly increased plasma and liver Fe at all three time points investigated. Spleen, heart, and kidney Fe were significantly increased only at the end of the study. However, on day 70, Fe concentration in the sternum had decreased significantly. No changes were found in skeletal muscle or femur Fe content. Mg deficiency led to increased intestinal absorption of Fe and decreased RBC counts, possibly as a result of increased fragility of the erythrocytes. Intestinal interactions between Fe and Mg, together with activation of erythropoiesis as a result of hemolysis, favored intestinal absorption of Fe. This situation gave rise to an increase in plasma Fe levels, which in turn favored Fe uptake and storage by different organs, especially the liver and spleen. However, despite the increased Fe content seen in the tissues of rats fed the Mg-deficient diet, these animals were unable to compensate for the hemolysis caused by this nutritional deficiency.