Increase In Osmotic Fragility Research Articles

Erythrocyte osmotic fragility and cation concentrations during experimentally induced bovine anaplasmosis

1. 1. The osmotic fragility, the concentrations of Na, K and Ca, the osmolality and the total ATPase activity of bovine erythrocytes from uninfected and Anaplasma marginale-infected bovines were studied in an attempt to correlate these parameters with the decrease in the cellular ATP concentration reported during bovine anaplasmosis. 2. 2. The osmotic fragility found in infected bovine erythrocytes, at 0.52% NaCl, was about two times greater than that observed in non-infected bovines. The increase in osmotic fragility was directly related to the increase in intra-erythrocytic parasitemia. 3. 3. The decrease in ATP concentration reported during bovine anaplasmosis could not be directly related to the increased fragility of these cells. The artificial depletion of erythrocytic ATP did not reproduce the same alteration in the osmotic response to NaCl. 4. 4. The plasmatic and cytoplasmatic concentrations of Na, K and Ca did not change significantly during bovine anaplasmosis, whereas the interior of the erythrocytes became hyperosmolal. 5. 5. A. marginale-infected bovine erythrocyte membranes showed an increased ATPase activity when compared to control bovines. Parasite-enriched fractions also presented ATPase activity.

Changes in the rate of haemolysis during the early stage after burns in the rabbit

The kinetics of erythrocyte haemolysis in blood samples taken from normal and burned rabbits were investigated by nephelometry. The rate of haemolysis decreased significantly from 30 min to 2 h after severe burns, when many abnormal red cell forms were found in blood films; there was also an increase in osmotic fragility. The results confirm that the rate of haemolysis is strongly related to structural changes of the red cell membrane but do not parallel osmotic fragility. Differences in haemolysis rate and osmotic fragility between erythrocytes taken from burned animals and those heated in vitro suggest that erythrocyte changes following burning injury involve complex kinetic processes in which factors other than the direct effect of heat play important roles.

The effect of the plasticizer di(2-ethylhexyl)phthalate on red cell deformability

Red cell concentrates (RCC) are stored for 35 to 42 days in plastic containers manufactured with the liquid plasticizer di(2- ethylhexyl)phthalate (DEHP). DEHP leaches from the polyvinylchloride (PVC) plastic bag, then binds to and stabilizes the RC membrane. This study was undertaken to determine the deformability of the RC membrane using an osmotic gradient ektacytometer and to relate these measurements to the concentration of DEHP in the stored RCC. Pooled RCC was aliquoted into PL146 (PVC), PL732 (polyolefin), and PL732 (with added DEHP) bags with samples removed weekly for analysis of osmotic fragility, deformability, and DEHP concentration. The adenosine triphosphate (ATP) content was also measured. The increase in osmotic fragility during storage was greater when RCC was stored without DEHP. In addition, there was a decrease in the maximum elongation index (El max) when there was decreased DEHP in the storage bag. The osmolarity (Omax) at which El max occurred, as well as the Omin, the osmolarity at which minimum elongation (El min) occurred was higher in the PL732 container than in the PL146 or in the PL732 to which DEHP had been added. These changes could be reversed by addition of DEHP at the beginning of the storage period, showing a direct correlation between DEHP concentration during storage and RC membrane flexibility. By a better understanding of the mechanism of DEHP protection, it might be possible to substitute a less toxic stabilizing compound.

The Effect of the Plasticizer Di(2-ethylhexyl)phthalate on Red Cell Deformability

Red cell concentrates (RCC) are stored for 35 to 42 days in plastic containers manufactured with the liquid plasticizer di(2-ethylhexyl)phthalate (DEHP). DEHP leaches from the polyvinylchloride (PVC) plastic bag, then binds to and stabilizes the RC membrane. This study was undertaken to determine the deformability of the RC membrane using an osmotic gradient ektacytometer and to relate these measurements to the concentration of DEHP in the stored RCC. Pooled RCC was aliquoted into PL146 (PVC), PL732 (polyolefin), and PL732 (with added DEHP) bags with samples removed weekly for analysis of osmotic fragility, deformability, and DEHP concentration. The adenosine triphosphate (ATP) content was also measured. The increase in osmotic fragility during storage was greater when RCC was stored without DEHP. In addition, there was a decrease in the maximum elongation index (El max) when there was decreased DEHP in the storage bag. The osmolarity (Omax) at which El max occurred, as well as the Omin, the osmolarity at which minimum elongation (El min) occurred was higher in the PL732 container than in the PL146 or in the PL732 to which DEHP had been added. These changes could be reversed by addition of DEHP at the beginning of the storage period, showing a direct correlation between DEHP concentration during storage and RC membrane flexibility. By a better understanding of the mechanism of DEHP protection, it might be possible to substitute a less toxic stabilizing compound.

Complement lysis of nucleated cells: Effect of temperature and puromycin on the number of channels required for cytolysis

We have previously shown that lysis of a nucleated mammalian cell requires several complement channels unlike lysis of erythrocytes and that this difference is due primarily to rapid elimination of channels from the plasma membrane. We have now investigated this problem further by studying the rate of channel elimination at low temp, the osmotic fragility of the cells, and the effectiveness of the membrane-associated ion pumps. When complement channels were formed for 3 min at 37°C, followed by prolonged incubation at 2°C, the C6 lytic dose-response curves indicated that a single channel was required for lysis of a cell, whereas multiple channels were required when the entire process was carried out at 30°C. The shift from multi- to one-hit lytic behavior can be explained by the drastic reduction in the rate of channel elimination at low temp. C6 lytic dose-response curves with puromycin-treated cells were also found to display one-hit behavior, but, in this case, the rate of channel elimination was reduced only about 35–40% (which would not suffice to explain the one-hit lytic characteristics). However, cell death was more extensive for puromycin-treated cells than normal cells after incubation in buffers of low ionic strength, suggesting that an increase in osmotic fragility may be a contributing factor in the shift from multi- to one-hit behavior. Blocking of the membrane-associated Na +/K +-ATPases with ouabain did not affect the multi-channel requirement. Presumably, this means that the ion pumping rate does not significantly influence the number of channels required for lysis.

The membrane of intact human erythrocytes tolerates only limited changes in the fatty acid composition of its phosphatidylcholine

(1) Using the phosphatidylcholine specific transfer protein from bovine liver, native phosphatidylcholine from intact human erythrocytes was replaced by a variety of different phosphatidylcholine species without altering the original phospholipid and cholesterol content. (2) The replacement of native phosphatidylcholine by the disaturated species, 1,2-dipalmitoyl- and 1,2-distearoylphosphatidylcholine, proceeded at a low rate and extensive replacement could only be achieved by repeatedly adding fresh donor vesicles. The replacement by disaturated molecules was accompanied by a gradual increase in osmotic fragility of the cells, finally resulting in hemolysis when 40% of the native PC had been replaced. Up to this lytic concentration, the replacement did not affect the permeability of the membrane for potassium ions. (3) Essentially, all of the PC in the outer monolayer of the membrane could be replaced by 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine. These replacements did not alter the osmotic fragility of the cells, nor the K + permeability of the membrane. (4) Increasing the total degree of unsaturation of the phosphatidylcholine species modified the properties of the membrane considerably. Replacement by 1,2-dilinoleoylphosphatidylcholine resulted in a progressive increase in osmotic fragility and hemolysis started to occur after 30% of the native PC had been replaced by this species. K + permeability was found to be slightly increased in this case. Cells became leaky for K + upon the introduction of 1-palmitoyl-2-arachidonoylphosphatidylcholine in the membrane. The increased permeability was also reflected by an apparent increase in the resistance of the cells against osmotic shock. (5) The conclusions to be drawn are that (i) 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine are species which fit most optimally into the erythrocyte membrane; (ii) loss of membrane stability results from an increase in the degree of saturation of phosphatidylcholine ( unsaturation index > 0.5 ) and (iii) the permeability is enhanced by increasing the content of highly unsaturated species ( unsaturation index > 1.0 ).

The relationship between the osmotic fragility of human erythrocytes and cell age

Erythrocytes in a normal blood sample are hemolyzed over a range of hypotonic salt concentrations. In order to investigate the relationship between the distribution of osmotic fragilities and the distribution of cellular ages, the osmotic fragility has been compared with three indices of cellular age. The activity of glutamic-oxalacetic transaminase (GOT) and the percentage hemoglobin A 1C were measured in samples hemolyzed in different hypotonic salt concentrations. The osmotic fragility curve was also obtained for cells of different density separated by centrifugation. These experiments indicate that the fragility distribution is not an accurate reflection of the distribution of cellular ages. The mean fragility for older cells is higher than that of younger cells. However, cellular aging does not produce a gradual increase in osmotic fragility. Instead, it seems to produce changes which can both increase and decrease the fragility, resulting in a broader distribution of fragilities with some of the older cells actually less fragile than the younger ones.

Increase in osmotic fragility of bovine erythrocytes induced by bacterial phospholipases C.

Bovine erythrocytes were treated with each of three bacterial phospholipases C; phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens, sphingomyelinase C (SMase) of Bacillus cereus and phosphatidylinositol-specific phospholipase C (PIase) of Bacillus thuringiensis. An increase in osmotic fragility was detected by means of a coil planet centrifugation (CPC) apparatus (Biomedical Systems Co., Tokyo) after the treatment with these enzymes. The peak of hemolysis normally observed in the untreated erythrocytes at the range between 50 and 100 mOsM shifted to 160 to 200 mOsM with the progress of sphingomyelin hydrolysis by phospholipase C of C. perfringens. Sphingomyelinase C of B. cereus showed two different effects on bovine erythrocytes: In the absence of divalent cations or in the presence of Ca2+ alone, the peak of hemolysis shifted to the region from 130 to 160 mOsM, without appreciable hydrolysis of sphingomyelin, while in the presence of Mg2+ or Mg2+ plus Ca2+, the peak of hemolysis further shifted to the region from 160 to 200 mOsM with the hydrolysis of sphingomyelin. Abrupt shift in osmotic fragility to a much higher region around 250 mOsM was produced by treatment with increasing amounts of phosphatidylinositol-specific phospholipase C. In this case, a significant amount of acetylcholinesterase was released from the erythrocyte membrane without hot or hot-cold hemolysis. The mechanism of alteration of osmotic fragility of bovine erythrocytes by treatment with phospholipases C seems to differ from case to case, depending upon the specific action of each enzyme toward the membrane phospholipids.

Does preincubation of the red blood cells contribute to the capability of the osmotic fragility test to detect very mild forms of hereditary spherocytosis?

During incubation for 24 h at 37 degrees C, erythrocytes from patients with hereditary spherocytosis (HS) undergo a greater increase in osmotic fragility than do normal cells, and this procedure has been recommended for differentiating more clearly between patients with very mild HS and normal subjects. The greater effect of preincubation on erythrocytes from patients with HS was confirmed, but, except in cases demonstrating a markedly increased osmotic fragility before incubation, this effect was outweighed by a simultaneous loss of test precision. It therefore seems that preincubation does not significantly contribute to the capability of the osmotic fragility test to detect very mild forms of HS.

Effect of parathyroid hormone on osmotic fragility of human erythrocytes.

The survival of erythrocytes (RBC) is shortened in uremia, and it has been shown that calcium influx into RBC evoked crenation and increased their rigidity. The high blood levels of parathyroid hormone (PTH) may augment entry of calcium into RBC and hence affect their integrity. We examined the effect of PTH on osmotic fragility of human RBC and investigated the mechanisms through which PTH interacts with RBC. Both the amino-terminal (1-34) PTH and the intact (1-84) PTH, but not the carboxy-terminal (53-84) PTH, produced significant increases in osmotic fragility. This effect was abolished by prior inactivation of the hormone. There was a dose-response relationship between both moieties of PTH and the increase in osmotic fragility. This action of PTH required calcium, was mimicked by calcium ionophore, and was partially blocked by verapamil. PTH caused significant influx of (45)Ca into RBC, which was not associated with potassium leak. The hormone did not affect water content of RBC. Scanning electron microscopy revealed that the incubation of RBC with PTH was associated with the appearance of membrane filamentous extensions, which anchor RBC together. Inhibition of glycolytic activity of RBC with NaF or inhibition of Na-K-activated ATPase with ouabain did not abolish the effect of PTH on osmotic fragility. PTH did not stimulate RBC Na-K-activated ATPase or Mg-dependent ATPase but caused marked and significant stimulation of Ca-activated ATPase. The basal activity of the RBC adenylate cyclase was low and PTH produced only a modest stimulation of this enzyme. Both cyclic AMP and dibutyryl cyclic AMP had no effect on osmotic fragility. THE DATA INDICATE THAT: (a) the RBC is a target organ for PTH, (b) the hormone increases osmotic fragility of RBC, and (c) this effect of PTH is due to enhanced calcium entry into RBC. We suggest that the increased calcium influx may affect the spectrin-actin of the cytoskeletal network of the RBC and may alter the stability and integrity of the cell membrane. This action of PTH on the RBC could be, at least in part, responsible for the shortened survival of RBC in uremia, and assign a new role for PTH in the pathogenesis of the anemia of uremia.

Riboflavin photosensitized hemolysis of rat erythrocytes in the presence of serum.

Rat erythrocytes incubated in photoactivated riboflavin system in the presence of serum caused a rapid hemolysis. During the course of illumination, a marked efflux of intracellular K+, increase in osmotic fragility and promotion of lipid peroxidation of the cell occurred prior to hemolysis indicating that riboflavin-induced photohemolysis is colloidosmotic process. In the absence of serum, however, no hemolysis was observed at any time of incubation, suggesting that membrane damages are enhanced by the illumination in the presence of serum. Furthermore, the illumination of cells in the photoactivated riboflavin system in the presence of serum under continued anaerobic conditions did not exhibit any significant hemolysis. These results indicate that the photohemolysis of cells is due to oxidative damages of cell membrane that initiated the hemolysis. Singlet oxygen (1O2) scavengers employed in this study strongly inhibited the photohemolysis, suggesting that cells are damaged oxidatively by 1O2 generated in photoactivated riboflavin system in the presence of serum. Furthermore, triplet quenchers and alpha-tocopherol suppressed this photohemolysis. A reference was made concerning a possible adverse effect of riboflavin on infant erythrocytes in phototherapy.

Increased Cholesterol and Decreased Fluidity of Red Cell Membranes (Spur Cell Anemia) in Progressive Intrahepatic Cholestasis

Progressive hemolytic anemia occurred in a 4 1/2-year-old girl with familial intrahepatic cholestasis; a peripheral smear contained bizarre spiculated "spur" red cells. Analysis of this patient's fresh red cells revealed a 59% increase in cholesterol content with a normal phospholipid content and therefore an increase in the cholesterol/phospholipid molar ratio to 1.35 (normal = 0.92). A similar abnormality of lipid composition was present in serum lipoproteins. The lipid abnormality in red cell membrane was associated with a decrease in membrane fluidity, as assessed by the fluorescence polarization of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene. Following incubation with patient's plasma, normal cells acquired a spur-shaped morphology with an associated decrease in osmotic fragility and a 25% increase in cholesterol content. The patient's cells, during incubation with normal plasma, acquired morphologic features of spiculated spherocytes with an increase in osmotic fragility and a 21% decrease in cholesterol content. Chenodeoxycholate and lithocholate were present in markedly elevated concentrations in serum. These studies show that a process identical to spur cell anemia in alcoholic cirrhosis may accompany severe liver disease in children with intrahepatic cholestasis.

Red cell cholesterol enrichment and spur cell anemia in dogs fed a cholesterol-enriched atherogenic diet

A diet supplemented with cholesterol and coconut oil is atherogenic in dogs. The purpose of the present study was to examine the effects of this diet on red cells in pure-bred beagles and greyhounds. Within 3 days after the initiation of this diet red cell cholesterol/phospholipid increased and membrane fluidity decreased, with maximum changes attained by 12 weeks. Serum lipoprotein cholesterol/phospholipid also increased, and serum from cholesterol-fed dogs transferred cholesterol to normal red cells. Significant abnormalities of liver function developed in all cholesterol-fed dogs. Hematocrit declined beginning at 6 weeks, with a parallel increase in osmotic fragility. Reticulocytes were elevated in beagles but normal in greyhounds. Red cell morphology resembled acanthocytes or spur cells. All red cell parameters returned to normal within 4 weeks after stopping the diet. These studies demonstrate that a cholestrol-enriched, atherogenic diet causes profound and reversible changes in the lipid composition, membrane fluidity, and morphology of red cells in dogs.

Sodium transport in red cells of patients with acute viral hepatitis

The red blood cell cation composition was determined in 25 patients with acute viral hepatitis and in 26 normal subjects. The potassium concentration was 83.4 ± 4.11 mmol/kg red cells (mean ± SD) in hepatitis red blood cells and 79.7 ± 4.74 mmol/kg red cells in the control samples (P < 0.005). There were no significant differences between the sodium content of normal and hepatitis red blood cells. 22Na-Efflux was determined in red blood cells of 17 patients with acute viral hepatitis and 18 normal subjects. The mean sodium efflux rate constant was 0.212 ± 0.036 in red cells of patients with hepatitis and 0.295 ± 0.046 in the control samples (P < 0.001). This difference resulted from a reduction in both the active and residual portions of the red blood cell sodium pump in the patients with viral hepatitis. Red blood cells of patients with acute viral hepatitis accumulated sodium at a significantly slower rate than normal red blood cells. The increase in osmotic fragility after 24 hr incubation at 37 °C in the absence of glucose was less prominent in hepatitis red blood cells than in normals. The findings of a reduced sodium efflux and slower accumulation of sodium during incubation, suggest that red blood cells from patients with viral hepatitis are less permeable to sodium, thereby leading to a new steady-state, characterized by a reduced sodium efflux.

Cross-linking of membrane proteins and protoporphyrin-sensitized photohemolysis.

Abstract— Irradiation of protoporphyrin‐sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross‐linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976).Facile oxidative hemolysis can be achieved without observable cross‐linking of membrane proteins upon incubation (37°C) of red cells containing membrane‐bound 3ß‐hydroxy‐5α‐hydroperoxy‐△6‐cholcstene. Thus, protein cross‐linking is not obligatory for oxidative lysis. Deoxygenation by Ar bubbling strongly retards the light‐induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin‐sensitized erythrocytes. However, similar reduction in oxygen concentration only partially inhibits cross‐linking of membrane proteins. These results suggest that membrane protein cross‐linking and photohemolysis are not coupled processes.

The susceptibility of red cell acetylcholinesterase to radiation-induced free radicals 1

Inactivation of red cell acetylcholinesterase has previously been reported in a number of experimental situations in which free radicals are believed to be present. The susceptibility of this red cell membrane enzyme has now been studied using a 4-MeV linear accelerator X-ray source to produce defined free radicals. Irradiation of a 0.2% red cell suspension gassed with nitrous oxide (a system in which the hydroxyl radical is the predominant radical species) resulted in a decrease in acetylcholinesterase activity to 38% of the initial level following a dose of 50 krad. Addition of bromide or thiocyanate ions to suspensions irradiated under nitrous oxide (systems in which hydroxyl radicals are converted to the radicals of Br 2 − or (SCN) 2 −) enhanced the extent of enzyme activity loss. In contrast, inclusion of iodide in the irradiation mixture, producing I 2 −, resulted in a reduced loss of acetylcholinesterase activity. Only a slight, not statistically significant, decrease in enzyme activity was observed under irradiation conditions in which the Superoxide anion radical was the predominant radical species. The extent to which the various radicals affected acetylcholinesterase activity was independent of the hemolytic response as measured by increase in osmotic fragility.

Changes of osmotic fragility of red blood cells due to repletion or depletion of cholesterol in human and rat red cells in vitro.

The relation of membrane lipids and osmotic fragility of red cells of human and rat were studied in vitro by repletion and depletion of cell cholesterol. A decrease in the red cell cholesterol corresponded to an increase in osmotic fragility. Conversely, a repletion of red cell cholesterol decreased the osmotic fragility, but the procedure did not significantly change the phospholipid contents of red cells. Accordingly, osmotic fragility of the red cells correlated highly with the cholesterol content of the cell membrane (r = -0.922, p < 0.001) and with the molar ratio of cholesterol to phospholipid in the red cells (r = -0.887, p < 0.001). Red cells incubated with plasma had decreased membrane cholesterol and increased osmotic fragility, but the change was prevented by the inactivation of lecithin cholesterol acyltransferase (LCAT) in the plasma. The above results confirm that membrane cholesterol stabilizes human and rat red cells in vitro as well as in vivo, and the content of red cell cholesterol is regulated by the exchange of plasma free cholesterol in vitro. LCAT activity in the plasma is an influential factor in controlling the cholesterol content of red cells.

Monocyte-induced increase in osmotic fragility of human red cells sensitized with anti-D alloantibodies.

The mechanism by which human monocytes increase the osmotic fragility of red cells sensitized with Rhesus alloantibodies anti-D was studied in vitro. Both the increase in osmotic fragility and the lysis of red cells by monocytes were enhanced by cytochalasin B and were inhibited by hydrocortisone. These effects were similar to the effects of these agents on lysosomal enzyme release by monocytes. However, hydrocortisone was completely ineffective when added 1 h after mixing monocytes and sensitized red cells. This indicates that the damage responsible for the fragility increase and lysis is completed within 1 h and suggests that it is due to lysosomal enzymes released by the monocytes. Since for the full expression of the osmotic fragility increase and lysis an incubation time much longer than 1 h is required, it appears that the latter phenomena are the non-specific sequelae of damage inflicted upon the red cell by released lysosomal enzymes.

The effect of phosphatidylcholine to sphingomyelin mole ratio on the dynamic properties of sheep erythrocyte membrane

Sheep red blood cells are shown to incorporate phosphatidylcholine when incubated in human plasma in the presence of EGTA. This treatment results in up to a 5-fold increase in mol ratio of phosphatidylcholine to sphingomyelin. By replacing EGTA with Ca 2+ the increase of phosphatidylcholine content is completely inhibited, due to the activation of the membrane bound lecithinase which rapidly degrades the incorporated phosphatidylcholine. Analogous treatments of the isolated erythrocyte membranes resulted in similar phosphatidylcholine incorporation but in the presence of Ca 2+ a residual phosphatidylcholine uptake was still observed. These results suggest that in the isolated membranes small amounts of phosphatidylcholine can be incorporated into an additional region which is unavailable for the membrane lecithinase. The increase in the phosphatidylcholine to sphingomyelin mol ratio in sheep red blood cells is concomitant with an increase in lipid fluidity, as well as increase in osmotic fragility.

ELEVATED CHOLESTEROL (CH)/PHOSPHOLIPID(PL) MOLAR RATIO IN RED CELL MEMBRANES-AN ACQUIRED DEFECT IN CIRRHOSIS WITH ARTERIOHEPATIC DYSPLASIA

Hemolytic anemia with cholesterol-rich, spur-shaped red cells has been described in adults with severe hepatocellular disease due to alcoholic cirrhosis. The red cell defect(elevated membrane CH content, without changes in PL content)is acquired from serum, and causes an increase in the microviscosity of membrane lipids. A 4½ year old girl with arteriohepatic dysplasia, intrahepatic cholestasis and cirrhosis developed progressive anemia(Hgb=5.8), reticulocytosis(21%), increasing jaundice (41 mgm/dl; 23 direct), and splenomegaly. Serum levels of iron, folate, Vit. B12 and E were normal. Peripheral smear showed fragmented cells and spur cells. Analysis of patient's fresh red cells revealed elevated CH(22.3 γ /108cells; N=14.5), normal PL(33.0γ/108cells; N=31.8), an increased CH/PL molar ratio (1.35; N=0.92), and an increase in microviscosity @ 37°(4.1 poise; N=3.0). Following incubation with the patient's plasma@ 37° for 24 hours, normal cells acquired the spur-shaped morphology with an associated decrease in osmotic fragility and a 25%increase in cholesterol content. Patient's cells incubated with normal plasma acquired morphological feature of spiculated spherocytes, with an increase in osmotic fragility and a 21% decrease in cholesterol content. CONCLUSION: These studies demonstrate that a process identical to spur-cell anemia in adults with alcoholic cirrhosis may accompany cirrhosis in children with intrahepatic cholestasis.