Osmotic Fragility Of Red Blood Cells Research Articles

Red Blood Cell Osmotic Fragility in Bufo arenarum Exposed to Lead

The effects on red blood cells of a single sublethal dose of Pb of 100 mg kg -1 administrated to adult Bufo arenarum were studied. The blood d-aminolevulinic acid dehydratase (d-ALAD) activity, the red blood cell (RBC) osmotic fragility (OF), and the hematocrit (Hct) were measured in control and lead poisoned toad. The enzyme d-ALAD is considered as a specific biomarker for human and animals lead exposure. In Bufo, lead also provoked a significant decrease in the d-ALAD activity without changes in the Hct. OF test was used to compare the impact of Pb on the extent of the RBC hemolysis produced by osmotic stress. Experimental data (absorbance of solubilized hemoglobin and [NaCl]) were fitted to the Orcutt et al. equation (1995) that allows a precise characterization of the parameters involved in OF. In blood from injected toads, the OF resulted significantly reduced. These changes were interpreted as a consequence of alterations in the composition and conformation of the RBC membrane due to Pb, as it was described for human erythrocytes.

Red Blood Cell Osmotic Fragility in Chronically Hemodialyzed Patients

Chronic renal failure induces anemia and a short erythrocyte life span. Red blood cell (RBC) osmotic fragility is the resistance of RBC hemolysis to osmotic changes that is used to evaluate RBC friability. To find the cause of shortened red cell survival in uremic patients, we evaluated the RBC osmotic fragility in 57 chronic hemodialyzed patients. Each patient had received 12 h of dialysis per week continuously prior to being enrolled in the study. Nineteen healthy volunteers served as a control group. Biochemistry, hemoglobin, electrolyte, osmolarity, β₂-microglobulin, and intact parathyroid hormone were examined before and after the dialysis session. To evaluate the osmotic fragility of RBC, blood samples were collected in heparinized test tubes. Fifty microliters of the RBC of each individual was then incubated in solutions containing a series of various concentrations of NaCl ranging from 0 to 0.6%. The concentration of NaCl at which 50% of RBCs were lysed was considered the median osmotic fragility (MOF). The results showed that the MOF was significantly greater in hemodialyzed patients before dialysis than in the control group (0.41 ± 0.03 vs. 0.39 ± 0.02%). The osmotic resistance to hemolysis was also recorded after dialysis (MOF 0.38 ± 0.03%). Correlation analysis showed that the MOF was significantly correlated with urea nitrogen, serum osmolarity, and intact parathyroid hormone level. In addition, the osmotic fragility was higher in patients who had a predialysis intact parathyroid hormone level >100 pg/dl. In conclusion, hemodialysis can improve the osmotic fragility. The mechanism underlying this improvement may be the removal of low molecular weight uremic toxins, resulting in normalization of serum osmolarity. Our results indicate that parathyroid hormone is probably a major factor influencing RBC osmotic fragility in chronic renal failure.

Supplementation with Vitamin C, Vitamin E or β-Carotene Influences Osmotic Fragility and Oxidative Damage of Erythrocytes of Zinc-Deficient Rats

Dietary zinc deficiency in rats causes increased osmotic fragility of their erythrocytes. In this study, the influence of supplementary antioxidants (vitamin C, vitamin E or β-carotene) on osmotic fragility, oxidative damage and components of the primary defense system of erythrocytes of zinc-deficient rats was investigated. Indicators of hemolysis in vivo were also examined. Five groups of 12 male rats were force-fed a zinc-adequate diet (control rats), a zinc-deficient diet or a zinc-deficient diet enriched with vitamin C, vitamin E or β-carotene. Compared with the control rats, the rats fed the zinc-deficient diet without supplementary antioxidants had greater red blood cell osmotic fragility, higher concentrations of thiobarbituric acid–reactive substances and alanine, higher glutathione S-transferase activity, lower concentration of glutathione and activity of glutathione peroxidase as well as lower activity of superoxide dismutase in plasma (P < 0.05). Supplementation with antioxidants generally improved osmotic fragility in zinc-deficient rats without influencing zinc concentration or alkaline phosphatase activity in plasma, indicators of zinc status. At some of the hypotonic saline concentrations tested, vitamin C and β-carotene significantly affected osmotic fragility. The zinc-deficient rats fed a diet without supplementary antioxidants had significantly higher concentrations of alanine in erythrocytes than the zinc-deficient rats supplemented with vitamin C, vitamin E or β-carotene and had significantly higher levels of thiobarbituric acid–reactive substances in erythrocytes than the rats supplemented with β-carotene. There was no indication of hemolysis in vivo in rats fed zinc-deficient diets. The results show that supplementary antioxidants decrease osmotic fragility and oxidative damage of erythrocytes in zinc-deficient rats.

Lactate/H+ uptake by red blood cells during exercise alters their physical properties.

We investigated whether uptake of lactate anions by red blood cells (RBC) during maximal exercise altered certain cellular physical properties including volume, density and osmotic fragility. Irrespective of whether individuals undertook 1 min of exercise at maximal effort or a progressive exercise test to exhaustion, whole-blood lactate concentration increased about tenfold relative to pre-exercise values (P < 0.001). The plasma and cellular concentrations of lactate increased uniformly, which maintained a constant plasma-to-cell gradient. Exercise-induced increases in RBC osmotic fragility (P < 0.001) and mean cell volume (MCV; P < 0.05), and decreases in mean cell haemoglobin concentration (MCHC; P < 0.05) and cell density (P < 0.005) also occurred. These exercise-induced cellular responses could be simulated in vitro by treating blood with L-lactic acid (15 mM), but not by low pH (7.1) or L-lactate alone. The uptake of lactate by RBCs during vigorous exercise alters certain cellular properties. Although the physiological implications of these changes are unclear, the traditional view that the lactate anion has no known harmful effects should be reconsidered.

Internalization and distribution of inositol hexakisphosphate in red blood cells.

Inositol hexakisphosphate (InsP6), an allosteric effector of haemoglobin, is able to modify the oxyhaemoglobin dissociation curve. The rightwards shift of the curve increases the in vivo oxygen delivery to tissues. Such an exogenous substance may be internalized into red blood cells (RBC) using a reversed lysis-resealing process following a hypoosmotic shock, resulting in InsP6-RBC with modified oxygen transport capacity. The efficacy of the process depends on various physicochemical parameters which can be fixed during the experimental protocol. The variability of InsP6 internalization from one sample to another appeared to be mainly due to the natural variation in osmotic fragility of RBC. This factor was also modified during the storage of RBC units before the lysis-resealing process. The separation of InsP6-RBC on a density gradient revealed a wide heterogeneity of internalized InsP6 concentration, varying with the degree of osmotic shock. The control of these various parameters will result in resealed InsP6-RBC in reproducible conditions suitable for in vivo use.

Hereditary xerocytosis: a report of six unrelated Spanish families with leaky red cell syndrome and increased heat stability of the erythrocyte membrane.

Hereditary xerocytosis (HX) is a rare haemolytic disease due to dehydrated red blood cells (RBCs). A unique feature of this syndrome is that affected members often show normal or near normal haemoglobin levels despite clinical and laboratory evidence of mild to moderate haemolysis. The diagnostic clue is the association of markedly increased RBC Na+ + K+ fluxes with low total cation (Na+ + K+) content. 11 patients of six unrelated families of Spanish origin with HX have been studied from clinical, genetical and biological points of view. In addition, we have investigated the sensitivity of RBC membrane to heat at three different incubation times (15, 30 and 60 min) and two different temperature values (46 degrees C and 49 degrees C). Under these conditions control RBCs (50 normal subjects) exhibited at 49 degrees C and 30 min a maximum of 30% fragmented RBCs. This value increased to 80% after 60 min of incubation. In contrast, patients with HX showed significantly lower percentages of fragmented RBCs at both 30 and 60 min of incubation (maximum 10% and 30%, respectively). In an attempt to determine if increased heat stability was unique to HX RBCs, several other congenital membranopathies with haemolytic anaemia were also studied. The degree of fragmentation, except in one case of HPP (which was strongly increased), did not differ from the control group. Electrophoretic studies of membrane proteins performed in RBCs of all the patients with HX did not explain any qualitative nor quantitative abnormality. In addition to its physiopathological interest, study of RBC heat stability, together with other haematological parameters (increased MCHC and decreased RBC osmotic fragility), may be useful for HX diagnosis, especially in laboratories which are not equipped to evaluate RBC membrane permeability.

Changes in the susceptibility of red blood cells to oxidative and osmotic stress following submaximal exercise

Red blood cell (RBC) susceptibility to oxidative and osmotic stress in vitro was investigated in cells from trained and untrained men before and after submaximal exercise. Whilst no significant change in peroxidative haemolysis occurred immediately after 1 h of cycling at 60% of maximal aerobic capacity (VO2max), a 20% increase was found 6h later in both groups (P < 0.05). The RBC osmotic fragility decreased by 15% immediately after exercise (P < 0.001) and this was maintained for 6h (P < 0.001). There was an associated decrease in mean cell volume (P < 0.05). Training decreased RBC susceptibility to peroxidative haemolysis (P < 0.025) but it did not influence any other parameter. These exercise-induced changes were smaller in magnitude but qualitatively similar to those found in haemopathological states involving haem-iron incorporation into membrane lipids and the short-circuiting of antioxidant protection. To explore this similarity, a more strenuous and mechanically stressful exercise test was used. Running at 75% VO2max for 45 min reduced the induction time of O2 uptake (peroxidation), consistent with reduced antioxidation capacity, and increased the maximal rate of O2 uptake in RBC challenged with cumene hydroperoxide (P < 0.001). The proportion of high-density RBC increased by 10% immediately after running (P < 0.001) but no change in membrane-incorporated haem-iron occurred. In contrast, treatment of RBC with oxidants (20-50 mumol.l-1) in vitro increased cell density and membrane incorporation of haem-iron substantially. These results showed that single episodes of submaximal exercise caused significant changes in RBC susceptibility to oxidative and osmotic stress. Such responses may account for the increase in RBC turnover found in athletes undertaking strenuous endurance training.

Genetic and Nongenetic Effects on Erythrocyte Osmotic Fragility in Lactating Holstein Cows and Its Association with Yield Traits

Blood samples from 262 lactating Holstein cows were analyzed to determine the resistance of erythrocytes to hypotonic NaCl solutions. The animal model contained stage of lactation, herd, parity, and season as fixed effects, milk yield as a covariate, and 520 random animal effects.A derivate-free algorithm of REML procedure was applied for estimation and prediction. The results suggested higher hemolysis rates and mean corpuscular fragility in first than in later lactations and decreased erythrocyte fragility in cows in late lactation. A negative linear regression of percentage of hemolysis on weekly milk yield was significant. Heritability estimates were .58±.14 for hemolysis rate at 111mM NaCl and .47±.18 for mean corpuscular fragility. Estimates for hemolysis rates decreased with increasing osmotic fragility at lower NaCl concentrations. Phenotypic correlations between erythrocyte fragility traits and 305-d milk, fat, and protein yields were all negative and low. Moderately negative genetic correlations between red blood cell osmotic fragility and fat yield and fat percentage and slightly positive genetic correlations between red blood cell osmotic fragility and protein yield and protein percentage suggested possible alterations in erythrocyte membrane stability if milk component traits were targets for genetic selection.

Human erythrocyte protein 4.2 deficiency associated with hemolytic anemia and a homozygous 40glutamic acid--&gt;lysine substitution in the cytoplasmic domain of band 3 (band 3Montefiore)

Red blood cell (RBC) protein 4.2 deficiency is often associated with a moderate nonimmune hemolytic anemia, splenomegaly, and osmotically fragile RBCs resembling, but not identical to, hereditary spherocytosis (HS). In the Japanese type of protein 4.2 deficiency (protein 4.2Nippon), the anemia is associated with a point mutation in the protein 4.2 cDNA. In this report, we describe a patient with moderate and apparently episodic nonimmune hemolytic anemia with splenomegaly, spherocytosis, osmotically fragile RBCs, reduced whole cell deformability, and abnormally dense cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the proposita's RBC membrane proteins showed an 88% deficiency of protein 4.2 and a 30% deficiency of glyceraldehyde-3-phosphate dehydrogenase (band 6). Structural and molecular analyses of the proposita's protein 4.2 were normal. In contrast, limited tryptic digestion of the proposita's band 3 showed a homozygous abnormality in the cytoplasmic domain. Analysis of the pedigree disclosed six members who were heterozygotes for the band 3 structural abnormality and one member who was a normal homozygote. Direct sequence analysis of the abnormal band 3 tryptic peptide suggested that the structural abnormality resided at or near residue 40. Sequence analysis of the proposita's band 3 cDNA showed a 232G-->A mutation resulting in a 40glutamic acid-->lysine substitution (band 3Montefiore). Allele-specific oligonucleotide hybridization was used to probe for the mutation in the pedigree, showing that the proposita was homozygous, and the pedigree members who were heterozygous for the band 3 structural abnormality were also heterozygous for the band 3Montefiore mutation. The band 3Montefiore mutation was absent in 26 chromosomes from race-matched controls and in one pedigree member who did not express the band 3 structural abnormality. In coincidence with splenectomy, the proposita's anemia was largely corrected along with the disappearance of most spherocytes and considerable improvements of RBC osmotic fragility, whole cell deformability, and cell density. We conclude that this hereditary hemolytic anemia is associated with the homozygous state for band 3Montefiore (40glutamic acid-->lysine) and a decreased RBC membrane content of protein 4.2. We speculate that band 3 structural abnormalities can result in defective interactions with protein 4.2 and band 6, and in particular, that the region of band 3 containing 40glutamic acid is involved directly or indirectly in interactions with these proteins.

Relationship of membrane fluidity, chemoprotection, and the intrinsic toxicity of butylated hydroxytoluene

In isolated rat hepatocytes, many chemicals elicit toxicity which is inhibitable by antioxidants such as butylated hydroxytoluene (BHT). Although BHT protection is evident at concentrations of less than about 50 nmol/mg protein, higher concentrations exhibit intrinsic concentration-dependent toxicity, which involves mitochondrial dysfunction. We evaluated the possibility that both chemoprotection and intrinsic toxicity could be explained by a common mechanism involving alterations in the physical properties of cellular membranes. In the red blood cell (RBC) osmotic fragility assay, BHT at less than 60 nmol/mg protein protected against osmotic fragility; however, BHT at higher concentrations enhanced osmotic fragility such that total osmolysis occurred at 135 nmol/mg. The BHT-mediated alterations in osmotic fragility correlated with changes in membrane fluidity, determined by fluorescence polarization of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene. Protection from osmolysis correlated with decreased fluidity, while enhanced RBC fragility correlated with increased fluidity. In rat hepatocyte suspensions, high BHT concentrations also permeabilized the plasma and mitochondrial membranes to enzyme leakage, and these effects were accompanied by enhanced membrane fluidity. Although other mechanisms may be operative, alterations in membrane fluidity appear to be, in part, responsible for the observed chemoprotective effects at low concentrations, and intrinsic toxicity at higher concentrations of BHT.

Copper deficiency anemia: altered red blood cell lipids and viscosity in rats

To investigate membrane lipid abnormalities and their possible role in copper deficiency anemia, we studied lipids, osmotic fragility, and viscosity of red blood cells (RBCs) of rats fed diets containing 1.1 mg Cu/kg (deficient, Cu-) and 6.0 mg Cu/kg (supplemented, Cu+) at age 3 wk for 9 wk. Data show an increase in cholesterol of 43% and in phospholipids of 38% per cell in Cu- compared with Cu+ rats. Thin-layer chromatography of RBC lipids showed an increased amount of phospholipid-malonyldialdehyde adduct in Cu- (0.70 +/- 0.35% [means +/- SD] of total phospholipids) compared with Cu+ (0.29 +/- 0.19%) rats, suggesting increased membrane lipid peroxidation in Cu- RBC. Osmotic fragility of RBCs from Cu- rats was lower. RBC viscosity was significantly higher in Cu- than in Cu+ rats at shear rates of 23, 11.5, and 5.75 shear/s and was similar at shear rates 115 and 46 shear/s. Increased cell viscosity caused by lipid loading and crosslinking of membrane components caused by lipid peroxidation may have a role in the reduced RBC survival and anemia in Cu deficiency.

Lack of relation between secondary hyperparathyroidism and red blood cell osmotic fragility in chronic renal failure.

The possible relationship between red blood cell (RBC) osmotic fragility and secondary hyperparathyroidism (HPT) in chronic renal failure was examined in 23 uremic patients on conservative therapy and in 42 patients on maintenance hemodialysis. Secondary HPT was evaluated by means of serum biochemistry (parathyroid hormone, calcium, phosphorus, and alkaline phosphatase) and radiographic examinations (X-ray films of the hand skeleton). This study showed increased RBC osmotic fragility in uremic patients when compared with controls, with no difference between those on conservative therapy and those on maintenance hemodialysis. No correlation between RBC osmotic fragility and the hematochemical changes associated with secondary HPT was found. No difference in RBC osmotic fragility was observed with regard to the activity (alkaline phosphatase) and the severity (X-ray findings) of secondary HPT. Effective treatment of secondary HPT by either pharmacological means (1,25-dihydroxycholecalciferol) or surgical removal was not associated with consequent improvement in RBC osmotic fragility. It is concluded that secondary HPT is probably not a major factor influencing RBC osmotic fragility in chronic renal failure.

Decreased Osmotic Fragility of Red Blood Cells of Eimeria adenoeides-Infected Turkeys

The osmotic fragility of red blood cells (RBC) from Eimeria adenoeides-infected turkey poults was compared with that of RBC from control and water-deprived poults. At different hypotonic NaCl concentrations, lysis of RBC from infected poults was 10 to 35% less on day 4 postinoculation (PI) and 50 to 65% less on day 7 PI than that of controls. Red blood cells of poults deprived of water for 3 days were also resistant to lysis; the percent lysis was roughly the same as that of RBC from infected poults at day 7 PI. Incubating control RBC in plasma from infected poults, in extracts of infected ceca, or at different pH levels did not increase their resistance to lysis, suggesting that neither a stabilizing factor in the plasma that had rapid effect on the RBC nor a transient shift in blood pH was involved. Mean RBC size differed little among infected, water-deprived, and control poults (14.0-14.2 X 8.0-8.1 X 3.8 microns). However, although 3.5% of RBC population of control and water-deprived poults were immature (mid to late polychromatic erythrocytes), only 0.4% of the RBC of infected poults were immature. The data suggest that reduced water intake as well as other factors may be involved in the decreased osmotic fragility of RBC from poults infected with E. adenoeides.

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.

Diagnosis of hereditary spherocytosis in newborn infants

The normal range of red blood cell osmotic fragility and autohemolysis was determined in venous blood of 32 healthy newborn infants. With these normal ranges as a reference, the diagnosis of hereditary spherocytosis was definitely possible in five newborn infants by demonstration of increased osmotic fragility of fresh and incubated red blood cells, moderately increased autohemolysis, and partial reduction of autohemolysis by the addition of glucose. Most previously studied newborn infants with hereditary spherocytosis have had atypical hyperbilirubinemia. Inconstant signs are anemia, reticulocytosis, erythroblastosis, spherocytes in the blood smear, and increased mean corpuscular hemoglobin concentration. In newborn infants with atypical hyperbilirubinemia in whom blood group incompatibilities are excluded, studies for hereditary spherocytosis should be done.

Preservation of human erythrocytes in the liquid state: biological results with a new medium.

Red blood cells (RBC) were collected with citrate-phosphate-dextrose (CPD) in a blood-pack optimal additive system. After concentration to 90% hematocrit they were diluted with saline-adenine-glucose medium (SAG-RBC), and stored for 35 days. In this work the RBC were stored in the presence of leukocytes. The SAG medium allows RBC conservation during 35 days at +4 degrees C. The adenosine triphosphate (ATP) level of RBC is compatible with their survival. During the first 2 weeks, hemolysis of SAG-RBC was not greater than in CPD blood. Nevertheless, hemolysis reached 1.49% on day 35, and there was a marked increase in RBC osmotic fragility. Scanning electron-microscopic studies of 35-day RBC showed that the majority of them became echinocytes. After incubation in fresh frozen plasma, the RBC recovered satisfactory osmotic resistance and normal disc shape. The post-transfusion viability was normal with greater than 70% recovery after 48 h. The in vivo restoration of 2,3-diphosphoglycerate (2,3-DPG) was rapid in the transfused SAG-RBC, 50% of the initial 2,3-DPG level being restored in 1 h. The in vivo studies proved that the functional quality of these RBC was compatible with their use in transfusion. The most important problem concerns the supernatant hemoglobin level of the SAG-RBC to be used for massive transfusion.

The Effect of Acupupcture on Osmotic-Fragility of Red Blood Cell

The Effect of Acupupcture on Osmotic-Fragility of Red Blood Cell

Changes in the red blood cell membrane in protein-calorie malnutrition1–3

Ten children with protein-calorie malnutrition (five with kwashiorkor, three with marasmus, and two with marasmic-kwashiorkor) were studied at hospital admission and during recovery for evidence of changes in red blood cell lipids, osmotic fragility, and chromium survival. On admission, the children had significantly higher-than-normal red blood cell membrane cholesterol and phospholipid, and an elevated cholesterol:phospholipid ratio. Both red blood cell osmotic fragility and red cell survivals were depressed. These measurements returned to normal limits with recovery.

Red Blood Cell Osmotic Fragility After Aortic Valve Replacement

Red blood cell osmotic fragility and autohemolysis studies were performed on 35 patients with aortic ball-valve prostheses. Osmotic fragility on fresh heparinized blood was normal. After 24 hours' incubation, it was increased in 17 patients. The most common abnormality was a tail of cells with increased fragility indicative of a heterogeneous population of cells. Sixteen patients with increased fragility had cloth-covered Starr-Edwards prostheses; the majority had intravascular hemolysis. Nine patients with normal fragility had Cutter or Magovern prostheses; the majority had minimal hemolysis. Autohemolysis after 48 hours' incubation with glucose was normal in all patients. Increased incubated osmotic fragility frequently reflects damage to red blood cells by prosthetic heart valves, and increased osmotic fragility is often associated with increased rates of hemolysis in vivo. The damage is greater with cloth-covered Starr-Edwards than with Magovern or Cutter prostheses.

Red blood cell osmotic fragility after aortic valve replacement

Red blood cell osmotic fragility after aortic valve replacement