Temperature effects on osmotic fragility, and the erythrocyte membrane

Results are reported on the temperature-dependence of intact-cell surface area, isotonic volume, hemolytic volume, and ghost steady-state surface area and volume, using several techniques of resistive pulse spectroscopy. Temperature was found not to alter the intact cell surface area permanently: the area remains constant at 130 +/- 1 micron 2, at temperatures ranging from 0 to 40 degrees C. Temperature does alter the steady-state volume of the cells, with a colder temperature inducing swelling by about 0.29 micron 3/deg. C. Such a temperature-induced volume change is sufficient to explain only approximately half of the fragility differences which result from temperature changes. The remainder was found to result from higher temperatures enabling a substantial transient increase in surface area of intact cells (up to at least 14% of 40 degrees C), with a corresponding increase in the cell's hemolytic volume (up to 21%). The hemolytic volume apparently increases linearly with temperature, since steady-state ghost volumes are found to increase linearly with the temperature at which the ghosts were produced. In the steady state (at high temperature), the membranes of electrically-impermeable resealed ghosts can remain extended by more than 10%, compared with membranes of the corresponding unhemolyzed, intact red cells.     

Effect of acetaminophen toxicity on erythrocyte osmotic fragility in the Fisher rat

The protective effect of propylthiouracil (PTU) pretreatment against acetaminophen-induced erythrocyte osmotic fragility was determined in the male Fisher rat. Hepatotoxicity was assessed for comparative purposes. PTU (0.15%) was fed in chow for a period of 12 days. Acetaminophen (1 g/kg body wt) was then administered orally by a stomach tube after an overnight fast. The rats were killed either 4 or 24 hr later. Erythrocyte osmotic fragility was determined by the extent of hemolysis in various concentrations of NaCl solutions. Hepatotoxicity was assessed by a rise in serum transaminases and by histological examination of hepatic tissue. PTU treatment when compared with control not only protected rats against acetaminophen-induced hepatotoxicity as reported before, but also protected against erythrocyte osmotic fragility. The time course of acetaminophen toxicity seems to be similar for liver and erythrocyte since both showed damage after 24 hr but not after 4 hr of acetaminophen administration. The data show that PTU pretreatment affords protection against acetaminophen-induced increased erythrocyte osmotic fragility even when their glutathione concentrations were not significantly different, suggesting that PTU per se has a protective effect.     

Comparison of erythrocyte osmotic fragility among ectotherms and endotherms at three temperatures

1. Comparison of erythrocyte osmotic fragility (EOF) between various ectotherms and endotherms was investigated at 5, 25, and 38 degrees C. 2. We hypothesized that ectotherms might possess erythrocytes whose osmotic fragility would be less affected by temperature than those of endotherms. 3. Ectotherm erythrocytes were much more osmotically resistant than those of endotherms. 4. The EOF of ectotherms and endotherms showed similar responses to temperature. 5. It does not appear that the osmotic fragility of erythrocytes from ectotherms in this study are adapted to be less affected by temperature than those of endotherms. The highly osmotic resistant erythrocytes of ectotherms may alleviate the need for further adaptation for osmotic resistance.     

Influence of whole-body gamma-irradiation upon rat erythrocyte: lipid peroxidation and osmotic fragility

The effects of whole-body gamma-irradiation of rats (8 Gy) on erythrocyte enzymes and biochemical components involved in lipid peroxidation were studied. Decreased superoxide dismutase and glutathione reductase activities, and lowered concentrations of reduced glutathione, were found to be the main factors responsible for the observed increase in lipid peroxidation in the erythrocytes of irradiated rats. This increased lipid peroxidation did not result in a greater tendency to hemolysis in hypotonic media; on the contrary, the mean osmotic fragility was decreased at days D + 1 and D + 3 after irradiation. The behavior of the erythrocyte populations towards hemolysis in hypotonic media appeared to be most homogeneous at days D + 4 and D + 8 after irradiation, which correspond to maxima of malonic dialdehyde concentrations in erythrocytes. Such a synchrony of variations suggests that crosslinking of primary amino groups of proteins or phospholipids by malonic dialdehyde might produce a rigidification in erythrocyte membranes, possibly leading to a more homogeneous behavior of the erythrocyte populations towards hemolysis in hypotonic media.     

The erythrocyte membrane site for the effect of temperature on osmotic fragility

The osmotic fragility of human erythrocytes is well known to decrease as the temperature is elevated. The cellular site for the temperature effect was studied by assessing possibles roles of hemoglobin and of membrane lipids and by taking advantage of the unique response of camel erythrocytes to temperature. It is concluded that the erythrocyte membrane is the site for the temperature effect on osmotic fragility. The human erythrocyte is likely to rupture in protein-lipid boundary regions in the membrane, from which cholesterol is apparently excluded.     

Seasonal changes in erythrocyte osmotic fragility and haematocrit in American plaice infected with Haemohormidium terranovae

American plaice held in captivity for a period of 8 months over winter exhibited increasing erythrocyte osmotic fragility and decreasing haematocrit values until December. In December, osmotic fragility parameters and haematocrit were strongly correlated, suggesting anaemia due to disruption of circulating erythrocytes. Intensity of infection with Haemohormidium terranovae increased through the winter months until March but was not correlated either with osmotic fragility or haematocrit. These results are explained in terms of compensatory haemopoetic changes occurring in plaice. Mortality was apparently unrelated to any of the parameters investigated.     

A computerized method for the determination of the osmotic fragility curve of erythrocytes

A computerized and precise method for the determination of the osmotic fragility curve of erythrocytes was developed. The pH and the temperature, the most important factors for the osmotic hemolysis, were controlled with an accuracy of 7.40 ± 0.01 and 25.0 ± 0.2°C, respectively. The method required an extremely minute amount of blood (about 5 μl). The fragility curve represented by the cumulative and first derivative curves as a function of salt concentration had excellent reproducibility for the mean corpuscular fragility and the slope of the fragility curve. The method was applied to erythrocytes treated with glutaraldehyde and to those with the various contents of cholesterol in the membrane.     

Interaction of chemical carcinogens with plasma membranes: the effect of dimethylaminoazobenzene on erythrocyte osmotic fragility

The capacity of the hepatocarcinogen dimethylaminoazobenzene and two of its analogs to protect erythrocytes against hemolysis is demonstrated. The aminoazo dye compounds exhibit stabilization at relatively low final concentrations, suggestive of an efficient partitioning of the dye molecules into the erythrocyte membrane. Methyl groups at either the 2 or 3′ position of dimethylaminoazobenzene confer even greater stabilization potential to the basic dye structure. The application of the erythrocyte osmotic hemolysis assay in analysis of carcinogen-membrane interactions is discussed.     

Membrane skeleton involvement in cell fusion kinetics: a parameter that correlates with erythrocyte osmotic fragility.

Spectrin levels in erythrocytes have been related to several biomechanical and biophysical membrane properties essential to the survival and function of the cell. Populations of erythrocytes display a natural and finite range of sensitivities to osmotic shock that has been directly correlated, in studies from other laboratories, to the presence of spectrin. We used a procedure to isolate subpopulations of 1) the osmotically most sensitive and 2) the osmotically most resistant erythrocyte membranes in an attempt to select for membranes enriched and depleted in spectrin (and/or a related component). The mechanical function of the spectrin-based membrane skeleton was further explored in these two subpopulations by searching for any effect on the time-dependent increase in fusion zone diameter in pairs of electrofused erythrocyte ghosts as a model for cell fusion. The results clearly show that the diameter expansions in fusions of membranes from osmotically resistant erythrocytes are faster in the early stage (up to 9 to 10 s after fusion) but do not thereafter expand as far as in fusions of membranes from osmotically sensitive membranes.     

Genetic control of red cell osmotic fragility

Among normal mouse strains, natural genetic variation offers the potential to investigate the structure and function of cell membranes. One such polymorphism between C57BL/6J and DBA/2J is a difference in erythrocyte sensitivity to osmotic lysis. The genetic basis for erythrocyte osmotic fragility differences between mouse strains C57BL/6 and DBA/2 was examined through analyses of their serial backcross progeny, recombinant inbred (ri) strains (BXD), and congenic C57BL/6 strains with allelic differences at Hbb or Fv-2. The data indicate that the fragility difference between C57BL/6 and DBA/2 is the result of allelic differences at a minimum of two segregating loci. One of these might be linked to, but is not identical with the gene encoding the beta chain of hemoglobin (Hbb). Allelic differences at Fv-2, a gene known to control the proportion of erythroid precursors in the S phase, and at Hba, the structural locus of hemoglobin alpha chain also appear to exert no major influence on red cell osmotic fragility. Furthermore, the fact that red cells from one of the RI strans (BXD-31) are strikingly more resistant than those from the resistant parental strain DBA/2 leads to the conclusion that the degree of resistance/susceptibility for either strain is determined by the combined contributions of gene effects not all of which act in the same direction. We also found that red cells from strans C57BL/6 and DBA/2 differ in their uptake of 51Cr. This result suggests the possibility that red cell osmotic fragility differences may be due in part to differences in ion metabolism or membrane transport.     

Hepatocytes from newborn and weanling rats in monolayer culture: Isolation by perfusion,fibronectin-mediated adhesion,spreading, and functional activities

Summary The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 3 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and α-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture. Part of this work was presented at the 30th Annual Meeting of the Tissue Culture Association, Seattle, June, 1979.     

Hepatocytes from newborn and weanling rats in monolayer culture: isolation by perfusion, fibronectin-mediated adhesion, spreading, and functional activities

The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 2 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and alpha-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.