Misuzu Sekiya

The decreased membrane fluidity of in vivo aged, human erythrocytes. A spin label study

The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane. In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown. On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.

Cell age-dependent changes in deformability and calcium accumulation of human erythrocytes.

The deformability of human erythrocytes was measured in a rheoscope, as a function of intracellular calcium content (varied with ionophore (A23187) and CaCl2) without complete ATP depletion and echinocytic transformation. Loading calcium into intact erythrocytes (calcium content: 16.8 mumol/1 packed cells = 1.48 amol per cell), the cell volume and energy charge gradually decreased. Further, the membrane fluidity of the lipid portion decreased without crosslinking of membrane proteins. A distinct transition from deformable to undeformable cells was observed by the rheoscope technique: i.e., 50% transition occurred at 40-50 mumol calcium/1 packed cells (= 3.5-4.0 amol per cell) and more than 90% above 100 mumol/1 packed cells (= 6.5 amol per cell) at a shear stress of 140 dyn/cm2. The deformable cells maintained their deformability to ellipsoidal disks independent of the average calcium content. The underformable cells, separated as high-density cells by density gradient centrifugation after calcium-loading, showed lower glucose-6-phosphate dehydrogenase activity than low-density-deformable cells; thus, the calcium-loaded, undeformable cells were presumably in vivo aged cells. The younger cells, fractionated as low-density cells from intact erythrocytes, were more deformable than aged cells. Upon calcium-loading, the younger cells restored their cell volume and deformability, while the aged cells, containing originally more calcium and less ATP, decreased their volume and became undeformable. Therefore, calcium accumulation by ionophore-CaCl2 takes place in preference to aged cells of lower energy metabolism, and leads to cellular dehydration and loss of deformability, due to condensed hemoglobin and altered membrane organization.

Rheological characteristics of desialylated erythrocytes in relation to fibrinogen-induced aggregation.

The effect of fibrinogen and sialic acid content of erythrocytes on the aggregation of erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. (1) The electrophoretic mobility of erythrocytes was proportional to the sialic acid content of erythrocytes (the surface potential of erythrocytes could be expressed by the sialic acid content). (2) The aggregation of erythrocytes was accelerated by increasing fibrinogen concentration in the medium (due to the increased bridging force among erythrocytes) or by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes). (3) An empirical equation expressing the velocity of aggregate formation (v, in micron2/min) by the concentration of fibrinogen (F, in g/dl) and the sialic acid content (S, in mumol/ml red blood cells), log v = -0.065 F-1.2S + 2.2 F0.35, was deduced. (4) The contribution of the bridging force of fibrinogen to the erythrocyte aggregation was much greater than that of the electrostatic repulsive force produced by sialic acid on the surface of erythrocytes.

Statistical determination of red cell adhesion to material surface, by varying shear force

Abstract A rapid, quantitative method for determining the red cell adhesion to a material surface has been developed, counting the adhered cell number as a function of the applied shear stress in several steps, by means of a rheoscope consisting of an inverted microscope and a transparent cone-plate viscometer. As a typical example, the experimental results on red cell adhesion to a polymethylmethacrylate plate are demonstrated. A theoretical treatment shows that the empirical relation between the number of adhered cells and the applied shear rates is converted to the distribution profile of the adhesive force. Thus the shear stress for 50% dissociation ( τ 50 ) gives an estimate of the median of the adhesive force. The inhibition of cell adhesion occurs by adding human albumin and fibrinogen, and is quantitatively expressed by the reduction of τ 50 as protein concentrations increase: fibrinogen shows a stronger inhibitory effect than does albumin. The influence of proteins on surface adhesion and on rouleau formation is discussed.

Contribution of glycoproteins to fibrinogen-induced aggregation of erythrocytes

The contribution of membrane glycoproteins to the velocity of fibrinogen-induced erythrocyte aggregation was examined using a rheoscope combined with a video camera, an image analyzer and a computer. The structure of glycoproteins was modified with proteolytic enzymes, trypsin or alpha-chymotrypsin. (1) Mild enzymatic treatment of erythrocytes decreased the velocity of erythrocyte aggregation, but more intense treatment increased the velocity remarkably. (2) The erythrocyte aggregation was affected not only by the density of surface negative charge of erythrocytes, but also by the structural changes of glycoproteins. (3) Erythrocyte deformability and the morphological characteristics were not altered by these enzymatic treatments. The physiological significance of glycoproteins of erythrocyte surface for the survival of erythrocytes and for the suspension stability of blood was discussed.

Effects of high α-linolenate and linoleate diets on erythrocyte deformability and hematological indices in rats

Rats were fed either a high α-linolenate diet or a high linoleate diet from weaning to 4 mon of age. Soybean oil was used as a control. Phospholipid compositions of erythrocytes from the three dietary groups were not significantly different. However, the difference in the α-linolenate (18∶3n−3)/linoleate (18∶2n−6) ratio of the diets was reflected in the n−3/n−6 ratios of the 20 and 22 carbon highly unsaturated fatty acids except for docosahexaenoic acid (22∶6n−3) in the phospholipids. Despite the significant differences in the fatty acid compositions of phospholipids, no measurable differences were detectable in erythrocyte deformability, whole blood viscosity and hematological indices of the three dietary groups. These results indicate that the beneficial effects of the high α-linolenate diet, as compared with the high linoleate diet, are exerted without significant changes in these parameters.

Increase of ATP level in human erythrocytes induced by S-adenosyl-l-methionine

The effect of S-adenosyl-L-methionine (SAM) on the ATP level, the morphology and the deformability of human erythrocytes was investigated and compared with that of adenosine. (i) Upon incubation with SAM, the ATP level increased considerably in fresh cells (in both young and old cells in similar extent) and in stored (partially ATP-depleted) cells. But the incubation with adenosine increased ATP level to a lesser extent. (ii) The incubation of stored cells with SAM hardly affected (or rather decreased) the IMP level, while that with adenosine remarkably increased IMP (and ITP). (iii) The morphology and the deformability of stored erythrocytes were well conserved in spite of the treatment with SAM, as compared with the treatment with adenosine. The echinocytic transformation was induced in old cells to some extent by SAM, while did not in young cells.

Protective Effect of α-Tocopherol on the Morphological and Rheological Changes of Rat Red Cells

The effect of α-tocopherol on the rheological properties of rat red cells was studied. The suspension viscosity of red cells increased, as the α -tocopherol content in red cells decr

Rheological and functional impairments in cholesterol-loaded human erythrocytes.

Decreased deformability, slightly increased suspension viscosity and retarded oxygen egress were observed in cholesterol-loaded, human erythrocytes. These functional changes resulted from the decreased membrane fluidity induced by cholesterol.

Rheological properties of erythrocytes in recombinant human erythropoietin-administered normal rat

Summary. Recombinant human erythropoietin [rhEPO (180 iu/μg); 1 or 10 μg polypeptide equivalent/kg] was intravenously administered daily to 6‐week‐old normal rats for 1 week. Rheologically, (1) blood viscosity at shear rate of 40–380 s−1 increased in a manner entirely dependent upon the haematocrit, (2) no change in erythrocyte deformability was detected by high shear rheoscopy, and (3) the velocity of rouleau formation in autologous plasma (at 7.5 s−1) decreased. Haematologically, rhEPO administration induced considerable polycythaemia with reticulocytosis in a dosedependent manner, accompanying increased cell volume and decreased intracellular haemoglobin concentration, thus the density distribution of erythrocytes shifted towards low specific gravity. Plasma viscosity and plasma protein composition were unaffected by rhEPO‐administration. In erythrocyte metabolism, no drastic alteration in the level of 2,3–DPG or ATP was detected.