Kazunori Kon

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.

Effect of nitric oxide on the oxygen transport of human erythrocytes

The oxygen dissociation curve of partially NO-liganded hemoglobin of human erythrocytes is measured. As the percentage of NO ligation increases, the affinity of nonliganded hemoglobin for oxygen increases and the heme-heme interaction decreases; furthermore, methemoglobin is formed. Therefore, NO affects the oxygen transport function of hemoglobin, decreasing the oxygen supply to peripheral tissues, because of (1) simple diminution of the available hemoglobin by the tightly bound NO, (2) the high affinity of hemoglobin for oxygen, and (3) the inevitable formation of methemoglobin.

A contribution of calmodulin to cellular deformability of calcium-loaded human erythrocytes.

The effect of intracellular calcium on the deformability of human erythrocytes was studied with a rheoscope, especially in relation to the dynamic structure of membrane cytoskeleton. The appropriate calcium-loading and calcium-depletion were performed to intact erythrocytes with A23187 in potassium buffer. The total calcium content was varied in the range of 0.25 to 3 times as much as normal content, without complete ATP depletion and shape change (the reduction of mean cell volume and the condensation of hemoglobin due to dehydration were avoided). Increasing the intracellular calcium content by about 1.5 times of normal, the deformability was distinctly decreased, while calcium depletion did not affect the deformability. Reduced deformability of the calcium-loaded erythrocytes was restored by the treatment with calmodulin inhibitors, W-7 or trifluoperazine. However, such an effect by calmodulin inhibitors was not detected on normal or calcium-depleted erythrocytes. In conclusion, the interaction between calcium-calmodulin complex and cytoskeletal proteins may affect the membrane stiffness which is regulated through the change of the cytoskeletal structure, and contributes to the deformability of erythrocytes.

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.

Boycott effect with vertical cylinder for paramagnetic red blood cells under the inhomogeneous magnetic field

Abstract The sedimentation rate of paramagnetic erythrocytes in a vertical cylinder increased with the application of an inhomogeneous magnetic field in the horizontal direction. This phenomenon is similar to the so-called Boycott effect, which produces increased sedimentation in an inclined cylinder or channel. The detailed mechanism of this effect has not been obtained. Since the direction of force acting on the particles in a suspension can be changed continuously by changing the magnetic field strength, our method may be used to control the sedimentation rate of small paramagnetic particles in a liquid.

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.