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This paper presents the studies on the erythrocyte aggregation of density-fractionated human erythrocytes, and demonstrates the accelerated aggregation of aged cells at low shear rate. Furthermore, the functional, biochemical and cell geometrical changes (e.g., cellular deformability, content of sialic acid in cell surface, electrophoretic mobility, cell size) are studied

Red blood cells (RBCs) generally deform to adopt a parachute-like, torpedo-like, or other configuration to align and flow through a capillary that is narrower than their major axis. As described herein, even in a narrow tube (25 microm) with diameter much larger than that of a capillary, flowing RBCs at 1 mm/s align axially and deform to a paraboloid shape in a viscous Newtonian fluid (505 kDa dextran medium) with viscosity of 23.4-57.1 mPa.s. A high-speed digital camera image showed that the silhouette of the tip of RBCs fits a parabola, unlike the shape of RBCs in capillaries, because of the longer distance of the RBC-free layer between the tube wall and the RBC surface ( approximately 8.8 microm). However, when RBCs are suspended in a "non-Newtonian" viscous fluid (liposome-40 kDa dextran medium) with a shear-thinning profile, they migrate toward the tube wall to avoid the axial lining, as "near-wall-excess," which is usually observed for platelets. This migration results from the presence of flocculated liposomes at the tube center. In contrast, such near-wall excess was not observed when RBCs were suspended in a nearly Newtonian liposome-albumin medium. Such unusual flow patterns of RBCs would be explainable by the principle; a larger particle tends to flow near the centerline, and a small one tends to go to the wall to flow with least resistance. However, we visualized for the first time the complete axial aligning and near-wall excess of RBCs in the noncapillary size tube in some extreme conditions.

Electrostatic repulsive force among human red blood cells (RBCs) was observed by macromolecules-induced RBC aggregation and RBC flow behavior in narrow tube, by reducing the sialic acid content of RBCs with neuraminidase. The electrophoretic mobility of the RBCs was proportional to the sialic acid content. (1) When the sialic acid content was reduced, the RBC aggregation observed with a low shear rheoscope was enhanced with fibrinogen (MW=340,000), Ig G (MW=160,000) or Dextran T-70 (MW=70,400), but did not without the macromolecules. In coexistence of normal cells, sialic acid-reduced cells settled significantly faster, possibly due to their preferential aggregation by the macromolecules. (2) Sialic acid-reduced RBCs accumulated more to flow axis in narrow tube (20-50 μm in inner diameter), especially at acidic pH, as evaluated by the thickness of marginal cell-free layer using an image processor. The thickness was further increased with Dextran T-70 due to the RBC aggregation. Conclusively, electrostatic repulsive force among RBCs provided by sialic acid is important for understanding hemorheological behavior of the RBCs and circulatory disturbances in various diseases.

The effects of the oxygenation-deoxygenation process on red blood cell (RBC) aggregation were examined in relation to morphological changes in RBCs and the contribution of CO2. A low-shear rheoscope was used to measure the rate of rouleaux (one-dimensional aggregate) formation in diluted autologous plasma exposed to gas mixtures with different Po2and Pco2. RBC indexes and RBC suspension pH were measured for the oxygenated or the deoxygenated condition, and the cell shape was observed with a scanning electron microscope. In the oxygenation-deoxygenation process, the rate of rouleaux formation increased with rising pH of the RBC suspension, which was lowered in the presence of CO2. The rate increased with increasing mean corpuscular hemoglobin concentration (thus the cells shrank), which increased with rising pH and decreased in the presence of CO2. With rising pH, cell diameter increased and cell thickness decreased (thus the cell flattened). In addition, slight echinocytosis was induced in the presence of CO2, and the aggregation was reduced by the morphological change. In conclusion, RBC aggregation in the oxygenation-deoxygenation process is mainly influenced by the pH-dependent change in the surface area-to-volume ratio of the cells, and the aggregation is modified by CO2-induced acidification and the accompanying changes in mean corpuscular hemoglobin concentration and cell shape.

The effects of erythrocyte aggregation on O2release were examined using O2-permeable fluorinated ethylenepropylene copolymer tubes (inner diameter, 25 μm; outer diameter, 100 μm). Measurements were performed using an apparatus built on an inverted microscope that contained a scanning-grating spectrophotometer with a photon count detector connected to two photomultipliers and an image processor through a video camera. The rate of O2release from the cells flowing in the narrow tube was determined based on the visible absorption spectrum and the flow velocity of the cells as well as the tube size. When the tube was exposed to nitrogen-saturated deoxygenated saline containing 10 mM sodium dithionite, the flowing erythrocytes were deoxygenated in proportion to the traveling distance, and the deoxygenation at a given distance increased with decreasing flow velocity and cell concentration (hematocrit). Adding Dextran T-70 to the cell suspension increased erythrocyte aggregation in the tube, which resulted in suppressed cell deoxygenation and increased marginal cell-free-layer thickness. The deoxygenation was inversely proportional to the cell-free-layer thickness. The relation was not essentially altered even when the medium viscosity was adjusted with Dextran T-40 to remain constant. The rate of O2release from erythrocytes in the tube was discussed in relation to the O2diffusion process. We conclude that the diffusion of O2from erythrocytes flowing in narrow tubes is inhibited primarily by erythrocyte aggregation itself and partly by thickening of the cell-free layer.

We investigated lipopolysaccharide (LPS)-induced nitric oxide (NO) production by rat microglia in neuron-microglia and astrocyte-microglia cocultures to evaluate the influence of neurons and astrocytes on microglial activity. Microglial cells solely cultured in medium devoid of serine (Ser), glycine (Gly) hardly expressed inducible NO synthase (iNOS), while those cocultured with neurons and astrocytes expressed iNOS. When microglial cells and astrocytes were separately cultured by using tissue culture inserts, which allowed the microglial cells to be exposed to only diffusible factors arising from astrocytes, NO production was significantly enhanced. On the other hand, neurons, when separated from microglial cells by the inserts, could not activate microglial cells possibly due to lacking of direct contact between neurons and microglial cells. NO production in pure microglial cultures was significantly enhanced in the presence of Ser/Gly at concentrations higher than 25 microM. Conditioned media obtained from microglia culture and neuron-microglia coculture contained less than 10 microM of Ser and Gly, while media from astrocyte culture and astrocyte-microglia coculture contained 33-41 microM Ser and 20-26 microM Gly. Accordingly, astrocytes modulate the activity of microglial cells by secreting Ser and Gly. The present study proposes a novel metabolic coupling between astrocytes and microglial cells via amino acids.

Changes in the distribution of flowing erythrocytes in a straight cylinder were studied under an inhomogeneous magnetic field. The magnetic field was applied perpendicular to a cylinder, which had a 90° side vessel at the end (oriented towards the magnetic field) to detect changes in the erythrocyte distribution within the cylinder. (1) The attraction of paramagnetic erythrocytes by the magnetic field was demonstrated by an increase in the concentration (or number) of erythrocytes drawn into the side vessel. The flow of diamagnetic, oxygenated erythrocytes was unaffected. (2) The degree of attraction of the paramagnetic erythrocytes was proportional to ``(magnetic susceptibility)'' and to ``(magnetic flux density) × (magnetic field gradient)'' up to 10 T2/m, but it saturated at high magnetic field. The onset of the saturation depended on the magnetic susceptibility of the erythrocytes. (3) The degree of attraction depended on the hematocrit of the flowing erythrocyte suspension, with a maximum value at a low hematocrit. These phenomena are explained on the basis of the balance between the paramagnetic attractive force of the magnetic field and the collision rate between erythrocytes.

The deformation of erythrocytes in microvessels less than 15 microns in inner diameter was analyzed using a microvascular bed isolated from rabbit mesentery. The deformation was compared with that found in glass capillaries.Human erythrocytes were perfused through two media: first, a microvascular-bed section isolated from rabbit mesentery; and second, a set of glass capillaries. Images of deformed erythrocytes were recorded on videotape under strobe light and analyzed with an image processor. The flow velocity of the erythrocytes was determined from the difference of their positions between video frames or by a dual-spot cross-correlation technique. Erythrocyte deformability was modified with diamide, diazene dicarboxylic acid bis[N,N-dimethylamide], by crosslinking spectrins.Symmetrical (parachute-like or slipper-like) deformation of erythrocytes was observed only in microvessels smaller than 13 microns in inner diameter. Erythrocytes in microvessels were less deformed than those in glass capillaries with corresponding diameters, and the marginal cell-free layer was narrower. The deformation increased by increasing the flow velocity of erythrocytes, and the cell-free layer became wider. Diamide-treated cells in microvessels were less deformed than normal cells and showed slightly narrower cell-free layers. Stronger stress in narrower microvessels induced further deformation of cells.Erythrocyte deformation in microvessels was essentially different from that in glass capillaries, and the effect of erythrocyte deformability on the flow dynamics of erythrocytes in microvessels was properly evaluated using an isolated microvascular bed.

The aggregation and sedimentation kinetics of human erythrocytes was studied by modifying the cellular properties and medium compositions simultaneously. Dextrans of average molecular weight 70400 and 494000 were used to provide suspending medium modifications, while diamide (diazene dicarboxylic acid bis(N,N-dimethylamide)) was used to alter the membrane structural properties. Laser scattering method was employed for this study, and it was compared with a kinetic method combined with a low-shear rheoscope and an image analyzer. From scattered light intensity profiles continuously obtained during aggregation of erythrocytes and sedimentation of the aggregates, characteristic kinetic parameters were computed. Kinetic parameters obtained from a phase of the one-dimensional aggregate formation and sedimentation corresponded well to the velocity of rouleaux formation obtained by the low-shear rheoscope technique. Dextrans accelerated the erythrocyte aggregation and the sedimentation, and diamide treatment suppressed the process by decreasing the erythrocyte deformability. The aggregating force by dextrans overcame the disaggregating force by the decreased deformability. However, the arrangement of erythrocytes as expressed in specific units for aggregates (i.e., rouleaux) became irregular by decreasing the erythrocyte deformability. In conclusion, the progression of erythrocyte aggregation and the structure of the aggregates were dependent on both erythrocyte properties and macromolecules in the medium.

The effect of an external inhomogeneous magnetic field on the flow of erythrocytes containing paramagnetic hemoglobin was studied systematically, with three experimental setups. (1) The attraction of a narrow stream of erythrocyte suspension towards stronger magnetic field, in a wide laminar flow, was found to be proportional to the magnetic susceptibility of erythrocytes χ, the product of the field strength and its spatial gradient B × d B /d z , and the reciprocal of flow velocity 1/ v , and also to the hematocrit h of the suspension. (2) A model flow of erythrocyte suspension in the vessel showed a small change in the radial distribution of erythrocytes arising from a magnetic field, which is proportional to χ, B × d d B /d z (up to 20 T 2 /m), 1/ v , and h ( B × d B /d z and h . (3) Acceleration of the sedimentation rate was detected for paramagnetic erythrocytes in an inhomogeneous magnetic field, but not with diamagnetic erythrocytes. In short, the paramagnetic attraction takes place with venous blood, and depends on the product of the field strength and its spatial gradient, the degree of deoxygenation, the flow velocity, and the hematocrit.

Relation between aggregating force (of fibrinogen and IgG) and disaggregating force (due to electrostatic repulsion among erythrocytes) in erythrocyte aggregation was investigated with a rheoscope combining a video camera, an image analyzer and a computer. (i) Erythrocyte aggregation was augmented with the increase of molecular weight of bridging macromolecules as far as examined for fibrinogen and the degradation products and IgG and the related macromolecules, and the augmentation seemed to be dependent on the molecular length of macromolecules. In accelerating the erythrocyte aggregation, fibrinogen was more effective than IgG, and some interaction between fibrinogen and IgG in their coexistence was suggested. (ii) The decrease of sialic acid content on the erythrocyte surface accelerated IgG-induced erythrocyte aggregation much greater than fibrinogen-induced one. (iii) Counteraction between aggregating force and disaggregating force in leading to erythrocyte aggregation was discussed relating to molecular length of bridging macromolecule and electrostatic repulsive force by sialic acid.

Effect of 2,3-diphosphoglycerate (2,3-DPG) on the deformability of human erythrocytes was examined with a rheoscope under shear stress of 8-82 dyn/cm2. With increasing 2,3-DPG in erythrocytes (from 5 to 15 mM/l cells) by incubating with inosine and pyruvate in isotonic 50 mM phosphate-buffered saline, erythrocyte deformability under uniform shear stress was remarkably impaired. But reduction of 2,3-DPG (from 5 to 2.2 mM/l cells) did not affect the deformability. In 2,3-DPG-enriched erythrocytes, increased intracellular hemoglobin concentration (MCHC), decreased intracellular pH, and increased contents of ATP and IMP (and ITP) were observed. (1) When the MCHC (i.e., the internal viscosity) was normalized by suspending in hypotonic medium, the deformability of 2,3-DPG-enriched erythrocytes was greatly improved, but still decreased. (2) The change of intracellular pH between 6.5 and 7.5 (as compared adjusting to same MCHC) did not alter the deformability. (3) The changes of purine nucleotides, ATP (0.6-2.1 mM/l cells), IMP (0-0.9 mM/l cells) and ITP (0-0.5 mM/l cells) did not alter the erythrocyte deformability. In conclusion, decreased deformability of erythrocytes induced by augmentation of 2,3-DPG is due mainly to the increased internal viscosity and due partly to the increased membrane viscoelasticity.

A phospholipid vesicle that encapsulates a concentrated hemoglobin (Hb) solution and pyridoxal 5′-phosphate as an allosteric effector [Hb vesicle (HbV) diameter, 250 nm] has been developed to provide an O2carrying ability to plasma expanders. The O2release from flowing HbVs was examined using an O2-permeable, fluorinated ethylenepropylene copolymer tube (inner diameter, 28 μm) exposed to a deoxygenated environment. Measurement of O2release was performed using an apparatus that consisted of an inverted microscope and a scanning-grating spectrophotometer with a photon-count detector, and the rate of O2release was determined based on the visible absorption spectrum in the Q band of Hb. HbVs and fresh human red blood cells (RBCs) were mixed in various volume ratios at a Hb concentration of 10 g/dl in isotonic saline that contained 5 g/dl albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The mixtures of acellular Hb solution and RBCs were also tested. Because HbVs were homogeneously dispersed in the albumin solution, increasing the volume of the HbV suspension resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of O2release from the HbV/RBC mixtures was similar to that of RBCs alone. On the other hand, the addition of 50 vol% of acellular Hb solution to RBCs significantly enhanced the rate of deoxygenation. This outstanding difference in the rate of O2release between the HbV suspension and the acellular Hb solution should mainly be due to the difference in the particle size (250 vs. 7 nm) that affects their diffusion for the facilitated O2transport.

The oxygen release from flowing erythrocytes under accelerational force (0-4 g) was examined using an oxygen-permeable, fluorinated ethylenepropylene copolymer tube (25 microm in inner diameter). The narrow tube was fixed vertically on the rotating disk of a new centrifuge apparatus, and erythrocyte suspension was perfused in the direction of Earth gravity. The accelerational force was applied perpendicularly to the flow direction of cells by centrifugation. The microscopic images of the flowing cells obtained at five different wavelengths were analyzed, and marginal cell-free layer and oxygen saturation of the cells were measured. By lowering oxygen tension around the narrow tube, erythrocytes were deoxygenated in proportion to their traveling distance, and the deoxygenation was enhanced with decreasing flow velocity and hematocrit. With increase of the g-value, the shift of flowing erythrocyte column to the centrifugal side was increased, the column was compressed, and the oxygen release from the cells was suppressed. Qualitatively, similar results were obtained by inducing erythrocyte aggregation with Dextran T-70 (MW = 70,400), without accelerational force. These results conclude that both the accumulation of erythrocytes under accelerational force and the enhancement of erythrocyte aggregation by macromolecules lead to the reduction of oxygen release from the flowing cells.

The review article deals with phenomena of the blood flow structure (structuring) in narrow microvessels-capillaries and the adjacent arterioles and venules. It is particularly focused on the flow behavior of red blood cells (RBCs), namely, on their specific arrangements of mutual interaction while forming definite patterns of self-organized microvascular flow. The principal features of the blood flow structure in microvessels, including capillaries, include axial RBC flow and parietal plasma layer, velocity profile in larger microvessels, plug (or bolus) flow in narrow capillaries, and deformation and specific behavior of the RBCs in the flow. The actual blood flow structuring in microvessels seems to be a most significant factor in the development of pathological conditions, including arterial hypertension, brain and cardiac infarctions, inflammation, and many others. The blood flow structuring might become a basic concept in determining the blood rheological properties and disorders in the narrow microvessels. No solid theoretical (biorheological) basis of the blood flow structuring in microvessel has been found, but in the future it might become a foundation for a better understanding of the mechanisms of these properties under normal and pathological conditions in the narrowest microvessels 5 to 25 microm large. It is also a topic for further biorheological research directed to find the background of actual physiopathological phenomena in the microcirculation.

Optical measurements of myoglobin (Mb) and cytochromes (Cyts) in the isolated rat hindlimb muscle perfused with cell-free medium were performed at 35 and 15 degrees C under various oxygen supply, in the relation to the oxygen metabolism. Molar ratio of Cyt a + a3, Cyt b, Cyt c + c1, and Mb, oxygen affinity of Mb (P50), and the thermodynamic parameter (delta H degree) of Mb oxygenation in cyanide-perfused muscle were similar to those reported. The "apparent P50" of Mb in cyanide-free muscle was almost two orders larger in magnitude than P50 in the presence of cyanide. O2 uptake by the perfused hindlimb muscle was constant above O2 supply of 0.73 mumol/(min.g muscle) (under a flow rate of 1.0 ml/(min.g muscle) at 35 degrees C). Below the value of O2 supply, the O2 uptake decreased and lactate/pyruvate ratio increased. The critical mean oxygen tension in tissue (estimated by Mb oxygenation) for O2 uptake at 35 degrees C was ca. 10 mmHg. It was found that oxidation level (%) of Cyt a + a3 was equivalent to oxygenation level (%) of Mb at 35 degrees C, while the oxidation level of Cyt a + a3 was higher than the oxygenation level of Mb at 15 degrees C. Based on the results, the uneven distribution of O2 in the muscle tissue and the intracellular O2 gradient were discussed.

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.

Physiologically, dynamics of oxygen release from erythrocytes in capillary to tissues is one of the most important phenomena. The major determinants of oxygen supply to tissues are blood flow to tissues, oxygen content in blood and the density of capillaries. The importance of capillary density has been well examined since the pioneering study of Krogh1.

Temperature effect on tissue oxygenation and metabolism is a major concern to activity and/or viability of tissues. In an ischemic state, supplement of oxygen and nutrients is impaired and metabolites are accumulated, thus tissue damage is induced reversibly or irreversibly. This deterioration of tissue accompanied by ischemia is mediated by chemical reactions, and the rates are directly dependent upon temperature. Rat hindlimb is well suited for the optical measurement of biochemicals that participate in oxygen transport (Tamura et al., 1987; Seiyama et al., 1988) as well as the biochemical investigation on the integral function of skeletal muscle (Shiota et al., 1986).

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.

The velocity of the aggregation of human erythrocytes was examined in the range of 5-43 degrees C with a rheoscope combined with a video camera, an image analyzer and a computer. (1) With increasing temperature, the velocity of erythrocyte aggregation induced by fibrinogen, immunoglobulin G and artificial macromolecules (dextran of 70 kDa and poly(glutamic acid) of 50 kDa) increased. However, the relationship between the velocity of erythrocyte aggregation and the temperature was different among these macromolecules. (2) In 70% autologous plasma, the velocity of erythrocyte aggregation was minimum at 15-18 degrees C, and increased at both higher and lower temperatures. (3) The shape of erythrocyte aggregates in 12 mumol/l fibrinogen (containing 770 mumol/l albumin) and in 70% autologous plasma was dependent on temperature: three-dimensional below 15-18 degrees C and one-dimensional (mainly rouleaux) above 15-18 degrees C. However, the shape of aggregates in 27 mumol/l immunoglobulin G (containing 770 mumol/l albumin) was three-dimensional in all temperature ranges. (4) The temperature dependency of erythrocyte aggregation was discussed in terms of the changes of medium viscosity, of erythrocyte properties and of bridging macromolecules.

Effects of an inhomogeneous magnetic field on narrow erythrocyte streams in a wide and transparent laminar buffer flow were studied. The stream line of erythrocytes containing paramagnetic hemoglobin showed distinct displacement toward the stronger magnetic field. The displacement increased in the order, oxygenated erythrocytes (no displacement), erythrocytes containing cyanomethemoglobin, deoxygenated erythrocytes, erythrocytes containing methemoglobin in the high spin state; more precisely the displacement was proportional to the square of the paramagnetic moment of hemoglobin contained in the erythrocytes. In addition, the displacement was proportional to the product of the magnetic flux density and its gradient, and approximately proportional to the hematocrit of the flowing-erythrocyte suspension, and was much larger than that calculated for a single erythrocyte. These phenomena could be successfully interpreted by the interaction of paramagnetic erythrocytes with the inhomogeneous magnetic field, the resistance force (Stokes Law) from the bulk water, and the hydrodynamic interaction between erythrocytes.

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.

The effect of albumin on the immunoglobulin G (IgG)-induced and fibrinogen-induced aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. (i) As albumin concentration in the medium was increased, the IgG-induced erythrocyte aggregation was inhibited, while the fibrinogen-induced erythrocyte aggregation was accelerated (albumin itself was not able to aggregate erythrocytes). These relations were empirically expressed by the equations, v = aG 1.8 /A and v = a′F 1.5 (A + b′) , respectively ( v , the velocity of erythrocyte aggregation; A, G and F , the concentrations of albumin, IgG and fibrinogen, respectively; a, a′ and b′ , constant). (ii) The IgG-induced erythrocyte aggregation was remarkably inhibited by the addition of poly(glutamic acid), but the fibrinogen-induced erythrocyte aggregation was not. (iii) A mechanism for the interaction of immunoglobulin G and fibrinogen with the surface of erythrocytes was proposed.

The effect of two cationic drugs (chlorpromazine and isoxsuprine) on the suspension viscosity of human erythrocytes were examined, comparing with the effect of anionic drugs. (1) As increasing the drug concentrations, the cationic drugs transformed the erythrocytes to stomatocytes, then to spherostomatocytes, while trinitrobenzene sulfonate, dehydroepiandrosterone sulfate and lysolecithin induced echinocytes, as well known. (2) The suspension viscosity decreased in parallel with the appearance of spherostomatocytes, but it increased in echinocytosis. (3) The membrane fluidity, measured by spin label method, was not a major determinant for the suspension viscosity in these cases, because of no systematic correlation. (4) The rheoscopic observation under shear force demonstrated that the spherostomatocytes deformed easily to ellipsoid with smooth cell surface, while the echinocytes less easily deformed to ellipsoid on which the small spikes persisted at higher shear. These distinct difference in deformed shape under high shear force could be related to the decreased suspension viscosity of spherostomatocytes. (5) In addition, the transformation to spherostomatocytes, thus the decreased viscosity, was primarily determined by the intramembraneous drug concentration.

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.

The effect of alpha-tocopherol on the rheological properties of rat red cells was studied. The suspension viscosity of red cells increased, as the alpha-tocopherol content in red cells decreased. The red cells gradually transformed to echinocytes in isotonic solution dependent on the alpha-tocopherol content. The unique correlations among the viscosity, the transformation, and alpha-tocopherol content were observed. It was shown that the increased suspension viscosity of alpha-tocopherol-deficient cells could have arisen from the difficulty of echinocytes to undergo high shear deformation. The possible mechanism is discussed.

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 (ν, in μm2/min) by the concentration of fibrinogen ( F , in g/dl) and the sialic acid content ( S , in μmol/ml red blood cells), log ν = −0.065 F −1.2 S + 2.2 F 0.35 , 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.

Oxygen transport to tissues depends on (i) the oxygen transporting capacity of blood (mainly the hemoglobin content and the functional properties of hemoglobin regulated by various factors such as H+, CO2, 2,3-diphosphoglycerate and temperature) and (ii) the blood flow to tissue. Blood viscosity is one of the important factors affecting blood flow, and is influenced by erythrocyte deformation in the high shear regions and erythrocyte aggregation in the low shear regions (Chien, 1975).

Erythrocyte aggregation is induced in blood flowing at low shear rates, especially in the stasis of blood, by macromolecular bridging between adjacent erythrocytes [1]. The process of erythrocyte aggregation is reversible; aggregates disintegrate at high shear rates. Since the facilitation of erythrocyte aggregation reduces the blood flow, the kinetic analysis of erythrocyte aggregation is very important for understanding the control of blood flow, rheologically. This paper describes a method for measuring the velocity of erythrocyte aggregation and the effect of immunoglobulin G and related macromolecules on the velocity.

Human erythrocytes, untreated and glutaraldehyde-treated, labeled with three kinds of fatty acid labels, and their electron spin resonance (ESR) spectra were studied in detail at various temperatures. 1. 1. The better spectral resolution could be obtained by packing the erythrocytes in a hematocrit capillary tube, because of the preferential parallel orientation of the cylindrical axes of erythrocyte-disc to the centrifugal axis. 2. 2. It was demonstrated by the incorporation and the release of the labels and the membrane possessed two kinds of the fatty acid “sites”: the tightly and weakly binding “sites” at the approximate molar ratio of 1 : 1. The rough estimates of the binding constants were obtained, which reproducibly varied with the blood donors over a period of a year. 3. 3. The temperature dependency of the ESR spectra revealed the presence of two distinct phases, perhaps the solid and fluid phases. With lowering of the temperature, the fluid phase became more solid but the solid phase unchanged. The pretreatment of the erythrocytes with glutaraldehyde increased the amount of the frozen phase, corresponding to the decrease of the membrane flexibility.

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.

The aggregation (especially the 'rouleau' formation) of human erythrocytes induced by polysaccharide and polyglutamic acid was quantitatively examined by using a low-shear rheoscope combined with a television image analyzer and a computer. (1) The morphological characteristics of rouleaux induced by these macromolecules are presented. (2) Polysaccharides with high molecular weights of 70 400 and 494 000 and poly(glutamic acids) with weights of 50 000 and 66 000 formed the rouleaux (then the three-dimensional aggregates). But polysaccharides with the low molecular weights of 10 300 and 42 500 and poly(glutamic acids) with weights of 8000 and 20 000 did not. The dependences of the velocity of rouleau formation on the macromolecule concentration and on the shear rate are shown. (3) The erythrocyte aggregation induced by high-molecular-weight polysaccharides was inhibited by low-molecular-weight polysaccharides and glucose, but was not affected by low-molecular-weight poly(glutamic acids). (4) The aggregation induced by high-molecular-weight poly(glutamic acids) was inhibited by poly(glutamic acid) with a molecular weight of 8000, but was accelerated by that of 20 000. The poly(glutamic acid)-induced aggregation was not affected by low-molecular-weight polysaccharides. (5) The stereochemical structure-dependent interaction (or the mode of bridging) of macromolecules with erythrocytes was stressed for the mechanism of erythrocyte aggregation.

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis (N,N- dimethylamide )) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking (‘% crosslinking’). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76–752 s−1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.