Theory of non-Newtonian viscosity of red blood cell suspension: effect of red cell deformation

The effects of the deformation of red blood cells on non-Newtonian viscosity of a concentrated red cell suspension are investigated theoretically. To simplify the problem an elastic spherical shell filled with an incompressible Newtonian fluid is considered as a model of a normal red cell. The equation of the surface of the shell suspended in a steady simple shear flow is calculated on the assumption that the deformation from a spherical shape is very small. The relative viscosity of a concentrated suspension of such particles is obtained based on the "free surface cell" method proposed by Happel. It is shown that the relative viscosity decreases as the shear rate increases.     

Ontogenic evolution of chicken red cell Fc receptor

The ontogenic evolution of chicken red cell Fc receptor was studied in red cells from different age chicken embryos, baby chicken, and adult chicken. The Fc receptor binding capacity for ligands, the number of Fc receptors by red cell, and the association constant between receptor and ligand were analyzed. The Fc receptor is expressed in the red cell surface of 6-day chicken embryo and its binding capacity for ligand—minimal at this moment—is increased in the 8-day chicken embryo red cells. The 12-day chicken embryo erythrocytes binding capacity is similar to the adult chicken red cells. The number of Fc receptors by red cell increase with the age of chicken embryo. After 9 days this number is not modified and it is the same as in adult chicken. Variations of Ko and binding capacity for ligands show a similar evolution in embryogenic development. From these data we suggest that although on Day 9 the number of receptors per cell is the same as in adult chicken, the receptors are not completely exposed at this time and as a consequence, their binding capacity for ligands is lower than in adult chicken erythrocytes.     

Iso-antibodies to red cell antigens in pigs' sera 3. The effect of maternal red cell iso-antibodies on the red cells of piglets

The effect of the red cell antibody, anti-Ea, on the red cells of piglets in four litters was studied. Although the antibody was absorbed from the colostrum by all the piglets, it had little effect on their packed cell volume, haemoglobin and red cell counts, and no differences were noted between Ea-positive and Ea-negative piglets in the same litter, despite the fact that red cells of the former were positive for the direct Coomb's test for up to a week after birth.
Anti-Ea was not detected in the serum of Ea-positive piglets, all of it apparently being taken up by their red cells, unlike the Ea-negative ones, in whose serum anti-Ea could be detected for up to fifteen weeks of age.     

Activity rhythms of enzymes in human red blood cell suspensions

Abstract

We have established the presence of a rhythm in the activity of 4 enzymes in in‐vitro cell suspensions of human red blood cells. Glucose 6‐phosphate dehydrogenase and glutamate oxaloacetate transaminase demonstrated semicircadian patterns of activity, while acid phosphatese and acetylcholine esterase exhibited circadian activity rhythms. The ratios between the highest to lowest activities varied from 2:1 to 10:1 among the various enzymes. The affinity of glucose 6 phosphate dehydrogenase to its substrate and coenzyme remained constant throughout the cycle. No evidence was obtained for the presence of a soluble inhibitor at the lower levels of the activity. Sonication of hemolysates with low glucose 6 phosphate dehydrogense activity yielded additional activity comparable to that of the peak activity. Sonication of hemolysates from the time of the peak activity did not change the original activity. The observations point to a role of the cell membrane in the biological clock.     

Haemoglobin and the red cell membrane.

The results presented here indicate that haemoglobin is an integral part of the red cell membrane. The haemoglobin content of the membrane is highly dependent on the Ca++ content of the membrane in health and disease. Changes in the red cell interior alter the whole organization of the membrane and are even reflected in the binding of immunoglobulins to the red cell surface. The preferential binding of Hb-s A2 and S to the membrane has been confirmed. This phenomenon cannot be explained by differences in the charge between these haemoglobins and Hb A.     

Fibrinogen inhibits red cell adhesion to glass

Studies of human erythrocyte adhesion to glass have demonstrated consistently greater adhesion with serum-containing media than with a comparable concentration of plasma. This serum-plasma difference is explained by the adhesion-inhibiting property of plasma fibrinogen. The fibrinogen effect is probably mediated through its firm binding to glass, since no adsorption onto the red cell surface could be demonstrated. The ability of more red cells to adhere to a foreign surface after plasma coagulation (the formation of serum from plasma) may be significant in the red cell surface interactions necessary for the formation of a fibrin-red cell thrombus.     

Biochemistry of red cell invasion

We are still far from an explicit understanding of the events that characterize the invasion of red cells by malarial parasites at the structural and biochemical levels. The nature of the interaction between the attached parasite and the host cell; the origin of the parasitophorous vacuole; the identity, disposition, and arrangement of the propulsive proteins of the parasite; the functions of the rhoptry organelles; and the rearrangement of the host cell membrane to permit entry of the parasite remain to be elucidated. A hypothetical scheme is presented that pursues the processes involved in invasion from the biochemical events generated by attachment of the parasite, to the steric rearrangement of red cell membrane proteins, which culminates in invasion.     

Further investigations of red cell deformability with nickel mesh

Although the filtration method has been widely employed in red cell deformability studies, the structural irregularity of the pores of a Nuclepore polycarbonate membrane has always been a major problem. Anegawa, T. et al. (Clin. Hemorheol., 7, 1987) obtained a higher reproducibility with the filtration method using a newly designed thin metal film with pores engraved by the photofabrication technique. We further studied the pressure - flow rate relationship of red cell suspension employing this nickel mesh. The filtration of red cell suspensions through the nickel mesh was not influenced by leukocytes contamination or added leukocytes up to a leukocyte count of 250 cells/mm3 within an experimental limitation. On the other hand, the flow was greatly influenced by leukocytes contamination when the polycarbonate membrane was used. The nickel mesh was found to be useful in detecting major determinants of red cell deformability, such as cell geometry and internal cellular viscosity, and in detecting abnormalities of red cell deformability in a patient with microangiopathic hemolytic anemia. In conclusion, the present study clearly shows that the nickel mesh is preferable for investigating red cell deformability to the polycarbonate membrane from a quantitative point of view. This material should contribute to the physiologic and clinical investigation of red cell deformability.     

Interaction between phloretin and the red blood cell membrane

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane.     

Role of hydrogen bonding in red cell aggregation.

The role of hydrogen bonding in red cell aggregation induced by dextran was studied with the use of urea, an inhibitor for hydrogen bonding. In order to avoid hemolysis of red cells by the high concentration of urea, the studies were performed on human red cells hardened in glutaraldehyde. The degree of red cell aggregation at Hct = 45% was estimated by the use of a coaxial cylinder viscometer. The viscometric aggregation index (VAI) was calculated from viscosity values at shear rates of 52 sec-1 (eta H) and 0.05 sec-1 (eta L); VAI = (eta L - eta H)/eta H. Red cells with surface charge intact and with charge removal by neuraminidase treatment were studied. Urea at high concentrations, e.g., 6 M, significantly inhibited red cell aggregation induced by dextran. These findings indicate that hydrogen bonding plays an important role in dextran-induced red cell aggregation. An understanding of the nature of the forces involved in red cell aggregation serves to establish the physicochemical principles of cell-to-cell interactions induced by macromolecules.     

Regulation of red cell acetylcholinesterase activity in diabetes mellitus

Despite the fact that the significance of red cell membrane acetylcholinesterase (AChE) is unknown, this enzyme of red cell assumes importance since many of its properties have been found to be similar to purified enzyme form of brain tissues. Our investigations on the effect of insulin-dependent diabetes mellitus on red cell AChE revealed that the activity of this enzyme is significantly decreased in diabetes. Insulin treatment restored the activity to the normal level. Solubilization of normal, diabetic and insulin treated diabetic red cell membranes with Triton X-100 (0.2% v/v) caused a general decline in AChE activity, however the per cent decline in activity of diabetic enzyme was lower as compared to normal and insulin treated conditions. From our results it is inferred that the decreased red cell AChE activity in diabetes is due to lesser number of active enzyme molecules and also due to altered membrane microenvironment.     

Developments in red cell rheology at the Institut de Pathologie Cellulaire

The present day rheological approximation, which has been used successfully to quantitate the deformability properties of red cells, is based on the view that the cell has a liquid interior encapsulated by a viscoelastic solid membrane shell. A review of historical developments in this field shows that determination of intrinsic red cell membrane properties has not come from simple mathematical analysis of experiments. On the contrary, considerable insight has been required to bring together physical and biological methods to rationalize the unique deformability characteristics of the red blood cell. Key developments at the Institut de Pathologie Cellulaire (IPC) in the early 1970s played a role in our improved understanding of red cell rheology. In this article, we describe the material concepts of the red cell membrane held before 1970, discuss the seminal developments at Bicetre, and, finally, outline the contemporary view of red cell deformability.     

Malaria, quinine and red cell lysis.

An hypothesis is presented to explain the red cell lysis which accompanies an acute malarial infection, as well as the mode of action of certain schizonticidal drugs in the quinoline and acridine series. Quinine and a number of other antimalarial drugs have been found to counteract the inhibition by protein of fatty acid-induced lysis, when tested in an in vitro system. It is suggested that these schizonticides exert their chemotherapeutic effect by inducing the premature lysis of the parasitized red cell, as a result of relieving the inhibition by protein of haemolysis.     

Modelling the structure of the red cell membrane

The red cell membrane has long been the focus of extensive study. The macromolecules embedded within the membrane carry the blood group antigens and perform many functions including the vital task of gas exchange. Links between the intramembrane macromolecules and the underlying cytoskeleton stabilize the biconcave morphology of the red cell and allow deformation during microvascular transit. Much is now known about the proteins of the red cell membrane and how they are organised. In many cases we have an understanding of which proteins are expressed, the number of each protein per cell, their oligomeric state(s), and how they are collected in large multi-protein complexes. However, our typical view of these structures is as cartoon shapes in schematic figures. In this study we have combined knowledge of the red cell membrane with a wealth of protein structure data from crystallography, NMR, and homology modelling to generate the first, tentative models of the complexes which link the membrane to the cytoskeleton. Measurement of the size of these complexes and comparison with known cytoskeletal distance parameters suggests the idea of interaction between the membrane complexes, which may have profound implications for understanding red cell function and deformation.     

Preservation of resuspended red cell concentrate. Release of vesicles from stored red cells

The effect of carbohydrates (sucrose, mannitol) and guanosine on red cell vesiculation was studied during storage of red cell concentrates (RCC) in glass bottles and plastic bags for 35 days. The course of vesicle release was followed by measuring acetylcholinesterase activity. It was found that sucrose and mannitol reduce the loss of membrane microvesicles. Preservation of red blood cells (RBC) in plastic bags results in a drastically retarded vesicle release.     

Human red cell acid phosphatase polymorphism

Red cell acid phosphatase phenotypes were determined in 401 unrelated persons from Southwestern Germany. The frequencies of genes P^a, P^b and P^c were estimated to be p=0.328, q=0.630 and r=0.042.Experiences in 101 cases of disputed paternity with 151 men involved are reported. 22 men could be excluded from paternity on the basis of red cell acid phosphatase phenotypes.In all 140 mother-child-combinations tested the distribution of red cell acid phosphatase variants was compatible with the genetic model suggested by Hopkinson et al.Usefulness of the system in forensic cases of disputed paternity is discussed.Data contained in this paper will also constitute part of the thesis of cand. med. Karl-Henning Lichte.     

NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions

NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions. Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms. The relaxation time of a red cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water. Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time. Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population. Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells.     

Changes in transferrin during the red cell replacement in amphibia

Transferrin, a plasma glycoprotein, carries iron from storage sites to immature erythroid cells for hemoglobin synthesis. The replacement of larval red cells by adult red cells, which occurs during metamorphosis in bullfrogs, requires extensive formation of hemoglobin and new red cells. Large changes in red cell iron storage also occur during the red cell replacement. Both the concentration and the level of iron saturation of plasma transferrin were measured during metamorphosis to determine if there were changes in plasma transferrin which coincided with the changes in red cell iron storage and ferritin content. Plasma transferrin concentrations increased from 0.96 to 2.6 mg/ml during the period when red cell storage iron and ferritin decreased. Plasma iron concentrations also increased when the transferrin concentration increased, suggesting that the additional transferrin may be involved in moving iron from the larval red cell stores. At the end of metamorphosis, the plasma iron concentration decreased to premetamorphic levels but the transferrin concentration remained high, resulting in a decrease in saturation to 18% compared to 45% in the larvae. In addition to differences in iron saturation, adult transferrin had different electrophoretic properties from larval transferrin. The results support the hypotheses that during early ontogeny plasma transferrin and red cell iron storage are coordinated to provide iron for the formation of the first generation of adult red cells and that transferrin may participate in the control of red cell ferritin synthesis.     

Molecular basis for red cell membrane viscoelastic properties.

An unusual combination of membrane properties allows the red cell to undergo extensive deformation without cell fragmentation, enabling it to effectively perform its function of oxygen delivery during its long life span in the circulation. These material properties are the consequence of a composite structure in which a plasma membrane envelope made up of amphiphilic surfactant molecules is anchored to a network of skeletal proteins through tethering sites (transmembrane proteins) in the bilayer. Explosive growth in our understanding of the primary structure of the various red cell membrane proteins, definition of specific mutations in various red phenotypes, and detailed biophysical characterization of membrane properties of normal and mutant red cells has enabled development of models of molecular and structural basis for red cell properties.