Protein Adsorption on Erythrocytic Membranes and Its Effect on Erythrocyte Rheology in Athletes during Competition Exercise

The adsorption of high-molecular-weight plasma proteins on erythrocyte membranes was studied in athletes after prolonged exercise under competition conditions. The adsorption of individual high-molecular-weight protein fractions depended on their concentration. The adsorption index changed biphasically at submaximum exercise. The adsorption of plasma high-molecular-weight protein fractions was associated with the fluidity of concentrated erythrocyte suspensions. The adsorbed high-molecular-weight globulins and fibrinogen had different effects on the parameters of erythrocyte rheology.     

Measurements of viscosity of synthetic erythrocyte suspensions

Measurements were made of the viscosity of suspensions of synthetic erythrocytes composed of hemoglobin solutions encapsulated in liposomes, as a function of shear rate, temperature, suspension concentration, lipid membrane composition, and the viscosity of the suspending medium. It was found that the viscous behavior of the synthetic erythrocyte suspensions was non-Newtonian and nearly the same as that of suspensions of natural erythrocytes prepared similarly, with the major difference being that synthetic erythrocyte suspensions are somewhat more viscous. Suspensions of Fluosol FC-43 prepared similarly were found to be essentially Newtonian fluids, and substantially different and more viscous than either erythrocyte suspension. The higher viscosity of synthetic erythrocyte suspensions probably accounts for the ability of these suspensions to maintain normal systemic vascular resistance in transfusion experiments, in spite of the fact that synthetic erythrocytes are smaller than natural erythrocytes.     

Application of some droplet sedimentation theories to layered erythrocyte suspensions

The applicability of some existing droplet sedimentation theories to erythrocyte suspensions was investigated using glutaraldehyde-fixed erythrocytes from horse, canine, pig, chicken, and human. The Svensson criteria were shown to underestimate the load supportable by a density gradient. The available theories on droplet formation times could not predict each other, and the data on erythrocyte suspensions, especially at high suspension concentrations. It was finally argued that, since the behavior of erythrocytes was not controlled by the diffusion process, the erythrocyte suspensions were not expected to exhibit lasting or absolute stability even when the particle load was small.     

Application of some droplet sedimentation theories to layered erythrocyte suspensions

The applicability of some existing droplet sedimentation theories to erythrocyte suspensions was investigated using glutaraldehydefixed erthrocytes from horse, canine, pig, chicken, and human. The Svensson criteria were shown to underestimate the load supportable by a density gradient. The available theories on droplet formation times could not predict each other, and the data on erythrocyte suspensions, especially at high suspension concentrations. It was finally argued that, since the behavior of erythrocytes was not controlled by the diffusion process, the erythrocyte suspensions were not expected to exhibit lasting or absolute stability even when the particle load was small.     

Effects of hemoglobin and cell membrane lipids on pulmonary surfactant activity

These experiments characterize the effects of hemoglobin and erythrocyte membrane lipids on the dynamic surface activity and adsorption facility of whole lung surfactant (LS) and a calf lung surfactant extract (CLSE) used clinically in surfactant replacement therapy for the neonatal respiratory distress syndrome (RDS). The results show that, at concentrations from 25 to 200 mg/ml, hemoglobin (Hb) increased the minimum dynamic surface tension of LS or CLSE mixtures (0.5 and 1.0 mumol/ml) from less than 1 to 25 dyn/cm on an oscillating bubble apparatus at 37 degrees C. Similarly, erythrocyte membrane lipids (0.5-3 mumol/ml) also prevented LS and CLSE suspensions (0.5-2.0 mumol/ml) from lowering surface tension below 19 dyn/cm under dynamic compression on the bubble. Surface pressure-time adsorption isotherms for LS suspensions (0.084 and 0.168 mumol phospholipid/ml) were also adversely affected by Hb (0.3-2.5 mg/ml), having a slower adsorption rate and magnitude. Significantly, these inhibitory effects of Hb and membrane lipids could be abolished if LS and CLSE concentrations were raised to high levels. In complementary physiological experiments, instillation of Hb, membrane lipids, or albumin into excised rat lungs was shown to cause a decrease in pressure-volume compliance. This decreased compliance was most prominent in lungs made partially surfactant deficient before inhibitor delivery and could be reversed by supplementation with active exogenous surfactant. Taken together, these data show that molecular components in hemorrhagic pulmonary edema can biophysically inactivate endogenous LS and adversely affect lung mechanics. Moreover, exogenous surfactant replacement can reverse this process even in the continued presence of inhibitor molecules and thus has potential utility in therapy for adult as well as neonatal RDS.     

In vitro effects of thyroxine on the mechanical properties of erythrocytes

In vitro effects of thyroxine on erythrocyte deformability and mechanical fragility were observed. Deformability of erythrocytes was improved in a dose dependent manner by thyroxine. Mechanical hemolysis was found to be lower if thyroxine was included in erythrocyte suspensions at concentrations close to the physiological levels (10(-9)M). These changes might be related to the alterations of intracellular calcium concentration, as in the erythrocyte suspensions containing 10(-9)M thyroxine, intracellular calcium concentration was found to be 30 times lower than the control suspensions which did not contain thyroxine. Thyroxine also reduced the mechanical hemolysis ratio in calcium loaded cells. These observations suggest that thyroxine might play some role in the regulation of the mechanical properties of erythrocytes which might be mediated via the effects on calcium metabolism.     

The pressure-flow relation in resting rat skeletal muscle perfused with pure erythrocyte suspensions

In spite of numerous investigations of erythrocyte rheology, there is limited information about the influence of erythrocyte suspensions on whole organ pressure-flow relationships. In this study, we present whole organ pressure-flow curves for resting vasodilated gracilis muscle of the rat, in which the microanatomy and vessel properties have been determined previously. For pure erythrocyte suspensions from donor rats, the organ resistance increases only mildly with perfusion time (less than a 5% shift over a one-hour perfusion time), while in contrast, erythrocyte suspensions containing leukocytes show an increases of resistance near 100% over a period of 25 min. Variation in pressure-flow curves in the muscle at the same arterial hematocrit between different rats is less than 15%. The pressure-flow relation for pure erythrocyte suspensions depends on hematocrit. Shear thinning is exhibited at high hematocrits, while Newtonian behavior is approached at arterial hematocrits below 15%. The whole organ apparent viscosity for pure erythrocyte suspensions (normalized by cell-free plasma resistance) is a non-linear function of hematocrit; at physiological pressures, it reaches values comparable to those of apparent viscosities measured in rotational viscometers or in in vitro tube flow (diameters greater than 0.8 mm). The apparent viscosities estimated from the whole organ experiments tend to be higher than those measured in straight tubes under in vitro conditions. The pressure-flow curves for pure erythrocyte suspensions are shifted towards lower pressures than the curves for mixed suspensions of erythrocytes at the same hematocrit and with leukocytes at physiological cell counts. These acute experiments show that pure erythrocyte suspensions yield highly reproducible resistances in the skeletal muscle microcirculation with dilated arterioles. Relative apparent viscosities measured in vivo are higher than those measured in straight glass tubes of comparable dimesions.     

Role of erythrocyte deformability during capillary wetting

Deformability of erythrocyte was found to fundamentally alter the wetting dynamics of red blood cell (RBC) suspensions during their invasion into capillaries. Normal RBC suspensions failed to penetrate more than 1 cm into a glass capillary when the capillary radius was smaller than a critical value that is dependent on the erythrocyte concentration (about 50 microm for whole blood). In contrast, suspensions of rigidified RBCs, after cross-linking with different concentrations of glutaraldehyde or incubating with 100 ng/mL of an endotoxin, could penetrate any capillary larger than the erythrocyte dimension. The effect of RBC deformability on penetration was attributed to the enhanced shear-induced migration of normal deformable RBCs toward the capillary centreline, which imparted a higher average velocity to the RBCs than the average plasma velocity. As a result, the erythrocytes advanced into the capillary faster than the wetting meniscus, packing behind it to form a concentrated slug. This tightly packed slug had a high hydrodynamic resistance that could arrest the penetrating flow of concentrated suspensions into the small capillaries.     

Aggregation of red blood cells studied by ultrasound backscattering

The erythrocyte aggregation phenomenon is an important factor in capillary circulation. This phenomenon can be evaluated by a number of methods (microscopic observations, viscometry, light measurements) which cannot be applied simply to in vivo measurements. In contrast, ultrasound which propagates through soft tissues allows measurement of the mechanical properties of red blood cell (RBC) suspensions which depend on the aggregation phenomenon. We devised an apparatus in order to measure in vitro the ultrasonic backscattering intensity of RBC suspensions. First, with latex particles of different sizes, the ultrasonic backscattering coefficient has been measured in order to evaluate the apparatus response. Then, the ultrasonic backscattering coefficient of different aggregated erythrocyte suspensions has been measured and correlated with the erythrocyte sedimentation rate. Finally, the size of RBC aggregates of different suspensions has been evaluated.     

Amperometric measurements of nitric oxide in erythrocytes

In the recent years, there has been an increase in the development of new biosensors that could be helpful in the study of various physiological processes. In this study, we report the development of a new in vitro experimental design for real-time nitric oxide (NO) amperometric measurements in erythrocyte suspensions. To achieve this, we employed human erythrocyte suspensions in sodium chloride 0.9%, pH 7 (haematocrit 0.05%). The production of NO by erythrocytes was measured with a commercial NO sensor during stimulation by L-arginine, acetylcholine, choline, atropine and velnacrine maleate (10 microM of final concentrations). We also measured the nitrite and nitrate concentrations produced by erythrocyte suspensions stimulated with the above effectors by means of the Griess reaction method. We observed that there was a direct relation between the electric current produced by the NO sensor, and the NO standard concentrations, thereby leading to a good calibration curve. The in vitro erythrocytes produced significant amperometric NO values in response to a wide range of effectors and these results have the same variation profile of the nitrites and nitrates results achieved with the Griess method. In conclusion, the amperometric NO sensor constitutes a reliable method for direct, and real-time measurement in vitro of the NO production of erythrocyte suspensions, As such, it offers a potential diagnostic technique for the evaluation of diseases, and the therapeutic progression of diseases, related to intracellular NO metabolism.     

Determination of the content of nonfilterable cells in erythrocyte suspensions as a function of the medium osmolality

The results of most filtration assays for deformability of erythrocytes do not distinguish whether the entire population or only its small fraction exhibits abnormal rheological properties. We developed a simple filtration method for determination of the percentage of nonfilterable cells in erythrocyte suspension using membrane filters with mean pore diameter of 3.1 microns. This method makes it possible to detect even minor abnormal subpopulations in erythrocyte suspensions. The flow rate of buffer depends on the number of free pores of a filter. The plot of the number of pores clogged by nonfilterable cells vs the total number of erythrocytes that were allowed to pass through the filter had a linear portion, with a slope representing the relative content, Z%, of nonfilterable cells in the suspension. We determined Z% for various medium osmolalities u and used the data to derive the distribution of erythrocytes in ucr (ucr is the maximum value of u at which an erythrocyte cannot pass through a pore of a given filter because of geometric limitations). The distribution of ucr in suspension of normal erythrocytes has a maximum of about 200 mOsm/kg and a half-width of about 20 mOsm/kg. The distributions of ucr are altered in normal erythrocyte suspensions at decreased pH values, in cryopreserved and ATP-depleted erythrocyte suspensions and in erythrocytes from a xerocytosis patient.     

Theory of sound attenuation in the blood and erythrocyte suspensions]

Mechanisms of the ultrasound attenuation in blood and erythrocyte suspension in the long wave range are examined. It is shown that the theory proposed for dilute suspension of structured microobjects has a good coincidence with the known experimental data, both on erythrocyte suspensions and on blood. The contribution of viscous losses to attenuation are decreased with frequency and reach 44% in water suspensions of erythrocytes and 24% in the whole blood at 1 MHz.     

Metabolism of leukotriene A4 by human erythrocytes. A novel cellular source of leukotriene B4

Human erythrocytes transformed leukotriene A4 into leukotriene B4. Metabolism was proportional to the erythrocyte concentration, even at subphysiological levels (0.08-4 X 10(9) erythrocytes/ml). Comparative metabolic studies excluded the possibility that leukotriene B4 originated from trace amounts of polymorphonuclear leukocytes or platelets present in the purified erythrocyte suspensions. For example, suspensions of isolated platelets (100-500 X 10(6) cells/ml) failed to convert leukotriene A4 into leukotriene B4; and conversion by suspensions of isolated polymorphonuclear neutrophils was insufficient to account for the amounts of leukotriene B4 formed by erythrocytes. Leukotriene B4 formation was maximal within 2 min and substrate concentration dependent. Enzymatic activity originated from a 56 degrees C labile nondialyzable (Mr greater than 30,000) soluble component in the 100,000 X g supernatant obtained from lysed erythrocytes. In contrast to the contemporary view, our results indicate that human erythrocytes are not metabolically inert in terms of eicosanoid biosynthesis. The role of human erythrocytes during inflammatory or pulmonary disorders deserves re-examination in this context.     

Standardized Viral Hemagglutination and Hemagglutination-Inhibition Tests. I. Standardization of Erythrocyte Suspensions

A spectrophotometric procedure for standardizing red blood cell suspensions for use in viral hemagglutination tests is described. The procedure is based on highly reproducible cyanmethemoglobin absorbance readings at 540 nm on any suitable spectrophotometer. Target values for milligrams of cyanmethemoglobin per 100 ml are given for the red cell suspensions of all mammalian and avian species employed in viral hemagglutination tests. By use of these values and a cyanmethemoglobin standard curve for a particular photometer, approximate 4% erythrocyte suspensions can be diluted to any lesser concentration. Coefficients of variation for the various diluted suspensions range from 4 to 7%.     

Ghost cells as sorption matrix for virus concentration from water

Pig erythrocyte membranes were used as adsorbent material for the concentration of bacteriophage phi x-174, MS-2, and f2 from 5 ml of saline solution. The adsorption was carried out at pH 3.5, and the elution was carried out at pH 7.0. Compared with adsorption on 3% beef extract, bacteriophage adsorption on erythrocyte ghost cells yielded 93 to 100%, and elution was 92 to 100%, whereas beef extract organic flocculation yielded adsorption of 0 to 98.8% and elution of 61 to 86.6%. The same procedure but with poliovirus LSC-1 gave 100% adsorption and 91 to 129% elution.     

Ghost cells as sorption matrix for virus concentration from water.

Pig erythrocyte membranes were used as adsorbent material for the concentration of bacteriophage phi x-174, MS-2, and f2 from 5 ml of saline solution. The adsorption was carried out at pH 3.5, and the elution was carried out at pH 7.0. Compared with adsorption on 3% beef extract, bacteriophage adsorption on erythrocyte ghost cells yielded 93 to 100%, and elution was 92 to 100%, whereas beef extract organic flocculation yielded adsorption of 0 to 98.8% and elution of 61 to 86.6%. The same procedure but with poliovirus LSC-1 gave 100% adsorption and 91 to 129% elution.     

Rosette formation of concanavalin A-treated erythrocytes around polymorphonuclear leucocytes and lymphocytes.

Concanavalin A (Con A) induces rosette formation of erythrocytes around polymorphonuclear leucocytes and lymphocytes in cell suspensions of autologous human blood cells. The effect which is most characteristic in a concentration between 25 and 50 microgram/ml is due to Con A bound on the erythrocyte membrane. A similar effect, although less pronounced, was observed with phytohaemagglutinin at concentrations of 10 and 25 microgram/ml. The treated erythrocytes showed a higher affinity to polymorphonuclears when compared with lymphocytes. At the contact area, the membrane of the erythrocyte became highly folded while its free surface was smooth and spherical. The effect of the local concentration and immunobilization of the lectin on the erythrocyte membrane and the similarity of the contact pattern to that of erythrophagocytosis are discussed.     

Results of a trial of a plague monoclonal antibody erythrocyte diagnostic agent

Plague antibody monoclonal erythrocyte diagnosticum was studied in serological tests simultaneously with commercial plague antibody erythrocyte diagnosticum prepared on the basis of hyperimmune horse serum and with commercial plague antigenic erythrocyte diagnosticum. In this investigation the suspensions of numerous strains of Yersinia pestis, other closely related and heterologous organisms, experimentally infected wild and laboratory animals, as well as samples of materials obtained from small rodents caught in several natural foci of plague, were studied. The monoclonal diagnosticum was, practically, not inferior to the similar commercial preparation with respect to the frequency of positive results and the activity of the materials under study in serological tests, but showed greater specificity, as it reacted strictly with Y. pestis capsular antigen.     

In vitro kinetics of oxygen transport in erythrocyte suspension or unmodified hemoglobin solution from human and other animals

Oxygen transport behavior in erythrocyte suspension or in hemoglobin solution was studied as a potential therapeutic model for the clinical treatment of blood loss, and this can also provide physiological data with which to evaluate blood substitutes. In the present project, we examined the in vitro kinetics of hemoglobin binding to and releasing oxygen, to provide detailed oxygen-flux measurements for unmodified hemoglobin solutions and erythrocyte suspensions in human, as well as other vertebrates. An in vitro method was used, based on a widely used artificial system, with the oxygen saturation level being detected in real time. Results from this study indicated that the kinetic curves of human erythrocyte suspensions and hemoglobin solutions were either S-shaped or hyperbolic, respectively. Based on these curves, the significance of T(50) emerged in our investigation, where T(50) is defined as the time needed for 50% hemoglobin to be saturated with oxygen, and reflects the efficiency with which hemoglobin carries oxygen. This parameter may be used to diagnose blood diseases, and could be a standard for evaluating blood substitutes. In this study, we also compared the T(50) of 4 species of vertebrates, and found that it shows a distinct efficiency of oxygen binding related to species, and potentially reveals the evolutionary function of hemoglobin and its possible adaptation to the environment.     

Interaction of erythrocytes with aminoalkylagarose

Some properties of aminoalkyl-agarose as a carrier for adsorption immobilization of erythrocytes were being studied. The effect of the hydrophobic spacer on the erythrocyte affinity for the matrix is shown. The erythrocyte adsorption is characterized by a positive cooperativity; the shorter the spacer, the higher is cooperativity.