Suspensions Of Human Erythrocytes Research Articles

Rheooptical transients in sheared suspensions: III. Effects of Brownian motion on oblate spheroids

The transient rheooptical response of a dilute suspension of oblate spheroids whose orientations are influenced by shear and Brownian torques was studied theoretically and experimentally using hardened human erythrocyte and kaolin suspensions. The results are given as a function of Br r which is a dimensionless measure of the ratio of shear rate to the rotary Brownian coefficient acting on the particles. As Br r decreases the damping of oscillations of the optical scattering cross section and hence the turbidity of the suspensions increases. Factors influencing this damping, as well as shifts in the apparent mean frequency of the transient oscillations, are analyzed in light of the existing theory.

Studies on the toxicity of chlorinated p-nitrobiphenyl ether : I—Methemoglobin formation in vitro and in vivo induced by nitroso and amino derivatives of chlorinated biphenyl ether

Methemoglobin formation by nitroso and amino derivatives of chlorinated biphenyl ether was investigated. All the nitroso derivatives used induced methemoglubin formation in suspensions of human erythrocytes. Ability to induce methemoglobin formation among the nitroso derivatives was the highest for nitrosobenzene, followed by p-nitrosobiphenyl ether, 4-chloro p-nitrosobiphenyl ether, 2,4-dichloro p-nitrosobiphenyl ether and then 2,4,6-trichloro p-nitrosobiphenyl ether. The monochloro p-nitrosobiphenyl ethers showed all the same activity. Therefore, the ability to induced methemoglobin formation depended on the number of chlorines rather than on the position of chlorine substitution on the phenoxy group in the molecule. Since lactate decreased but glucose and xylitol increased the methemoglobin level produced by the nitroso compounds, this reaction would appear to require an NADPH-dependent enzyme system in erythrocytes. All of the amino derivatives used induced methemoglobin formation in suspensions of human erythrocytes with rat liver homogenates. Ability to induced methemoglobin formation among the amino derivatives was the highest for p-aminobiphenyl ether, followed by 4-chloro p-aminobiphenyl ether, 2,4,-dichloro p-aminobiphenyl ether, 2,4,6-trichloro p-aminobiphenyl ether, and then aniline. Methemoglobin formation bychlorinated p-aminobiphenyl ether was examined by intraperitoneal and oral administration to rats. After i.p. injection, methemoglobinemia was induced by p-aminobiphenyl ether, aniline, and 2,4-dichloro p-aminobiphenyl ether but not by 2,4,6-trichloro p-aminobiphenyl ether over the period tested. The order of methemoglobin formation was p- aminobiphenyl ether ≧ 4- chloro p- aminobiphenyl ether > aniline ≧ 2,4- dichloro p-aminobiphenyl ether. Oral administration gave almost the same result as i.p. injection. The maximum methemoglobin levels produced by the amino derivatives were highest for 4-chloro p-aminobiphenyl ether, followed by p-aminobiphenyl ether, 2,4-dichloro p-aminobiphenyl ether, aniline, and then 2,4,6-trichloro p-aminobiphenyl ether. However, the methemoglobin level produced by 2,4,6-trichloro p-aminobiphenyl ether was very low and not significantly greater than the background level.

Limitations on the uptake of mercury vapour by human erythrocytes in a closed exposure system.

Previous in vitro studies using the exposure of suspensions of normal human erythrocytes to mercury vapour (Hg0) in Warburg vessels revealed a broad variation of the cellular Hg uptake in the presence of hydrogen peroxide and furnished evidence for an oxidation of Hg0 by the catalase-H2O2 pathway. This report describes the uptake of Hg0 in the same exposure system as it depends on haematocrit, H2O2 supplementation and exposure time. The results indicate that the uptake of vapour by the whole suspensions was virtually independent of haematocrit and of the H2O2 supplementation at high rates of H2O2 infusion, whereas at low rates it was directly proportional to the amount of peroxide added. Since the retention of mercury by the medium is negligible, the haemoglobin-related (specific) uptake is inversely proportional to the haematocrit at high rates of H2O2 infusion. These results are consistent with the conclusion that the incorporation of Hg0 by erythrocytes is controlled by two processes: transfer of vapour to the cells and enzymic oxidation. The net rate of uptake is limited by the oxidizing capacity at low, and by the vapour transfer at high H2O2 supplementation. As a practical consequence of these findings the spread of the specific uptakes reported in the literature is considerably reduced if the values observed when transfer becomes rate limiting are related to the haematocrit.

The effect of hydroxyethyl starch on the rheological properties of human erythrocyte suspensions.

The effect of hydroxyethyl starch on the rheological properties of human erythrocyte suspensions.

Effects of inhibition of RBC HCO3-/Cl- exchange on CO2 excretion and downstream pH disequilibrium in isolated rat lungs.

To determine the effects of the speed of the erythrocyte membrane chloride shift on pulmonary gas transfer, CO2 exchange and the kinetics of pH equilibration were measured with isolated rat lungs perfused with 20% suspensions of human erythrocytes. The lungs were ventilated with room air, and the inflowing perfusate blood gases were similar to those in mixed venous blood in vivo. All experiments were performed at 37 degrees C. Rates of CO2 excretion were determined by measuring the fraction of CO2 in mixed expired gas in a steady state. The time-course of extracellular pH equilibration in the effluent perfusate was measured in a downstream stopflow pH electrode apparatus. CO2 excretion was reduced by approximately 16% when the lungs were perfused with suspensions containing erythrocytes whose HCO-3/Cl- exchange rates was inhibited, compared with CO2 excretion when the lungs were perfused with normal erythrocyte suspensions. A fall of 0.06 in effluent perfusate extracellular pH was noted during perfusion with inhibited erythrocyte suspensions, in contrast to no observable downstream pH change during perfusion with normal erythrocyte suspensions. These results are in close agreement with the predictions of a theoretical model. Our observations suggest that CO2 transfer in capillary beds will be adversely affected in vivo when the rate of the erythrocyte HCO-3/Cl- exchange is abnormally low. Since a number of commonly used drugs are known to inhibit the chloride shift in human erythrocytes, these findings may have important clinical implications, especially in patients with impaired lung function.

Decreased viscosity of human erythrocyte suspension induced by chlorpromazine and isoxsuprine

The effects of two cationic drugs, chlorpromazine and isoxsuprine, on the suspension viscosity of human erythrocytes were studied. Chlorpromazine and isoxsuprine reduced the suspension viscosity, the former being more effective than the latter (i.e. a concentration of isoxsuprine about fifteen times higher than of chlorpromazine was needed to obtain similar effects). The cells transformed to stomatocytes and then to spherostomatocytes, depending on the drug concentration. The decrease in suspension viscosity paralleled the appearance of spherostomatocytes. Membrane fluidity, monitored by the motion of fatty acid spin labels, was increased by chlorpromazine at high concentrations, but was unaffected by isoxsuprine. Quantitative data of drug uptake by erythrocytes and ghosts revealed that the drugs were incorporated into both membrane and cytoplasm (partly bound to hemoglobin, as shown by equilibrium dialysis). The above drug effects appeared to depend primarily on the drug concentration in the membrane. In short, the transformation to spherostomatocytes, induced by drug incorporation into the erythrocyte membrane, was essential for the decrease in suspension viscosity.

Measurement of transmembrane pH gradients in human erythrocytes using 19F NMR

A new high-sensitivity method has been described for measuring transmembrane pH gradients in vesicular systems using 19F NMR. The 19F resonance of trifluoroethylamine has been shown to have a large pH-dependent chemical shift and the position of the resonance was measured with high precision and sensitivity. In suspensions of human erythrocytes, trifluoroethylamine distributed itself across the membrane and separate 19F resonances were obtained from the trifluoroethylamine inside and outside of the cells. The pH in each compartment was calculated from the resonance positions.

Effect of cell geometry, internal viscosity, and pH on erythrocyte filterability.

AbstractA simple filtration method has been developed for measuring the passage time of dilute erythrocyte suspensions through 2.8-um cylindrical pores relative to the passage time of the suspending buffer. This method of determining relative filtration time (RFT) is shown to be sensitive to the major determinants of overall red cell deformability by demonstration of the effects of variations in internal viscosity (intracellular hemoglobin concentration), cell geometry (volume, surface area-to-volume ratio), and membrane properties. These factors were examined by varying the buffer pH at constant osmolality and by varying the buffer osmolality at constant pH. In addition, the influence of cell volume alone was investigated by measuring RFT of suspensions of rat, rabbit, and human erythrocytes, of naturally different volumes but of the same basic biconcave shape. The results of these studies indicate that: (a) RFT is significantly increased by decreases in the surface area-to-volume ratio produced by osmot...

Identification of proteolytic activities in the cytosolic compartment of mature human erythrocytes.

Individual lysates from human erythrocyte suspensions, completely deprived of leucocytes and were assayed for a number of proteolytic activities using both naturally occurring and synthetic substrates. Removal of hemoglobin by batchwise DEAE-cellulose chromatography did not modify the complement of the various proteolytic activities which were then fractionated by means of chromatography on a column of DEAE-cellulose, followed by conventional techniques such as gel chromatography and preparative electrophoresis. This procedure allowed a number of proteinases to be identified in the erythrocyte cytosol while providing a tool for their selective though partial separation. The following peptidases were found to be present in the soluble fraction of mature human erythrocytes: (a) a neutral endopeptidase having an approximate molecular weight of 110 000; (b) three acidic endopeptidases, with pH optima between 2.5 and 3.5, showing molecular and functional properties almost identical with those of the three proteinases previously purified from solubilized erythrocyte membranes [Pontremoli et al. (1979) Biochem. J. 181, 559--568]; (c) two dipeptidylaminopeptidases whose molecular weights are around 80 000 and tentatively identified as dipeptidyl aminopeptidases II and III, respectively, on the basis of their substrate specificities and pH optima; (d) presumably two aminopeptidases, having an approximate molecular weight of 80 000 and classified as an aminopeptidase with broad substrate specificity and an aminopeptidase B, respectively. No evidence for any carboxypeptidase activity was found in the cytosolic compartment of mature human erythrocytes.

Phagocytic funnel-like pseudopodia in lectin-treated gastropod hemocytes

L. R. Renwrantz, T. Yoshino, T. Cheng, and K. Auld (Zoo/. Jb. Physiol. 83, l12, 1979) have described a phagocytic mechanism of Crassostrea virginica (Mollusca: Pelecypoda) hemocytes by which rat erythrocytes could be internalized through a single funnel-shaped pseudopodium. These structures were similar to those seen in vertebrate leukocytes (J. M. Orenstein and E. Shelton, Lab. Invest. 36, 363-374, 1977). This paper reports that hemocytes from two gastropods, Biomphalaria glabrata and Bulinus truncatus, will form funnel-like pseudopodia by which human erythrocytes may be phagocytosed. These pseudopodia may also form in the absence of erythrocyte-sized particles, but after treatment with a lectin. Certain lectin treatments may increase the rate of endocytosis in short-term experiments (D. A. Schoenberg and T. C. Cheng, Devel. Comp. Zmmunol., in press). and sealed with nail polish. In preparations requiring incubation with living human erythrocytes, a hypertonic CBSS (HS, 276 mosM) (D. A. Schoenberg and T. C. Cheng, lot. cit.) was substituted during lectin treatment. A 5 x 10’ cell/ml suspension of human erythrocytes in Dulbecco’s phosphate-buffered saline (DPBS, R. Dulbecco and M. Vogt, J. Exp. Med. 99, 167184, 1954) was added to the lectintreated hemocytes, and incubated for 30 min, rinsed, and incubated in DPBS for an additional 30 min. This was followed by fixation and staining.

PH equilibration in human erythrocyte suspensions.

A stopped-flow rapid reaction apparatus was used to study the rate of pH equilibration in human red cell suspensions. Flux of OH- or H+ was determined over a wide range of extracellular pH (4-11) in CO2-free erythrocyte suspensions. In these experiments, an erythrocyte suspension at pH 7.3 is rapidly mixed with an equal volume of NaCl solution at 3.0 greater than pH greater than 11.5. The pH of the extracellular fluid of the mixture changes rapidly as OH- or H+ moves across the red cell membrane. Flux and velocity constants can be calculated from the initial dp H/dt using the known initial intra- and extracellular pH. It was found that the further the extracellular pH is from 7.3 (in either direction from 4-11), the greater the absolute valute of total OH- and/or H+ flux. Pretreatment with SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), a potent anion exchange inhibitor, greatly reduces flux over the entire pH range, while exposure to valinomycin, a potassium ionophore, has no measurable effect. These data suggest that (i) both H+ and OH- may be moving across the red cell membrane at all pH; (ii) the species dominating pH equilibration is probably dependent on the extracellular pH, which determines the magnitude of the driving gradient for each ion; and (iii) the rapid exchange pathway of the erythrocyte membrane may be utilized for both H+ and OH- transport during CO2-free pH equilibration.

Statistical evaluation of biological rhythms in short, non‐synchronous time series

Abstract A statistical method or analysing the rhythmicity of several short, non‐synchronous time series in biological systems is discussed. As a first step the single series are analysed by techniques based on linear regression and variance methods. As a second step, some test statistics are proposed for combining the results of the single curves. The statistical procedure is demonstrated by two examples: fluorescence intensity of dye‐labelled Drosophila salivary gland cells in vitro and acetylcholinesterase activity in human erythrocyte suspensions.

Photometric detection of ATP release from human erythrocytes exposed to ultrasonically activated gas-filled pores

Suspensions of human erythrocytes in saline containing Luciferase/Luciferin (an enzyme preparation from fireflies which emits visible light in the presence of ATP) were exposed to 1.6 MHz ultrasound in the presence of stable gas bodies trapped within the holes in hydrophobic Nuclepore membranes. Ultrasonic intensities greater than 20–30 mW/cm 2 (spatial peak) resulted in the emission of light, apparently indicating that some erythrocytes were being disrupted. No effects could be observed at these low intensities in the absence of the gas bodies.

Effects of microwave radiation on erythrocyte membranes

Suspensions of rabbit, human, and dog erythrocytes were irradiated by S‐band microwaves in a WR‐284 waveguide chamber for a period of 20 minutes or three hours. The release of K+ and hemoglobin, and the osmotic fragility of the cells, were assayed to determine the effects of microwave and conventional heating on erythrocyte membranes. Conventional heating and microwave radiation resulted in sample temperatures that ranged from 25 to 44°C. There were no statistically significant differences between freshly prepared cells that were irradiated and those heated in a temperature‐controlled water bath at energy absorption rates near 200 mW g−1 for three‐hour exposures at a frequency of 3.0 GHz, and near 80 and 100 mW g−1, respectively, for frequencies of 2.45 and 3.95 GHz. Freshly prepared suspensions of rabbit erythrocytes and ouabain‐treated suspensions were also exposed to 3.0‐GHz microwaves at an absorption rate of 170 mW g−1 for 20 minutes or for three hours. No statistically signficant differences were detected between irradiated and water‐bath‐heated samples. In addition, effects of 3.0‐GHz microwaves on rabbit erythrocytes that had been held for seven days at 4°C prior to three hours of exposure were studied at a range of absorption rates to 170 mW g−1; no statistically significant effects of microwave irradiation were observed compared with the water‐bath‐heated controls although both modes of heating induced permeability changes relative to room‐temperature controls.

Capillary pore rheology of erythrocytes: V. The glass capillary array—Effect of velocity and haematocrit in long bore tubes

In earlier reports (P. S. Lingard, 1974, Microvasc. Res. 8 , 53–63; 1977, Microvasc. Res. 13 , 29–58; 1977, Microvasc. Res. 13 , 59–77), the resistance-velocity and resistance-concentration behaviour of human erythrocyte suspensions was described for Nuclepore filters. A potential disadvantage of Nuclepore filter geometry was the shortness (only 10–15 μm) of the capillary pores. This paper describes the hydrodynamic resistance-velocity and-concentration behaviour for erythrocyte suspensions in Glass Capillary Arrays (GCAs) of length-to-radius ratio 200 and diameter 5 μm. Two-limbed linear relations between pressure drop and velocity are found as for Nuclepore filters, showing a region of constant resistance at velocities below a critical value followed by a region in which resistance decreases in proportion to the inverse of velocity. This finding eliminates hypothetical entrance effects as possible explanations for the nonlinear velocity behaviour described before. Although less than with the Nuclepore filter, resistance levels are consistent with tight-fitting conditions for the cells (lubricant film thickness 0.15 μm). The falling resistance with velocity is again consistent with changing clearance due to distortion of the cells. Haematocrit-dependent relative resistance behaviour is again found, weaker at concentrations below 60% than seen before, but strengthening markedly between 50 and 80% as before. Possible evidence of “seize-up” at high concentrations is now reported and evidence for the absence of virtual shunting (considered for Nuclepore filters) is noted at low concentrations.

The Effect of External ADP on Red Cell Nucleotide Levels

SummaryIt has previously been suggested that ADP added to human erythrocyte suspensions may enter the cells and perform a metabolic function. Incubation of human red cells with radioactive and nonradioactive ADP failed to provide any evidence of the entry of this nucleotide into the erythrocytes.

Temperature dependence of the velocity of sound in suspensions of erythrocytes

Measurements of the velocity of sound in suspensions of human erythrocytes were made using a Panametrics 5054 ultrasonic time intervalometer. Data was taken using the pulse echo overlap method for sound velocity at a frequency of 2.25 MHz. The erythrocyte suspensions had a hematocrit of 45% and were made using either 0.75, 0.9, or 1.1% saline solutions. The erythrocyte samples were placed in a chamber maintained at a specified temperature for 10 min to allow thermal equilibrium to be reached and stirred throughout the measurement procedure. The velocity measurements were made by having the pulse of sound pass through a buffer region and observing the echoes received from the front and rear interfaces of the sample chamber as well as a target rod placed in the chamber. For all suspensions studied the velocity of sound increases in a nonlinear manner by about 40 m/s in the temperature range from 25° to 60°C. Details of this variation depend upon the suspending medium and age of the cells. [This work was supported in part by the National Science Foundation.]

Adriamycin and daunomycin generate reactive oxygen compounds in erythrocytes

Adriamycin and daunomycin produce dose-related cardiac toxicity that may be related to oxygen radicals. Addition of these compounds to human erythrocyte suspensions resulted in stimulation of hexose monophosphate shunt activity that was markedly impaired in the absence of oxyhemoglobin. Evidence for generation of hydrogen peroxide by these compounds was provided by oxidation of reduced glutathione, by 14C- formate oxidation, and by the catalase-aminotriazole trapping technique. These experiments indicate that Adriamycin and daunomycin interact with oxyhemoglobin to generate reactive oxygen metabolites. A similar interaction with oxymyoglobin may occur in the heart and produce oxygen radicals that injure cardiac myocytes.

Cold-induced hemolysis in a hypertonic milieu.

Suspension of human erythrocytes at 37 degrees C in an environment made hypertonic by increasing concentrations of sodium chloride and sucrose was followed by hemolysis when the temperature was lowered to 0 degrees C. Two distinct stages were involved in this hemolytic phenomenon, the first being incubation with hypertonic solute at some temperature above 20 degrees C with an increasing effect up to 45 degrees C, and the second stage consisting of lowering the temperature below 15 degrees C with increasing hemolysis down to 0 degrees C. The rate of cooling was not an important factor, but the presence of ions reduced the extent of cold-induced hemolysis in hypertonic sucrose. No significant release of membrane phospholipid and cholesterol accompanied this hemolysis. The solubilization of membrane protein components was investigated, with some differences appearing on sodium dodecyl sulfate polyacrylamide gel electrophoresis between hypertonic and isotonic supernatants. Spectrin could not be identified in solubilized form. Correlation of the temperatures of note in these studies with results from the literature on other biological effects of temperature-induced phase transitions in membrane lipids strongly points to the conclusion that such transitions are involved in the mechanism of cold-induced hypertonic hemolysis. It is postulated that the hypertonic milieu has resulted in membrane-protein alteration damage which prevents normal adaption to the new physical state of the membrane lipids during cooling.

Voltage-induced pore formation and hemolysis of human erythrocytes

Isotonic suspensions of human erythrocytes were exposed to single electric pulses of intensity at a few kV/cm and duration in microseconds. Upon pulsation, the cell membranes became permeable to Na + and K +, and the erythrocytes eventually hemolysed through the colloid osmotic effect of hemoglobin. The enhanced permeability is attributed to the formation of pores in the cell membranes. These pores are formed within a fraction of a microsecond, once the transmembrane potential induced by the applied electric field reaches a critical value of 1.0 V. Increased field intensity and pulse duration, or pulsation at low ionic strengths all expand the pore size, leading to an accelerated hemolysis reaction. In contrast to this expansion process, the initial step of pore formation is governed solely by the magnitude of the transmembrane potential: the critical value of the potential stays essentially constant in media of different ionc strengths, nor does it change appreciably with varying pulse duration. An abrupt increase in membrane permeability at a transmembrane potential around 1 V has been observed in many cellular systems. It is suggested that a similar mechanism of pore formation may apply to these systems as well.