Geoffrey V.F. Seaman

The effect of neutral polymers on the electrokinetic potential of cells and other charged particles: I. Models for the zeta potential increase

Abstract Electrokinetic measurements have shown that in many cases the apparent zeta potential of cells and charged particles is raised when dextran or other neutral polymers are included in the suspending medium. A consideration of artifacts which could be introduced by the presence of polymer suggests that the experimental results reflect a true increase in the electrokinetic potential. The dependence of the relative zeta potential increase on the type of cell or particle used implicates polymer adsorption in the explanation of the effect. Previously suggested explanations involving a rearrangement of the surface region to expose additional charge groups or a dielectric constant change in the double layer region are shown not to apply in general. Furthermore, although dextran increases the solubility of NaCl in aqueous solution, electrophoretic evidence strongly suggests that ion binding to an adsorbed polymer layer is not responsible for the effect. No satisfactory explanation for the zeta potential increase appears to have been offered.

Electrostatic and electrokinetic potentials in two polymer aqueous phase systems

Abstract Two polymer aqueous phase systems have proven useful as partition media for biological cells, the partition coefficients obtained being dependent on cell surface properties. One such property appears to be the surface charge density of cells which prompted this detailed examination of electrostatic potential distributions in dextran/poly(ethylene glycol) two phase systems. The partition coefficients of potassium sulfate and potassium chloride in the phase systems were measured and correlated with the Donnan potential difference between the phases, measured with salt bridges to reversible electrodes. Thermodynamic predictions of the relationship between the two measured quantities were confirmed. The preference of sulfate for the dextran-rich bottom phase, which linearly increases with increasing poly(ethylene glycol) (PEG) concentration, is due to the greater exclusion of sulfate by PEG than by dextran, as indicated by equilibrium dialysis of the polymer against different salt concentrations. Electrokinetic studies of droplets of one phase suspended in the other phase revealed relatively large electrophoretic droplet mobilities which increased linearly with the drop diameter and supralinearly with the sulfate concentration. The sign of the mobility depended on the phase the droplet originated from, but the implied surface charge sign was opposite to that anticipated from the difference in potential between the bulk phases. Assuming the mobility and zeta potential are of the same sign, the simplest potential profile which is consistent with these observations is one in which a dipole potential is present at the phase boundary oriented in such a way as to locally reverse the potential gradient. Levines partial theory for the electrophoresis of systems of this type was tested and found to be consistent with our results. The correlations obtained suggest that the magnitude of the dipole potential is related to the anion partition coefficient in these systems.

The effects of salts on the interfacial tension of aqueous dextran poly(ethylene glycol) phase systems

Abstract The use of the rotating drop technique to measure the interfacial tension, γ, of aqueous poly(ethylene glycol) (PEG)/dextran phase systems was studied and compared to the pendant drop method. The dependence of γ on the concentrations of the polymers, sodium phosphate, and sodium chloride was investigated. The difference in polymer concentrations between the phases plotted against γ on a loglog scale yields straight parallel lines for the different salt concentrations. The tension tie line length relationship therefore obeys a power law. The influence of temperature on γ was unusual in that for equal tie line lengths the tension is lower at 6°C than at 22°C in systems containing 0.11 M phosphate.

The partition of sodium phosphate and sodium chloride in aqueous dextran poly(ethylene glycol) two-phase systems

Abstract It is known that the addition of phosphate buffer to two polymer aqueous phase systems has a strong effect on the partition behavior of cells and other particles in such mixtures. The addition of sodium phosphate to aqueous poly(ethylene glycol) dextran phase systems causes a concentration-dependent shift in the binodial on the phase diagram, progressively lowering the critical conditions for phase separation as the phosphate concentration is increased. Sodium chloride produces no significant shift in the critical point relative to the salt-free case. Accurate determinations of the phase diagram require measurements of the density of the phases; data are presented which allows this parameter to be calculated from polarimetric measurements of the dextran concentrations of both phases. Increasing polymer concentrations in the phase systems produce increasing preference of the phosphate for the dextran-rich bottom phase. Equilibrium dialysis experiments showed that poly(ethylene glycol) effectively rejected phosphate, and to a lesser extent chloride, but that dextran had little effect on the distribution of either salt. Increasing ionic strength via addition of 0.15 M NaCl to phase systems containing 0.01 M phosphate produces an increased concentration of phosphate ions in the bottom dextran-rich phase.

Surface alterations of erythrocytes with cell age: rat red cell is not a model for human red cell.

Summary The charge-related surface properties of human and rat young and old erythrocytes were examined by partitioning in an aqueous dextran-poly(ethylene glycol) phase system having an electrostatic potential difference between the phases and by electrophoresis. It was found (a) that while rat red cells undergo changes in surface charge-related properties as a function of cell age which are detectable by partitioning, human erythrocytes do not; (b) that analytical particle electrophoresis after countercurrent distribution confirms both the alteration in rat and its absence in human erythrocytes; and (c) that particle electrophoresis cannot detect the age-related charge-associated alteration in rat red cells without prior cell partitioning.

Surface properties of human lymphocytes

Abstract Cell electrophoresis of freshly isolated human lymphocytes revealed the following surface properties including (1) a positive branch of the pH mobility curve at low pH and, (2) an increase in negative charge after aldehyde treatment, indicating the presence of surface positive charges, (3) the presence of a relatively high surface p K of about 3.1 and, (4) the comparatively low surface concentration of N -acetylneuraminic acid, (5) lymphocytes separated from stored blood showed surface characteristics that deviated from fresh lymphocytes: the electrophoretic mobility of these cells was lower, not pH reversible and showed considerable variability.

Column Electrophoresis on the Apollo-Soyuz Test Project

INTRODUCTION Electrokinetic separation techniques have been widely used for the analysis and characterization of charged materials of biological origin.3,4 Under terrestrial conditions preparative methods based on zone electrophoresis, isotachophoresis, and isoelectric focusing are prominent in the purification of charged macromolecules and small particles but have only been of limited usefulness in the separation of biological cells and larger particles. The major difficulties in the latter applications arise primarily from In the Apollo 16 experiment, a mixture of polystyrene latices was processed in an improved version of the apparatus used on Apollo 14. Whereas with such a device it is not possible to conduct the processing under terrestrial conditions without the appearance of extensive thermal convection which destroys the separation bound- aries, under microgravity conditions flight photographs showed no boundary deterioration, but did indicate that electroosmosis was a major factor in producing ov...

Electrochemical features of platelet interactions

Abstract Platelets participate in hemostasis and thrombus formation. The physicochemical basis at the molecular level for platelet adhesion, cohesion to form aggregates and the resultant viscous metamorphosis is not well understood. Under physiological conditions, blood platelets possess a net negative charge and decreases or changes in this charge have been implicated in both their adhesion and aggregation. The peripheral zone of the platelet contains glycoproteins and glycolipids. A variety of charged groups are present including positively charged amino groups and at least three types of negatively charged groups. The carboxyl group of terminal sialic acid residues is considered to be a major contributor to the negative charge of platelets. Removal of as little as 10 percent of the peripheral zone sialic acid by neuraminidase results in a shortened platelet lifespan in vivo . The electrokinetic properties of blood platelets are changed by interaction with a great variety of agents. In those cases where specific adsorption occurs it is possible to study the relationship between adsorption, change in surface charge, and the process of aggregation. It should be noted that in physiological media only those charge groups within about 8 A of the electrophoretic surface of the platelet will contribute to a significant extent to the electrophoretic properties. The electrophoretic mobility of platelets has been determined for a variety of animal species and in humans for a wide spectrum of disease situations. The measurement of platelet electrophoretic mobilities before and after treatment with adenosine diphosphate and norepinephrine has been introduced as a test of platelet function. However, so many variables may influence the surface properties of blood platelets including method of preparation, time, temperature, pH, contact phenomena, shear conditions, ionic strength and composition of suspending media, conditions for mobility measurements, etc., that considerable apparent disagreement exists in the literature regarding platelet electrokinetics.

Red Cell Electrophoretic Mobility Test for Early Diagnosis of Multiple Sclerosis

We have confirmed the red cell electrophoretic mobility-unsaturated fatty acid test for multiple sclerosis, which involves measuring the change in electrophoretic mobility of erythrocytes in Medium 199 upon the addition of linoleic acid. Using defibrinated blood samples, the average decrease in the mobility of red cells upon the addition of linoleic acid of 57 multiple sclerosis patients was 6.7 +/- 2.1% and that of 23 normal control subjects was .09 +/- 1.2% (p less than 0.001). In addition, blood anticoagulated with trisodium citrate was found to give similar test results, and such preparations were found to be simpler and quicker to use.

Increased vascular resistance due to a reduction in red cell deformability in the isolated hind limb of swine

This study investigated whether red cells with reduced deformability impeded flow through the microcirculation. Red cells were made less deformable in their normal biconcave disc shape by incubation with 2% formaldehyde (fRBCs). The blood supply to the right hind limb was isolated in 26 swine and the femoral artery was instrumented with two fine catheters, a flow probe, and an inflatable occluder. Flow was measured over a range of different perfusion pressures during adenosine-induced vasodilation under control conditions (C) and during an infusion of fRBCs at 1 ml/kg per minute (not to exceed 20 ml) into the femoral artery. At the same perfusion pressure (P), flow was significantly reduced 5 min after the fRBC infusion: Flow at P = 20 mm Hg, C = 41 ml/min vs fRBC = 10 ml/min; at P = 40, C = 160 ml/min vs fRBC = 79 ml/min; and at P = 60, C = 278 ml/min vs fRBC = 147 ml/min, with P less than 0.02 for all comparisons. Flow was still significantly reduced 15 min after the fRBCs, but by 30 min, it had returned to the control value. Chromium-51-labeling of red cells revealed that about one-third of fRBCs was trapped in the microcirculation compared to less than 3% of normal cells. This reduction in flow with fRBC infusion was not altered by alpha blockade, indicating that adrenergically mediated spasm was not responsible for the reduced flow. Aspirin 35 mg/kg iv completely prevented the reduction in flow despite an absence of change in the percentage of fRBCs trapped. Thus, red cells with reduced deformability infused into the circulation caused a significant, but transient, reduction in flow. The reduction in flow was not primarily related to entrapment of the abnormal RBCs, but may be mediated through platelet aggregation or release of potent vasoconstrictor substances from platelets or endothelial cells.