Study of the relationship between electrophoretic mobility of the diabetic red blood cell and hemoglobin A1c by using a mini-cell electrophoresis apparatus.

A mini-cell electrophoresis system using capillary tubing (50 microm inner diameter, length ca. 10 mm and volume ca. 20 nL) was applied to measure the electrophoretic mobility of red blood cells of 89 patients with diabetes on single cell level. A significant negative correlation was observed between hemoglobin A1c and the average electrophoretic mobility, with a correlation coefficient of 0.793. By statistically processing the electrophoretic mobility of each single red blood cell, it became clear that the reduction of the average value of electrophoretic mobility was caused by the reduction of the relative frequency of the red blood cell with high mobility. The cause of the average reduction was not the shift of electrophoretic mobility of all red blood cells to the lower mobility.     

Blood compatibility of particulate carriers investigated by cell electrophoresis and particle electrophoresis: a preliminary report

Polyethylene glycol (PEG) and dextran were covalently coupled, or only adsorbed, to the surface of three kinds of inorganic particles in order to shield their surface and reduce their nonspecific binding to red blood cells. Surface modification as well as interaction of particles with red blood cells was followed up by particle electrophoresis. This allowed a quick evaluation of the efficiency of polymer coupling. Moreover, the nonspecific binding of particles to red blood cells was easily investigated with cell electrophoresis, showing the inhibitory effect of immobilized PEG-5000 or dextran. The electrophoretic mobility analysis presented here may be used for screening blood compatibility of particulate drug carriers and could be helpful in formulating long-living circulating particles.     

Covalent binding of polyethylene glycol to the surface of red blood cells as detected and followed up by cell electrophoresis and rheological methods

Cyanuric chloride activated polyethylene glycol (PEG)-5000 was covalently coupled to murine and human red blood cells (pegylated RBC). Our purpose was to camouflage RBC receptors, which is necessary for parasite invasion, a process essential to sustain parasitemia. Cell electrophoretic mobility analysis (CEM) of pegylated RBC distinguished a new population of cells bearing characteristic CEM. Pegylation of RBC also modified their rheological properties, which were documented by evaluation of cell deformability (based on cell transit time through calibrated micropores) and cell aggregation (as measured by ultrasonic interferometry). Homologous transfusion of pegylated RBC into murine malaria-infected mice had no significant effect on the cerebral malaria death rate in Plasmodium berghei-infected mice, but it reduced the peripheral blood parasitemia by a factor 2 while in Plasmodium yoelii infected mice, the parasitemia was dramatically reduced by a factor of 4. These experiments demonstrate that transfusion of pegylated RBC may inhibit peripheral parasitemia. Cell electrophoresis appears to be a useful tool to allow in vivo detection and to investigate the fate of transfused pegylated RBC.     

Effect of dextran polymer on glycocalyx structure and cell electrophoretic mobility

We determined the electrophoretic mobility of human red blood cells suspended in physiological dextran solution for polymer samples with different polydispersity indexes. The electrophoretic mobility of cells is shown to depend mainly on the physical and hydrodynamical characteristics of the porous glycocalyx coating the membrane bilayer. We then propose a new model for the observed increases in effective mobility when cells are exposed to neutral polymers. The extension of glycoproteins under the action of weakly adsorbed chains is shown to reduce the frictional interaction of the surface coat on the flowing liquid. Such a structural rearrangement of the outer membrane improves the penetration of the electro-osmotic flow within the glycocalyx and increases the effective cell mobility.     

Influence of buffer composition on the capillary electrophoretic separation of carbon nanoparticles

Carbon nanoparticles obtained from the flame of an oil lamp were examined by means of capillary electrophoresis. The influence of buffer composition on the separation of the mixture of negatively charged carbon nanoparticles was studied by varying buffer selection, pH, and concentration. The electrophoretic pattern was affected by both the co- and counter-ion in the buffer solution, influencing selectivity and peak shape. The capillary electrophoretic separations at different pH revealed species with large electrophoretic mobilities under a wide range of pH. The mobility of selected species in the mixture of nanoparticles showed a strong dependence upon the solution ionic strength. The mobility of these nanoparticles as a function of ionic strength was compared to classical electrokinetic theory, suggesting that under the experimental conditions utilized, the species are small, highly charged particles with appreciable zeta potentials, even at low pH.     

High-voltage capillary zone electrophoresis of red blood cells

The high-voltage wide-bore capillary zone electrophoresis of red blood cells was investigated. The reproducibility of the retention time (electrophoretic mobility) is excellent and the differentiation among various species is good. The peaks in the electropherogram describe the distribution of the size and/or surface charge of the cells and are therefore broad. The relationship between the peak height and the number of cells injected is good, with linear correlation coefficients better than 0.98. Details of the preparation of cell suspensions and support electrolytes are given, which is essential for obtaining reproducible results. The inner surface of FEP capillary tubing is degraded by the application of high voltage and a pause is necessary between successive experiments if good and reproducible peak shapes are to be obtained. The length of the pause increases with the number of experiments made, and finally the tubing becomes useless. Inspection of the inner surface by X-ray photoelectron spectroscopy showed the breakdown of CHF bonds, but the actual mechanism is not known.     

人红细胞的显微毛细管电泳法研究

近年来,利用毛细管电泳的诸多优点[1]进行完整细胞的电泳分析已有许多报道[2～6].但由于细胞自身特点,电泳结果(特别是电泳峰形)有很大不同,给分析带来很多困难.为解决上述问题,我们设计并建立了毛细管电泳-紫外检测-显微成像(CE-UV-MVI)联用分析系统,并以人红细胞为对象进行了研究.观察到尖峰与细胞聚集有关,并进行了细胞淌度的高速测定,直观地研究了不同聚集状态细胞的迁移情况,特别是细胞吸附情况.1实验部分1.1试剂与仪器所用药品均为分析纯,购自北京市化学试剂公司.缓冲生理盐水(PBS)由质量分数为0.85%的NaCl和10mmol/L磷酸盐组成…     

Multistage electrophoresis II: treatment of a kinetic separation as a pseudoequilibrium process

An electrophoresis device is described which separates cells, particles, proteins and other separands by collecting samples having decreasing electrophoretic mobility in a train of inverted cavities while an electric field is applied between the inverted cavities and a sample cuvette containing a mixture of cells, particles, proteins or other separands. A circular plate is provided for the inverted cavities, and this circular plate is rotated to collect fractions. The system utilizes an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles, and proteins in useful quantities at high concentrations. Most multistage processes are based on equilibrium separations, but electrophoresis is a kinetic separation; therefore, a pseudoequilibrium paradigm was developed for use in optimizing separation parameters including number of stages and electrophoresis time per stage. This paradigm allows the application of McCabe-Thiele type analysis, and it was calculated, for example, that two separands differing by 20% in electrophoretic mobility can be purified to 95% purity with acceptable yield in about seven stages. Laboratory experiments demonstrated a 95% purification in four stages of a separand originally present at 4% when electrophoretic mobilities differed by 80%.     

Measurement of the electrophoretic mobility of sheep erythrocytes using microcapillary chips

Cell electrophoretic mobility (EPM) can be used to characterize individual cells. The purpose of this study is to establish reproducible and reliable cell EPM values obtained using microcapillary electrophoresis (microCE) chips. We studied cell electrophoresis on microCE chips through the comprehensive measurement of EPM and zeta potential. The inner wall of microchannels in microCE chips was coated with three kinds of reagents, namely bovine serum albumin (BSA), gelatin, and 2-methacryloyloxyethylphosphorylcholine (MPC) polymer to prevent nonspecific adhesion and interaction between cells and the inner wall. Electrophoresis was conducted in phosphate-buffered saline (pH 4-9) using erythrocytes extracted from sheep whole blood. Electroosmotic flow (EOF) mobility was measured using noncharged particles, and then the true EPM was calculated by subtracting the EOF mobility from the electromigration. MPC polymer coatings in microCE chips reduced the zeta potential of the inner wall and fully prevented nonspecific adhesion. EPM data obtained using microCE chips were almost the same and reproducible over a wide range of pH irrespective of the coating reagent used. In conclusion, reliability in the measurement of cell EPM using microCE chips was realized.     

Antigen-specific electrophoretic cell separation for immunological investigations

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non-capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen-positive and antigen-negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence-labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen-positive cells in the fractions of electrophoresis. Carrier-free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti-IgM-antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate-labelled anti-IgG resulted in the large-scale separation of high pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen-specific induction and regulation of antibody synthesis in vitro. These separate lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell subpopulations. We studied the influence of the anesthetic thiopental on separated human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B-Cells, T-helper and T-suppressor cells were equally affected and showed the same dose response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of host lattice on antigenicity of glycophorin in membranes

The influence of the host lattice on the antigenicity of glycophorin in membranes was confirmed by complement-dependent immune lysis of liposomes with two rabbit antisera, which were prepared by immunization with either human red blood cells or isolated glycophorin A. The immune lysis by either antiserum depended on the kind of phospholipid in the liposomes. Anti-glycophorin antiserum more strongly recognized glycophorin in egg-lecithin membranes than in dipalmitoyl-lecithin membranes, as did anti-red blood cell antiserum. Cholesterol in the liposomal membranes influenced the antigenicity of glycophorin. The relationship between the state of glycophorin in membranes and recognition by antibody is discussed.     

Mathematical representation of electrophoretic mobility of basic drugs in ternary solvent buffers in capillary zone electrophoresis

The electrophoretic mobilities of two beta-blocker drugs, i.e., labetalol and atenolol, have been determined in a mixed solvent background electrolyte system containing sodium acetate+acetic acid as buffering agent and different volume fractions of water, methanol and ethanol using capillary electrophoresis. The produced data and three other sets collected from a recent work are employed to study the accuracy and prediction capability of a mathematical model to calculate the electrophoretic mobility with respect to the volume fractions of the solvents in the mixture. The results show that the proposed model is able to correlate/predict the mobility within an acceptable error range and it is possible to use the model in industry to achieve the optimum solvent composition for the buffer where using a ternary solvent system is required. The average percentage deviations (APDs) obtained for correlated and predicted data points are 0.71-2.48 and 1.72-4.39%, respectively. The accuracy of the proposed model is compared with that of a mixture response surface method and the results show that the proposed model is superior from both correlation and prediction points of view. The possibility of calculation of the mobility of chemically related drugs in water-methanol-ethanol mixtures using the proposed model is also shown and the produced prediction APD is approximately 8%.     

Electrophoretic mobility of human erythrocytes in the presence of poly(styrene sulfonate)

The adsorption and depletion of the anionic polymer poly(styrene sulfonate) (PSS) on fresh human red blood cells (RBC) were investigated by measuring RBC electrophoretic mobility as a function of polymer molecular mass (48-2610 kDa), ionic strength (15 and 150 mM NaCl) and polymer concentration (相似文献   

Integration of nanoporous membranes for sample filtration/preconcentration in microchip electrophoresis

Microfluidic devices integrating membrane-based sample preparation with electrophoretic separation are demonstrated. These multilayer devices consist of 10 nm pore diameter membranes sandwiched between two layers of PDMS substrates with embedded microchannels. Because of the membrane isolation, material exchange between two fluidic layers can be precisely controlled by applied voltages. More importantly, since only small molecules can pass through the nanopores, the integrated membrane can serve as a filter or a concentrator prior to microchip electrophoresis under different design and operation modes. As a filter, they can be used for separation and selective injection of small analytes from sample matrix. This has been effectively applied in rapid determination of reduced glutathione in human plasma and red blood cells without any off-chip deproteinization procedure. Alternatively, in the concentrator mode, they can be used for online purification and preconcentration of macromolecules, which was illustrated by removing primers and preconcentrating the product DNA from a PCR product mixture.     

Particle electrophoresis as a tool to understand the aggregation behavior of red blood cells

Red blood cell (RBC) electrophoresis measurements in polymer solutions have recently been introduced as a promising approach for investigating polymer-cell interactions near the RBC surface. A polymer-poor depletion layer near the RBC has been demonstrated: for depletion layers thicker than the double layer, viscosity within the depletion layer, rather than suspending medium viscosity, affects cell mobility. Using a well-documented model of sepsis in rats, we have induced RBC membrane damage, and then measured the electrophoretic mobility of rat RBC from control and septic animals. Mobility measurements were carried out for cells suspended in polymer-free buffer and in 0.5-2% solutions of dextran 500 (500 kDa molecular mass); RBC aggregation in autologous plasma and in dextran 500 was also studied. Our results indicate: (i) as anticipated from prior studies, the aggregation of RBC from septic animals is markedly enhanced (p<0.001) in plasma and in 0.5-1% dextran; (ii) the mobility of septic RBC in polymer-free buffer was identical to control, whereas cells from septic animals had lower mobilities in 0.5% dextran; (iii) Over the range studied (0.5-2%), the mobility of RBC from septic animals was less sensitive to increases of dextran concentration and hence medium viscosity. These mobility-aggregation findings can be partially interpreted in terms of a depletion model for RBC aggregation; alterations of RBC surface charge and the hydrodynamic friction within the cell's glycocalyx may also be involved. In overview, we believe that these results suggest the merits of microelectrophoresis for exploring protein or polymer behavior near biological particles and the potential value of future studies for understanding cell-cell interactions.     

SPECIES DIFFERENCES IN RESPONSE TO PHOTOHEMOLYTIC AGENTS

Species differences in red blood cell susceptibility to the photohemolytic agents chlorpromazine, menadione and tetracycline were examined in mouse, rat, dog, and human blood. Menadione and tetracycline (25 microM) hemolyzed mouse but not dog, rat, or human red blood cells (RBC) when irradiated with UV light but not in the dark. Chlorpromazine (25 microM) produced a photohemolytic response in all four species with mouse and rat RBC lysing fastest followed by human then dog cells. Investigations into the nature of these species differences suggested that the size of mouse RBC may contribute to its high sensitivity to photohemolytic agents. An investigation of the effect of UV light on key antioxidant enzymes revealed species differences in enzyme inactivation. These data suggest that mouse RBC may be particularly vulnerable to phototoxic agents, especially those compounds which produce active oxygen species and, therefore, may prove more useful than human RBC as a model for predicting phototoxic potential of some chemical entities.     

药物与细胞膜相互作用的毛细管电泳方法研究

以细胞膜在毛细管内构成假固定相,建立一种基于毛细管电泳测定药物与细胞膜相互作用参数的方法．以西酞普兰和兔红细胞膜为相互作用模型,以不同浓度的细胞膜混悬液为电泳缓冲液,采用峰漂移法,并结合Scatchard分析,测得西酞普兰与兔红细胞膜的结合常数为0．977g^-1·L．该方法简单、快速,为研究药物与细胞膜的相互作用提供了新的技术手段,为高通量筛选药物膜通透性和活性,以及评价药物在体内吸收提供一种新的方法．     

Influence of a soluble anionic polymer on the electrophoresis of proteins

The influence of a soluble anionic polymer on electrophoresis of proteins was studied in relation to the nonspecific ionic effect of an affinophore on application to affinophoresis. Zone electrophoresis of proteins was carried out in agarose gel in the presence of succinyl-poly-L-lysine (degree of polymerization, 120) by using three electrophoresis buffers differing in ionic strength (0.06, 0.12 and 0.18) and pH (7.0 and 7.9). Proteins migrated as distinct single bands even in the presence of the polymer. The mobility of cationic proteins towards the cathode was first decreased and then increased towards the anode as the polymer concentration increased, while that of anionic proteins was not affected. The dependence of the apparent mobility changes of the proteins on the concentration of the polymer was treated quantitatively in the same way as affinity electrophoresis. The extent of the ionic interaction between a cationic protein and the polymer could be estimated as an apparent dissociation constant. It greatly depended on the ionic strength of the electrophoresis buffer. Except for the extremely cationic proteins such as lysozyme, the ionic interaction with up to 0.1 mM of the polymer could be practically suppressed by the use of 0.1 M sodium phosphate buffer (pH 7.0).     

Analysis of the Apollo-Soyuz Test Project Experiment on the Electhophoretic Separation of Fixed Red Blood Cells

The Apollo-Soyuz Test Project (ASTP) carried out in July, 1975 included an electrophoretic separation experiment of biological cells (MA-011). The nature of these experiments was to determine if biological separations could be carried out in space under microgravity conditions and with better resolution than obtainable on earth. The experiments consisted of a series of static and isotacho electrophoresis experiments in which mixtures of aldehyde-fixed red blood cells, B and T lymphocytes, and urokinase producing kidney cells were separated. The separation results of the aldehyde-fixed rabbit, human and horse red blood cells, which were taken in the form of photographs taken at three-minute intervals in columns 1 and 5, are the subject of this report.     

Oxidation of human catalase by singlet oxygen in myeloid leukemia cells

Catalases are oxidized by singlet oxygen giving rise to more acidic conformers detected in zymograms after electrophoresis in polyacrylamide gels. This shift in catalase mobility can be indicative of singlet oxygen production in vivo. Catalase from human cells, as from many organisms, is susceptible to in vitro modification by singlet oxygen. Human myeloid leukemia (U937) cells were treated under different stress conditions and catalase activity and its electrophoretic mobility was monitored. The U937 cells were found to have high levels of catalase activity, as compared to cultured fibroblasts, and to be very resistant to oxidative stress. Hydrogen peroxide did not modify the electrophoretic mobility of catalase, even at doses that produced cell damage. Conditions that primarily generate superoxide, such as treatment with paraquat or heat shock, also failed to modify the enzyme. In contrast, photosensitization reactions using rose Bengal gave rise to a more acidic conformer of catalase. Singlet oxygen quenchers prevented catalase modification by rose Bengal and light. The growth medium had a photosensitizing activity. Catalase was not modified in cells illuminated in phosphate buffer but was modified in cells illuminated in phosphate buffer containing riboflavin. Intense light per se also generated a slight shift in the electrophoretic mobility of catalase. Ultraviolet light (350 or 366 nm) did cause a change in catalase, but to a less acidic catalase conformer, indicating other modifications of the enzyme. The main effect of photosensitization with methylene blue was crosslinking of the enzyme, although some shift to acidic conformers was observed at a low concentration of the photoactive compound. Results indicate that catalase can be modified by singlet oxygen generated intracellularly, even though the enzyme is predominantly inside peroxisomes. Under some photosensitization conditions, catalase modification can be used as a marker to detect intracellular singlet oxygen.