Parameters of bioimpedance spectroscopy in young and old erythrocytes

The parameters of bioimpedance spectroscopy (BIS) were studied in suspensions of young and old erythrocytes. The separation of erythrocytes according to age in a density gradient was performed. The BIS parameters, including the extracellular (Re) and intracellular (Ri) fluid resistances, characteristic frequency (Fchar), cell membranes’ capacitance (Cm), and the Alpha parameter of concentrated suspensions of young and old erythrocytes (n = 6) were measured using an ABC-01 Medass bioimpedance analyzer in the frequency range 5–500 kHz. Re (300.4 ± 30.0 and 261.2 ± 21.8 Ω in old and young erythrocytes, respectively, p < 0.05), Ri (86.6 ± 9.1 Ω and 73.4 ± 7.3 Ω in old and young erythrocytes, respectively, p < 0.001), and Alpha (0.305 ± 0.003 and 0.302 ± 0.001 in old and young erythrocytes respectively, p < 0.05) were greater in the suspension of old erythrocytes than in the suspension of young erythrocytes; and Fchar (308.3 ± 42.0 kHz and 347.4 ± 48.0 kHz in old and young erythrocytes, respectively, p < 0.05) and Cm (99.3 ± 10.1 pF and 112.8 ± 6.3 pF in old and young erythrocytes, respectively, p < 0.01) were less in the suspension of old erythrocytes than in the suspension of young erythrocytes. These differences between the BIS parameters of old and young erythrocytes were possibly due to the structural change in erythrocytes during aging (an increase in the concentration of intracellular hemoglobin, the change in the shape of the erythrocyte, their converging due to the decrease in cellular negative surface charge, and an increase in membrane permeability to ions). Thus, the BIS parameters depend on the erythrocyte age composition.     

Measurement of intracellular Ca2+ in young and old human erythrocytes using 19F-NMR spectroscopy.

Elevated cell calcium has been implicated in functional changes with human erythrocyte aging. However, until recently it has been difficult to measure free ionic intracellular calcium in red cells. We have made use of a fluorinated calcium chelator probe (5,5'-difluoroBAPTA) and fluorine nuclear magnetic resonance (19F-NMR) techniques to measure changes of intracellular Ca2+ concentrations ([Ca2+]i) with cell aging. We have demonstrated in these studies that human erythrocyte [Ca2+]i is significantly elevated as a function of in-vivo aging. Young cells, the least dense fraction of density-separated erythrocytes, contained an average of 62 (+/- 4) nM Ca2+ (+/- S.E.), whereas the oldest, most dense cell fraction contained 221 nM Ca2+ (+/- 25). Mechanisms by which intracellular [Ca2+] increases with in-vivo aging are currently under investigation.     

Clearance of senescent erythrocytes: wheat germ agglutinin distribution on young and old human erythrocytes

To add an additional aspect to the process of recognition and removal of senescent human erythrocytes from the circulation, the binding of wheat germ agglutinin (WGA) to separated young, old and sialidase-treated human erythrocytes is evaluated with the immune-electron microscopical method. WGA/gold conjugate binding to old erythrocytes was lower (27%) than to young erythrocytes and even lower following treatment with sialidase (82%), exhibiting a clustered, non-continuous labeling pattern in all three erythrocyte populations, thus showing a possible redistribution of WGA binding sites. The decrease in bound WGA/gold particles correlates well with the previously reported decrease in surface sialic acid on old erythrocytes. The binding of WGA/gold are indicative of the changes occurring on erythrocyte membrane surfaces when interacting with different agglutinins.     

Membrane glycopeptides from old and young human erythrocytes (Short Communication)

Glycopeptides were extracted by papain digestion from old and young human erythrocyte membranes and fractionated on DEAE-Sephadex A-25. Chemical characterization of the unfractionated samples and of the main peak eluted from the column indicates that glycoproteins of the erythrocyte membrane undergo significant decreases in sialic acid and galactosamine content with aging.     

Red cell ageing: phagocytosis and life-span of young and old erythrocytes fractionated by centrifugation

Young and old red blood cells, separated by centrifugation on the basis of differences in cell density, were submitted to phagocytosis by either autologous human alveolar macrophages or syngeneic murine bone-marrow macrophages. Young cells adhere to macrophages, but to a much smaller extent than old ones. The influence of both type and quality of the separation procedure on the differences observed between the two erythrocyte subpopulations is discussed in the light of the half-life times of murine young and old red blood cells. Fractionation according to age was obtained following the method of Murphy (1973) and glutamate oxalo-acetate transaminase activity was measured and used as an indicator of both cell age and separation.     

Flicker spectroscopy of erythrocytes

Frequency analysis of thermally excited surface undulations of erythrocytes leading to the flicker phenomenon is applied to determine biochemically and physically induced modulations of the membrane curvature elasticity. Flicker spectra of individual cells fixed to the window of a flow chamber by polylysine are taken by phase contrast microscopy, enabling investigations of the reversibility of the structural modifications. The spectra may be approximated by Lorentzian lines in most cases. By measuring the amplitude (at zero frequency) and the line width, effects of the structural changes on the curvature elastic constant, K _c , and the wavelength distribution of the undulations may be studied separately. Effect of physically induced modifications: The temperature dependence of the flicker spectra are taken from 10°C to 37°C. Above 20°C, K _c decreases with increasing temperature whereas the reverse holds below this limit. The latter anomalous behaviour is explained in terms of a conformational change associated with protein and lipid lateral phase separation. The bending stiffness increases when the cells swell osmotically, owing to surface tension effects. The dependence of the flicker spectra on the viscosity of the suspension medium agrees with the theoretical prediction. Biochemically and drug induced modifications: 5 vol of ethanol leads to a pronounced and reversible suppression of the long wavelength undulations without altering the discoid cell shape and without affecting the bending stiffness appreciably. Adsorption of dextran to the glycocalix increases K _c by a factor of 1.6 at saturation. The bending stiffness is increased by a factor of 1.3 after cross-linking the proteins with the SH-oxidizing agent diamid. Injection of Ca⁺⁺ into the cell via ionophores evokes (within 10 min) the formation of fine — probably spectrin free — spicules. This leads to an increase in K _c by a factor of 1.3 which is explained in terms of a lateral condensation of the spectrin/actin network. The spicule formation and K _c change is completely reversible (within 2 min) after perfusion with Ca⁺⁺-free buffer. Cholesterol depletion leads first to a continuous increase in K _c without change of the cell shape whereas a sudden discocyte- to echinocyte transformation sets in below a critical steroid content. The latter transition is also observed in cell suspensions and is reminiscent of a phase transition. The anti-tumor drug actinomycin D evokes an increase in the bending stiffness K _c by a factor of two, suggesting that its effect is at least partially due to a modulation of the membrane structure. The -receptor agonist leads to a remarkable increase in K _c (by about 25%) at 10^-4 M but the effect is not reversed by the -antagonist prazosin, suggesting that the agonist exerts a non-specific effect.A new technique, dynamic reflection interference contrast microscopy, is introduced by which absolute values of the amplitudes of the surface undulations and therefore K _c can be determined. The value obtained: K _c =5·10^-13 erg is about a factor of two larger then the bending stiffness of pure lipid bilayers. We suggest that the surface undulations may also be determined by lateral fluctuations of the quasi-two-dimensional spectrin/actin network.     

Aging human erythrocytes. Differential sensitivity of young and old erythrocytes to hemolysis induced by peroxide in the presence of thyroxine.

Human erythrocytes were separated according to cell age using albumin density gradients. In the presence of glucose (100 mg%), young cells were able to effectively protect themselves against thyroxine-peroxide induced hemolysis; old cells exhibited less protection. Hemolysis in heterogeneous populations is preceded by lipid peroxidation, K⁺ leak and decreased filtrability of the cells. Hydroxy radical scavengers partially inhibited hemolysis while superoxide dismutase had no effect. It is postulated that the differential sensitivity of young and old erythrocytes to thyroxine-peroxide induced metabolic and morphological alterations may play a role in the recognition and removal of senescent cells from the circulation.     

Mathematical analysis of the effects of geometric parameters and mechanical properties of erythrocytes on the filterability of nonuniform suspensions

Approaches to determination of the pattern of erythrocytes distribution with regard to the rates of their passage through pores (3 microm in diameter) of a membrane filter by processing the data on changes in the flow rates of erythrocyte suspensions with time (filtration curves) are discussed. We considered the case when the suspension consisted of two subpopulations of erythrocytes differing in a single parameter. Using a model describing the erythrocyte passage through a pore and a model describing filtration of a nonuniform suspension, we analyzed the dependences of filtration kinetics of such suspensions on the relative contents of the subpopulations and their rheological characteristics. It has been shown that the filtration rates of the major subpopulation and the minor abnormal subpopulation, and their relative contents can be determined from the analysis of filtration curves. This can be done when the filtration rate of cells from the minor subpopulation is at least one order of magnitude lower than the filtration rate of cells from the major subpopulation. Thus we can register the presence of the minor subpopulation in the range of 0.5-1%. If filtration rates are recorded at different osmolalities, their analysis makes it possible to determine the surface area, intracellular viscosity, and membrane rigidity of cells of the major subpopulation and, in certain cases, the same parameters for the cells of the minor subpopulation.     

Non-linear dielectric spectroscopy of microbiological suspensions

Background  

Non-linear dielectric spectroscopy (NLDS) of microorganism was characterized by the generation of harmonics in the polarization current when a microorganism suspension was exposed to a sinusoidal electric field. The biological nonlinear response initially described was not well verified by other authors and the results were susceptible to ambiguous interpretation. In this paper NLDS was performed to yeast suspension in tripolar and tetrapolar configuration with a recently developed analyzer.     

Comparative study on the main membrane-surface sialoglycopeptides released from young and old human erythrocytes with trypsin

1. The amount of sialoglycopeptide (SGP) mixture released from young (Y) erythrocytes by trypsin treatment was significantly larger than that from old (O) ones. 2. The two main membrane-surface sialoglycopeptides (Y-SGP and O-SGP) were isolated in yields of about 45% and 42% from the mixtures by a combination of Sephadex G-50 superfine gel filtration with DEAE-cellulose chromatography, respectively. Molecular weights of 12,000 and 12,100, respectively, were found for the Y-SGP and the O-SGP. 3. The Y-SGP showed a greater proportion of sialic acid than the O-SGP. The contents of galactose, N-acetylglucosamine and N-acetylgalactosamine of the Y-SGP were significantly lower than those of the O-SGP. 4. Both the SGPs exhibited the same reactivities against the MN reagents, but the reactivities against the lectins of Limulus polyphemus and Phaseolus vulgaris (E) were stronger in the Y-SGP than in the O-SGP. Neither the Y-SGP nor the O-SGP showed the reactivity to Arachis hypogaea (anti-T) lectin.     

Antioxidant enzymes in erythrocytes from old and diet restricted old rats

Diet restriction, prolonging the lifespan of rodents, represents an interesting model for gerontological studies. We analyzed the activity of antioxidant enzymes, Superoxide Dismutase, Catalase and Glutathione Peroxidase in erythrocytes from young, old and old food restricted Wistar rats. Diet restriction was applied feeding the animals on every-other-day schedule starting from the age of 3.5 months. The age-dependent decrease of Catalase and Glutathione Peroxidase activities was prevented by food restriction, whereas Superoxide Dismutase activity was not influenced either by aging and dietary intervention. Present results support the hypothesis that diet restriction increases the protection of cell structure against the peroxidative damage, preserving the activity of antioxidant enzymes.     

Impedance spectroscopy of alpha-beta tubulin heterodimer suspensions

Impedance spectroscopy is a technique that reveals information, such as macromolecular charges and related properties about protein suspensions and other materials. Here we report on impedance measurements over the frequency range of 1 Hz to 1 MHz of alpha-beta tubulin heterodimers suspended in a buffer. These and other polyelectrolyte suspensions show enormous dielectric responses at low frequencies, due both to the motion of charges suspended in the medium and to an electrical double layer that forms at each electrode-medium interface. We propose an equivalent circuit model to minimize electrode polarization effects and extract the intrinsic response of the bulk medium. At megaHertz frequencies, the conductivity increases with concentration below the critical concentration of approximately 1 mg/ml for microtubule polymerization, above which the conductivity decreases. This suggests that such measurements can be used to monitor the dynamics of microtubule polymerization. Finally, we obtain the net charge number per tubulin dimer of /Z/ = 306 in the saline buffer, which, if maintained as the dimers polymerized, would yield a linear charge density of 3.8 e/angstroms for the assembled microtubules. These results are potentially important for fundamental electrostatic processes in biomolecules and suggest the possibility of developing future bioelectronic applications.