Bioelectrochemistry (v.73, #2)

Preface by Ingolf Bernhardt; Clive Ellory; Robert Wilkins (83).

Morphology of small aggregates of red blood cells by S. Svetina; P. Ziherl (84-91).
Blood can be considered a two-phase liquid composed of plasma as well as cells and cell aggregates. The degree of cell aggregation is an important determinant of blood rheology: The size and shape of the aggregates affect blood viscosity. The microscopic mechanisms of red blood cell adhesion involve a complex interplay of electrostatic, van der Waals, and a range of specific biochemical inter-membrane interactions. Here we use an effective model of these interactions combined with the membrane elasticity theory to calculate the equilibrium shape of a red blood cell doublet and compare it with the experimentally observed red blood cell aggregates both in vitro and in vivo. Special attention is devoted to the shape of doublets formed by dissimilar cells. A possible effect of doublet shape on pathways of the formation of multicellular aggregates is discussed. Red blood cell rouleau formation is expected to take place at intermediate adhesion strengths where the outer doublet surfaces are either concave or flat, whereas in the strong-adhesion regime where the outer doublet surfaces are convex the cells should form rounded clump-like aggregates.
Keywords: Erythrocyte; Aggregation; Doublets; Rouleau;

Application of digital holographic microscopy to investigate the sedimentation of intact red blood cells and their interaction with artificial surfaces by Ingolf Bernhardt; Lyubomira Ivanova; Patrik Langehanenberg; Bjoern Kemper; Gert von Bally (92-96).
Red blood cells are able to undergo shape change from the “normal” discocyte to either echinocytes or stomatocytes depending on a large variety of membrane and cytoplasmic parameters. Such shape changes can be relatively fast (within seconds) during the sedimentation of the cells in suspension or after the cells are getting in contact with artificial surfaces. High resolution digital holographic microscopy has been applied to study these processes. This method represents a new set-up allowing a contact-less and marker-free quantitative phase-contrast imaging of living cells under conventional laboratory conditions. With the applied technique we were able to detect and analyse fast shape changes of red blood cells.
Keywords: Red blood cell; Erythrocyte; Shape change; Digital holographic microscopy; Cell surface interaction; Artificial surface;

Quantum mechanical calculations of elastic scattering cross sections for some permeant ions crossing the human red blood cell and resting axolemma squid axon membranes have been carried out using the three-dimensional spherically symmetric square potential well. Making the assumption that the permeability coefficient is inversely proportional to scattering cross section, we obtain the order of membrane selectivity for the ions as well as values for the permeability coefficients. Despite the relatively simple method used, good agreement between calculated permeability coefficients and data available in the literature is obtained. We suggest that elastic scattering cross section measurements for ions in various membranes would be valuable not only because they give a precise idea about the permeability ratios between ions but they also determine the form of the potential the ions are moving in.
Keywords: Potential well; Elastic scattering cross section; Permeability coefficient; Human red blood cell; Resting axolemma squid axon;

Thermally induced transformation of mammalian red blood cells during hyperthermia by Nikolay V. Repin; Elena N. Bobrova; Svetlana V. Repina (101-105).
The structural and transport characteristics of membranes are mainly determined by the state of the cytoskeleton. The characteristic changes in morphology of human (adult donor and cord) and rat Red Blood Cells (RBC) and of their membrane, induced by hyperthermia (46–51 °C) have been analyzed. Two different types of morphological changes have been observed to take place during hyperthermia in all studied RBC groups. We have observed either formation and exfoliation of spiculas from membrane, resulting in the formation of large (4–5 μm) sphere-like cell body and small (0.5–1.5 μm) vesicles or cell fragmentation with formation of large (3–3.5 μm) vesicles. The two distinct phenomena are likely to be determined by the heterogeneity of the RBC population in terms of cell age. There was noted the difference of cord RBC from the donor ones in temperature value of transformation beginning, as well as the character of deformation and vesicle formation, that may testify to their less thermoresistance. The ultrastructure of the membrane, studied with the freeze-fracturing technique, testifies to an irreversible character of membrane changes. The aggregation of intramembrane particles (IMPs) as a continuous network testifies to the strengthening of the interactions between denatured spectrin and bilayer integral components.
Keywords: Red blood cell; Hyperthermia; Membrane; IMPs aggregation; Vesiculation;

Peculiarities of RBCs resistance to acid hemolysis in hibernating mammals by Svetlana V. Repina; Nikolay V. Repin (106-109).
The binding of acids and alkalis, formed in tissues by metabolism, along with oxygen and CO2 transport are recognise as the principal functions of red blood cells (RBCs). Decreases in internal environment pH may result in activation of potential endogenous cytotoxic metabolites, OH˙ and oxidant formation and, as a consequence, result in oxidative damage of cell membrane leading to hemolysis. The characteristics of acid hemolysis in hibernating mammals have been determined in this study. Parameters of HCl-hemolysis, such as the average time for RBC hemolysis and the population distribution of the response, have been investigated. Measurements were performed within 40–5 °C temperature range. The resistance of hibernator RBCs to increased acidity, determined according to the acid hemolysis parameters, was found to reflect whether the animal was in the summer active period or in hibernation, with differences also apparent at different points with the hibernation season itself. It was demonstrated that hemolysis parameters of naturally cold-tolerant organisms are altered by decreases in temperature. We discuss a role of cytoskeletal–membrane interactions as a fast-acting switch of the structural and functional state of hibernator RBCs as an adaptation mechanism to acidosis arising from hypothermia and hypermetabolism.
Keywords: RBCs; HCl-hemolysis; Mammalian hibernation; Temperature decrease; Cytoskeletal–membrane interaction;

Agglutination of like-charged red blood cells induced by binding of β 2 -glycoprotein I to outer cell surface by Maruša Lokar; Jasna Urbanija; Mojca Frank; Henry Hägerstrand; Blaž Rozman; Malgorzata Bobrowska-Hägerstrand; Aleš Iglič; Veronika Kralj-Iglič (110-116).
Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that β 2 -glycoprotein I (β 2 -GPI) may induce RBC discocyte–echinocyte–spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed β 2 -GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of β 2 -GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound β 2 -GPI molecules. The suggested β 2 -GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound β 2 -GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.
Keywords: Red blood cells; Liposomes; Thrombosis; β 2 -glycoprotein I; Electric double layer; Bridging force;

The effect of the presence of charged amphiphiles during the incubation of human erythrocytes in a sucrose-substituted low-Cl solution on the shift of the osmotic resistance profile and the net K+ efflux was investigated. Osmotic fragility was determined by fitting the complementary error function to the haemolysis resistance curve. K+ efflux was calculated from the increase in the K+ concentration in supernatant measured by inductively coupled plasma atomic emission spectrometry (ICP-AES). The cationic amphiphile hexadecyltrimethylammonium bromide (CTAB) at 14 μM decreases, whereas the anionic amphiphile sodium dodecyl sulfate (SDS) at 50 μM increases the shift of the haemolysis resistance curve of erythrocytes incubated in isotonic sucrose by 0.069 and 0.079 %NaCl, respectively. Both the positively and the negatively charged amphiphile caused a significant change in the K+ efflux into isotonic sucrose solution: CTAB decreased and SDS increased K+ efflux by about 40%. In view of the lack of effect of the investigated compounds on the haemolysis resistance curve and K+ efflux from human erythrocytes incubated in isotonic NaCl solution, these results suggest that the insertion of charged amphiphiles into the erythrocyte membrane modulates the properties of the K+ transport pathway which is activated under low ionic strength (LIS) conditions.
Keywords: Human erythrocyte; Low ionic strength; Charged amphiphile; Potassium efflux; Osmotic fragility;

Dielectrophoretic sorting on a microfabricated flow cytometer: Label free separation of Babesia bovis infected erythrocytes by Elisabete M. Nascimento; Nuno Nogueira; Tiago Silva; Thomas Braschler; Nicolas Demierre; Philippe Renaud; Abel G. Oliva (123-128).
Dielectrophoresis is a method that has demonstrated great potential in cell discrimination and isolation. In this study, the dielectrophoretic sorting of normal and Babesia bovis infected erythrocytes was performed using a microfabricated flow cytometer. Separation was possible through exploitation of the dielectric differences between normal and infected erythrocytes, essentially due to the higher ionic membrane permeability of B. bovis infected cells. Sorting experiments were performed inside a microchip made from Pt microelectrodes and SU-8 channels patterned on a glass substrate. Optimum cell separation was achieved at 4 MHz using an in vitro culture of B. bovis suspended in 63 mS/m phosphate buffer and applying a sinusoidal voltage of 15 V peak-to-peak. Normal erythrocytes experienced stronger positive dielectrophoresis (pDEP) than B. bovis infected cells, moving them closer to the microelectrodes. Under these conditions it was possible to enrich the fraction of infected cells from 7 to 50% without the need of extensive sample preparation or labelling. Throughout the experiments very few microliters of sample were used, suggesting that this system may be considered suitable for integration in a low-cost automated device to be used in the in situ diagnostic of babesiosis.
Keywords: Dielectrophoresis; Babesia bovis; Infected erythrocytes; Microfabricated flow cytometer; Label-free sorting;

Further characterization of cation channels present in the chicken red blood cell membrane by Franck Lapaix; Guillaume Bouyer; Serge Thomas; Stéphane Egée (129-136).
In this paper, we provide an update on cation channels in nucleated chicken erythrocytes. Patch-clamp techniques were used to further characterize the two different types of cation channels present in the membrane of chicken red blood. In the whole-cell mode, with Ringer in the bath and internal K+ saline in the pipette solution, the membrane conductance was generated by cationic currents, since the reversal potential was shifted toward cations equilibrium when the impermeant cation NMDG was substituted to small cations. The membrane conductance could be increased by application of mechanical deformation or by the addition of agonists of the cAMP-dependent pathway. At the unitary level, two different types of cationic channels were revealed and could account for the cationic conductance observed in whole-cell configuration. One of them belongs to the family of stretch-activated cationic channel showing changes in activity under conditions of membrane deformation, whereas the second one belongs to the family of the cAMP activated cationic channels. These two channels could be distinguished according to their unitary conductances and drug sensitivities. The stretch-activated channel was sensitive to Gd3+ and the cAMP-dependent channel was sensitive to flufenamic acid. Possible role of these channels in cell volume regulation process is discussed.
Keywords: Red blood cells; Ionic channels; Patch-clamp; Chicken; Cation;

New analytical tools and epidemiological data for the identification of HbA2 borderline subjects in the screening for beta-thalassemia by Andrea Mosca; Renata Paleari; Renzo Galanello; Carla Sollaino; Lucia Perseu; Franca Rosa Demartis; Cristina Passarello; Antonino Giambona; Aurelio Maggio (137-140).
The increase of HbA2 is the most important feature in the identification of beta-thalassemia carriers. However, some carriers are difficult to identify, because the level of HbA2 is not in the typical range. Few data are available concerning the prevalence of such unusual phenotypes, and knowing their expected prevalence could be helpful in detecting systematic drifts in the analytical systems for HbA2 quantification.In this study we report a retrospective investigation in two centres with high prevalence of beta-thalassemia. The prevalence of borderline subjects was found to be 2.2 and 3.0%, respectively. The genotypes of a subgroup of these subjects were then analyzed and in about 25% of cases a mutation in the globin genes was identified. We conclude that the occurrence of HbA2 borderline phenotypes is not a rare event.In order to obtain more accurate HbA2 measurements the development of an international reference measurement system for HbA2, based on quantitative peptide mapping, has been recently started. We believe that the innovative approach of our method could also be used as a model to develop accurate quantitative methods for other red cell proteins relevant to the biodynamic properties and the surface electrochemistry of erythrocytes.
Keywords: Beta-thalassemia; HbA2; HPLC; Mass spectrometry; Reference systems;

Permeability of rat and rabbit erythrocyte membranes for a series of amides by O.I. Gordiyenko; G.V. Kovalenko; I.F. Kovalenko; V.S. Kholodnyy; T.P. Linnik; E.O. Gordiyenko (141-144).
Permeability coefficients of rat and rabbit erythrocyte membranes for a series of amides, as well as for erythrocytes treated with p-chloromercuribenzenesulfonic acid monosodium salt (pCMBS) have been determined at 25 and 37 °C. Directly proportional dependence of the pCMBS treated erythrocyte permeability for investigated substances and their partition coefficients between the hydrophobic phase and water as well as the values of activation energy of this process indicate that penetration of small hydrophilic molecules is realized by passive diffusion through the lipid bilayer. The results obtained indicate that penetration of small hydrophilic molecules of formamide through lipids is determined by the existence of a free space between hydrocarbon chains that arises from kink formation. The differences in permeability between rat and rabbit erythrocyte membranes could arise in particular as a result of the differences in lipid composition.
Keywords: Erythrocyte; Permeability; Amides;

Evidence for a random entry of Ca2+ into human red cells by Maria Baunbæk; Poul Bennekou (145-150).
Under the influence of a Gardos channel activator, NS309, acting through an increase of the channels Ca2+ sensitivity, it is found that the single population behavior of a suspension of human red cells, showing normal distributed osmotic resistance and density, after addition of NS309 in a time dependent manner changes to a two population distribution, with an increasing fraction of cells having high osmotic resistance or high density.The increase with time of the high resistance fraction can be fitted to an exponential, with a time constant corresponding to about 50 min. Since the ‘remaining’ cell fraction is practically unchanged, this points to a sudden random activation of the individual cells, caused by Ca2+ entry through a channel like pathway.
Keywords: Erythrocytes; Gardos channel; Ca2+-influx; Random; Fragility;

Changes in the intracellular Ca2+ content in human red blood cells in the presence of glycerol by Olga A. Kofanova; Nina G. Zemlyanskikh; Lyubomira Ivanova; Ingolf Bernhardt (151-154).
Changes of the intracellular Ca2+ content in human red blood cells (RBCs) in glycerol-containing solutions and after freeze–thawing the cells with glycerol and subsequent deglycerolization were investigated with the Ca2+-sensitive fluorescent dye fluo-4 using fluorescence microscopy. In the glycerol-containing solutions the Ca2+ content increased when compared with a physiological medium (Hepes buffered saline solution (HBSS)). This effect was most likely a result of an inhibition of the Ca2+ pump. After inhibiting the Ca2+ pump using o-vanadate, the Ca2+ uptake was not significantly different in the cells in glycerol-containing and physiological medium. Freeze–thawing and deglycerolization of RBCs resulted in a more pronounced increase in the Ca2+ content. Also in this case, the Ca2+ pump seemed to play a major role.
Keywords: Red blood cells; Intracellular Ca2+; Fluo-4; Glycerol; Freeze–thawing;

Structural and functional changes in the membrane and membrane skeleton of red blood cells induced by peroxynitrite by Maria N. Starodubtseva; Amanda L. Tattersall; Tatyana G. Kuznetsova; Nicolai I. Yegorenkov; J. Clive Ellory (155-162).
The changes in passive ion permeability of the red blood cell membrane after peroxynitrite action (3 µM–3 mM) have been studied by biophysical (using radioisotopes of rubidium, sodium and sulphur (sulphate)) and electrophysiological methods. The enhancement of passive membrane permeability to cations (potassium and sodium ions) and the inhibition of anion flux through the anion exchanger in peroxynitrite-treated red blood cells were revealed. In patch-clamp experiments the whole-cell conductance after peroxynitrite (80 μM) treatment of red blood cells increased 3–3.5-fold with a shift in the reversal potential from − 7.0 ± 1.5 mV to − 4.3 ± 0.9 mV (n  = 7, p  = 0.005). The addition of cobalt and nickel ions to red blood cell suspensions before peroxynitrite treatment had no effect on the peroxynitrite-induced cation flux but zinc ions in the same condition decreased cation flux about 2-fold. Using atomic force microscopy methods we revealed an increase in red blood cell membrane stiffness and the membrane skeleton complexity after peroxynitrite action. We conclude that the peroxynitrite-induced water and ion imbalance and reorganization in membrane structure lead to crenation of red blood cells.
Keywords: Peroxynitrite; Red blood cell; Passive cation and anion transport; Membrane reorganization; Atomic force microscopy;