Dielectric spectroscopy of red blood cells in sickle cell disease

Hypoxia-induced polymerization of sickle hemoglobin and the related ion diffusion across cell membrane can lead to changes in cell dielectric properties, which can potentially serve as label-free, diagnostic biomarkers for sickle cell disease. This article presents a microfluidic-based approach with on-chip gas control for the impedance spectroscopy of suspended cells within the frequency range of 40 Hz to 110 MHz. A comprehensive bioimpedance of sickle cells under both normoxia and hypoxia is achieved rapidly (within ∼7 min) and is appropriated by small sample volumes (∼2.5 μL). Analysis of the sensing modeling is performed to obtain optimum conditions for dielectric spectroscopy of sickle cell suspensions and for extraction of single cell properties from the measured impedance spectra. The results of sickle cells show that upon hypoxia treatment, cell interior permittivity and conductivity increase, while cell membrane capacitance decreases. Moreover, the relative changes in cell dielectric parameters are found to be dependent on the sickle and fetal hemoglobin levels. In contrast, the changes in normal red blood cells between the hypoxia and normoxia states are unnoticeable. The results of sickle cells may serve as a reference to design dielectrophoresis-based cell sorting and electrodeformation testing devices that require cell dielectric characteristics as input parameters. The demonstrated method for dielectric characterization of single cells from the impedance spectroscopy of cell suspensions can be potentially applied to other cell types and under varied gas conditions.     

Red blood cell ghosts and intact red blood cells as complementary models in photodynamic cell research

Recent research on erythrocytes as model cells for photodynamic therapy showed differing behaviour of certain photosensitisers in erythrocytes compared to other cells. Differences of dye accumulation in the cell membrane were proposed to be the reason for the distinct photodynamic effects. Using pheophorbide a as an example, the combination of erythrocyte ghosts as models to follow the dye accumulation in the cell membrane and intact erythrocytes as model cells to show the photodynamic damage is provided. Evidence for the correctness of the combination of erythrocyte ghosts and intact erythrocytes as a functioning model system in photodynamic cell research is provided using the confocal laser scanning microscopy on intact, pheophorbide a loaded erythrocytes.     

Penetration of laser light through red blood cell ghosts

Hemoglobin is the main absorber of visible light in blood and blood-perfused tissues. However, hemoglobin is released from a red blood cell (RBC) during hemolysis. Hemolysis may be caused by a large number of medical conditions, including photodynamic therapy (PDT) and this subsequently can affect passage of light through the treated biological structures. The purpose of the present study was to determine the penetration of a laser beam through a suspension of hemoglobin-free human red blood cells (RBCs) - ghosts. Although hemoglobin has been efficiently removed from the samples used in our experiments, our measurements show that the samples still effectively attenuate the radiant power of penetrating laser light. We established penetration depths of 12.6mm and 15.4mm for two different laser light wavelengths, 532nm and 630nm, respectively. The penetration depth of laser light was about one order of magnitude higher for hemoglobin-free RBC ghosts as compared to intact RBCs [8,10,12]. These results can be important in case of phototherapy or biostimulation, since all photons that penetrate in a biological object may interact with it and evoke biological response.     

Oxygen sensitivity of red cell membrane transporters revisited

In this paper, we provide an update on O2-dependent membrane transport in red cells. O2-sensitive membrane transport was compared in nucleated (chicken) and enucleated (human) red cells, to investigate effects on organic (glucose transporter [GLUT]) and inorganic (K(+)-Cl- cotransporter [KCC]/Na(+)-K(+)-2Cl- cotransporter [NKCC]) transporters, to study the response of so-called "housekeeping" transporters (Na+/K+ pump and anion exchanger [AE]) and, finally, to compare O2 sensitivity in normal human red cells with those from sickle cell patients. The Na+/K+ pump showed no change in activity between oxygenated and deoxygenated cells in any of the samples. KCC in normal human red cells had the greatest O2 sensitivity, being stimulated some 20-fold on oxygenation. It was more modestly stimulated by O2 in chicken red cells and HbS cells. By contrast, NKCC was stimulated by deoxygenation in all cases. GLUT showed little response to O2 tension, other than a small stimulation in deoxygenated chicken red cells. Finally, AE1 was stimulated by oxygenation in HbA cells, but this stimulation by O2 was absent in HbS cells and pink ghosts prepared from HbA cells. The significance of these findings is discussed.     

On the temperature dependence of the dielectric membrane properties of human red blood cells

Electrorotation (ER) spectra of human red blood cells (HRBCs) have been recorded in the frequency range from 10 kHz to 250 MHz in a 4-electrode microchip chamber. The cells were suspended at conductivities in the range from 0.02 to 3.00 S/m (corresponding to an ionic strength range from 1.6 to 343 mM) at temperatures between 10 degrees C and 35 degrees C. Generally, the characteristic frequencies as well as the rotation speeds of the first (membrane-dispersion) and second ER peaks increased with temperature. The rotation speed increase was largely correlated to the temperature dependence of the medium viscosity. Standard temperature dependencies were assumed for the conductivities and permittivities of cytoplasm, membrane, and external solution to explain the frequency shifts, starting from the cell parameters of Gimsa et al. [Gimsa et al., 1996, Biophys. J. 71: 495-506.]. The membrane capacitance was assumed to be temperature independent, based on the permittivity of alkyl-chains. Under these assumptions, the spectra could be well fitted only in a narrow temperature range around 20 degrees C. The temperature dependence of the first characteristic frequency was much stronger than predicted. In addition, around 15 degrees C, an anomalously high rotation speed was observed for the first peak at low external conductivities. Interestingly, this finding corresponds to the change in the chloride transport rate described by Brahm [Brahm, 1977, J. Gen. Physiol. 70: 283-306.].     

Inhibitors of the K+(Na+)/H+ exchanger of human red blood cells

The effect of substances as possible inhibitors of the K+(Na+)/H+ exchanger in the human red cell membrane has been tested on the (ouabain+bumetanide+EGTA)-resistant K+ influx in both physiological (HIS) and low ionic strength (LIS) solution with tracer kinetic methods. It is demonstrated that high concentrations of quinacrine (1 mM) and chloroquine (2 mM) inhibit the residual K+ influx in LIS solution to 60% and 85%, respectively, but activate it in HIS solution. Thus, chloroquine suppressed the 10-fold LIS-induced activation of the flux nearly completely. Amiloride derivatives were able to inhibit the K+ influx in both HIS and LIS solution. EIPA (75 microM) reduced the flux by about 20% and 55% in HIS and LIS solution, respectively. Newly developed drugs (HOE 642, 1 mM; HOE 694, 0.5 mM) designed to inhibit Na+/H+ exchanger isoforms showed an inhibition of the residual K+ influx of 40% and 33% in HIS and 65% and 44% in LIS solution, respectively, without haemolysis. The inhibitory effect of HOE 642 persisted in HIS (24%) and LIS (48%) solutions when Cl- was replaced by CH3SO4-. The K(+)-Cl- cotransport inhibitor DIOA (100 microM) stimulated the residual K+ influx in both solutions. It is, therefore, concluded that the K(+)-Cl- cotransporter does not contribute to the residual K+ influx both in HIS and LIS media. Okadaic acid decreased the residual K+ influx by 40% and 25% in HIS and LIS solution, respectively, showing that the residual K+ influx is affected by phosphatases like other ion transport pathways. The results show that the residual K+ influx can be decreased further by inhibiting the K+(Na+)/H+ exchanger. It remains still unclear to what extent the K+(Na+)/H+ exchanger is inhibited by the different substances used. However, the ground state membrane permeability for K+ is much smaller than assumed so far.     

Oxygen dependence of two-photon activation of zinc and copper phthalocyanine tetrasulfonate in Jurkat cells

Abstract Photodynamic therapy (PDT), the use of light-activated drugs, is a promising treatment of cancer as well as several nonmalignant conditions. However, the efficacy of one-photon (1-gamma) PDT is limited by hypoxia, which can prevent the production of the cytotoxic singlet oxygen ((1)O(2)) species, leading to tumor resistance to PDT. To solve this problem, we propose an irradiation protocol based on a simultaneous, two-photon (2-gamma) excitation of the photosensitizer (Ps). Excitation of the Ps triplet state leads to an upper excited triplet state T(n) with distinct photochemical properties, which could inflict biologic damage independent of the presence of molecular oxygen. To determine the potential of a 2-gamma excitation process, Jurkat cells were incubated with zinc or copper phthalocyanine tetrasulfonate (ZnPcS(4) or CuPcS(4)). ZnPcS(4) is a potent (1)O(2) generator in 1-gamma PDT, while CuPcS(4) is inactive under these conditions. Jurkat cells incubated with either ZnPcS(4) or CuPcS(4) were exposed to a 670 nm continuous laser (1-gamma PDT), 532 nm pulsed-laser light (2-gamma PDT), or a combination of 532 and 670 nm (2-gamma PDT). The efficacy of ZnPcS(4) to photoinactivate the Jurkat cells decreased as the concentration of oxygen decreased for both the 1-gamma and 2-gamma protocols. In the case of CuPcS(4), cell phototoxicity was measured only following 2-gamma irradiation, and its efficacy also decreased at a lower oxygen concentration. Our results suggest that for CuPcS(4) the T(n) excited state can be populated after 2-gamma irradiation at 532 nm or the combination of 532 and 670 nm light. Dependency of phototoxicity upon aerobic conditions for both 1-gamma and 2-gamma PDT suggests that reactive oxygen species play an important role in 1-gamma and 2-gamma PDT.     

High-throughput biophysical measurement of human red blood cells

This paper reports a microfluidic system for biophysical characterization of red blood cells (RBCs) at a speed of 100-150 cells s(-1). Electrical impedance measurement is made when single RBCs flow through a constriction channel that is marginally smaller than RBCs' diameters. The multiple parameters quantified as mechanical and electrical signatures of each RBC include transit time, impedance amplitude ratio, and impedance phase increase. Histograms, compiled from 84,073 adult RBCs (from 5 adult blood samples) and 82,253 neonatal RBCs (from 5 newborn blood samples), reveal different biophysical properties across samples and between the adult and neonatal RBC populations. In comparison with previously reported microfluidic devices for single RBC biophysical measurement, this system has a higher throughput, higher signal to noise ratio, and the capability of performing multi-parameter measurements.     

Influence of PAMAM dendrimers on human red blood cells

Polyamidoamine (PAMAM) dendrimers with different concentrations (1 nM-1 mM) (generations 2, 3, and 4) impact on human red blood cell morphology, and membrane integrity is studied. Erythrocyte shape changes from biconcave to echinocytic in dendrimers. Cell aggregation occurs. Polymers cause also concentration- and generation-dependent haemolysis.     

Redox-switched complexation/decomplexation of K(+) and Cs(+) by molecular cyanometalate boxes

The reaction of [N(PPh(3))(2)][CpCo(CN)(3)] and [Cb*Co(NCMe)(3)]PF(6) (Cb* = C(4)Me(4)) in the presence of K(+) afforded {K subset[CpCo(CN)(3)](4)[Cb*Co](4)}PF(6), [KCo(8)]PF(6). IR, NMR, ESI-MS indicate that [KCo(8)]PF(6) is a high-symmetry molecular box containing a potassium ion at its interior. The analogous heterometallic cage {K subset[Cp*Rh(CN)(3)](4)[Cb*Co](4)}PF(6) ([KRh(4)Co(4)]PF(6)) was prepared similarly via the condensation of K[Cp*Rh(CN)(3)] and [Cb*Co(NCMe)(3)]PF(6). Crystallographic analysis confirmed the structure of [KCo(8)]PF(6). The cyanide ligands are ordered, implying that no Co-CN bonds are broken upon cage formation and ion complexation. Eight Co-CN-Co edges of the box bow inward toward the encapsulated K(+), and the remaining four mu-CN ligands bow outward. MeCN solutions of [KCo(8)](+) and [KRh(4)Co(4)](+) were found to undergo ion exchange with Cs(+) to give [CsCo(8)](+) and [CsRh(4)Co(4)](+), both in quantitative yields. Labeling experiments involving [(MeC5H4)Co(CN)(3)]- demonstrated that Cs(+)-for-K(+) ion exchange is accompanied by significant fragmentation. Ion exchange of NH(4+) with [KCo(8)](+) proceeds to completion in THF solution, but in MeCN solution, the exclusive products were [Cb*Co(NCMe)(3)]PF(6) and the poorly soluble salt NH(4)CpCo(CN)(3). The lability of the NH(4+)-containing cage was also indicated by the rapid exchange of the acidic protons in [NH(4)Co(8)](+). Oxidation of [MCo(8)](+) with 4 equiv of FcPF(6) produced paramagnetic (S = 4/2) [Co(8)](4+), releasing Cs(+) or K(+). The oxidation-induced dissociation of M(+) from the cages is chemically reversed by treatment of [Co(8)](4+) and CsOTf with 4 equiv of Cp(2)Co. Cation recognition by [Co(8)] and [Rh(4)Co(4)] cages was investigated. Electrochemical measurements indicated that E(1/2)(Cs(+))--E(1/2)(K(+)) approximately 0.08 V for [MCo(8)](+).     

Relation between K+ leakage and damage to band 3 in photodynamically treated red cells

Potassium leakage is one of the first events that appear after photosensitization of red blood cells. This event may subsequently lead to colloid osmotic hemolysis. The aim of our study was to determine which photodynamically induced damage is responsible for increased membrane cation permeability. This was done by studying the effect of dimethylmethylene blue (DMMB)-mediated photodynamic treatment (PDT) on different membrane transport systems. Inhibition of band 3 activity (anion transport) showed a comparable light dose dependency as PDT-induced potassium leakage, whereas glycerol transport activity was inhibited only at higher light doses. Dipyridamole (DIP), an inhibitor of anion transport, protects band 3 against DMMB-induced damage, and prevents the increase in cation permeability of the membrane. Damage to glycerol transport was partially reduced when PDT was performed in the presence of DIP. Because DIP has no affinity for the glycerol transporter, this protection might result from the reduced photodamage to band 3. These results support the hypothesis that band 3 might be involved in glycerol transport. Glucose transport was not affected by DMMB-mediated PDT. The present results are the first to show a causal relationship between DMMB-mediated photodamage to band 3 and increased cation permeability of red blood cells.     

Spectrophotometric determination of manganese in human plasma and red cells with benzohydroxamic acid

A spectrophotometric method for the determination of manganese in human plasma and red cells with benzohydroxamic acid is presented, Interfering ions are removed by an anion exchange method, using Dowex-1, 8X resin. The manganese concentration found for plasma and red cells was about 0.1 and 0.2 p.p.m , respectively.     

Role of physiologic autoantibody in the removal of senescent human red cells.

The mechanism by which mononuclear phagocytes distinguish mature "self" from senescent "self" was investigated. Evidence is presented indicating that human mononuclear phagocytes distinguish senescent RBC from mature RBC on the basis of selective Ig attachment to the membranes of senescent cells. This Ig, eluted from senescent human RBC, was shown to be IgG and free of other Igs by immunodiffusion, immunoelectrophoresis, and polyacrylamide gel electrophoresis. The IgG was polyclonal with respect to light chains. The eluted IgG reattaches to homologous stored RBC, but not to mature autologous or allogeneic RBC, via the Fab region. It then initiates phagocytosis of these stored RBC by mononuclear phagocytes. Evidence suggests that the IgG is directed against altered membrane receptors. Thus, this IgG may be a "physiologic" autoantibody and contribute to the maintenance of homeostasis by performing regulatory function.     

Voltage activation and hysteresis of the non-selective voltage-dependent channel in the intact human red cell

Suspension of intact human red cells in media with low chloride and sodium concentrations (isotonic sucrose substitution) results in strongly inside positive membrane potentials, which activate the voltage-dependent non-selective cation (NSVDC) channel. By systematic variation of the initial Nernst potentials for chloride (degree of ion substitution) as well as the chloride conductance (block by NS1652), and by exploiting the interplay between the Ca(2+)-permeable NSVDC channel, the Ca(2+)-activated K+ channel (the Gárdos channel) and the Ca(2+)-pump, a graded activation of the NSVDC channel was achieved. Under these conditions, it was shown that the NSVDC channels exist in two states of activation depending on the initial conditions for the activation. The hysteretic behaviour, which in patch clamp experiments has been found for the individual channel unit, is thus retained at the cellular level and can be demonstrated with red cells in suspension.     

Effects of protein kinase and phosphatase inhibitors and anti-L antisera on K+ transport in LK sheep red cells

We investigated the role of protein phosphorylation in the action of anti-L on low potassium (LK) sheep red cells. Anti-L stimulated the Na/K pump by four- to fivefold, but Na/K pump activity in anti-L-stimulated or in control cells was unaffected by protein kinase/protein phosphatase (PK/PP) inhibitors. KCl co-transport activity was inhibited by anti-L (about 50%). Co-transport was stimulated by staurosporine; and inhibited by calyculin A, okadaic acid, tyrphostin B46 and genistein; with a similar pattem in both control and anti-L-treated cells. O2 sensitivity of KCl co-transport was similar in control and anti-L-treated cells. Neither control nor anti-L-stimulated Na/K pump activities were O2 sensitive. Incubation with urea stimulated KCl co-transport in both control and anti-L-treated cells. Inhibition of co-transport by anti-L was unaffected by low concentrations of urea but was reduced at higher urea concentrations. Na/K pump activity of control cells was unaffected by incubation with urea, but that in cells stimulated by anti-L was reduced, though not significantly. Under high hydrostatic pressure, KCl co-transport was stimulated, and the inhibitory effects of PP inhibition (okadaic acid), anti-L or combinations of the two were reduced. Results suggest that anti-L does not affect K+ transport in LK sheep red cells via protein phosphorylation.     

Determination of cocaine in human urine, plasma and red blood cells by gas-liquid chromatography

This paper describes a sensitive and reliable method for the determination of cocaine in human urine, plasma and red blood cells. Cocaine is extracted into cyclohexane from the biological materials at slightly alkaline pH, reduced with lithium aluminium hydride, acylated with pentafluoropropionic anhydride and detected by an electron capture detector. When compared with a gas chromatography-mass spectrometry method the results of cocaine determination correlated highly (r = 0.986). When cocaine was given intravenously to volunteer subjects only 0.2-1.4% of the administered dose was excreted as unmetabolized cocaine in the first 9 h after administration. Plasma and red blood cell levels of cocaine were also determined by this method after intravenous administration.     

Dielectrophoretic detection of electrical property changes of stored human red blood cells

The ability to transport and store a large human blood inventory for transfusions is an essential requirement for medical institutions. Thus, there is an important need for rapid and low-cost characterization tools for analyzing the properties of human red blood cells (RBCs) while in storage. In this study, we investigate the ability to use dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant, and stored in a blood bank refrigerator for a period of 20 days. Cells were removed from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction to “freeze” cells at their ionic equilibrium at that point in time and prevent ion leakage during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to measurements taken with a 2D quadrupole electrode array. The DEP analysis confirms that RBC electrical property changes occur during storage and are only discernable with the use of the cell crosslinking reaction above a glutaraldehyde fixation concentration of 1.0 w/v%. In particular, cytoplasm conductivity was observed to decrease by more than 75% while the RBC membrane conductance was observed to increase by more than 1000% over a period of 20 days. These results show that the presented combination of chemical crosslinking and DEP can be used as rapid characterization tool for monitoring electrical properties changes of human RBCs while subjected to refrigeration in blood bank storage.     

Analysis of enzymopathies in the human red blood cells by constraint-based stoichiometric modeling approaches

The human red blood cell (RBC) metabolism is investigated by calculating steady state fluxes using constraint-based stoichiometric modeling approaches. For the normal RBC metabolism, flux balance analysis (FBA) is performed via optimization of various alternative objective functions, and the maximization of production of ATP and NADPH is found to be the primary objective of the RBC metabolism. FBA and two novel approaches, minimization of metabolic adjustment (MOMA) and regulatory on-off minimization (ROOM), which can describe the behavior of the metabolic networks in case of enzymopathies, are applied to observe the relative changes in the flux distribution of the deficient network. The deficiencies in several enzymes in RBC metabolism are investigated and the flux distributions are compared with the non-deficient FBA distribution to elucidate the metabolic changes in response to enzymopathies. It is found that the metabolism is mostly affected by the glucose-6-phosphate dehydrogenase (G6PDH) and phosphoglycerate kinase (PGK) enzymopathies, whereas the effects of the deficiency in DPGM on the metabolism are negligible. These stoichiometric modeling results are found to be in accordance with the experimental findings in the literature related to metabolic behavior of the human red blood cells, showing that human RBC metabolism can be modeled stoichiometrically.