Deduction of intrinsic mechanical properties of the erythrocyte membrane from observations of tank-treading in the rheoscope

Measurements of the dimensions and membrane rotational frequency of individual erythrocytes steadily tank-treading in a Rheoscope are used to deduce the surface shear viscosity (eta m) and the shear elastic modulus (mu m) of the membrane. Previously published algorithms (Trans-Son-Tay et al., Biophys. J. 46: 65, 1984, and 51: 915, 1987) plus an assumed area-conserving membrane velocity field (Secomb and Skalak, Q. J. Mech. Appl. Math. XXXV 2: 233, 1982) are applied to calculate eta m as a function of the second invariant of the surface strain rate and mu m as a function of the second invariant of membrane strain. The results indicate density-related increases in membrane stiffness and viscosity, shear-thinning viscous behavior, and strain-stiffening elastic behavior.     

On the measurement of shear elastic moduli and viscosities of erythrocyte plasma membranes by transient deformation in high frequency electric fields.

We present a new method to measure the shear elastic moduli and viscosities of erythrocyte membranes which is based on the fixation and transient deformation of cells in a high-frequency electric field. A frequency domain of constant force (arising by Maxwell Wagner polarization) is selected to minimize dissipative effects. The electric force is thus calculated by electrostatic principles by considering the cell as a conducting body in a dielectric fluid and neglecting membrane polarization effects. The elongation A of the cells perpendicular to their rotational axis exhibits a linear regime (A proportional to Maxwell tension or to square of the electric field E2) at small, and a nonlinear regime (A proportional to square root of Maxwell tension or to the electric field E) at large extensions with a cross-over at A approximately 0.5 micron. The nonlinearity leads to amplitude-dependent response times and to differences of the viscoelastic response and relaxation functions. The cells exhibit pronounced yet completely reversible tip formations at large extensions. Absolute values of the shear elastic modulus, mu, and membrane viscosity, eta, are determined by assuming that field-induced stretching of the biconcave cell may be approximately described in terms of a sphere to ellipsoid deformation. The (nonlinear) elongation-vs.-force relationship calculated by the elastic theory of shells agress well with the experimentally observed curves and the values of mu = 6.1 x 10(-6) N/m and eta = 3.4 x 10(-7) Ns/m are in good agreement with the micropipette results of Evans and co-workers. The effect of physical, biochemical, and disease-induced structural changes on the viscoelastic parameters is studied. The variability of mu and eta of a cell population of a healthy donor is +/- 45%, which is mainly due to differences in the cell age. The average mu value of cells of different healthy donors scatters by +/- 18%. Osmotic deflation of the cells leads to a fivefold increase of mu and 10-fold increase of eta at 500 mosm. The shear modulus mu increases with temperature showing that the cytoskeleton does not behave as a network of entropy elastic springs. Elliptic cells of patients suffering from elliptocytosis of the Leach phenotype exhibit a threefold larger value of mu than normal discocytes of control donors. Cross-linking of the spectrin by the divalent S-H agents diamide (1 mM, 15 min incubation) leads to an eightfold increase of mu whereas eta is essentially constant. The effect of diamide is reversed after treatment with S-S bond splitting agents.     

Tank-treading dynamics of red blood cells in shear flow: On the membrane viscosity rheology

In this article, extensive three-dimensional simulations are conducted for tank-treading (TT) red blood cells (RBCs) in shear flow with different cell viscous properties and flow conditions. Apart from recent numerical studies on TT RBCs, this research considers the uncertainty in cytoplasm viscosity, covers a more complete range of shear flow situations of available experiments, and examines the TT behaviors in more details. Key TT characteristics, including the rotation frequency, deformation index, and inclination angle, are compared with available experimental results of similar shear flow conditions. Fairly good simulation-experiment agreements for these parameters can be obtained by adjusting the membrane viscosity values; however, different rheological relationships between the membrane viscosity and the flow shear rate are noted for these comparisons: shear thinning from the TT frequency, Newtonian from the inclination angle, and shear thickening from the cell deformation. Previous studies claimed a shear-thinning membrane viscosity model based on the TT frequency results; however, such a conclusion seems premature from our results and more carefully designed and better controlled investigations are required for the RBC membrane rheology. In addition, our simulation results reveal complicate RBC TT features and such information could be helpful for a better understanding of in vivo and in vitro RBC dynamics.     

Influence of preparative procedures on the membrane viscoelasticity of human red cell ghosts

The effects of systematic variations in the preparative procedures on the membrane viscoelastic properties of resealed human red blood cell ghosts have been investigated. Ghosts, prepared by hypotonic lysis at 0 degrees C and resealing at 37 degrees C, were subjected to: measurement of the time constant for extensional recovery (tc); measurement of the membrane shear elastic modulus (mu) via three separate techniques; determination of the membrane viscosity (eta m) via a cone-plate Rheoscope. Membrane viscosity was also determined as eta m = mu X tc. Compared to intact cells, ghosts had shorter tc, regardless of their residual hemoglobin concentration (up to 21.6 g/dl). However, prolonged exposure to hypotonic media did increase their recovery time toward the intact cell value. The shear elastic modulus, as judged by micropipette aspiration of membrane tongues (mu p), was similar for all ghosts and intact cells. This result, taken with the tc data, indicates that ghosts have reduced membrane viscosity. Rheoscopic analysis also showed that eta m was reduced for ghosts, with the degree of reduction (approx. 50%) agreeing well with that estimated by the product mu p X tc. However, flow channel and pipette elongation estimates indicated that the ghost membrane elastic modulus was somewhat elevated compared to intact cells. We conclude that: ghosts have reduced membrane viscosity; ghosts have membrane rigidities close to intact cells, except possibly when the membrane is subjected to very large strains; the reduction in eta m is not directly related to the loss of hemoglobin; prolonged exposure of ghosts to low-ionic strength media increases the membrane viscosity toward its initial cellular level. These data indicate that the mechanical characteristics of ghost membranes can be varied by changing the methods of preparation and thus have potential application to further studies of the structural determinants of red cell membrane viscoelasticity.     

Erythrocyte filtrability measurement by the initial flow rate method

A new filtration technique, based on the initial filtration rate of a diluted RBC suspension through 5 mu Nucleopore filter is described. As only a few hundreds RBCs traverse each pore and as the measurement are made in a few seconds, the method is by large insensitive to filter plugging and to sedimentation effects. The results are given as a filtration index IF which is, as a first order approximation, independent of the filter conductance and of the suspending medium viscosity. The filtration times are measured electronically. The filters are re-used many times. The influence on the results reproducibility of RBC washing, of the anticoagulant, of the blood sample and the suspension storage times are considered. With our technical procedure, the relative incertitude on the measurement of I.F. is about +/- 10%. The filtration index is shown to be an intrinsic RBC filterability property.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Red cell extensional recovery and the determination of membrane viscosity.

A theory of membrane viscoelasticity developed by Evans and Hochmuth in 1976 is used to analyze the time-dependent recovery of an elongated cell. Before release, the elongated cell is the static equilibrium where external forces are balanced by membrane elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of the viscoelastic solid model. It is shown that the model describes the time-dependent recovery process very well for a time constant in the range of 0.1-0.13 s. The time constant is the ratio membrane surface viscosity eta:membrane surface elasticity mu. Measurements for the shear modulus mu of 0.006 dyne/cm give a value for the surface viscosity of red cell membrane as a viscoelastic solid material of eta = mu tc = (6-8) X 10(-4) poise . cm.     

Action of hydroxyethyl starch on the flow properties of human erythrocyte suspensions

Hydroxyethyl starch (HES) has often been used as a plasma expander, but questions still remain concerning the mechanisms by which it produces changes in the rheological properties of blood and erythrocyte (RBC) suspensions under various flow conditions. The present investigation has shown that the dynamic viscosity of HES (232,000 and 565,000 daltons) solutions rises in a nonlinear fashion with increasing HES concentration, and for a given concentration of HES exhibits Newtonian behavior at shear rates between 0.15 to 124 sec-1. At low (less than 0.9 sec-1) shear rates the apparent viscosity of a 40% RBC suspension increases with lower concentrations of HES because of RBC aggregation. At higher concentrations of HES, increases in suspension viscosity are due to an increase in the viscosity of the continuous phase since the RBC are largely disaggregated. At high (greater than 36 sec-1) shear rates the relative viscosity (eta/eta O) of RBC suspensions slowly decreases with increasing HES concentration. At low shear rates eta/eta O increases and then decreases with increasing HES concentration. Evidence of the concentration-dependent effects of HES on RBC aggregation is provided not only by the viscometric analysis but also from measurements of erythrocyte sedimentation rate (ESR) and the zeta sedimentation ratio (ZSR). HES is a more potent aggregating agent in phosphate buffered saline (PBS) than it is in plasma. Polymer size has only a slight effect on the extent of RBC aggregation produced, but does have a significant effect on the concentration of polymer at which maximum aggregation occurs. The viscosity-corrected electrophoretic mobility of RBC in HES rises monotonically with the concentration of HES in the suspending medium. Decreases in the extent of RBC aggregation with increasing polymer concentrations probably result from an increase in the electrostatic repulsive forces between the cells.     

Modulation of red blood cell aggregation and blood viscosity by the covalent attachment of Pluronic copolymers

Despite many years of research, the physiologic or possible pathologic significance of RBC aggregation remains to be clearly determined. As a new approach to address an old question, we have recently developed a technique to vary the aggregation tendency of RBCs in a predictable and reproducible fashion by the covalent attachment of nonionic polymers to the RBC membrane. A reactive derivative of each polymer of interest is prepared by substitution of the terminal hydroxyl group with a reactive moiety, dichlorotriazine (DT), which covalently bonds the polymer molecule to membrane proteins. Pluronics are block copolymers of particular interest as these copolymers can enhance or inhibit RBC aggregation. Pluronics exhibit a critical micellization temperature (CMT): a phase transition from predominantly single, fully hydrated copolymer chains to micelle-like structures. The CMT is a function of both copolymer molecular mass and concentration. This micellization property of Pluronics has been utilized to enhance or inhibit RBC aggregation and hence to vary low-shear blood viscosity. Pluronic-coated RBCs were prepared using reactive DT derivatives of a range of Pluronics (F68, F88, F98 and F108) and resuspended in autologous plasma at 40% hematocrit. Blood viscosity was measured at a range of shear rates (0.1-94.5 s(-1)) and at 25 and 37 degrees C using a Contraves LS-30 couette low shear viscometer. RBC aggregation and whole blood viscosity was modified in a predictable manner depending upon the CMT of the attached Pluronic and the measurement temperature: below the CMT, RBC aggregation was diminished; above the CMT it was enhanced. This technique provides a novel tool to probe some basic research questions. While certainly of value for in vitro mechanistic studies, perhaps the most interesting application may be for in vivo studies: typically, intravital experiments designed to examine the role of RBC aggregation in microvascular flow require perturbation of the suspending plasma to promote or reduce aggregation (e.g., by the addition of dextran). By binding specific Pluronics to the surface, we can produce RBCs that intrinsically have any desired degree of increased or decreased aggregation when suspended in normal plasma, thereby eliminating many potential artifacts for in vivo studies. The copolymer coating technique is simple and reproducible, and we believe it will prove to be a useful tool to help address some of the longstanding questions in the field of hemorheology.     

Effects of intracellular Ca2+ and proteolytic digestion of the membrane skeleton on the mechanical properties of the red blood cell membrane

Intracellular Ca2+ at concentrations ranging from 0 to 10 mumol/l increases the shear modulus of surface elasticity (mu) and the surface viscosity (eta) of human red blood cells by 20% and 70%, respectively. K+ selective channels in the red cell membrane become activated by Ca2+. The activation still occurs to the same extent when the membrane skeleton is degraded by incorporation of trypsin into resealed red cell ghosts, suggesting that the channel activation is not controlled by the proteins of the membrane skeleton and is independent of mu and eta. Incorporation of trypsin at concentrations ranging from 0 to 100 ng/ml into red cell ghosts leads to a graded digestion of spectrin, a cleavage of the band 3 protein and a release of the binding proteins ankyrin and band 4.1. These alterations are accompanied by an increase of the lateral mobility of the band 3 protein which, at 40 ng/ml trypsin, reaches a plateau value where the rate of lateral diffusion is enhanced by about two orders of magnitude above the rate measured in controls without trypsin. Proteolytic digestion by 10-20 ng/ml trypsin leads to a degradation of more than 40% of the spectrin and increases the rate of lateral diffusion to about 20-70% of the value observed at the plateau. Nevertheless, mu and eta remain virtually unaltered. However, the stability of the membrane is decreased to the point where a slight mechanical extension, or the shear produced by centrifugation results in disintegration and vesiculation, precluding measurements of eta and mu in ghosts treated with higher concentrations of trypsin. These findings indicate that alterations of the structural integrity of the membrane skeleton exert drastically different effects on mu and eta on the one hand and on the stability of the membrane on the other.     

Tank treading of optically trapped red blood cells in shear flow

Tank-treading (TT) motion is established in optically trapped, live red blood cells (RBCs) held in shear flow and is systematically investigated under varying shear rates, temperature (affecting membrane viscosity), osmolarity (resulting in changes in RBC shape and cytoplasmic viscosity), and viscosity of the suspending medium. TT frequency is measured as a function of membrane and cytoplasmic viscosity, the former being four times more effective in altering TT frequency. TT frequency increases as membrane viscosity decreases, by as much as 10% over temperature changes of only 4°C at a shear rate of ∼43 s⁻¹. A threshold shear rate (1.5 ± 0.3 s⁻¹) is observed below which the TT frequency drops to zero. TT motion is also observed in shape-engineered (spherical) RBCs and those with cholesterol-depleted membranes. The TT threshold is less evident in both cases but the TT frequency increases in the latter cells. Our findings indicate that TT motion is pervasive even in folded and deformed erythrocytes, conditions that occur when such erythrocytes flow through narrow capillaries.     

Microscopic photometric quantification of stiffness and relaxation time of red blood cells in a flow chamber

The Microscopic Photometric Monolayer Technique provides a tool to measure red blood cell (RBC) stiffness (resistance to elongation) and relaxation time. It combines many of the advantages of flow channel studies of point-attached RBCs with the simplicity, sensitivity and accuracy of photometric light transmission measurement This technique allows the study of the effects of physicochemical factors on the elongation and relaxation time of the same cells within an average of four to five thousand cells adhered as a monolayer to glass. Further, the time course of physicochemical effects on cell membrane and wash-in/wash-out kinetics of interactions can be followed. An automated version of this technique was developed. A dense monolayer of point-attached RBCs was prepared at the bottom of a flow-chamber. A steady-state flow, with stepwise increases of flow rate, induced the RBC elongation. The light transmission perpendicular through the monolayer plane was measured photometrically. Photomicrographs compared with photometric results showed that the flow-induced bending and curvature change of RBC membrane was associated with the increase of light transmission. There was a linear correlation between the photometric index of elongation and the elongation taken from photomicrographs for shear stresses up to 0.75 Pa. A stiffness parameter, S (in Pa), was defined as the ratio of shear stress and elongation at a shear stress of 0.25 Pa. Following a sudden flow stoppage, the RBCs returned to their resting shape and the RBC relaxation time was measured. The stiffness-relaxation time product, V (in mPas), was calculated to provide an estimate of viscosity. Diamide treatment, known to stiffen RBCs, did result in dose-dependent decreases of elongation and relaxation time. With increasing temperature, the relaxation time decreased at a rate of −2.96 ms/K; the stiffness increased significantly at a rate of 0.0038 Pa/K, and the stiffness-relaxation time product decreased with −2.95 mPas/K, reflecting an inverse relationship between RBC viscosity and temperature. Using the automated version of this technique (Elias-c-) to test RBCs of 36 healthy subjects, we found the inter-individual coefficients of variation to be 8.6% for stiffness, 7.9% for relaxation time and 12.4% for stiffness-relaxation time product.     

Quasi-elastic light scattering studies of membrane motion in single red blood cells.

Studies of red blood cells (RBCs) and RBC ghosts, using a quasi-elastic light scattering (QELS) microscope spectrometer, have identified the membrane as the primary source of the light scattering signal. This is the first report in which motion of the cell membrane has been demonstrated to be the primary source of the QELS signal from a cell. Cytoplasmic changes induced in the RBC by varying the osmotic strength of the medium were also detected using this technique. Comparison of the data from white blood cells (WBCs) with the RBC data demonstrated significant differences between different types of cells.     

Alteration of red cell membrane viscoelasticity by heat treatment: effect on cell deformability and suspension viscosity

The membrane shear elastic modulus (mu) and the time constant for extensional shape recovery (tc) were measured for normal, control human red blood cells (RBC) and for RBC heat treated (HT) at 48 degrees C. Three separate methods for the measurement of mu were compared (two used a micropipette and one employed a flow channel), and the membrane viscosity (n) was calculated from the relation n = mu. tc. The deformability of HT and control cells was evaluated using micropipette techniques, and the bulk viscosity of RBC suspensions at 40% hematocrit was measured. The shear elastic modulus, or "membrane rigidity", was more than doubled by heat treatment, although both the absolute value for mu and the estimate of the increase induced by heat treatment varied depending on the method of measurement. Heat treatment caused smaller increases in membrane viscosity and in membrane bending resistance, and only minimal changes in cell geometry. The deformability of HT cells was reduced: 1) the pressure required for cell entry (Pe) into 3 micrometers pipettes was increased, on average, by 170%; 2) at an aspiration pressure (Pa) exceeding Pe, longer times were required for cell entry into the same pipettes. However, when Pa was scaled relative to the mean entry pressure for a given sample (i.e, Pa/Pe), entry times were similar for control and HT cells. Bulk viscosity of HT RBC suspensions was elevated by approximately 12% on average (shear rates 75 to 1500 inverse seconds). These findings suggest that alteration of RBC membrane mechanical properties, similar to those induced by heat treatment, would most affect the in vivo circulation in regions where vessel dimensions are smaller than cellular diameters.     

Nitric oxide red blood cell membrane permeability at high and low oxygen tension.

Red blood cell (RBC) encapsulated hemoglobin in the blood scavenges nitric oxide (NO) much more slowly than cell-free hemoglobin would. Part of this reduced NO scavenging has been attributed to an intrinsic membrane barrier to diffusion of NO through the RBC membrane. Published values for the permeability of RBCs to NO vary over several orders of magnitude. Recently, the rate that RBCs scavenge NO has been shown to depend on the hematocrit (percentage volume of RBCs) and oxygen tension. The difference in rate constants was hypothesized to be due to oxygen modulation of the RBC membrane permeability, but also could have been due to the difference in bimolecular rate constants for the reaction of NO and oxygenated vs deoxygenated hemoglobin. Here, we model NO scavenging by RBCs under previously published experimental conditions. A finite-element based computer program model is constrained by published values for the reaction rates of NO with oxygenated and deoxygenated hemoglobin as well as RBC NO scavenging rates. We find that the permeability of RBCs to NO under oxygenated conditions is between 4400 and 5100 microm s(-1) while the permeability under deoxygenated conditions is greater than 64,000 microm s(-1). The permeability changes by a factor of 10 or more upon oxygenation of anoxic RBCs. These results may have important implications with respect to NO import or export in physiology.     

Mercury leads to abnormal red blood cell adhesion to laminin mediated by membrane sulfatides

Exposure to mercury is associated with numerous health problems, affecting different parts of the human body, including the nervous and cardiovascular systems in adults and children; however, the underlying mechanisms are yet to be fully elucidated. We investigated the role of membrane sulfatide on mercuric ion (Hg²⁺) mediated red blood cell (RBC) adhesion to a sub-endothelial matrix protein, laminin, using a microfluidic system that mimics microphysiological flow conditions. We exposed whole blood to mercury (HgCl₂), at a range of concentrations to mimic acute (high dose) and chronic (low dose) exposure, and examined RBC adhesion to immobilized laminin in microchannels at physiological flow conditions. Exposure of RBCs to both acute and chronic levels of Hg²⁺ resulted in elevated adhesive interactions between RBCs and laminin depending on the concentration of HgCl₂ and exposure duration. BCAM-Lu chimer significantly inhibited the adhesion of RBCs that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C, while it did not prevent the adhesion of 3 h and 24 h Hg²⁺-treated RBCs. Sulfatide significantly inhibited the adhesion of RBC that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C and 0.5 μM of HgCl₂ solution for 24 h at room temperature (RT). We demonstrated that RBC BCAM-Lu and RBC sulfatides bind to immobilized laminin, following exposure of RBCs to mercuric ions. The results of this study are significant considering the potential associations between sulfatides, red blood cells, mercury exposure, and cardiovascular diseases.     

Visualization study of motion and deformation of red blood cells in a microchannel with straight,divergent and convergent sections

The size of red blood cells (RBC) is on the same order as the diameter of microvascular vessels. Therefore, blood should be regarded as a two-phase flow system of RBCs suspended in plasma rather than a continuous medium of microcirculation. It is of great physiological and pathological significance to investigate the effects of deformation and aggregation of RBCs on microcirculation. In this study, a visualization experiment was conducted to study the microcirculatory behavior of RBCs in suspension. Motion and deformation of RBCs in a microfluidic chip with straight, divergent, and convergent microchannel sections have been captured by microscope and high-speed camera. Meanwhile, deformation and movement of RBCs were investigated under different viscosity, hematocrit, and flow rate in this system. For low velocity and viscosity, RBCs behaved in their normal biconcave disc shape and their motion was found as a flipping motion: they not only deformed their shapes along the flow direction, but also rolled and rotated themselves. RBCs were also found to aggregate, forming rouleaux at very low flow rate and viscosity. However, for high velocity and viscosity, RBCs deformed obviously under the shear stress. They elongated along the flow direction and performed a tank-treading motion.     

Effects of cellular morphology on the viscoelastic behavior of high hematocrit RBC suspensions

Using a constant-amplitude (+/- 1 degree) oscillatory Couette viscometer (f = 0.01-1.0 Hz), we have measured the viscous (eta') and elastic (eta") components of the complex viscosity at 25 degrees C for shape-transformed human RBC suspended in isotonic buffer at 80% hematocrit. Morphology-altering drugs employed were: ECHINOCYTIC AGENT 2,4-dinitrophenol (DNP, 0.1-5 mM); STOMATOCYTIC AGENT chlorpromazine hydrochloride (CPZ, 0.01-0.1 mM). All suspensions exhibited decreasing eta' and eta" with increasing frequency. Compared to biconcave, control RBC suspensions, salient effects of shape transformation included: 1) for DNP, a dose-related elevation of both eta' and eta", with a 850% increase in eta' and a 2500% increase in eta" at 5 mM and the lowest frequency; 2) for CPZ, a dose-related elevation of both eta' and eta", with a 170% increase in eta' and a 280% increase in eta" at 0.1 mM and the lowest frequency; 3) for both DNP and CPZ, the elevations of eta' and eta" were inversely related to frequency. Using 2 mM DNP and various concentrations of CPZ, both eta' and eta" could be returned to control with 0.08 mM CPZ; further increases of CPZ at constant DNP led to elevations of both components. Comparisons of eta' and eta" to steady shear viscometric data indicated that neither a nominal shear rate approach nor a RMS complex viscosity technique was able to completely reconcile these data; a modified Kelvin-Voigt model proved useful in evaluating cellular versus membrane contributions to eta". These results indicate that RBC morphology is an important determinant of the oscillatory behavior of RBC suspensions and suggest the usefulness of the technique for studies of drug-membrane interactions.     

Red Blood Cells: Centerpiece in the Evolution of the Vertebrate Circulatory System

All vertebrates except cold-water ice fish transport oxygenvia hemoglobin packaged in red blood cells (RBCs). VertebrateRBCs vary in size by thirtyfold. Differences in RBC size havebeen known for over a century, but the functional significanceof RBC size remains unknown. One hypothesis is that large RBCsare a primitive character. Agnathans have larger RBCs than domammals. However, the largest RBCs are found in urodele amphibianswhich is inconsistent with the hypothesis that large RBCs areprimitive. Another possibility is that small RBCs increase bloodoxygen transport capacity. Blood hemoglobin concentration ([Hb])and mean RBC hemoglobin concentration (MCHC) increase from Agnathato birds and mammals. However, the changes in [Hb] and MCHCdo not parallel changes in RBC size. In addition, RBC size doesnot affect blood viscosity. Thus, there is no clear link betweenRBC size and oxygen transport capacity. We hypothesize thatRBC size attends changes in capillary diameter. This hypothesisis based on the following observations. First, RBC width averages25% larger than capillary diameter which insures cell deformationduring capillary flow. Functionally, RBC deformation minimizesdiffusion limitations to gas exchange. Second, smaller capillariesare associated with increased potential for diffusive gas exchange.However, smaller capillaries result in higher resistances toblood flow which requires higher blood pressures. We proposethat the large capillary diameters and large RBCs in urodelesreflect the evolutionary development of a pulmonary vascularsupply. The large capillaries reduced systemic vascular resistancesenabling a single ventricular heart to supply blood to two vascularcircuits, systemic and pulmonary, without developing high pressureson the pulmonary side. The large RBCs preserved diffusive gasexchange efficiency in the large capillaries.     

Effects of gender and age on thixotropic properties of whole blood from healthy adult subjects

The thixotropic parameters of whole blood from 314 healthy subjects (154 women, 160 men) were measured with our modified method by Low shear 30 Rheometer and calculated according Huang's equation. This communication offered the reference range of thixotropic parameters from man and woman group. The results demonstrated that no significant differences existed in the plasma viscosity and fibrinogen between man and woman group. Man group had statistically higher values in HCT, yield stress (tau 0), Newtonian contribution of viscosity (mu), non-Newtonian contribution of viscosity (eta s--mu), apparent viscosity at 2.37 sec-1 (eta s), the equilibrium value of the structural parameter (A) and apparent kinetic rate constant of rouleaux breakdown (ARC) than those in woman group. The man and woman groups could be separately divided into five subgroups in terms of age. It was found that the levels of fibrinogen and plasma viscosity had a tendency of increasing with aging. In the old subgroup (greater than 60 years) of men and women HCT, tau v, mu, eta s, (eta s--mu) and A had significant lower values than those in young and middle-age subgroups. However, it was very interested that there were differences of ARC versus age between man group and woman group, i.e. ARC in the man subgroup II, IV had lower and the woman subgroup II, III, IV had higher values than their respective older subgroup did.