Effects of ethanol on red blood cell rheological behavior.

Consumption of red wine is associated with a decreased risk of several cardiovascular diseases (e.g., coronary artery disease, stroke), but unfortunately literature reports regarding ethanol's effects on hemorheological parameters are not concordant. In the present study, red blood cell (RBC) deformability was tested via laser ektacytometry (LORCA, 0.3-30 Pa) using two approaches: 1) addition of ethanol to whole blood at 0.25%-2% followed by incubation and testing in ethanol-free LORCA medium; 2) addition of ethanol to the LORCA medium at 0.25%-6% then testing untreated native RBC in these media. The effects of ethanol on deformability for oxidatively stressed RBC were investigated as were changes of RBC aggregation (Myrenne Aggregometer) for cells in autologous plasma or 3% 70 kDa dextran. Significant dose-related increases of RBC deformability were observed at 0.25% (p < 0.05) and higher concentrations only if ethanol was in the LORCA medium; no changes occurred for cells previously incubated with ethanol then tested in ethanol-free medium. The impaired deformability of cells pre-exposed to oxidative stress was improved only if ethanol was in the LORCA medium. RBC aggregation decreased with concentration at 0.25% and higher for cells in both autologous plasma and dextran 70. Our results indicate that ethanol reversibly improves erythrocyte deformability and irreversibly decreases erythrocyte aggregation; the relevance of these results to the health benefits of moderate wine consumption require further investigation.

An automated tube-type blood viscometer: validation studies.

The technical complexity of previous rheometers has tended to limit the availability of blood viscosity data obtained over a wide range of shear rates. However, an automated tube-type viscometer, the Rheolog, has been developed; it employs a disposable flow assembly and less than five minutes are required to obtain blood viscosity results over a shear rate range of 1-1500 s(-1). We have carried out validation studies of the Rheolog using normal human blood and have compared these results with those obtained by cone-plate and Couette viscometers; storage time and temperature effects were also evaluated. Replicate measurements indicated mean CV levels less than 5%, and were independent of hematocrit and shear rate. Rheolog blood viscosity data agreed closely with those from other viscometers: average Rheolog differences from mean cone-plate and Couette values were -0.3% at 28% hematocrit, -1.4% at 41% hematocrit (i.e., native), and 1.0% at 56% hematocrit. Storage at room temperature up to 8 hours and at 4 degrees C up to 4 days had minimal effects whereas notable changes were observed when stored for 3 hours at 37 degrees C. Our results indicate that, within the hematocrit and shear rate limits employed herein, the Rheolog provides rapid, accurate and reproducible blood viscosity data, and suggest its usefulness for both basic science and clinical studies.

The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation.

The effects of nonionic polymers on human red blood cell (RBC) aggregation were investigated. The hydrodynamic radius (Rh) of individual samples of dextran, polyvinylpyrrolidone, and polyoxyethylene over a range of molecular weights (1,500-2,000,000) were calculated from their intrinsic viscosities using the Einstein viscosity relation and directly measured by quasi-elastic light scattering, and the effect of each polymer sample on RBC aggregation was studied by nephelometry and low-shear viscometry. For all three polymers, despite their different structures, samples with Rh <4 nm were found to inhibit aggregation, whereas those with Rh >4 nm enhanced aggregation. Inhibition increased with Rh and was maximal at approximately 3 nm; above 4 nm the pro-aggregant effect increased with Rh. For comparison, the Rh of 12 plasma proteins were calculated from literature values of intrinsic viscosity or diffusion coefficient. Each protein known to promote RBC aggregation had Rh >4 nm, whereas those with Rh <4 nm either inhibited or had no effect on aggregation. These results suggest that the influence of a nonionic polymer or plasma protein on RBC aggregation is simply a consequence of its size in an aqueous environment, and that the specific type of macromolecule is of minor importance.

Decreased red blood cell aggregation subsequent to improved glycaemic control in Type 2 diabetes mellitus.

AIMS: Reports of rheological changes following intensification of metabolic control are limited and not concordant. The present study was designed to test the hypothesis that intensification of management of Type 2 diabetes (T2DM) with diet, exercise and insulin improves haemorheological behaviour by reducing red blood cell (RBC) aggregation. METHODS: Blood was sampled from 55 subjects before and following 14 +/- 3 weeks of intensified management. RBC aggregation was measured in vitro for cells in plasma or in an aggregating 70 kD dextran solution. Plasma viscosity and whole blood viscosity were also measured. RESULTS: During treatment, fasting glucose fell 27%, HbA1c fell 21%, and serum triglycerides and total cholesterol fell 28% and 12%, respectively (P < 0.0001 for each). The extent and strength of RBC aggregation in plasma fell by 10-13% (P < 0.002). Similar decreases of RBC aggregation were seen for cells suspended in dextran (P < 0.002). Plasma viscosity decreased by 3% (P < 0.02) and high shear blood viscosity by 6-7% (P < 0.0001). Changes of RBC aggregation in plasma and in dextran were significantly correlated, supporting a cellular rather than a plasmatic origin for these changes. However, there were no significant correlations between RBC aggregation changes and changes of fasting glucose, HbA1c, serum triglycerides, serum cholesterol, or plasma fibrinogen. CONCLUSIONS: Intensified metabolic control results in a reduction of RBC aggregation that appears to be intrinsic to RBC. Since increased RBC aggregation can impair microcirculatory flow, it is possible that haemorheological factors may contribute to the reduction of microvascular complications resulting from improved metabolic control in T2DM.

Regional differences of H+, HCO3-, and CO2 diffusion through native porcine gastroduodenal mucus.

Gastroduodenal mucus may play a critical role in defending the epithelium from luminal acid and in the creation of a microenvironment suitable for H. pylori. We measured transmucus permeation of H+, HCO3-, and CO2 with an in vitro perfusion chamber through freshly harvested or partially purified porcine gastric mucin. pH and CO2 concentrations were measured with selective ion electrodes; HCO3- and CO2 concentrations were derived. Viscosity was measured by rotational microviscometry. Mucin viscosity was directly related to concentration. There was a large variation in viscosity among native mucus from antrum, corpus, and duodenum. The highest viscosity was found in the antral mucus; duodenal mucus had the lowest. Diffusion coefficients of duodenal mucus for H+ and HCO3- were significantly lower than those from corpus and antrum. CO2 diffusion coefficients were invariant. In conclusion, despite large variations in viscosity, antral and corpus gastric mucus were similar in terms of ion diffusion. Surprisingly, the low viscosity duodenal mucus was a more potent barrier to ion diffusion than was gastric mucus. Consequently, duodenal mucus may play a more important role in inhibiting ion diffusion than its gastric counterpart.

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.

Inhibition of polymer-induced red blood cell aggregation by poloxamer 188.

Previous reports have suggested that non-ionic poloxamer surfactants of appropriate molecular mass and composition can reduce red blood cell (RBC) aggregation in whole blood and in RBC-plasma suspensions. We have thus evaluated this phenomenon for RBC aggregated by several water-soluble polymers, using poloxamer 188 (P188), a non-ionic, tri-block molecule (total molecular mass of 8.40 kDa, 80% polyoxyethylene). Human RBC were washed, then re-suspended in isotonic solutions of dextran 70 (70.3 kDa), dextran 500 (476 kDa), PVP (360 kDa) or P-L-GLU (61.2 kDa); density-separated RBC were also studied. RBC aggregation was quantitated via a computerized Myrenne Aggregometer (extent, strength) and by the Microscopic Aggregation Index (MAI) method. Over the range of 0.5 to 5 mg/ml, poloxamer 188 inhibited both the extent and strength of aggregation in a dose-dependent manner, with the magnitude of the decrease related to polymer type (e.g., at 5 mg/ml, 62% decrease for dextran 70 vs. 14% decrease for P-L-GLU); MAI results with dextran 70 also showed a dose-dependent decrease. Poloxamer 188 at 5 mg/ml was more effective with younger, less-dense cells. Based upon the depletion model for polymer-induced aggregation, these findings suggest that poloxamer 188 acts by penetrating the depletion layer near the glycocalyx, thereby reducing the osmotic gradient between the intercellular gap and the suspending medium. Regardless of the specific mechanism(s) of action, poloxamers appear to offer interesting approaches for future basic science and clinical studies, and thus the possibility for greater insight into RBC aggregation.

Determination of particle sedimentation rate by ultrasonic interferometry: role of particle size, density and volume fraction.

The sedimentation rate (SR) of non-aggregated spherical particles in suspension was determined using an ultrasonic interferometry technique (Echo-Cell); this method is based on A-mode echography and measures the rate of formation of a sediment on a solid plate during settling. The particle accumulation rate, which is related to SR, is obtained from the interference of two waves reflected by two interfaces: one between the plate and the sediment and the other between the sediment and the suspension. Studies were carried out at 25 degrees C using latex spheres of different diameters (7 to 20 micron) and densities (1.062 to 1.190 g/cm3) suspended in distilled water at various volume fractions (1% to 5%). As anticipated by the Stokes model, linear relations were found between SR and both particle density and the square of particle radius. Experimental SR values decreased with increasing suspension particle concentration; these concentration effects were in good agreement with those predicted by the Steinour model. Our results thus serve to validate the theoretical aspects of the Echo-Cell method and suggest its usefulness as a tool for studies of RBC interaction and RBC aggregation.

Pulse shape analysis of RBC micropore flow via new software for the cell transit analyser (CTA).

The Cell Transit Analyser (CTA) provides a means to rapidly measure the deformability of large numbers of individual cells. It combines many of the advantages of micropipette studies with the simplicity and speed of filtrometry methods by measuring the duration of each resistive pulse generated as a cell passes through one of 30 identical micropores in a membrane. However, in our opinion, the potential of the system is limited by the microcomputer and software supplied for data analysis. We have therefore written new software for a more-powerful microcomputer to examine the shape of each resistive pulse rather than just the duration. Seven new parameters are derived, which provide additional information regarding the passage of cells through the pores. In particular, the contribution of the entry and exit phases of the cell transit are evident in the rise time and fall time of the pulses. The software is user-friendly and allows the analysis of each pulse to be reviewed, which aids understanding of the system and helps to avoid errors in interpreting the data.

Nimodipine and the evolution of hemorheological variables after acute ischemic stroke.

We studied the effects of the calcium channel antagonist nimodipine on the evolution of hemorheological variables during the first 3 weeks following ischemic stroke. We studied 13 patients and found that, compared to baseline levels, plasma fibrinogen concentration and low-shear whole-blood viscosity rose significantly in patients receiving placebo but not in those receiving nimodipine. Red blood cell aggregation rose in both groups but less so in nimodipine-treated patients. Hematocrit, high-shear whole-blood viscosity, and red blood cell deformability did not change significantly in either group. In conclusion, the use of nimodipine appears to alter the evolution of some hemorheological variables following acute ischemic stroke.

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes. Relevance to their fractionation by centrifugation.

Rats were injected with 59Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2-7.4 in the former case and 6.4-6.7 in the latter. Red cells were then centrifuged (5) and approximately 7-10% of the packed cells from the top and 7-10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2-7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4-6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4-6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.

Red cell aggregation and viscoelasticity of blood from seals, swine and man.

RBC aggregation and viscoelasticity parameters were determined for 40% suspensions of washed cells in autologous plasma from elephant seals (ES), Mirounga angustirostris, ringed seals (RS), Phoca hispida, and swine, (SS), Sus scrofa. Interspecific comparisons including human (HS) blood data revealed unusual rheological properties of seal blood relative to that from pigs or man: 1) RBC aggregation extent, rate and sedimentation were lower for seals (AI = 0, ZSR = .40, ESR = 0 for RS blood) relative to humans; 2) Viscous (n') and elastic (n") components of complex viscosity (OCRD) were lower for both seal species relative to SS blood, but only at shear rates less than or equal to 10 sec-1 (P less than 0.05), while n"/n' ratios for RS blood were lower than HS blood at all shear rates (P less than 0.01); 3) Blood viscosity measurements for RS and SS blood from rotational viscometry (Contraves) were consistent with OCRD data; 4) Seal plasma fibrinogen levels were low compared to pigs or humans (RS fibrinogen = -43% v. HS and -57% v. SS; ES fibrinogen = -58% v. HS and -69% v. SS). Electrophoretic mobility of RS red cells was +25% relative to those of humans. These results demonstrate differences in hemorheological indices among mammalian species and suggest the value of comparative rheologic studies.

Determination of erythrocyte transit times through micropores. II. Influence of experimental and physicochemical factors.

A new red blood cell filtration system, termed the Cell Transit Time Analyzer (CTTA), has been developed in order to measure the individual transit times of a large number of cells through cylindrical micropores in special "oligopore" filters; the system operates on the electrical conductometric principle and employs special computer software to provide several measures of the resulting transit time histogram. Using this system with filters having pore diameters of 4.5 or 5.0 microns and length to diameter ratios of 3.0 to 4.7, we have evaluated the effects of several experimental factors on the flow behavior of normal and modified human RBC. Our results indicate: 1) linear RBC pressure-flow behavior over a driving pressure range of 2 to 10.5 cm H2O with zero velocity intercepts at delta P = 0, thus suggesting the Poiseuille-like nature of the flow; 2) resistance to flow or "apparent viscosities" for normal RBC which are between 3.1 to 3.9 cPoise and are independent of driving pressure and pore geometry; 3) increased flow resistance (i.e., increased transit times) for old versus young RBC and for RBC made less deformable by DNP-induced crenation or by heat treatment at 48 degrees C; 4) increased mean transit time and poorer reproducibility when using EDTA rather than heparin as the anticoagulant agent. Further, using mixtures of heat-treated and normal RBC and various percentile values of the transit time histogram, we have been able to demonstrate the presence of sub-populations of rigid cells and thus the value of measurements which allow statistical analyses of RBC populations.

Determination of erythrocyte transit times through micropores. II-- Influence of experimental and physicochemical factors.

A new red blood cell filtration system, termed the Cell Transit Time Analyzer (CTTA), has been developed in order to measure the individual transit times of a large number of cells through cylindrical micropores in special "oligopore" filters: the system operates on the electrical conductometric principle and employs special computer software to provide several measures of the resulting transit time histogram. Using this system with filters having pore diameters of 4.5 or 5.0 cm and length to diameter ratios of 3.0 to 4.7, we have evaluated the effects of several experimental factors on the flow behavior of normal and modified human RBC. Our results indicate : 1) linear PBC pressure - flow behavior over a driving pressure range of 2 to 10.5 cm H2O with zero velocity intercepts at delta P = 0, thus suggesting the Poiseuille - like nature of the flow; 2) resistance to flow or "apparent viscosities" for normal RBC which are between 3.1 to 3.9 cPoise and are independent of driving pressure and pore geometry; 3) increased flow resistance (i.e., increased transit times) for old versus young RBC and for RBC made less deformable by DNP-induced crenation or by heat treatment at 48 degrees C; 4) increased mean transit time and poorer reproducibility when using EDTA rather than heparin as the anticoagulant agent. Further, using mixtures of heat-treated and normal RBC and various percentile values of the transit time histogram. We have been able to demonstrate the presence of sub-populations of rigid cells and thus the value of measurements which allow statistical analyses of RBC populations.