Protective effect of quercetin against in vitro erythrocyte rheology alterations produced by arsenic.

Humans are exposed to heavy metals such as arsenic (As), through contaminated food and drinking water. The effect of As on RBC membrane is one of the most important biological effects. In a previous work, we have studied the AsVin vitro effect on erythrocytes biophysical properties discovering alterations regarding aggregability deformability, cell morphology, membrane fluidity and osmotic response. We have also observed that the presence of the metal produces an oxidative stress in RBCs that might be the origin of rheological impairment. In the present work we analyzed RBCs rheological properties associated with membrane fluidity and lipid peroxidation in presence of As and quercetin (Qc). From our results we can conclude that RBCs treatment with Qc is efficient to prevent the impairment of the mechanical properties of the cell membrane produced by the As, through oxygen reactive agents in the membrane structure, mainly on the lipids. This protective effect is observed in the preservation of the erythrocytes rheological properties and consequently in the maintenance of an appropriate blood flow, specially in the small vessels in the peripheral circulation.

Effects of ascorbate fatty ester derivatives on erythrocyte membrane lipoperoxidation.

6-O-alkyl ascorbic acid esters (ASC(n)) are amphiphilic molecules that behave as surfactants in aqueous solution. ASC(n) have shown some physical and rheological properties that suggest a potential utility as drug carriers. The present paper aims to evaluate the effect of ASC(n) on erythrocyte properties in order to get information regarding the relationship between osmotic fragility, erythrocyte deformability and membrane lipoperoxidation process. The assays were performed at the following concentrations: the critical micelar concentration (CMC), producing 10% hemolysis (CH(10)) and producing 50% hemolysis (CH(50)). We observed that ASC(n) (ASC(8), ASC(10) and ASC(12)), at concentration nearby CMC, neither affected cell deformability nor produced lipoperoxidation. Nevertheless, at higher concentrations (CH(10) and CH(50)), the RBCs incubated with ASC(n) were affected by a significant and progressive loss of deformability, simultaneously with an increase of osmotic fragility and membrane lipoperoxidation.

Arsenic intoxication, a hemorheologic view.

Arsenic (As) is a toxic semi-metal of wide distribution in nature. People living in regions where drinking water contains large quantities of arsenic, have an unusually high likelihood of developing blood-vessel diseases, but little is known about the mechanisms involved, i.e. the blood rheologic alterations that would contribute to the circulatory obstruction. Erythrocytes are the main target cells for arsenic compounds systemically absorbed and their cell membrane is the first place against the toxic. In this paper we have examined the in vitro effect of arsenic (As(V)) on the rheologic properties of human erythrocytes in relation with membrane fluidity and internal microviscosity. According to our present results, As(V) treatment produces oxidative degradation of membrane lipids and alteration of internal microviscosity. These red blood cells (RBCs) membrane and cytoplasmic structural damage consequently alters RBCs rheologic properties: an alteration of the RBCs discoid shape to stomatocytes, a diminution of erythrocyte deformability and an enhancement of osmotic fragility and cell aggregability. These effects impaired blood fluid behaviour that contribute to obstruct peripheral circulation and provides anemia, both clinic evidences typical of arsenic cronic intoxication.

In vitro and ex vivo effect of hyaluronic acid on erythrocyte flow properties.

BACKGROUND: Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology. METHODS: Erythrocyte deformability (by filtration through nucleopore membranes) and erythrocyte aggregability (EA) were tested on blood from healthy donors additioned with purified HA. EA was measured by transmitted light and analyzed with a mathematical model yielding two parameters, the aggregation rate and the size of the aggregates. Conformational changes of cytoskeleton proteins were estimated by electron paramagnetic resonance spectroscopy (EPR). RESULTS: In vitro, erythrocytes treated with HA showed increased rigidity index (RI) and reduced aggregability, situation strongly related to the rigidization of the membrane cytoskeleton triggered by HA, as shown by EPR results. Also, a significant correlation (r: 0.77, p < 0.00001) was found between RI and serum HA in RA patients. CONCLUSIONS: Our results lead us to postulate the hypothesis that HA interacts with the erythrocyte surface leading to modifications in erythrocyte rheological and flow properties, both ex vivo and in vitro.

Erythrocyte aggregation in rheumatoid arthritis: Cell and plasma factor's role.

UNLABELLED: Increase in erythrocyte aggregation (EA) is pathognomonic for rheumatoid arthritis (RA), and its estimation through erythrocyte sedimentation rate (ESR) is part of DAS 28-4 activity diagnosis, with low correlation with EA and that does not discriminate the contribution of cell factors that increase aggregation. OBJECTIVE: To analyse cell and plasma factors that might be involved in EA increase, to understand how RA affects blood components, thus modifying blood fluid behavior. METHODOLOGY: One hundred women presenting active RA were compared with age-matched controls (C). EA was measured by transmitted light, obtaining two parameters: 2k2n0, characterizing the aggregation process kinetics and s0/n0, estimating aggregates size. Cell factors assays: erythrocyte deformability, by filtration through nucleopore membranes, cell shape, by microscopy, and membrane fluidity by EPR. Plasma: total proteins and CRP, albumin, fibrinogen (Fb), by gravimetry, and IgG and IgM by single radial immuno-diffusion. RESULTS: AR and C (x+/-SE). 2k2n0: 31.83+/-2.84, 23.75+/-1.91; s0/n0: 0.92+/-0.05, 0.87+/-0.04. Rigidity index (RI): 14.79+/-4.71, 6.92+/-1.31. Morphological index: 0.28+/-0.03, 0.30+/-0.05, n.s. Fb (mg/dl): 382+/-80, 299+/-70. IgG (mg/dl): 1580+/-219, 1296+/-158; IgM (mg/dl) 233+/-28, 183+/-23; albumin (g/dl) 3.84+/-0.44, 3.77+/-0.51 n.s. p<0.05 accepted. Correlations: 2k2n0 vs. Fb r=0.66; s0/n0 vs. Fb r=0.51; 2k2n0 vs. Igs r=0.65; s0/n0 vs. Igs r=0.56. 2k2n0 vs. RI r=-0.59; s0/n0 vs. RI=-0.52, p<0.05. CONCLUSIONS: Plasma factors, Igs and Fb increased aggregation, since RI is altered, this reduces the process efficiency regarding aggregation. Patients with active RA present an increased EA, with values modifications associated with the activity index DAS 28-4, thus becoming an RA activity indicator.

In vitro effect of aluminium upon erythrocyte membrane properties.

The link between aluminium (Al(III)) and a range of disorders in organisms (plants and animals including human beings) has been stated in diverse studies. As regards as human beings in particular, there are numerous studies on this metal's toxicity in relation to pathological processes. Only few references to the metal's effect upon cell rheological properties can be found. In this study, we present evidence for alterations in the rheological properties of cells as consequence of the Al(III)'s interaction with human red blood cell membrane. Al(III) could damage membrane functions of the red blood cell by favouring lipid peroxidation reactions due to the presence of Fe(II) as an initiator. The metal's effect on lipid bilayer, and probably on the cytoskeleton as well, would constitute the cause for the impaired erythrocyte rheology.

Study on membrane fluidity and erythrocyte aggregation in equine, bovine and human species.

The aim of the present paper is to analyze whether membrane fluidity can be predicted from its lipid composition and to assay the possible relationship between such variable and the aggregating properties of erythrocytes from equine, bovine and human species due to the widely acknowledged differences in their tendency to form aggregates. The main difference between phospholipids from plasma membrane in these species lies in the concentration levels of sphyngomyelin (SM) and phosphatidilcoline (PC); more precisely, in the external hemilayer of the lipid bilayer. Membrane fluidity was estimated by the fluorescence polarization method, while erythrocyte aggregation was assessed by an optical method. According to our results, bovine erythrocytes containing high SM and low PC levels, presented the highest anisotropy value as well as an imperceptible aggregation value. Equine erythrocytes, which contain a considerable PC percentage and scarce SM levels, showed the lowest anisotropy value and the highest values of the aggregation parameters. Human erythrocytes presented intermediate values for both properties. Our hypothesis claims that the phospholipid composition would constitute one of the factors determining erythrocyte membrane fluidity and also taking part in the different aggregation tendency shown by equine, bovine and human species.

Acute training in racing horses at two different levels of effort: A haemorheological analysis.

During acute exercise several significant cardiovascular alterations take place, along with possible physiopathological consequences, such as Exercise Inducted Pulmonary Haemorrhage (EIPH). Given the relevance of blood rheology in the determination of flow resistance and its supposed participation in the stated events, the present paper focuses on the analysis of modifications in haematocrit, blood viscosity and erythrocyte deformability in Thoroughbred horses caused by two different levels of effort (6 to 9 m/s and 13 to 16 m/s), in the same track they train in and with their own jockeys. The results obtained show a deep modification in those parameters, as consequence of the exercise. However, no significant discrepancies were observed between the different levels of effort in which the training was performed.

Effect of ascorbic acid based amphiphiles on human erythrocytes membrane.

6-O-alkyl ascorbic acid esters (ASCn) are amphiphilic molecules that behave as surfactants in aqueous solution. These compounds show physico-chemical and aggregation properties that depend on the alkyl chain length, pH and temperature. It must consider that ASCn have shown some physical and rheological properties that suggest a potential utility as drug carriers. The present paper aims to evaluate the effects of these surfactants on human erythrocyte membranes. The membrane properties studied were: osmotic resistance in hypotonic media, shape transformation, and vesicle release at lytic concentration. According to our results, all properties depended on the length of the hydrophobic chain and they did not evolve monotonically. Finally, the study of ASCn interaction with erythrocyte membrane allowed us to postulate the crucial influence that the molecular structure exerts upon the manner in which amphiphiles interact with biological membranes and the effects involved in them.

Study of intrinsic flow properties at the normal pregnancy second trimester.

During normal pregnancy the cardiovascular system undergoes extensive changes. In a previous work we demonstrated the role of the haemorheological profile as predictor of hypertensive gestational disorders through a retrospective study. In an attempt to clarify the rheological characteristics during normal gestation, blood and plasma viscosity, erythrocyte deformability and aggregation, and plasma fibrinogen levels were measured at second trimester of pregnancy. The interrelationships of different haemorheologic parameters, were analysed by Pearson correlation coefficient. The results showed decreased erythrocyte deformability, and increased plasma viscosity and erythrocyte aggregation due to increased fibrinogen in pregnant women. Despite these modifications blood viscosity did not increase, but there was a decrease in relative blood viscosity, therefore, a profile in accordance, from the haemorheological point of view, with the classical concept stating that haemodilution is of the utmost importance to maintain an adequate microcirculation in the uteroplacental unit during normal gestation.

Haemorheological variables as a rheumatoid arthritis activity indicator.

OBJECTIVE: To investigate if blood hyperviscosity in RA patients is due to a reduced erythrocyte deformability and, therefore, turning it into a reliable activity indicator, as well as a therapy follow-up marker for this pathology. METHODS: (1) The haemorheological profile consisting of erythrocyte deformability, blood and plasma viscosity, and erythrocyte membrane fluidity was determined in 24 AR patients and 17 healthy controls. (2) A 4 year follow-up was carried on in 16 patients monitoring blood viscosity, erythrocyte deformability and biochemical variables in relation to clinical assessment of disease activity (Disease Activity Score "DAS 28-4"). RESULTS: Erythrocyte deformability and membrane fluidity were impaired in RA patients compared to controls (p<0.001). Blood viscosity was significantly increased and correlated with the cell rigidity index (r=0.85, p<0.0000) in RA patients. The follow-up showed a good correlation between haemorheological parameters and DAS 28-4 during disease evolution. CONCLUSION: our results support the hypothesis that in RA, blood hyperviscosity is determined by deformability loss, which in turn is due to a membrane rigidization. This could evidenced that a widespread cell membrane damage is expressed through an impaired erythrocyte deformability, turning haemorheological parameters into reliable tools to study disease evolution. The follow-up study enabled us to confirm that erythrocyte deformability is an efficient indicator of rheumatoid arthritis activity.

Proposal of a haemorheological profile for early detection of hypertensive gestational disorders.

The objective of this study was to emphasise the potential power of simple and inexpensive haemorheological tests as predictors of hypertensive gestational disorders through a retrospective study. Blood samples of 195 primigravids with gestational age 18-23 weeks were studied. For data processing pregnant women were allocated into 3 groups, based on difference of SBP and DBP values measured at first and last consultation, and presence of proteinuria and oedema: Normotensive pregnants (n=149), hypertensive pregnants; n=26 and preeclamptic pregnants; n=20. Whole blood viscosity, plasma viscosity, erythrocyte rigidity and aggregability, haemoglobin, hematocrit, total protein, albumin and fibrinogen were assayed. Increased relative viscosity (eta(r)) (p<0.01), decreased erythrocyte deformability (p<0.05), lower O(2) release (p<0.01) and birth weight (p<0.01), showed a negative correlation with filterability index in preeclamptics. There was a decrease of erythrocyte deformability in hypertensive women. Erythrocyte deformability and blood viscosity could be an early indicator in preeclamptics, and erythrocyte deformability in hypertensive ones, therefore they could be considered alert factors in order to decide a thorough control in these patients to prevent further complications.

Effect of hormone replacement therapy upon haemorheological variables.

It is already admitted that hormone replacement therapy (HRT) decreases the risk of developing cardiovascular disease, although its mechanism is not clear yet. In the present work, the effect of the HRT upon cellular and plasmatic haemorheological factors determining blood flow properties: blood viscosity, plasma viscosity, plasma fibrinogen, rigidity erythrocyte index and erythrocyte aggregation rate was studied. Menopausal women were followed through a whole year of HTR. Results demonstrate that after six months of treatment there is a diminution in relative blood viscosity and erythrocyte rigidity, with constant values along the second semester. Erythrocyte aggregation, plasmatic and blood viscosity diminution observed during the treatment can be explained by the simultaneous plasma fibrinogen decrease. Modified cellular and plasmatic rheology could produce beneficial effects on blood flow, particularly in microcirculation, presenting a possible mechanism by which HTR decreases the risk of cardiovascular disease development during menopause.

Haemorheological variables in a rat model of hypertriglyceridaemic obesity and diabetes.

Obesity is a heterogeneous condition of variable aetiology, generally associated with pathologies such as arterial hypertension, hyperlipidaemia, diabetes and cardiac disease. These conditions, either themselves or because of the various treatments used, may further modify blood rheology in an arbitrary manner. Therefore, analyses of changes in the blood rheology induced by obesity in humans have had differing and controversial results. In our laboratory, a model of hypertriglyceridaemic obesity is provided by an inbred rat strain; the beta genotype from the IIMb/Fm strain, presenting a syndrome of moderate obesity with apparent peripubertal onset, associated with hypertriglyceridaemia and glucose intolerance that turns into diabetes. The alpha genotype, originated from the same IIM/Fm stock, represents the control. The present study describes a comparative analysis of the variables determining the rheological behaviour of the blood in obese and control strains. Our results, agreeing with some other studies performed in humans, confirmed the haemorheological changes associated with obesity, and the fact that these changes became more evident in the presence of pathologies such as diabetes. It appears that triglyceridaemia. cholesterolaemia and hyperglycaemia may influence the rheological behaviour of the cell membrane and this damage may provoke a decrease in erythrocyte deformability and, consequently, hyperviscosity of the blood.

Influence of adsorbed plasma proteins on erythrocyte rheological properties: in vitro and ex vivo studies.

The influence of plasma protein adsorption on the mechanical properties characterizing erythrocyte behaviour under flow was studied in human and rats. The deformability index, elastic modulus and surface viscosity were measured by laser diffractometry. In in vitro studies, human and rat erythrocytes were washed to remove their original protein coating, and then incubated in saline-diluted plasma media. For erythrocytes incubated in the most diluted solutions (plasma/saline 1:3, v/v), the deformability index increased 30% for both species (human, P<0.01 and rat, P<0.2); the elastic modulus decreased 20% (human, P<0.05) and 60% (rat, P<0.01); and surface viscosity decreased 20% (human, P<0.05) and 40% (rat, P<0.01), relative to values for erythrocytes incubated in pure plasma. Ex vivo experiments were performed using rats. Plasma proteins were diluted replacing 15% volemic plasma by saline in three consecutive plasmapheresis steps. The rheological properties of erythrocytes, tested after each step, followed the general trends of the in vitro pattern. These results suggest that the decrease in plasma protein concentration affects blood rheology in two ways. The first is the well known decrease in plasma viscosity, and the second is an improvement of erythrocyte deformability, as has been shown in this work. Thus, a new argument supporting the benefits of normovolemic hemodilution in patients with poor peripheral perfusion is provided.

Influence of plasma proteins on erythrocyte aggregation in three mammalian species.

The aggregation capacity of human erythrocytes lies between that of the non-aggregating bovine erythrocytes and the remarkably aggregating equine ones. As the ability to aggregate is attributed to cell factors and the composition of the plasma proteins, the role that plasma proteins play in the aggregation process in these three species was studied. Washed erythrocytes were suspended in phosphate-buffered saline (PBS; pH 7.4, 300 mOsm/L) plus polyvinylpyrrolidone (PVP) in a suitable concentration to obtain an average intensity of aggregation (control media). The superimposed effect of replacing 80% of the medium by either autologous plasma, serum or albumin solution was studied. The plasma proteins appeared to enhance aggregation by human and equine erythrocytes, but impaired this process in bovine erythrocytes. Some evidence was obtained supporting the existence of serum factors capable of reducing aggregation of erythrocytes in cattle and it was concluded that the non-aggregating behaviour of bovine erythrocytes may be due to the cells interacting particularly with the macromolecules in the serum.

Effect of tetracaine chlorhydrate on the mechanical properties of the erythrocyte membrane.

Pharmacologically active agents that locate in the cell membrane are useful tools to investigate the interactions taking place between its molecular components. In the present work, the effect of tetracaine chlorhydrate (Tc) on the membrane mechanical properties of intact and desialated erythrocytes was studied. Our results evince the complex interaction between the drug and the membrane structures. The effect of Tc on erythrocyte shape suggests that this drug locates in the inner hemilayer of the lipid bilayer. Since Tc also modifies osmotic fragility and mechanical properties ascribed to the cytoskeleton, it can be inferred that the lipid bilayer has an effect on the rheology of the membrane, in a direct or indirect way, in this case through the close interaction with the structural proteins. Moreover, our results support the hypothesis of a second localization of the drug in the membrane, i.e., as monovalent cations intercalated among the glycocalix sialic endings, where it generates an effect superimposed on that produced from its typical site in the lipid bilayer.

Kinetic model for erythrocyte aggregation.

It is well known that light transmission through blood is the most widely utilized method for the study of erythrocyte aggregation. The curves obtained had been considered empirically as exponential functions. In consequence, the process becomes characterized by an only parameter that varies with all the process factors without discrimination. In the present paper a mathematical model for RBC aggregation process is deduced in accordance with von Smoluchowski's theory about the kinetics of colloidal particles agglomeration. The equation fitted the experimental pattern of the RBC suspension optical transmittance closely and contained two parameters that estimate the most important characteristics of the aggregation process separately, i.e., (1) average size of rouleaux at equilibrium and (2) aggregation rate. The evaluation of the method was assessed by some factors affecting erythrocyte aggregation, such as temperature, plasma dilutions, Dextran 500, Dextran 70 and PVP 360, at different media concentrations, cellular membrane alteration by the alkylating agent TCEA, and decrease of medium osmolarity. Results were interpreted considering the process characteristics estimated by the parameters, and there were also compared with similar studies carried out by other authors with other methods. This analysis allowed us to conclude that the equation proposed is reliable and useful to study erythrocyte aggregation.

Red blood cell shape as a function of medium's ionic strength and pH.

Glycocalyx, the characteristic first line of interaction between membrane and environment, can be visualized as a polyelectrolyte anchored to a bending-resistant matrix. This structure has an amazing resemblance with the ionized monolayers, in which, the cohesion among hydrocarbon chains is counteracted by the repulsion among similarly charged ionic heads, and thus the balance determines the curvature of the membrane. Likewise, it could be assumed that in biological membranes, repulsion among similarly charged groups in the glycocalyx could generate different curving trends. Hence, the factors directly influencing the electrostatic interaction among surface charged groups were studied, assessing the effect of the medium's ionic strength (mu) and pH, in an extensive range of values around the physiological one. The results point out mu variations inducing different shapes, depending on whether mu values were lower or higher than the physiological ones; which could be explained by the polyelectrolyte theory. The occurrence of more invaginated shapes as the medium's pH decreases, and the opposite event, when the pH increases, could be attributed to the coupling between the dissociation of the glycocalyx ionic groups and the H+ concentration. The behavior of the cells with reduced surface charges (by neuraminidase degradation) supports the hypothesis that the observed mu and the pH effect on erythrocyte shape could be mediated by glycocalyx charged groups.

Effects of an amphipathic drug on the rheological properties of the cell membrane.

Sodium thiopental, as other amphiphilic molecules, interacts with the membrane by inserting into the lipid bilayer and causing alterations of the membrane properties such as curvature and hypotonic lysis resistance. But can it modify the mechanical properties of the membrane? In the present work it was observed that sodium thiopental affected the membrane rheological properties by improving erythrocyte deformability; this effect resulted from a reduction of both the elastic modulus and surface viscosity. In erythrocytes devoid of sialic acid after treatment with neuraminidase, sodium thiopental membrane concentration was significantly higher than in normal cells, suggesting that drug access to the lipid bilayer be facilitated by the absence of the steric and electrostatic barrier of the glycocalyx negative charges. From a rheological point of view, desialated and normal cells showed the same response to the anesthetic as regards elastic modulus but in opposite direction if surface viscosity was considered. This finding supports the hypothesis that sodium thiopental molecules enter the bilayer of desialated cells in a higher proportion, as compared to the normal erythrocyte, promoting a disorganization that results in a greater inner friction. The changes in the rheological parameters, triggered by sodium thiopental, could be attributed to the bilayer contribution to the membrane mechanical properties, either directly or through interaction between the bilayer and the cytoskeleton.