Hemorheological in vitro action of propofol on erythrocytes from healthy donors and diabetic patients.

Drugs used during anesthesia might induce disturbance on microcirculation due to their systemic cardiovascular actions and to direct hemorheological effects. A comparative investigation of the hemorheological alterations related to in vitro propofol treatment of red blood cells (RBCs) from healthy and diabetic volunteers is presented here. Viscoelasticity and aggregation of RBCs from type 2 diabetic patients (DBT) and healthy donors (HD) were studied from RBCs incubated with propofol near steady-state concentration. 'S parameter', which measures the aggregation degree, was obtained using digital analysis of microscopic images. Erythrocyte viscoelasticity parameters were determined using an Erythrocyte Rheometer. Results obtained from DBT samples showed an increase of 10% or more in aggregation due to the propofol action. The phase shift between erythrocyte response and oscillating shear stress applied at 1 Hz was altered by propofol treatment of erythrocyte from HD and DBT. Propofol could produce slight alterations in the rheological behavior of erythrocyte from HD and DBT, at concentrations near those of steady state. Moreover, this anesthetic could induce an adverse effect in DBT, particularly on erythrocyte aggregation. The observed hemorheologic alteration would increase the possibility of microcapillary obstruction. Hence, this type of study [0] would prove relevant to avoid possible postoperative complications.

Pfaffia paniculata extract improves red blood cell deformability in sickle cell patients.

The aim of the present study was to test the effects of Pfaffia paniculata (PP) extract on the red blood cell (RBC) rheological properties of patients with sickle cell disease (SCD) and healthy (AA) individuals. Blood from 7 SCD and 4 AA individuals were collected in EDTA tubes. Washed RBCs were incubated with various concentration of PP extract: 0.0, 0.2 or 0.5 mg/ml of PP solution for 5 hrs at 37°C. RBC deformability was measured by ektacytometry at 9 shear stresses ranging from 0.3 to 30 Pa, and RBC aggregation properties were determined by laser-backscattered techniques. Because RBCs from SCD patients are fragile, a stability test was also performed to test for the fragility of RBC exposed to a constant shear stress (70 Pa) for 10 min. While RBC deformability was not improved by the use of PP extract in AA, we noted an improvement of this parameter in patients with SCD between the 0.0 and 0.5 mg/ml conditions. In contrast to AA RBCs, the fragility of SCD RBCs was not affected by PP extract. In conclusion, this study demonstrates the beneficial effects, in-vitro, of PP extract on the RBC deformability of SCD patients, notably at high shear stress (a shear stress condition usually found in capillaries).

PMCA activity and membrane tubulin affect deformability of erythrocytes from normal and hypertensive human subjects.

Our previous studies demonstrated formation of a complex between acetylated tubulin and brain plasma membrane Ca(2+)-ATPase (PMCA), and the effect of the lipid environment on structure of this complex and on PMCA activity. Deformability of erythrocytes from hypertensive human subjects was reduced by an increase in membrane tubulin content. In the present study, we examined the regulation of PMCA activity by tubulin in normotensive and hypertensive erythrocytes, and the effect of exogenously added diacylglycerol (DAG) and phosphatidic acid (PA) on erythrocyte deformability. Some of the key findings were that: (i) PMCA was associated with tubulin in normotensive and hypertensive erythrocytes, (ii) PMCA enzyme activity was directly correlated with erythrocyte deformability, and (iii) when tubulin was present in the erythrocyte membrane, treatment with DAG or PA led to increased deformability and associated PMCA activity. Taken together, our findings indicate that PMCA activity is involved in deformability of both normotensive and hypertensive erythrocytes. This rheological property of erythrocytes is affected by acetylated tubulin and its lipid environment because both regulate PMCA activity.

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.

Effect of Ligaria cuneifolia catechin- and quercetin-enriched fractions on hemorheology and plasma cholesterol.

UNLABELLED: We tested the in vivo and the in vitro effects of both Ligaria cuneifolia catechin- and quercetin-enriched fractions on erythrocyte shape and deformability, and on plasma cholesterol level. For in vivo studies, adult male Wistar rats were randomized in three experimental groups which received intraperitoneally, once a day, 3 days: CONTROL: saline solution (C; n = 6); catechin from L. cuneifolia, 0.60 mg/100 g body weight (CLc; n = 6), or quercetin from L. cuneifolia, 2.3 mg/100 g body weight (QLc; n = 6). For in vitro studies, blood samples obtained from male Wistar rats were divided into three fractions, which were incubated with saline solution (C), catechin (CLc; n = 5) and quercetin (QLc; n = 5), in a concentration equivalent to 0.60 mg/100 g body weight, and 2.3 mg/100 g body weight, respectively. CLc significantly reduced the rigidity index due to a diminished mean concentration volume. QLc induced erythrocyte rigidization (less deformability), thus increasing blood viscosity. Neither of the two treatments produced any changes in plasmatic or biliary excretion of cholesterol. Opposite results were observed in rigidity index with CLc and QLc. In vitro studies showed an interaction of both CLc and QLc with the erythrocyte membrane, which induced changes in the erythrocyte shape from discocyte to stomatocyte.

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.

Effect of disodium monofluorphosphate on plasma and blood viscosity in the rat.

Alpha macroglobulins (AM) are plasma proteins whose main function is to inactivate proteinases, protecting the tissues from the action of these enzymes. AM have influence on plasma viscosity (PV) and binds monofluorophosphate (MFP), which disturbs its homeostasis. The aim of this work was to evaluate whether the administration with MFP could modify blood viscosity. AM levels (micromol/l), PV (mPa.s), viscosity of red blood cells suspensions in NaCl 9 g/l (VES) and in autologue plasma (VEP) were measured in fifty-day old rats after a single dose of 80 micromol MFP or after 30 days of treatment with 80 micromol of MFP. Relative viscosity (RV) was calculated as the ratio VEP/PV. AM and PV increased significantly after 30 min of an oral dose of MFP. Controls (n=6), AM: 19.65+/-0.85, PV: 1.39+/-0.01, treated (n=6), AM: 22.88+/-0.75 (p<0.05), PV: 1.76+/-0.14 (p<0.05). After 30 days of treatment with MFP, AM and PV increased significantly. Controls (n=6), AM: 10.76+/-1.33, PV: 1.19+/-0.04, treated (n=6), AM: 17.66+/-1.27 (p<0.05), PV: 1.38+/-0.03 (p<0.05). The treatment with MFP modifies neither the VEP nor the RV. These results would indicate that AM and/or MFP did not interact with erythrocyte membrane and did not modify erythrocyte deformability.

Maximal exercise and lactate do not change red blood cell aggregation in well trained athletes.

Exercise and lactate usually change blood rheology, particularly red blood cell (RBC) deformability. The effect of lactate on RBC aggregation is unknown. The present study tested the in vivo effects of exercise on both lactate and RBC aggregation and the in vitro effects of lactate on RBC aggregation. Thirteen well trained athletes performed a progressive and maximal exercise test during which blood was sampled at rest, at 50% of maximal exercise, and at maximal exercise. RBC aggregation was assessed with the Myrenne aggregometer which gives two indexes of RBC aggregation: "M" (aggregation during stasis after shearing at 600 s(-1)) and "M1" (facilitated aggregation at low shear rate after shearing at 600 s(-1)). A part of the resting blood sample was also reserved to test the in vitro effects of three lactate concentrations (2, 4 and 10 mM). The lactate solutions were described in a previous study (P. Connes, D. Bouix, G. Py, C. Préfaut, J. Mercier, J.F. Brun and C. Caillaud, Opposite effects of in vitro lactate on erythrocyte deformability in athletes and untrained subjects, Clin. Hemorheol. Microcirc. 31 (2004), 311-318). The results demonstrated that M and M1 were unchanged with exercise and lactate. Therefore, lactate is able to change RBC deformability but not RBC aggregation.

Effect of the vehicle polyvinylpyrolidone and the Methanolic Fraction of Ligaria cuneifolia (Argentine Mistletoe) extract on hemorheological properties and biliary secretion in rats.

Ligaria cuneifolia (R et P) Tiegh. (Loranthaceae) (Lc) aqueous extract-treated rats by via intraperitoneal (i.p.) show increased blood viscosity and decreased plasma cholesterol (Chol) levels. In this work, we analize the effect of the vehicle polyvinylpyrrolidone (PVP) and that of the Methanolic Fraction of the extract of Lc (MFLc) on hemorrheological properties in vivo and in vitro and on biliary excretion. For in vivo conditions, adult male Wistar rats were divided in five experimental groups (n=5 each one) which were injected, every 24 hr during 3 days by via i.p., with: (1) saline solution (Control); (2) PVP 0.47 mg/100 g bw; (3) MFLc 0.95 mg/100 g bw plus PVP 0.47 mg/100 g bw; (4) PVP 12.5 mg/100 g bw; and (5) MFLc 23.0 mg/100 g bw plus PVP 12.5 mg/100 g bw. Intended for in vitro conditions, blood samples obtained by heart puncture were divided into three fractions, which were incubated with: saline solution (Control), PVP 12.5 mg%, and MFLc 25 mg% plus PVP 12.5 mg%. We demonstrated a direct effect of PVP alone and of MFLc "per se" on the erythrocyte membrane resulting in a cell shape change from dyscocyte to spherostomatocyte (MI more negative) as well as a decrease in erythrocyte deformability (increased RI). These changes induce an increase in blood viscosity. Decreased plasma Chol is a consequence of an increased bile salts biliary excretion.

Nifedipine effect on red cell rheological properties in patients with systemic scleroderma.

Systemic scleroderma is an autoimmune disease, due to a connective tissue alteration characterized by extracellular matrix increase in the skin and internal organs. It is already known that the Raynaud's phenomenon and the microcapillary obliteration lead to ischemia and peripheral tissue injury. The ischemia-reperfusion phenomenon releases free radicals, that react with red blood cells (RBCs) membrane components originating lipid peroxidation and impairment of the ATP-Ca(++) pump, two possible mechanisms responsible of disease pathogenesis. Nifedipine is a Ca(++)-channel antagonist that has been used for a long time in Raynaud's phenomenon treatment. In the present study we were able to demonstrate that erythrocyte deformability and two other related variables such as membrane fluidity and osmotic fragility improve significantly with nifedipine therapy. It is likely that nifedipine inhibiting cytoplasmic calcium accumulation could restore some red blood cell membrane properties.

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 hemorheological parameters following partial hepatectomy in rats with chronic aluminium intoxication.

The aim of our work was to analyze the hemorheological parameters following partial hepatectomy in rats with chronic Al-intoxication (Al). Male Wistar rats were randomly assigned into four experimental groups (n=6 each one): Sham (rats subjected to simulated surgery); Al+Sham; Partial Hepatectomy (animals subjected to 65% liver resection) and Al+Partial Hepatectomy. Our results show that both Partial Hepatectomy and Al treatment produce a decrease of plasma cholesterol level, which showed a negative association with Rigidity Index increase (r(s)=-0.6475, p<0.05). The increase of Rigidity Index observed in Partial Hepatectomy, Al+Sham and Al+Partial Hepatectomy could be related to the increase of the proportion of non-discocytic erythrocytes, particularly stomatocytes, which determines a diminution of the Morphological Index. In the Altreated groups, greater changes in Rigidity Index and Morphological Index were observed. The relative viscosity of blood at a standard haematocrit of 40% was increased in Partial Hepatectomy, Al+Sham and Al+Partial Hepatectomy as compared to Sham, due to erythrocyte rigidity. On the other hand, we observed that the increase of plasma fibrinogen concentration correlates with augmentation of plasma viscosity (r(s)=0.689, p=0.004) for all the experimental groups studied. The results indicate that both administration of Al and Partial Hepatectomy induce microcytic hypocromic anaemia in the rats reflected by a significant decrease of haematocrit, mean corpuscular volume and mean corpuscular haemoglobin concentration. From these results, we conclude that in partially hepatectomized, Al-overloaded rats the decrease in erythrocyte deformability may be an important factor leading to the installation of anaemia.

A dynamic and stationary rheological study of erythrocytes incubated in a glucose medium.

A higher than normal glucose concentration in a suspending medium may produce non-enzymatic glycosylation of erythrocyte proteins. This process can modify the viscoelastic properties of erythrocytes. In this paper, we studied the possible relationship between glucose concentration in a suspending medium and erythrocyte rheological parameters. Human venous blood was obtained from the antecubital veins of 10 healthy volunteers. Blood samples were anticoagulated with EDTA and centrifuged. Red blood cells (RBCs) were washed and subsequently divided in aliquots, which were incubated in vitro with glucose solutions of different concentrations. Dynamic and stationary viscoelastic parameters of RBCs were determined by laser diffractometry in an Erythrodeformeter. Aggregate shape parameter (ASP) of the RBCs was determined by digital image processing. Significant changes were observed both in ASP and in rheological parameters when the glucose concentration in the medium was increased, demonstrating that a glucose concentration as low as 1% induces alterations in the mechanical properties of RBCs.

Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease.

The deformability of erythrocytes is a critical determinant of blood flow in microcirculation. By capturing red blood cells (RBC) with optical tweezers and dragging them through a viscous fluid we were able to measure their overall elasticity. We measured, and compared, the RBC deformability of 15 homozygous patients (HbSS) including five patients taking hydroxyurea (HU) for at least 6 months (HbSS/HU), 10 subjects with sickle cell trait (HbAS) and 35 normal controls. Our results showed that the RBC deformability was significantly lower in haemoglobin S (HbS) subjects (HbSS and HbAS), except for HbSS/HU cells, whose deformability was similar to the normal controls. Our data showed that the laser optical tweezers technique is able to detect differences in HbS RBC from subjects taking HU, and to differentiate RBC from normal controls and HbAS, indicating that this is a very sensitive method and can be applied for detection of drug-response in sickle cell disease.

Hydration of sickle erythrocytes using a herbal extract (Pfaffia paniculata) in vitro.

Pfaffia paniculata (PP) is a perennial wild plant that grows in South America. Its root powder has been used by South American Indians for a variety of ailments and has been reported to have a salutary effect on sickle cell disease in Brazil. Its mechanism of action, however, is unknown. In this report, we present experimental data showing that PP improves the deformability of sickle cells, increases their Na+ content and their mean corpuscular volume (MCV). These findings indicate that PP functions as a sodium ionophore on sickle cells and improves their hydration status and rheological properties.

Morphologic and functional alterations of erythroid cells induced by long-term ingestion of aluminium.

Anaemia has been associated with aluminium (Al) accumulation in plasma and/or bone tissue in patients with chronic renal insufficiency. Nevertheless, in previous works, we have found shortened red-cell life span, increased osmotic resistance and inhibition of colony-forming units-erythroid (CFU-E) development in Al-overloaded rats with normal renal function. To elucidate further the action of Al on in vivo erythropoiesis, aluminium citrate was provided to Sprague Dawley rats (n = 18) in the drinking water for 8 months. Significant decreases in haematocrit (38.8 +/- 4.29 versus 43.1 +/- 3.58%, p < 0.05) and blood haemoglobin concentration (137 +/- 10.1 versus 148 +/- 8.5 g/l, p < 0.05), reticulocytosis (1.8/1.3-4.2 versus 1.2/0.4-3.7%, p < 0.05), and severe inhibition of CFU-E growth (670/120-950 versus 1530/810-2440 CFU-E/2 x 10(5) cells, p < 0.005) were found. Anysocytosis, poikilocytosis and schistocytosis were detected in peripheral blood stained films. Scanning electron microscopy revealed the presence of erythrocytes with abnormal shape, including crenated and target cells. Aluminium was localised specially inside the schistocytes by EDAX analysis. Decreased haptoglobin concentration (107/83-127 versus 139/89-169 mg/l, p < 0.05) supports the assumption of haemolytic nature of the anaemia. Rats were not iron depleted, as plasma iron concentration and total iron binding capacity were found in the range of control values, and sideroblasts and haemosiderin deposits were observed in bone marrow smears. Total 59Fe uptake and 59Fe incorporated to haem by the bone marrow cells were found decreased. In conclusion, the erythropoiesis impairment induced by Al may be a combined effect of direct action on circulating erythrocytes and interference with the cellular iron metabolism in erythroid progenitors.

Effect of complement on the rheological properties of the erythrocyte membrane.

The effect of complement (C) on the erythrocyte deformability was investigated. Sheep erythrocytes (E) were sensitized with specific antibodies (A) and treated with various doses of human C. Gravity filtration of unlysed EAC showed a C dose-dependent decrease in whole cell deformability, which was shown to be due to an impairment of the membrane rheological properties. As the fluidity of the lipid bilayer, sensed by a spin label, was not modified, the observed effects should be related to an interaction between C and the cytoskeleton. The influence of C3b and C3d in these processes seems unlikely, as similar hemagglutination titers were found, respectively, for anti-C3c and anti-C3d antisera against the varying EAC. The study of the degree of spontaneous hemolysis of EAC in buffers with solutes of different Stokes radii showed that differing-sized functional C lesions were produced, and that the formation of larger-sized lesions was favored by incubation with higher C doses. These results suggest that the insertion of proteins from the membrane attack complex, C5b-9 (MAC), into the erythrocyte membrane may be responsible for the effect of C on the rheological properties of the erythrocyte membrane.

Influencia del colesterol en membrana sobre la deformabilidad y la fragilidad osmótica eritrocitarias / Variation of erythrocyte deformability and osmotic fragility with cholesterol

La incorporación de colesterol (hemisuccinato de colesterol) a la membrana eritrocitaria afecta sus propiedades reológicas. Esta influencia ha sido evaluada a través de dos parámetros reológicos característicos de la membrana celular: la deformabilidad celular y la fragilidad osmótica. Para ello se aplicaron dos técnicas ambas basadas en el fenómeno de difracción láser, cada una de las cuales presenta diferente sensibilidad de respuesta. Se aplicaron dos protocolos de incubación (12 h a 25ºC el primero y 12 h a 25ºC + 12 h a 37ºC el segundo); en cada uno de ellos se fue variando la cantidad de colesterol incorporado, expresado como Tasa Colesterol/Proteína (Tcp = 0,18 a 0,83). La alteración de las propiedades reológicas de la membrana eritr

Complex viscoelasticity of normal and lectin treated erythrocytes using laser diffractometry.

A new method to find directly complex viscoelastic parameters (CVP) of red blood cells (RBC) is presented in this paper. Experimental determinations were carried out in an Erythrodeformeter (Rasia et al., 1986) operating in oscillating mode (0.5 to 3.5 Hz). The Erythrodeformeter performs direct determination of CVP of erythrocytes undergoing sinusoidal shear stresses by laser diffractometry. The measurements lead to the determination of mean values of the four components of erythrocyte complex viscoelasticity. The influence of the alterations induced on erythrocyte membrane by vegetable lactins (Ulex europaeus, wheat germ agglutinin and Enterolobium contorticilicum seeds) was analyzed to verify the sensitivity of this method. Differences observed between the CVP parameters of treated cells and the ones corresponding to control samples (non treated cells) are analyzed. Results obtained from cells treated with wheat germ agglutinin agree with observations published by Smith and Hochmuth (1982). Determinations of RBC complex viscoelasticity carried out by laser diffractometry could become an important tool to understand the influence of the factors associated with alterations of the rheologic properties of RBC membrane, which can affect the in vivo blood flow.