The effect of malonyldialdehyde on erythrocyte deformability

The addition of malonyldialdehyde (5-20 micro M) to human erythrocytes results in a marked decrease in cellular deformability as measured with a counter-rotating, cone plate Rheoscope when low shear forces (2.5-25 dynes/sq cm) are applied. At high shear forces (125-500 dynes/sq cm), malonyldialdehyde at 5 micro M had no effect on deformability, although at concentrations of 10 and 20 micro M a small but statistically significant decrease was evident. These effects of this crosslinking agent are observed in the absence of alterations in cell volume and intracellular viscosity. The results obtained are in accord with the view that the polymerization of membrane constituents may contribute to the events that lead to the removal from the circulation of either aging cells or cells exposed to peroxidation-initiating agents.     

Red blood cell deformability in iron deficiency anaemia

In patients with iron deficiency anaemia (IDA) it has been suggested that the shortened erythrocyte lifespan may be in part due to decreased erythrocyte deformability. In order to know whether erythrocyte deformability is decreased in IDA patients, we have determined the erythrocyte Elongation Index (EI) by means of ektacytometric techniques (Rheodyn SSD, Myrenne Gmbh, Germany), in 50 IDA patients and 100 well age and sex matched healthy controls. At the three shear stresses tested, 12, 30 and 60 Pa, IDA patients show statistically lower EI than controls (37.4+/-6.7 vs 48.6+/-2.9; 45.0+/-6.0 vs 54.5+/-2.8; 48.7+/-5.8 vs 57.0+/-2.9 mPa.s, respectively; p<0.001). A statistically significant correlation was found between EI at 12, 30, and 60 Pa and the hematimetric indices (MCV, MCH and MCHC), suggesting that the alteration in surface/volume ratio (shape) which characterizes this kind of microcytic hypocromic anaemia, accounts in part for the decreased EI. Rheodyn SSD, as an ektacytometric technique, is very sensitive to alterations in red blood cell geometry, for what seems to be a useful tool for detecting diminished erythrocyte deformability in IDA patients.     

The influence of temperature on red cell deformability.

This study was undertaken to examine the influence of temperature on physical properties of red cell membranes. Red cells adhering to cover slips were subjected to fluid shear stress in a rotating disc apparatus for 1 min or for 10 min at temperatures ranging from 2 degrees to 50 degress C. They were fixed while subject to shear stress by addition of glutaraldehyde and then processed for examination and photography by reflected-light microscopy. Cell dimensions were obtained with a computerized planimeter. At shear stresses under 2 dynes/sq cm, cells changed shape from biconcave discs to tear drops, the dimensions of which were influenced very little by temperature or duration of shear stress. Above 2 dynes/sq cm, filamentous processes or "tethers" developed at attachment points of cells to cover slips. Tether length and the percentage of cells possessing tethers increased markedly with increasing temperature and duration of shear stress. At approximately 48 degrees C, a dramatic change occurred over a narrow temperature range such that cells were markedly elongated and irregularly deformed by a shear stress of 1 dyne/sq cm or less. These observations demonstrate that elongation of human red cells subjected to fluid shear stress in a rotating disc system is markedly influenced by temperature as well as by magnitude and duration of shear stress. They also indicate that significant increases in red cell membrane fluidity occur between 2 degrees and 24 degrees-37 degrees C and again between 48 degrees and 50 degrees C.     

Effect of stress on erythrocyte deformability, influence of gender and menstrual cycle

This study aimed to investigate the effect of acute mental stress on erythrocyte deformability (ED) in women during different phases of the menstrual cycle and to compare the results with men. For this purpose, healthy males (n=10) and females (n=10) (during follicular and luteal phases) underwent Stroop color-word interference and cold pressor tests. Hemoglobin, hematocrit and leukocyte counts before and after this stress test revealed no difference in either group; erythrocyte sedimentation rate was significantly lower in the post-test samples in all. In all groups erythrocyte filtration time was significantly higher and thus ED was significantly lower after the stress test (mean+/-SEM, PRE-TEST: follicular: 3.08+/-0.05; luteal: 2.07+/-0.05; men: 2.9+/-0.05) (POST-TEST: follicular: 4.5+/-0.07; luteal: 4.1+/-0.07; men: 4.39+/-0.1). ED was appreciably influenced by gender and menstrual cycle. Women at the luteal phase had better ED compared to both women at the follicular phase and men, the effect being especially pronounced in the pre-test samples. Our results suggest that stress may induce cardiovascular diseases by lowering ED in both genders. The effect of stress on ED varies with gender and during different phases of the menstrual cycle, which may be explained by variations in the sex hormones.     

Influence of P. vivax malaria on erythrocyte aggregation and deformability

Malaria due to Plasmodium vivax affects blood flow in cardiovascular system. The present work is aimed to determine the variability of erythrocyte deformability and aggregation in malaria patients in comparison with that of normal subjects. Blood samples of malaria patients (n=16), selected based on occurrence of parasitaemia, are categorized into low (LP), medium (MP) and high parasitaemia (HP), which represent increasing levels of the disease severity. For measurement of aggregation and deformability, by laser aggregometer and optical hemorheometer, the erythrocyte suspensions are prepared in plasma and physiological saline at hematocrit 5%. The results show that the erythrocyte deformability is significantly reduced (p<0.001) with the increasing level of parasitaemia. Despite this decrease in deformability, the aggregation parameters are significantly increased, indicating the occurrence of hyper-aggregation, particularly in MP and HP, due to involvement of various factors. These changes may partly be contributing in the observed changes in blood flow in the microcirculation.     

Influence of calcium permeabilization and membrane-attached hemoglobin on erythrocyte deformability.

The present study was designed to evaluate the influence of intracellular calcium [Ca]i regulated membrane attached hemoglobin (Hbm) on the deformability of human RBC and ghosts. [Ca]i of RBC was elevated via the ionophore A23187 (10 microM); the deformability of RBC and resealed ghosts was determined via measuring RBC and ghost transit times through 5 microns diameter pores with the Cell Transit Analyzer (CTA). Salient results included: (1) significantly increased RBC levels of Hbm following ionophore treatment; (2) elevated Hbm with increasing lysing medium calcium concentration (0-5 mM); (3) decreased deformability of both intact RBC and ghosts with increasing Hbm and significant (P less than 0.02 or better) linear relationships between Hbm and RBC or ghost transit times; and (4) an increased sensitivity to ionophore treatment/membrane attached hemoglobin for the higher percentiles of the CTA transit time distribution (i.e., for more rigid subpopulations). Our results thus indicate that calcium-induced interaction of hemoglobin with the RBC membrane produces cellular rheological changes; in addition, they demonstrate the usefulness of the CTA system in measuring both average RBC rheologic behavior and the distribution of cellular rheologic properties within an erythrocyte population.     

The importance of circadan rhythm alterations in erythrocyte deformability

The hormone melatonin, secreted from the pineal gland at night and suppressed during the day, provides a circadian and seasonal signal to the organism. The impact of pharmacological doses of melatonin on erythrocyte deformability was investigated by our group in several studies in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of alterations in the physiological melatonin levels via the circadian rhythm on erythrocyte deformability. 50 male rats weighing 250-300 g were divided in 5 groups. The rats were subjected to 12/12, 24/0, 0/24, 16/8 and 8/16 h of Light/Dark (L/D) cycle, respectively. The elongation indexes (EI) of the erythrocytes were measured by a laser diffractometer (Myrenne Rheodyne SSD) by using 30 microl of whole blood suspended in 2 ml of Dextran 60. There was no significant difference in the EI of the 24/0 h L/D group compared to the control (12/12), whereas the decrease of EI was statistically important in the 0/24 h L/D group (p = 0.009). This decrease in EI was also significant when this group was compared to the 24/0 h L/D group (p = 0.05). Furthermore, the EI was affected significantly by alterations in the circadian rhythm, compared to control (16/8, 8/16 h L/D; p = 0.05 and p = 0.007, respectively). As a result, the alterations in physiological melatonin levels via different circadian rhythms have significant impacts on the deformability of erythrocytes, which therefore may cause important cardiovascular implications in the people who are exposed to different light dark cycles. Furthermore, these data represents a new and a quite crucial open-field to be investigated and taken into account in in vivo hemorheological studies.     

肝硬化患者红细胞变形性及其影响因素

用 L BY- BX红细胞变形聚集仪检测 ED,采用 SDS-聚丙烯酰胺凝胶电泳分离红细胞膜蛋白并分析带3蛋白含量 ;取适量患者红细胞检测红细胞丙酮酸 - Cl- 交换速率常数。以红细胞 ATP和 2 ,3-二磷酸甘油酸 (2 ,3-DPG)含量作为衡量红细胞能量代谢状态的指标 ,观察 ATP和 2 ,3- DPG含量的变化 ,并与正常人比较。结果 :肝硬化患者 ED明显降低 ;凝胶电泳显示患者组带 3蛋白分布较窄 ,着色减弱 ,扫描图谱显示带 3蛋白的浓度峰明显低于对照组 ;患者组红细胞丙酮酸 - Cl- 交换速率常数明显低于对照组 ;肝硬化患者 ATP含量正常而 2 ,3- GPD含量明显增加。认为红细胞膜带 3蛋白含量减少及功能降低是肝硬化患者 ED降低的重要原因     

Modulation of erythrocyte deformability by PKC activity

The interactions between membrane, peripheral and cytoskeleton proteins are responsible for the maintenance of erythrocyte deformability (EEI) and some of these interactions are modulated by PKC activity. Protein band 3 of the erythrocyte membrane is phosphorylated by phosphotyrosine kinases (PTK) and dephosphorylated by phosphotyrosine phosphatase (PTP). It was previously described by us a signal transduction mechanism that describes a possible pathway connecting an erythrocyte external membrane protein, acetylcholinesterase (AChE), with protein band 3. So how does PKC activity modulate EEI when protein band 3 is phosphorylated or dephosphorylated in absence or presence of AChE effectors?To answer this we used phorbol 12-myristate 13-acetate (PMA) as an activator and chelerythrine chloride as inhibitor of PKC and also band 3 modulators of band 3 phosphorylation degree, in presence and absence of AChE effectors in order to measure in whole blood samples EEI. Our results showed that erythrocyte deformability was significantly (i) decreased by inhibition of PKC, in absence and presence of AChE inhibitor velnacrine (ii) increased with PMA in absence and presence of ACh and (iii) decreased in presence of calpeptin in absence and presence of either chelerythrine or PMA. These results establish dependence between cytoskeleton proteins, PKC activity, band 3 phosphorylation degrees and EEI. Better understanding of those proteins interactions on transduction mechanisms might trigger possible targets for drug action that would modulate EEI.     

Effect of anti-spectrin antibody and ATP on deformability of resealed erythrocyte membranes.

Deformability of resealed erythrocyte membranes was measured by using an ektacytometer. Divalent anti-spectrin antibody, but not monovalent anti-spectrin Fab fragments, decreased membrane deformability. Membranes resealed with MgATP were more deformable than those without MgATP. Exogenous alkaline phosphatase, which dephosphorylates spectrin, decreased membrane deformability. These results suggest that spectrin is an essential component of the system that determines erythrocyte deformability. They are consistent with the view that the role of ATP in membrane deformability is mediated through phosphorylation of the spectrin.     

The clinical importance of erythrocyte deformability,a hemorrheological parameter

Summary Hemorrheology, the science of the flow behavior of blood, has become increasingly important in clinical situations. The rheology of blood is dependent on its viscosity, which in turn is influenced by plasma viscosity, hematocrit, erythrocyte aggregation, and erythrocyte deformability. In recent years it has become apparent that the shape and elasticity of erythrocytes may be important in explaining the etiology of certain pathological situations. Thus, clinicians have become increasingly interested in hemorrheology in general and erythrocyte deformability in particular. In the course of time, many clinical studies have been performed, but no concise review has thus far been published. This article encompasses a review of the clinically based literature on this subject.     

Some factors affecting erythrocyte deformability and rupture

Human erythrocytes suspended in a viscous isotonic saline medium may be ruptured in vitro by the application of uniform hydrodynamic shear stresses. For any given combination of exposure parameters, cellular fragility is a measure of cellular deformability; i.e., it is related to the cellular dimensions that result from the competition between those forces that tend to extend the cell and those forces--particularly those associated with the resistance to bending of the membrane--that tend to restore the cell to its original biconcave shape. The pharmacologically active agent nicotinic acid increases cellular fragility in our experimental system and so has presumably increased the deformability of the human erythrocyte.     

Erythrocyte deformability and erythrocyte aggregation in preeclampsia

One of the features of preeclampsia is impaired blood rheology due to altered erythrocyte aggregation and erythrocyte deformability. We investigated these two parameters which affect the viscosity of blood, along with serum and intraerythrocytic magnesium concentrations, immunoglobulin titres and fibrinogen concentration in 12 preeclamptic women. Eighteen (18) other non-preeclamptic, gestation-matched women acted as controls. Erythrocyte deformability, expressed as elongation index (EI), and erythrocyte aggregation expressed as aggregation half-time (t 1/2) were measured with the Laser-assisted Optical Rotational Cell Analyser (LORCA). Serum and intraerythrocytic magnesium concentrations were analysed by atomic absorption spectrometry, immunoglobulin titres by radial immunodiffusion and fibrinogen concentration by a clot weight technique. There was no statistically significant difference in these parameters between preeclamptics and controls suggesting that erythrocyte deformability and aggregation as well as serum and intraerythrocytic concentrations, fibrinogen levels and immunoglobulin titres are not altered in preeclampsia. Further investigations are required in severe preeclampsia and in preeclamptic women taking magnesium sulphate supplement.     

Changes in erythrocyte deformability in Alzheimer's disease

In a previous work we showed an alteration of erythrocyte filtration ability in patients with Alzheimer's disease according to their age and illness duration. This study has for aim to find a criteria of deformability that would be constant in all Alzheimer patients and would show a modification of red cell membranes. The erythrocyte filterability was studied in this present paper, in accord to Reid and Dormandy method using two values of depression (5 and 0 cm of water). These depressions correspond to the physiological values of blood pressure at the level of precapillary and capillary systems. The ratio between the result obtained at 5 cm and the result at 0 cm is constant in normal patient without organic disease and it is independent of age. At the opposite, this ratio increase very significantly in all Alzheimer patients, and this is not correlated to the initial value of filtrability. This ratio could be an index of the alteration of red cell membranes.     

Microrheologic investigation of erythrocyte deformability in diabetes mellitus

This study was undertaken to determine whether diabetes alters the viscoelastic properties of erythrocytes. The oldest and youngest 10% fractions of circulating red cells were separated by centrifugation of freshly drawn blood obtained from ten diabetics with disease of one to 20 years' duration and from an equal number of age- and sex-matched control subjects. Cells from each fraction were suspended in phosphate- buffered saline, and their rheologic behavior was examined in a rheoscope. The elongation of cells, the percentage of cells that tank- treaded in response to shear stress, tank-treading frequency, and the rate of recovery of cell shape upon cessation of shear stress were determined in the oldest and youngest 10% of cells for diabetics as well as for controls. All four parameters were virtually identical for diabetics and controls. Additional aliquots of cells were taken for assessment of nonenzymatic glucosylation of hemoglobin and cell membrane protein. The absence of any measurable difference in rheologic behavior of cells from diabetic and control subjects, despite substantial differences in nonenzymatic glucosylation of hemoglobin and cell membrane proteins, suggests that the magnitude of glucosylation observed in these cellular constituents does not alter the viscoelastic properties of the cells. The implication of these observations is that microvascular complications of diabetes are not attributable to altered deformability of red cells.     

Role of erythrocyte deformability in erythrocyte transit time and bioavailability of O2

The bioavailability of oxygen and its transfer towards all of the cells of the body essentially depends on the flow rate of red blood cells in the capillaries, which often have a smaller diameter than the red cell itself. The authors discuss the results obtained for the mean transit time of erythrocytes, based on microfiltration through artificial polycarbonate capillaries with a diameter of 5 microns, a length of 13 microns and a density of 4.10(5) pores/cm2.     

Techniques to evaluate erythrocyte deformability in diabetes mellitus

Abstract. Using several rheological techniques, we examined erythrocyte deformability in different groups of diabetic subjects. The macrorheological techniques used for this evaluation were respectively whole-blood filtration, filtration of erythrocyte suspensions, polyviscosimetry and diffractometry. Whole-blood filterability, at a negative pressure of 20 cm water, was decreased in type 2 diabetics; no difference was evident at a negative pressure of 10 cm water. The filtration of erythrocyte suspensions at low haematocrit (5%) did not show differences between normal and diabetic subjects. Polyviscosimetry, which explores the filterability of erythrocyte suspensions at high haematocrit (80%) through wide pores, demonstrated an impaired behaviour especially in type 2 diabetics. Diffractometry, which measures erythrocyte elongation induced by a defined shear stress through the diffraction pattern of a laser beam, showed an alteration in type 1 diabetic subjects. The microrheological methods employed for this evaluation were those based on fluorescence spectroscopy. Labeling intact red blood cells with fluorescent probes, we determined the membrane dynamic properties and using these techniques we found a reduction of erythrocyte membrane fluidity and a decrease of red cell membrane protein lateral mobility.