RESUMO
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.
Assuntos
Eritrócitos/metabolismo , Ácido Hialurônico/farmacologia , Viscossuplementos/farmacologia , Artrite Reumatoide/sangue , Viscosidade Sanguínea , Proteínas do Citoesqueleto/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Deformação Eritrocítica , Membrana Eritrocítica/metabolismo , Eritrócitos/citologia , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Artrite Reumatoide/sangue , Agregação Eritrocítica , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Deformação Eritrocítica , Feminino , Fibrinogênio/análise , Humanos , Imunoglobulinas/sangue , Pessoa de Meia-Idade , Fator Reumatoide/sangue , Índice de Gravidade de DoençaRESUMO
Microcirculatory alterations would explain focal lesions found in Chagas' cardiomyopathy. Trypanosoma cruzi (T. cruzi) infection induces host blood properties modifications and defensive responses capable of producing blood hyperviscosity, an ischemic risk factor able to affect microvascular blood flow. We studied whole blood viscosity (eta(b)) and plasmatic and cellular factors influencing it in rats, 7 and 14 days after experimental infection with T. cruzi. Increased plasma viscosity (eta(p)) was found in infected versus control rats and it was correlated with high blood parasite levels at 7 days and enhanced gamma-globulin fraction concentration at 14 days. The hematocrit, mean corpuscular volume (MCV) and eta(b) were higher in 14 days infected rats vs. 7 days and control animals. Also, electron microscopy observation showed morphological changes in red blood cells (RBC) at 7 and 14 days post-infection, with increased proportion of echinocyte and stomatocyte shapes transformation. In our rat model of Chagas' disease, BPL, increased plasmatic protein concentration, enhanced MCV and RBC shapes transformation would determine blood hyperviscosity, cause of microvascular blood flow abnormalities.
Assuntos
Viscosidade Sanguínea , Sangue/parasitologia , Doença de Chagas/sangue , Trypanosoma cruzi/metabolismo , Animais , Forma Celular , Modelos Animais de Doenças , Índices de Eritrócitos , Eritrócitos/parasitologia , Eritrócitos/ultraestrutura , Hematócrito , Isquemia , Masculino , Microcirculação , Microscopia Eletrônica de Varredura , Parasitemia/sangue , Ratos , Fatores de Risco , gama-Globulinas/metabolismoRESUMO
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.
Assuntos
Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Viscosidade Sanguínea , Adulto , Estudos de Casos e Controles , Deformação Eritrocítica , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/fisiologia , Feminino , Seguimentos , Testes Hematológicos , Humanos , Fluidez de Membrana , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Proteínas Sanguíneas/farmacologia , Eritrócitos/fisiologia , Hemorreologia/efeitos dos fármacos , Adsorção , Animais , Elasticidade , Deformação Eritrocítica , Hematócrito , Humanos , Masculino , Ratos , Ratos Wistar , ViscosidadeRESUMO
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.
Assuntos
Proteínas do Sistema Complemento/farmacologia , Deformação Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Eritrócitos/ultraestrutura , Humanos , Fluidez de Membrana/efeitos dos fármacos , OvinosRESUMO
The hypothesis that the internal viscosity of erythrocytes is governed by the intracellular hemoglobin (Hb) concentration is examined. Here viscosity is determined by labeling of the cytoplasmic reduced glutathione with the spin label maleimido-Tempo. Erythrocyte populations with different Hb concentrations in isosmotic conditions were obtained through incomplete lysis, followed by cell resealing, and discontinuous density gradient separation. This procedure maintains normal cell shape and volume. Microviscosity of membrane-free Hb solutions was measured by addition of spin labeled glutathione. It was found that microviscosity values are similar for the erythrocyte cytoplasm and for Hb solutions of equivalent concentrations, showing that the erythrocyte membrane does not have any influence on internal microviscosity. The dependence of the microviscosity on the concentration of Hb solutions was compared with results of macroscopic viscosity obtained by other authors. It is concluded that microviscosity is sensitive to individual properties of the Hb molecule (intrinsic viscosity), but that it is not sensitive to intermolecular interactions. As the microviscosity behavior as a function of Hb concentration is the same in Hb solutions as in the erythrocyte cytoplasm, the inferences regarding macroscopic viscosity in Hb solutions could be translated to the rheological properties of the erythrocyte cytoplasm. Thus, these properties could be predicted from the values of the mean corpuscular Hb concentration.