RESUMEN
Although many RBC rheological properties have been previously described in detail, the biochemical mechanisms leading to premature destruction of red blood cells are less clear. However, several biochemical processes have been suggested as possible mechanisms for membrane structural alterations (e.g., crosslinking of membrane proteins, oxidant damage, binding of cytoplasmic proteins, and altered intracellular ion composition). We have carried out a series of studies aimed at evaluating the effects of calcium-regulated membrane-bound hemoglobin (Hbm) on RBC and derived ghost rheologic behavior. Intracellular calcium was elevated by 10 microM A23187, with cell deformability determined via the Cell Transit Analyzer (CTA). Our results indicate: 1) Linear, positive correlations between Hbm and average RBC rigidity and 2) a marked influence of heterogeneous calcium concentration on both Hbm and rheologic properties for various subpopulation. These findings therefore suggest the importance of hemoglobin-membrane interactions as a determinant of erythrocyte deformability, and may be relevant to RBC aging as well as to diseases such as sickle cell anemia, hereditary spherocytosis and thalassemia.
Asunto(s)
Calcio/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/fisiología , Calcimicina/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Deformación Eritrocítica/efectos de los fármacos , Deformación Eritrocítica/fisiología , Membrana Eritrocítica/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Hemoglobinas/metabolismo , Hemorreología , Humanos , Técnicas In Vitro , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Ionóforos/farmacologíaRESUMEN
Guidelines and recommendations to conduct clinical trials have been defined for Europe since 1st July 1991 to ensure high quality standards. Thus, cost and speed of clinical trials are becoming more and more important in clinical research. The article tries to summarize specific requirements to the investigator site considering the aspects of quality and data evaluation. Furtheron a model is proposed to optimize realization of clinical trials in hospital institutions. Realization of "Good Clinical practice" (GCP) at the sponsor and the investigator site will improve the quality of clinical trials for the sake of the patients and finally leads to the acceptance of European clinical trials abroad.
Asunto(s)
Ensayos Clínicos como Asunto/normas , Ensayos Clínicos como Asunto/economía , Industria Farmacéutica , Unión Europea , Hospitales , Humanos , Modelos Teóricos , Pacientes , Control de CalidadRESUMEN
Neonatal red blood cells (RBC) show large variations in size, density, and deformability, with a relatively high percentage of neonatal RBC being extremely dense, almost spherical, and poorly deformable. Previous reports suggest that loss of membrane and oxidation of fetal Hb might account for the generation of the dense, rigid RBC in neonates and the shortened life-span of neonatal RBC. To test whether the dense RBC population is particularly fragile and which mechanical properties are responsible for the rigidity of these cells, the following measurements were made for the top (least dense) and bottom (most dense) 3% fractions of density-separated neonatal and adult RBC: cellular deformability (rheoscope); RBC geometry (micropipette system); elasticity, fragility, and viscosity of RBC membrane (micropipette system); Hb solution viscosity (cone-plate viscometer); and selected biochemical parameters. Fetal Hb of neonatal RBC decreased with increasing cell density. When the bottom fractions were compared with the top fractions, neonatal RBC showed a greater reduction in glutamic oxalacetic transaminase activity (72% versus 53%), potassium (39% versus 19%), volume (32% versus 19%), and surface area (42% versus 21%), and a greater rise in density (3.5% versus 1.9%) and mean corpuscular Hb concentration (42% versus 23%) than adult RBC. Cellular deformability in the rheoscope (shear stress 5 Pa) decreased by 24% in adults and by 41% in neonates. Membrane extensional and bending elastic moduli (i.e. membrane deformability) and membrane fragility of neonatal and adult RBC did not significantly change with increasing cell density.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Deformación Eritrocítica/fisiología , Eritrocitos/citología , Eritrocitos/fisiología , Adulto , Aspartato Aminotransferasas/sangre , Fenómenos Biomecánicos , Separación Celular , Índices de Eritrocitos , Membrana Eritrocítica/fisiología , Humanos , Recién Nacido , Masculino , Fluidez de la Membrana , Potasio/sangreRESUMEN
Several investigators have demonstrated that red blood cell (RBC) deformability decreases progressively with increasing cell density and proposed that reduction in deformability plays a role in the senescence process of normal RBCs. Transient erythroblastopenia of childhood (TEC) results from temporary cessation of erythropoiesis. Since no new RBCs are produced for some time, the circulating RBCs are relatively old. RBS density (phthalate-oil method) and RBC deformability (RBC elongation in a counter-rotating rheoscope) were studied in seven children with TEC and in 10 control children. The mean values of MCHC, RBC density and RBC deformation were not significantly different between TEC and control children. Compared to controls, the frequency distribution of RBC density in TEC was slightly shifted to higher values. The percentage of RBCs with extremely low densities (< 1.090 g/ml) was 0.9 +/- 1.2% in the patients and 5.6 +/- 2.3% in the controls (P < 0.001). The percentage of RBCs with high density (> 1.106 g/ml) was 6.4 +/- 2.1% in the patients and 4.9 +/- 1.8% in the controls (P > 0.10). The reduction of RBCs with low density in TEC suggests that RBCs with low density are relatively young. Since the percentage of RBCs with high density increased only slightly in TEC, we conclude that only a fraction of dense RBCs is old. In TEC, the frequency distribution of RBC elongation was slightly shifted to lower values. 5% of the RBCs studied in the control children had RBC elongation values above 0.39 (TEC 1.2%) and 5% had elongation values below 0.16 (TEC 6.7%). Thus, only a small fraction of highly deformable RBCs was diminished in TEC. These data suggest that a decrease in deformability is not a significant part of the normal ageing process of human RBCs.
Asunto(s)
Eritroblastos , Envejecimiento Eritrocítico/fisiología , Deformación Eritrocítica , Preescolar , Recuento de Eritrocitos , Deformación Eritrocítica/fisiología , Índices de Eritrocitos , Humanos , LactanteRESUMEN
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.
Asunto(s)
Calcio/farmacocinética , Deformación Eritrocítica/fisiología , Membrana Eritrocítica/fisiología , Hemoglobinas/fisiología , Adulto , Calcimicina/farmacología , Calcio/fisiología , Permeabilidad de la Membrana Celular , Separación Celular , Citoesqueleto/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Deformación Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Eritrocitos/fisiología , Eritrocitos/ultraestructura , Filtración/métodos , Hemoglobinas/análisis , Hemoglobinas/metabolismo , Humanos , ReologíaRESUMEN
The basal and calmodulin-stimulated activity of the human erythrocyte Ca2(+)-ATPase was determined in 56 healthy individuals of different ages in order to set control values. The basal activity of all 56 healthy subjects was 0.805 +/- 0.525 mumole hydrolyzed ATP/mg protein/hr, while the calmodulin-stimulated activity gave an average of 2.437 +/- 0.785 mumole hydrolyzed ATP/mg protein/hr. There was no significant difference in basal Ca2(+)-ATPase activity in males and females; however, significantly increased levels of the stimulated red cell calcium pump was seen in females. Based on age, the basal activity for newborns and children up the age of 7 years was higher than that for adults and a distinct increase in activation by calmodulin was observed with a maximum at the age range of 8-13 years. It was demonstrated that for the investigation of Ca2(+)-ATPase activities in children different normal values must be taken into consideration than for those already reported for adults. Our data may serve as controls for Ca2(+)-ATPase activities for comparison purposes and for further studies from different diseases in childhood where increased intraerythrocytic calcium levels are reported.
Asunto(s)
Envejecimiento/sangre , ATPasas Transportadoras de Calcio/sangre , Calmodulina/farmacología , Eritrocitos/enzimología , Adolescente , Adulto , Niño , Preescolar , Activación Enzimática/efectos de los fármacos , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/enzimología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Valores de Referencia , Factores Sexuales , Estimulación QuímicaRESUMEN
Two newborn infants with severe alloimmunothrombocytopenia due to maternal immunization against fetal PlA1-positive platelets were treated by a combination of i.v.-immunoglobulin (400 mg/kg b.w.) and a transfusion of maternal PlA1-negative platelets. In one infant this regimen was successful; the other one had to be retreated by i.v. immunoglobulin application (400 mg/kg b.w.) over a period of five days.
Asunto(s)
Antígenos de Plaqueta Humana , Transfusión Sanguínea , Eritroblastosis Fetal/terapia , Inmunización Pasiva , Isoantígenos/inmunología , Transfusión de Plaquetas , Trombocitopenia/terapia , Plaquetas/inmunología , Terapia Combinada , Femenino , Humanos , Recién Nacido , Integrina beta3 , Masculino , Trombocitopenia/inmunologíaRESUMEN
1. A method for determining free Ca2+-ions in the erythrocyte is described, using a commercially available ORION-Ca-electrode and calomel reference electrode assembly, where changes in free Ca2+-ion concentration upon addition of 0.01% digitonin could be measured. 2. The average value found for fresh cells from 20 healthy donors at 37 degrees C (pH = 7.4) was 0.20 +/- 0.04 mumol/L referred to a haematocrit of 10%. 3. Decrease of the simultaneously determined adenosinetriphosphate (ATP) concentration indicates that ATP is presumably needed to activate the Ca-ATPase. 4. In vitro addition of the calcium antagonists pentoxifylline and nifedipine, respectively, induced a normalization of the intraerythrocytic Ca2+-ion concentration after previous increase with the ion carrier ionophore A23187. 5. The advantages and possible clinical applications of this method are discussed.
Asunto(s)
Calcio/sangre , Eritrocitos/metabolismo , Nifedipino/farmacología , Pentoxifilina/farmacología , Teobromina/análogos & derivados , Adenosina Trifosfato/farmacología , Calcimicina/farmacología , Calcio/metabolismo , Eritrocitos/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Ionóforos/farmacología , TemperaturaRESUMEN
Ca2+ ions were transported into the cell by incubation of the erythrocyte suspension with ionophore A23187, a lipophil electric neutral ion complexing substance. Erythrocyte aggregation could be increased twice, when doubling the intracellular Ca2+-ion concentration. Our measurements lead to the suggestion that an increase of the cytoplasmatic Ca2+ ion changes the physical and/or biochemical properties of the aggregation receptors on the membrane surface, i.e., cell-protein interactions are regulated by alteration of the intracellular Ca2+-ion concentration.