大鼠力竭运动诱导的氧化应激损伤对红细胞变形性的影响

目的:探讨一次性力竭运动诱导的氧化应激反应对大鼠红细胞的抗氧化能力和细胞变形性的影响。方法:大鼠分为3组(n=10):对照组(Control)、适度运动组(MRE)和力竭运动组(ERE)。力竭运动组大鼠运动的前20 min保持5%的坡度和20 m/min的速度,20 min后调整为15%的坡度和25 m/min的速度,直至运动力竭。适度运动组大鼠在5%的坡度和20 m/min的速度下跑40 min。检测各组大鼠红细胞的抗氧化能力,并对氧化应激反应诱导的红细胞膜蛋白巯基水平、膜脂质过氧化水平和膜蛋白SDS-Page电泳条带变化进行了分析。通过激光衍射法对不同运动组大鼠红细胞变形性进行了检测。结果:力竭运动条件下大鼠红细胞受到严重的氧化应激损伤,红细胞内抗氧化能力下降。导致膜脂质过氧化损伤和膜蛋白巯基交联为主的蛋白聚簇化,形成高分子聚合物(HMW)。力竭组大鼠红细胞变形性(0.314±0.013 at 3 Pa and 0.534±0.009 at 30 Pa)显著低于对照组(0.41±0.01 at 3 Pa and 0.571±0.008 at 30 Pa;P0.05 and P0.01,respectively)和适度运动组。结论:力竭运动诱导的氧化损伤导致了红细胞变形能力(EI)的显著下降,使红细胞在微循环的转运受到限制,导致组织缺血缺氧进而引起休克、死亡等运动性疾病。     

Calcium,magnesium, and other elements in the red blood cells and hair of normals and patients with premenstrual syndrome

This study compared the levels of 18 red cell elements and 22 hair elements in 46 patients (median age: 36.2 yr) diagnosed with PMS (premenstrual syndrome) to 50 normals (median age: 37.7 yr). Significantly lower amounts of calcium, chromium, copper, and manganese were found in the blood of patients with PMS. The ratios of Mg/Ca and K/Na and toxic metals such as lead, arsenic, and germanium were significantly elevated in the PMS patients. In hair, mercury and the Zn/Cu ratio were significantly greater in the PMS patients than the controls, but iron, potassium, and the Mg/Ca ratio were lower. The highly significant Mg/Ca ratio in blood cells may be indicative of a more complex relationship between PMS and magnesium and calcium than either element alone. The significantly lower blood cell calcium level found in these studies may provide additional evidence that PMS may be related to a calcium-deficiency state or a metabolic defect involving calcium.     

In vitro effects of high glucose concentrations on membrane protein oxidation, G-actin and deformability of human erythrocytes

The object of this study was to examine the effect of elevated in vitro glucose concentrations on protein modification and functional changes in human erythrocytes. Groups were exposed to 5-45 mM glucose concentrations. The time effect of any changes was also evaluated. In erythrocyte ghosts, protein glycation and oxidation were evaluated using spectrophotometric methods. G-actin was measured by a DNase I inhibition assay in cell lysates. Erythrocyte deformability was assessed using a cell transit analyser. At 24 h, a significant protein oxidation (at 25 and 45 mM glucose; p < 0.05), and G-actin increase was observed for all concentrations (p < 0.05). At 48 h, a significant increase in glycation (25 and 45 mM glucose; p < 0.05), protein oxidation (p < 0.05), and G-actin (p < 0.05) was observed in all groups. A significant positive correlation was observed between glucose /protein oxidation, glucose/G-actin and protein oxidation/G-actin at 24 and 48 h. Our findings show that the oxidative effect of glucose on erythrocytes depends on concentration and incubation time. We also present the first evidence of increased G-actin in human erythrocytes exposed to high glucose concentrations as a diabetes model.     

Serum magnesium, copper, and zinc alterations following spinal fusion

Changes in serum magnesium, copper and zinc values were evaluated in spinal fusion patients at four monitorings. For magnesium and copper individually, a significant difference was found between the mean values at each monitoring (p<0.05), whereas the changes of zinc values between four monitorings were insignificant. There was no statistical difference between the changes of each trace element values and gender, age, operative time, intraoperative blood loss, blood replacement, number of the vertebral levels fused, and antibiotic type used. As a result, magnesium can be suggested to be more important than the other two elements in the postoperative period. Alterations of serum magnesium, copper, and zinc values do not have any correlation with the fusion of the spinal column, either with the width of the fused area or operative time and blood loss. This study cannot confirm the exact reason for this entity and the etiology remains speculative. There is no need for magnesium, copper, or zinc supplementation during the surgical period for the patients. It will be worthy to evaluate the patients who were sent to the intensive care unit after spinal surgery and compare their results with the other intensive care patients.     

Signaling mechanisms in red blood cells: A view through the protein phosphorylation and deformability

Intracellular signaling mechanisms in red blood cells (RBCs) involve various protein kinases and phosphatases and enable rapid adaptive responses to hypoxia, metabolic requirements, oxidative stress, or shear stress by regulating the physiological properties of the cell. Protein phosphorylation is a ubiquitous mechanism for intracellular signal transduction, volume regulation, and cytoskeletal organization in RBCs. Spectrin-based cytoskeleton connects integral membrane proteins, band 3 and glycophorin C to junctional proteins, ankyrin and Protein 4.1. Phosphorylation leads to a conformational change in the protein structure, weakening the interactions between proteins in the cytoskeletal network that confers a more flexible nature for the RBC membrane. The structural organization of the membrane and the cytoskeleton determines RBC deformability that allows cells to change their ability to deform under shear stress to pass through narrow capillaries. The shear stress sensing mechanisms and oxygenation-deoxygenation transitions regulate cell volume and mechanical properties of the membrane through the activation of ion transporters and specific phosphorylation events mediated by signal transduction. In this review, we summarize the roles of Protein kinase C, cAMP-Protein kinase A, cGMP-nitric oxide, RhoGTPase, and MAP/ERK pathways in the modulation of RBC deformability in both healthy and disease states. We emphasize that targeting signaling elements may be a therapeutic strategy for the treatment of hemoglobinopathies or channelopathies. We expect the present review will provide additional insights into RBC responses to shear stress and hypoxia via signaling mechanisms and shed light on the current and novel treatment options for pathophysiological conditions.     

Effects of excessive copper intake on hematological and hemorheological parameters

Copper plays an important role in the structure and function of metalloproteins and in the absorption of iron. The present study deals with the effects of excessive copper intake on hematological and hemorheological parameters. Drinking water containing 250 μg/mL copper for a period of 9 wk, Wistar albino rats showed increased erythrocyte count, blood viscosity, and hematocrit values (p<0.05) and lower hemoglobin (p<0.05) than controls fed a normal diet. The two groups also had differences in the erythrocyte deformability index. The results suggest that excessive copper intake results in hematological and hemorheological changes affecting both the protein content of the erythrocyte membrane and heme synthesis.     

Effects of the calcium-mediated enzymatic cross-linking of membrane proteins on cellular deformability

Summary Excess calcium binding affects the shape and dynamics of cellular deformation of human erythrocytes. It may be hypothesized that incorporation of calcium may modify cellular deformability by processes which include specific cross-linking of membrane proteins with resultant changes in cell shape and deformability. Since previous studies indicate that accumulation of calcium ions causes development of -glutamyl--lysine bridges in membrane proteins, under control of a membrane transamidating enzyme which specifically requires calcium ions for activation, experiments were devised to examine the relationship between cross-linking and deformability and to determine the effects of specific inhibitor of membrane protein cross-linking on the calcium-dependent modification of erythrocyte to the echinocytic shape. The elastic shear modulus of the membrane was not significantly affected by calcium-induced cross-linking, indicating that induced shape change, not altered elasticity, causes the observed reduction in cellular deformability. These findings support the interpretation that Ca⁺⁺-induced and transamidase-catalyzed cross-linking of membrane proteins contributes to fixation of altered cellular shape and decreased cellular deformability.     

Elastic properties of the red blood cell membrane that determine echinocyte deformability

The natural biconcave shape of red blood cells (RBC) may be altered by injury or environmental conditions into a spiculated form (echinocyte). An analysis is presented of the effect of such a transformation on the resistance of RBC to entry into capillary sized cylindrical tubes. The analysis accounts for the elasticity of the membrane skeleton in dilation and shear, and the local and nonlocal resistance of the bilayer to bending, the latter corresponding to different area strains in the two leaflets of the bilayer. The shape transformation is assumed to be driven by the equilibrium area difference (A₀, the difference between the equilibrium areas of the bilayer leaflets), which also affects the energy of deformation. The cell shape is approximated by a parametric model. Shape parameters, skeleton shear deformation, and the skeleton density of deformed membrane relative to the skeleton density of undeformed membrane are obtained by minimization of the corresponding thermodynamic potential. Experimentally, A₀ is modified and the corresponding discocyte–echinocyte shape transition obtained by high-pressure aspiration into a narrow pipette, and the deformability of the resulting echinocyte is examined by whole cell aspiration into a larger pipette. We conclude that the deformability of the echinocyte can be accounted for by the mechanical behavior of the normal RBC membrane, where the equilibrium area difference A₀ is modified.     

Dietary zinc effects on zinc, calcium, and magnesium content in bones of growing rats

The aim of the present study was to assess dietary zinc effects on femur weight and mineral content in growing rats. For this purpose, 70 weanling Sprague-Dawley rats were divided into four groups. Each group was subject to a diet containing 2 (BZ), 5 (DZ), 10 (MZ), and 30 (CZ) ppm zinc. The calcium and magnesium content in all diets was 5 g/kg and 507 mg/kg, respectively. The animals were kept on this regime for 28 d and then sacrificed and their femurs were removed for analysis using atomic absorption spectrophotometry. The weights of the BZ and DZ groups were significantly different from the MZ and CZ groups (38.5±10.5, 89.9±13.7, 118.6±13.6 and 134±19.9 g, p<0.01) respectively. There were no differences between the MZ and CZ groups. Femur weight also varied with dietary zinc, as it was significantly different among all groups (BZ, 265±49 mg; DZ, 380±40 mg; MZ, 452±54 mg; CZ, 735±66 mg; p<0.01). The femur zinc content varied with diets, following a different pattern than the above parameters. Femur zinc from the BZ group (51.5±5.4 ppm) was significantly different from the MZ and CZ groups (115.9±14.2 and 175.0±13.5 ppm, respectively), whereas the DZ group (62.5±11.3 ppm) did not differ from the other three groups. The femur content of calcium (BZ, 83.2±9.8 mg/g; DZ, 88.0±9.2 mg/g; MZ, 90.2±13.6 mg/g; CZ, 83.1±14.7 mg/g) and magnesium (BZ, 1.82±0.13 mg/g; DZ, 1.98±0.09 mg/g; MZ, 1.93±14 mg/g; CZ, 1.83±0.19 mg/g) were not significantly different among the groups, nor was the calcium-magnesium ratio. These results suggest that although dietary zinc deficiency retards growth and causes bone fragility, bone deposition of calcium and magnesium and its ratio are not affected.     

Nitric oxide influences red blood cell velocity independently of changes in the vascular tone

Abstract

Nitric oxide (NO) plays a key role in regulation of vascular tone and blood flow. In the microcirculation blood flow is strongly dependent on red blood cells (RBC) deformability. In vitro NO increases RBC deformability. This study hypothesized that NO increases RBC velocity in vivo not only by regulating vascular tone, but also by modifying RBC deformability. The effects of NO on RBC velocity were analysed by intra-vital microscopy in the microcirculation of the chorioallantoic membrane (CAM) of the avian embryo at day 7 post-fertilization, when all vessels lack smooth muscle cells and vascular tone is not affected by NO. It was found that inhibition of enzymatic NO synthesis and NO scavenging decreased intracellular NO levels and avian RBC deformability in vitro. Injection of a NO synthase-inhibitor or a NO scavenger into the microcirculation of the CAM decreased capillary RBC velocity and deformation, while the diameter of the vessels remained constant. The results indicate that scavenging of NO and inhibition of NO synthesis decrease RBC velocity not only by regulating vascular tone but also by decreasing RBC deformability.     

Role of zinc in mitigating the toxic effects of chlorpyrifos on hematological alterations and electron microscopic observations in rat blood

The present study determined the protective potential of zinc in attenuating the toxicity induced by chlorpyrifos in rat blood. Male Sparque Dawley (SD) rats received either oral chlorpyrifos (13.5 mg/kg body weight) treatment every alternate day, zinc alone (227 mg/l in drinking water) or combined chlorpyrifos plus zinc treatment for a total duration of 8 weeks. The effects of different treatments were studied on various parameters in rat blood including haemoglobin (Hb) levels, total leukocyte count (TLC), differential leukocyte count (DLC), zinc protoporphyrins (ZPP), serum trace elemental concentrations and Scanning Electron Microscopic (SEM) observation of the blood cells. Chlorpyrifos treatment to normal control animals resulted in a significant decrease in TLC and ZPP concentration after 4 and 8 weeks. Chlorpyrifos treated animals also showed significant neutrophilia and lymphopenia after 8 weeks of toxicity. In addition, a significant decrease in serum zinc and iron concentrations were observed following chlorpyrifos intoxication, however, these animals responded with increased serum copper levels following the toxic treatment with this organophosphate. SEM studies of the red blood cells from chlorpyrifos treated animals indicated marked alterations in the topographical morphology of the various cell types, with the prominent feature being common aniscocytosis of the erythrocytes. Oral zinc treatment to the chlorpyrifos treated animals significantly improved the total leukocyte, neutrophil and lymphocyte counts, as well as the otherwise reduced concentrations of ZPP and the levels of various serum trace elements. Protective effects of zinc were also evident in the electron microscopic observations where most blood cell types depicted reverted to a close to the normal appearance. Based upon these data, the present study is first of its kind and suggests that zinc treatment considerably attenuates chlorpyrifos induced toxicity induced in restoring the altered hematological indices and morphological changes.     

Effect of carbonyl compounds on red blood cells deformability

The effect of Maillard reaction on red blood cells (RBC) deformability was investigated. Exposure of RBC to carbonyl compounds (dl-glyceraldehyde, glyoxal, glycolaldehyde, 3-deoxyglucosone, and d-glucose) leading to Maillard reaction caused a marked decrease in RBC deformability even at 1 mM level. The decrease rate depended on the kind of carbonyl compounds, in which both dl-glyceraldehyde and glyoxal significantly decreased the RBC deformability (p < 0.05). In addition, the decrease rate also differed among volunteers tested, indicating that the sensitivity against carbonyl compounds varies among them. In order to elucidate the mechanism of the decrease in RBC deformability, RBC was exposed to carbonyl compounds in the presence of aminoguanidine which is the inhibitor of AGE formation in Maillard reactions. Aminoguanidine inhibited the decrease in RBC deformability by dl-glyceraldehyde and glyoxal. When Hb which has a high reactivity with carbonyl compounds was incubated with those carbonyl compounds, dl-glyceraldehyde and glyoxal showed the high reactivity with Hb compared with other carbonyl compounds. These results indicate that Maillard reaction between RBC proteins and carbonyl compounds leads to the decrease in RBC deformability. On the other hand, generated by carbonyl compounds involved in lowering the deformability only to a negligible level.     

The influence of magnesium ions on pyruvate kinase-deficient red blood cells.

The effect of intravenous and oral administration of magnesium containing drug (Magnesium Diasporal, Protina, München) on the erythrocyte pyruvate kinase (PK) activity in two cases of congenital nonspherocytic hemolytic anemia associated with PK deficiency has been examined. Intravenous injection of the drug has been followed by transient increase in magnesium content. Positive correlation between erythrocyte magnesium ions concentration and PK activity has been found. The authors propose a hypothesis that the observed correlation may be due to the erythrocyte accumulation of some PK activators or to the elimination of some PK inhibitors. The authors did not, however, succeed to maintain the high erythrocyte magnesium concentration inducing the increase in PK activity during the longer period of time.     

In vitro hemolytic activity ofPlasmodium berghei on red blood cells

A suspension ofPlasmodium berghei obtained by lysis with saponin of red blood cells from an infected rat showed high hemolytic activity, when incubatedin vitro with normal rat red blood cells. The hemolysis was a temperature-dependent process and was dependent on the concentration of the parasite. Plasma ofPlasmodium berghei infected albino rats also possessed lytic activity.     

Effect of some nickel compounds on red blood cell characteristics

The effect of certain inorganic and coordinated nickel compounds on the resistance to different destructive substances, rheological properties, and functional activity of healthy human red blood cells (RBC), was investigated. It is shown that nickel compounds affect the erythrocyte membrane lipid bilayer, as well as membrane proteins to various extents, depending on the type of compounds used. In general, the acceleration of erythrocyte aging was observed to be more pronounced in young erythrocytes. The observed results suggest that nickel compounds decrease water permeability across erythrocyte membranes. Almost all the investigated nickel compounds decrease erythrocyte thermostability, deformability, and the rate of O₂ release by erythrocytes.     

In vitro studies on the entry of polyamines into normal red blood cells

Polyamines are mainly transported in the blood by erythrocytes: Putrescine, spermidine and spermine can be taken up in vitro by red blood cells (RBC); their entry is greater in the presence of serum than in the presence of plasma, and spermine entry is lower than that observed for the two other polyamines. In the presence of serum, the affinity of RBC for spermidine is 30 fold greater than that for putrescine. The majority of RBC polyamines are present in the hemolysate and are not complexed to high molecular weight material. At + 4 degrees C the polyamine uptake is considerably reduced and for putrescine and spermine practically non existent, but it seems that it is internalization rather than binding which constitutes the dependent step. Though intracellular spermidine and spermine levels reflect differences in uptake rather than in outward flux across the cell membrane, the values of putrescine appear to be the resultant of influx and efflux. The presence of specific receptor sites for polyamines visualized by SEM on the surface of RBC using latex-putrescine spheres, confirms the results obtained with labelled polyamines. Therefore, only the understanding of the polyamine repartition inside the blood compartments would permit the clinical use of those molecules as non statistical tumor markers.     

Concentations of copper, Zinc, and magnesium in sera from patients with idiopathic dilated cardiomyopathy

Trace elements are known to have a key role in myocardial metabolism. The accumulation (cobalt, arsenic, copper) or deficiency (selenium, zinc) of trace elements may be responsible for idiopathic dilated cardiomyopathy. We investigated the trace element concentrations (Cu, Zn, Mg) in sera from patients with dilated cardiomyopathy by atomic absorption spectrophotometry. We observed that patients with dilated cardiomyopathies have higher copper and lower zinc concentrations in serum than healthy controls. The magnesium concentrations of patients did not differ significantly from that of control subjects.     

The influence of temperature and incubation time on deformability of human erythrocytes

Human erythrocytes have been heated and stressed in a novel and controlled manner using rectangular microcapillaries. Heated cells attached to the capillary wall were stressed by liquid flow. Under particular conditions of stress, temperature and incubation time the body of the cell could be pulled in the flow, retaining a connection with the glass by means of a narrow process or tether. The tethers appear as: regularly beaded, irregularly beaded or without beads depending upon the incubation conditions. We have outlined the incubation regimes necessary to achieve these different responses in the temperature range 48–55°C. The cells become less deformable as the incubation is continued beyond an optimum time. The behaviour of the tether is compared with that of a viscoelastic liquid. Circular dichroism studies of ghost membranes show that the denaturation of membrane proteins is partially reversible when incubation times are similar to those required to bring about a loss of deformability.     

Zinc uptake by blood cells of rats in zinc deficiency and inflammation

In zinc deficiency, the function of leukocytes is impaired. However, the results of studies on the zinc concentration of blood cells in zinc deficiency are conflicting, probably in part because of technical and analytical problems. The aim of this study was to investigate, under standard conditions, the uptake of⁶⁵Zn-labeled zinc by blood cells, taken from zinc-deficient rats and from rats in which an inflammation is induced. In both conditions, the serum zinc concentration is reduced. In clinical practice, this makes it difficult to determine whether the decrease in serum zinc is the result of a real or an apparent zinc deficiency. In stress, like an inflammatory disease, the decrease of zinc reflects an apparent zinc deficiency because of redistribution of serum zinc into the liver and because of decrease in serum albumin concentration. Over 70% of the serum zinc is bound to albumin. Blood cells from zinc-deficient and control rats were isolated using a discontinuous Percoll gradient and incubated under nearly physiological conditions in a⁶⁵Zn-containing medium. A significant increase in the in vitro uptake of⁶⁵Zn-labeled zinc by the blood cells of zinc-deficient rats was seen: erythrocytes 1.3, mononuclear cells 2.0, and polymorphonuclear cells 2.6 times the control values. During inflammation, no change in⁶⁵Zn-labeled zinc uptake by erythrocytes and mononuclear cells was demonstrated after 2 d, although the serum zinc and albumin concentrations were decreased, but a small but significant increase in zinc uptake by polymorphonuclear cells was observed. This study of⁶⁵Zn uptake in vitro under standard conditions may prove of value for distinguishing in patients real zinc deficiency from apparent zinc deficiency owing to, e.g., stress, although additional experiments should be performed. A part of this study has been presented at the Meeting of The American Gastroenterological Association on May 12–18, 1990, San Antonio, TX, and has been published in abstract inGastroenterology 98 suppl., A423.