Bivariate and Multivariate Analyses of the Influence of Blood Variables of Patients Submitted to Roux-en-Y Gastric Bypass on the Stability of Erythrocyte Membrane against the Chaotropic Action of Ethanol

The stability of the erythrocyte membrane, which is essential for the maintenance of cell functions, occurs in a critical region of fluidity, which depends largely on its composition and the composition and characteristics of the medium. As the composition of the erythrocyte membrane is influenced by several blood variables, the stability of the erythrocyte membrane must have relations with them. The present study aimed to evaluate, by bivariate and multivariate statistical analyses, the correlations and causal relationships between hematologic and biochemical variables and the stability of the erythrocyte membrane against the chaotropic action of ethanol. The validity of this type of analysis depends on the homogeneity of the population and on the variability of the studied parameters, conditions that can be filled by patients who undergo bariatric surgery by the technique of Roux-en-Y gastric bypass since they will suffer feeding restrictions that have great impact on their blood composition. Pathway analysis revealed that an increase in hemoglobin leads to decreased stability of the cell, probably through a process mediated by an increase in mean corpuscular volume. Furthermore, an increase in the mean corpuscular hemoglobin (MCH) leads to an increase in erythrocyte membrane stability, probably because higher values of MCH are associated with smaller quantities of red blood cells and a larger contact area between the cell membrane and ethanol present in the medium.     

Alterations in chloride transport during differentiation of Friend virus-transformed cells.

Friend erythroleukemic cells can be induced by a variety of agents to synthesize hemoglobin and to exhibit other characteristics suggesting erythroid maturation. Upon induction of hemoglobin synthesis with dimethylsulfoxide (DMSO), the chloride flux in Friend cells gradually increases, until after five days of exposure to DMSO (when the hemoglobin content of the cells approaches that of the mature erythrocyte) the flux is three times the value in non-induced cells. A similar flux increase is observed in the presence of a different type of inducer, hypoxanthine, but no increase in flux is seen in the mutant cell line, TG-13, which does not synthesize hemoglobin after DMSO treatment. Thus, the flux increase seems to be associate d with the induction process, rather than being a direct effect of the inducing agent. After DMSO treatment, the sulphate flux decreases and the chloride/sulphate selectivity increases, aswould be expected if the cells were becoming more like red cells. On the other hand, the sensitivity of the chloride flux to the inhibitor, furosemide, and to temperature is the same in the induced as in the non-induced Friend cells, and different from that of the mature red cell. Thus, the anion transport properties of the induced Friend cell are different from those of both the non-induced Friend cell and the mature erythrocyte. Either the system in the induced cell represents an intermediate stage in the development of the mature red cell characteristics, or else the maturation of transport function in the Friend cell differs from that in normal erythrocyte precursors.     

The structural organization of skeletal proteins influences lipid translocation across erythrocyte membrane

In order to define the influence of skeletal protein organization on transmembrane phospholipid movement in erythrocyte membranes, we measured the translocation rate of lysophosphatidylcholine in pathologic red cells. A simple method based on the differential extraction of lysophosphatidylcholine from the red cell membrane by saline and albumin solutions was used to quantitate the translocation rate. Two groups of pathologic red cells were chosen for these studies: red cells with quantitative deficiencies of the skeletal proteins, spectrin and protein 4.1, and sickle erythrocytes in which controlled reorganization of the membrane was induced by hemoglobin polymerization. Marked increase in lipid translocation rate was seen in red cells having quantitative deficiencies of spectrin and protein 4.1. The magnitude of the increase in translocation rate in spectrin-deficient red cells was related to the magnitude of protein deficiency. Translocation rate in sickle erythrocyte membranes increased by 50% upon deoxygenation as a result of sickle hemoglobin polymerization. No increase in translocation rate was seen in normal cells upon deoxygenation. By manipulating the extent of membrane reorganization that occurred following deoxygenation of sickle cells, we have been able to show that skeletal reorganization induced by hemoglobin polymerization and not hemoglobin polymerization per se is responsible for the increase in translocation rate. Together, these findings imply that the structural organization of membrane skeletal proteins plays an important role in regulating the rate of transbilayer movement of lipids across the erythrocyte membrane.     

Changes in the concentration of metalloproteins in the blood plasma in hypoxia and hyperbaric oxygenation

Dynamics of hemoglobin, ceruloplasmin concentration, the changes of chemiluminescence in blood plasma and kinetics of rat erythrocyte heat denaturation during consequent exposition of high altitude hypoxia and hyperbaric oxygenation have been studied. Severe hypoxia causes the decrease of extraerythrocyte hemoglobin and oxidase activity of ceruloplasmin. Reoxygenation results in significant increase of blood plasma chemiluminescence with simultaneous increase of extraerythrocyte hemoglobin level and with modification of surface structure of the erythrocyte membranes. Possible pathways of activation of oxygen-dependent of free-radical processes during reoxygenation are discussed.     

The red blood cells in growing guinea pigs. (Cavia cobaya). II. Communication: erythropoiesis and red blood cells in the postnatal development period

Bone-marrow smears of 175 guinea pigs aged 1-27 days and venous blood samples of 351 animals aged 1-25 days were prepared for cell counting. A significant increase of erythroblasts were found between life day 1 and 2; normoblasts increased in number synchronously with a decrease of erythroblasts after the 5th day. The percentage of the erythroid bone marrow increased from 10 to 14 during the developmental period. Beyond the perinatal period the red blood picture is characterized by the following changes: a decrease of erythrocyte count, hematocrit, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin; a constant mean corpuscular hemoglobin concentration; an increase of the reticulocyte count. The decrease of the red cell count is compensated by a decreasing oxygen affinity attained by an important increase of 2,3-DPG. Nevertheless, the stimulus for a raising erythropoiesis remains constant which can be shown by the growing percentage of erythroid cells and reticulocytes. The difference between the human postnatal development and that of the guinea pig becomes obvious. Cell counts in dependence of body masses in postnatally growing guinea pigs, veil the perinatal finding of the increase in erythrocytes up to the 5th day and the decrease of the mean corpuscular volume after the 3rd day.     

Blood viscosity parameter correlation with types of leukemia.

We investigated the hemorheological, hematological and biochemical parameters in 30 cases of acute lymphocytic leukemia (ALL), 21 cases of acute myelogenous leukemia (AML) and 30 cases of chronic myelogenous leukemia (CML). The parameters studied include whole blood viscosity, plasma viscosity, erythrocyte sedimentation rate (ESR), red cell filterability, hematocrit, platelet count and aggregation, fibrinogen, hemoglobin, leucocyte count, bleeding time and lactate dehydrogenase activity (LDH). In the cases of ALL we observed significant decrease in whole blood viscosity, hemoglobin, hematocrit and platelet count but an increase in plasma viscosity, fibrinogen, bleeding time and LDH activity. In the cases of AML, we observed increase in whole blood viscosity, plasma viscosity, ESR, fibrinogen, leucocyte count, bleeding time and LDH activity but decrease in the hemoglobin, hematocrit and platelet count. In the cases of CML, we observed an increase of whole blood viscosity, plasma viscosity, ESR, fibrinogen elevation but decreases in bleeding time. In all cases, red cell filterability was unaffected.     

Effect of the level of ATP and of the state of spectrin on osmotic properties of bovine erythrocytes

The effect of the intracellular level of ATP and of the state of spectrin on the critical cell volume of bovine erythrocyte was studied. The state of spectrin was changed by thermal denaturation, which for the bovine red cell took place at similar temperature as for the human erythrocyte. The increase of the ATP level and the spectrin denaturation increased the critical cell volume, while metabolic starvation decreased it. The changes of the ATP level did not influence the critical volume after the denaturation of spectrin. The results suggest that the ATP-dependent effect on the critical cell volume was caused by an alteration of the membrane extensibility due to the change of the membrane skeleton-lipid bilayer interaction(s).     

能量补充对红细胞膜通透性的影响

本文报道利用亚硝酸钠(NaNo_2)穿透红细胞膜与血红蛋白(Hb)作用生成高铁血红蛋白(MetHb),测定MetHb可作为红细胞膜通透性的指标,研究在红细胞悬液中补充葡萄糖,萄葡糖-6-磷酸(G-6-P),3-磷酸甘油(3-PG),ATP后红细胞膜通透性的变化。萄葡糖,G-6-P,ATP均可不同程度地提高通透性,而3-PG有一定的抑制作用,可改善由血卟啉衍生物或γ-线(1000 rad)照射小鼠后引起的红血球通透性增加的作用,使恢复到接近正常水平。     

Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extra-erythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases.     

Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.     

Asymmetric transport of a fluorescent glucose analogue by human erythrocytes

A fluorescent glucose analogue, 6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose (NBDG), was synthesized and its interactions with the hexose transport system of the human red blood cell were investigated. NBDG entry is inhibited by increasing concentrations of d-glucose (K_i = 2 mM). However, NBDG exit is unaffected by d-glucose in red blood cells. Cytochalasin B was found to inhibit both NBDG entry and exit. NBDG accumulates in the red blood cell above the theoretical equilibrium concentration. Accumulation of NBDG is temperature-sensitive and is due to the binding of NBDG to some intracellular substance. The binding of NBDG to purified hemoglobin suggests that accumulation of NBDG by erythrocytes is due to the intracellular binding of NBDG to hemoglobin. NBDG does not accumulate in pink erythrocyte ghosts, while its rate of uptake is still inhibited by d-glucose and cytochalasin B. Although there was no apparent d-glucose inhibition of NBDG exit by intact red blood cells, d-glucose was able to inhibit NBDG exit by pink erythrocyte ghosts. The differing properties of NBDG influx and efflux support the interpretation that the hexose transport system of the human red blood cell appears asymmetric although it may be intrinsically symmetric.     

A novel device for the non-invasive measurement of free hemoglobin in blood bags.

The production of red blood cell concentrates from human donors is a very expensive procedure and human resources are in short supply. Under perfect storage conditions at a temperature of 2-6 degrees C, a blood bag must be used within 35-49 days (in Germany). Visual inspection of the bag for apparent hemolysis by a blood bank physician is a crucial but subjective quality control assessment. Since an interruption of the cold chain cannot be definitely ruled out, bags are often disposed of prematurely for safety reasons. There is currently no method of testing a closed blood bag with respect to hemolysis for its suitability to be used in a transfusion. The proposed optical measuring device is a hemoglobin sensor which determines the free hemoglobin in standard erythrocyte concentrates without opening the bag. The optical measurements are done on the flexible tube connected to the main bag. The optical measurements were evaluated using standard hemoglobin solutions with an accuracy of 0.005 g/dL. These investigations show that in the future each blood bag can be tested non-invasively for its content of free hemoglobin. This will contribute to decreasing the wastage rate of red blood cell concentrates.     

Blood parameter changes during stopover in a long-distance migratory shorebird, the bar-tailed godwit Limosa lapponica taymyrensis

Bar-tailed godwits migrate from West African wintering sites to breeding areas in northern Russia with only one stopover. We compared hematocrit (Hct), blood hemoglobin concentration (Hb), and mean cell hemoglobin concentration (MCHb; a measure of the relative proportion of Hb in the cellular blood fraction) between arriving godwits lured to land 60 km short of the stopover site and godwits during subsequent refueling. The Hct and Hb of arriving godwits was low when compared to that of refueling birds. On the stopover site, Hct and Hb correlated positively with size-corrected body mass. In addition, Hb and MCHb reached peak levels in the last days of stopover. We explored the possibility of regenerative anemia in arriving godwits by comparing the fraction of reticulocytes (young red blood cells) between arriving and refueling birds. No differences were found. Therefore, we suggest that the increase in Hct, Hb, and MCHb during refueling is not in response to a severe anemic state at arrival. Rather, we suggest that the increase in blood parameters may anticipate the increased aerobic requirements of impending migratory flight and possibly satisfy heightened oxygen demands of the larger body mass of fattened birds. The Hct increase on the stopover site may also serve to buffer the red blood cell population against possible red blood cell breakdown during long-distance flight.     

Increase in hematocrit, hemoglobin and red cell mass in normal mice after treatment with cyclic AMP (38543).

Chronic treatment of normal mice with either dibutyryl cyclic AMP or erythropoietin produced elevations in the hematocrit, hemoglobin concentration and red cell mass when compared to these same hematological parameters in untreated mice. Dibutyryl cyclic AMP increased red cell mass by 46% while ESF treatment resulted in a 56% increase in red cell mass. These studies confirm earlier reports of the effects of cyclic AMP in increasing radioactive iron incorporation into red cells and further indicate that this change is associated with an absolute increase renal cyclic AMP concentrations probably stimulate erythropoiesis as a consequence of increased kidney production of erythropoietin.     

Cholesterol ester increases the permeability of erythrocyte membrane

The effect of cholesteryl palmitate on erythrocyte membrane permeability for K+ and hemoglobin was studied. Cholesterol ether was incorporated into the erythrocyte membrane by liposomes containing cholesteryl palmitate and lecithin or by dispersion of cholesteryl palmitate. It was shown that cholesteryl palmitate considerably increases permeability of the erythrocyte membrane for K+ and hemoglobin. The leakage of K+ and hemoglobin from red blood cells is not accompanied by cell destruction.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decrease membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC) ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipoproteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80-85% of abetalipoproteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity (eta) between 5 and 42 degrees C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively at 10 degrees C; 4.63 vs 4.04 poise at 37 degrees C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Effect of nitrite-induced methemoglobinemia on the kinetics of blood deoxygenation

Kinetics of blood deoxygenation was studied during acute hypoxia induced by subcutaneous administration of sodium nitrite using polarographic method. Plasma oxygen tension remained unaltered as the dose of sodium nitrite increased, while the dynamics of oxygen release was dose-dependent. The constant of oxyhemoglobin deoxygenation rate proved to vary with blood deoxygenation. The nitrite-induced deceleration of oxyhemoglobin deoxygenation was due to the inactivation of a fraction of hemoglobin as well as to the increased hemoglobin oxygen affinity and possible changes in the oxygen permeability of erythrocyte membranes during acute methemoglobinemia.     

四种苏丹红染料对小鼠体内血液的影响

目的探讨苏丹红对小白鼠血液和组织器官的影响。方法取小鼠连续12 d腹腔注射不同剂量苏丹红Ⅰ、Ⅱ、Ⅲ、Ⅳ（0、40、80、160 mg/kg）,末次给药24 h后采集血样,用血细胞分析仪检测血常规指标,毛细血管法测定凝血时间,剖腹取主要器官称重,计算脏器系数。结果苏丹红Ⅰ和Ⅱ组血液红细胞数明显减少,而苏丹红Ⅲ和Ⅳ组不明显,苏丹红不同剂量之间比较,低剂量苏丹红Ⅰ和高剂量苏丹红Ⅱ组比苏丹红Ⅲ和Ⅳ组血液红细胞数变化明显;苏丹红Ⅰ和Ⅱ组血液血红蛋白含量明显降低,而苏丹红Ⅲ和Ⅳ组血红蛋白含量变化不明显。四种不同剂量苏丹红组小鼠血液中血小板数量减少,随着剂量增加小鼠血液凝固时间延长。随着苏丹红剂量增加,苏丹红I和II组小鼠血液白细胞数比对照组明显增加,苏丹红Ⅲ和Ⅳ组白细胞数有增加趋势但差异不显著。与对照组比较,苏丹红组小鼠血液白细胞数明显增加、而淋巴细胞和单核细胞显著减少。随着苏丹红注射剂量增加,肾脏和脾脏脏器系数较对照组明显增大,肝脏脏器系数变化不明显。结论苏丹红对小鼠血液细胞、血红蛋白、血小板和凝血时间以及组织器官都有不同程度影响。     

Is red blood cell survival limited by the activity of a critical enzyme?

Erythrocyte glutathione reductase is responsible for generating reduced glutathione, which has been implicated in maintaining the integrity of the red blood cell.Erythrocytes from peripheral blood were separated into fractions of increasing age and the activity of glutathione reductase and aspartate amino transferase determined in each fraction.The age-related decline in activity of both enzymes was confirmed, but with detailed resolution of the cells by age a significant secondary rise in only glutathione reductase activity was found in very old cells. As red blood cells from the same cohort survive in the circulation for varying periods they must vary in some way from one another. It is postulated that glutathione reductase is a critical enzyme which limits erythrocyte survival and that the rate of decline in activity varies from cell to cell. A simple mathematical model based on this postulate accounted quantitatively for both the pattern of glutathione reductase activity and the erythrocyte survival curve. In addition, a simplified model of the passage of erythrocytes through the circulation was designed and run. The predicted erythrocyte survival curve and pattern of glutathione reductase activity were very similar to observed patterns. This model may be useful in other situations where a finite resource is degraded at different rates by random passages through different pathways.