NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions

NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions. Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms. The relaxation time of a red cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water. Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time. Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population. Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells.     

Macromolecular crowding and volume perception in dog red cells

To differentiate whether the primary volume signal in dog red cells arises from a change in cell configuration or the concentration and dilution of cell contents, we prepared resealed ghosts that had the same surface area and hemoglobin concentration as intact cells but less than 1/3 their volume. Shrinkage of both intact cells and resealed ghosts triggered Na/H exchange. Activation of this transporter in the two preparations correlated closely with cytosolic protein concentration but not at all with volume. The Na/H exchanger was more sensitive to shrinkage in albumin-loaded resealed ghosts than in intact cells or ghosts containing only hemoglobin. Similar results were obtained for the swelling-induced [K-Cl] cotransporter. We believe perception of cell volume originates with changes in cytoplasmic protein concentration. We think the kinases and phosphatases that control the activation of membrane transporters in response to cell swelling or shrinkage are regulated by the mechanism of macromolecular crowding.     

Zinc content of normal human serum and its correlation with some hematic parameters

Numerous studies have demonstrated that several diseases and stress conditions are associated with changes in the levels of zinc in the blood plasma and cellular elements. In this research the association between serum zinc concentrations and other hematic parameters of diagnostic interest has been evaluated. Quantitative determinations of zinc, total plasmatic proteins, albumin, hemoglobin and calculation of mean corpuscular volume were performed on blood samples from 58 males aged 20–61 years. Concentrations measured in our sample are comparable with reference values. Statistically significant correlation coefficients were found between age and albumin (r = - 0.562, P < 0.001), serum zinc and albumin (r = 0.328, P < 0.05), serum zinc and hemoglobin (r = 0.291, P < 0.05), and total plasmatic proteins and albumin (r = 0.463, P < 0.001). These correlation coefficients were significant even after adjustment for age effect. The determination of serum zinc concentration may be useful in the assessment of clinical scenarios. Particularly, it may provide additional information for the diagnosis of specific pathologies, such as hepatic malfunctions. It could also be useful in the identification of different stages of anemia.     

Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells.

Chloride-dependent K transport ([K-Cl] cotransport) in dog red cells is activated by cell swelling. Whether the volume signal is generated by a change in cell configuration or by the dilution of some cytosolic constituent is not known. To differentiate between these two alternatives we prepared resealed ghosts that, compared with intact red cells, had the same surface area and similar hemoglobin concentration, but a greatly diminished volume. Swelling-induced [K-Cl] cotransport was activated in the ghosts at a volume (20 fl) well below the activation volume for intact cells (70 fl), but at a similar hemoglobin concentration (30-35 g dry solids per 100 g wet weight). Ghosts made to contain 40% albumin and 60% hemoglobin showed activation of [K-Cl] cotransport at a concentration of cell solids similar to intact cells or ghosts containing only hemoglobin. [K-Cl] cotransport in the resealed ghosts became quiescent at a dry solid concentration close to that at which shrinkage-induced Na/H exchange became activated. These results support the notion that the primary volume sensor in dog red cells is cytosolic protein concentration. We speculate that macromolecular crowding is the mechanism by which cells initiate responses to volume perturbation.     

Hematology of neonatal Japanese quail.

The hematologic development of Japanese quail was studied as a function of time through the first 49 hr post-hatching. Packed cell volumes, hemoglobin levels, erythrocyte and leukocyte numbers, as well as differential percentages of lymphocytes, heterophils, basophils, eosinophils, and monocytes were determined. Additionally, the mean corpuscular volumes, mean corpuscular hemoglobins, and mean corpuscular hemoglobin concentrations were calculated. Of particular interest are the increasing mean corpuscular volume and the increasing number of erythrocytes which were observed as the birds aged. Between the 13th and 19th hr after hatching, an almost 2-fold increase in the erythrocyte number was detected with a concomitant decrease in the mean corpuscular volume. These findings indicate that the state of the hematologic system in quail is highly dependent on the stage of post-embryonic maturation.     

Preparation and characterization of hormone-sensitive, resealed erythrocyte ghosts.

A method for preparing resealed turkey erythrocyte ghosts is described which utilizes hypotonic lysis and resealing following restoration of isotonicity. The resealed ghosts are isolated above 55% sucrose. The resealed ghosts are shown to be capable of maintaining high intracellular K+ concentrations in the presence of a low K+ extracellular environment. When ATP and an ATP-regenerating system are included during the resealing stage, (R)-(-)-epinephrine- and NaF-stimulated cyclic AMP accumulation, which is linear for 20 min, can be demonstrated. The concentration of (R)-(-)-epinephrine producing a half-maximal response in resealed ghosts is 1.0 +/- 0.4 X 10(-6) M. This is the same as that for (R)-(-)-epinephrine in the intact erythrocyte. The resealed ghosts are impermeable to Ca2+, but Ca2+ inhibition of cyclic AMP accumulation is noted if the divalent cation ionophore. A-23187, is present or if Ca2+ is included during the resealing stage.     

Effects of preparative procedures on the volume and content of resealed red cell ghosts

The effects of variations in preparative procedures on the volume and content of resealed red cell ghosts have been investigated. Following hypotonic lysis at 0 degrees C, and after a variable delay time (td), concentrated buffer was added to restore isotonicity; resealing was then induced by incubation at 37 degrees C for one hour. Using this procedure, both the resealed ghost volume and the residual hemoglobin (Hb) content decreased for increasing td. If ghosts were maintained at 0 degree C (i.e., no 37 degrees C incubation), they remained nearly spherical until isotonicity was restored. Their volume then fell abruptly, but subsequently increased toward an intermediate level. The fall in volume was greater and the final level achieved was smaller for longer delay times. At 0 degree C, return to isotonicity also halted the otherwise gradual loss of residual Hb from unsealed ghosts. In addition, ghosts with internal osmolality of 40 to 300 mosmol/kg were prepared by adding different amounts of concentrated buffer before resealing for one hour at 37 degrees C. Under these conditions, the final ghost volume was inversely related to the resealing osmolality (i.e., lower osmolality yielded a larger volume). Ghost volume also increased, along with Hb content, if the quantity or concentration of the red cell suspension added to the lysing medium was increased. We conclude that resealed ghost volume is influenced by the ratio of lysate to resealing medium osmolality and by the colloid osmotic pressure of the residual ghost Hb. These data indicate methods by which ghosts with desired characteristics can be prepared, and have potential application for studies of ghost mechanical and biophysical behavior.     

Transmembrane distribution of sterol in the human erythrocyte

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.     

A fluorimetric assay for the effects of cytolytic toxins on the transport properties of resealed erythrocyte ghosts.

We prepared resealed erythrocyte ghosts loaded with SPQ and chloride. We demonstrated that these membranes were still functional, as they were capable of exchanging anions, most probably through the band-3 protein. When cytolytic toxins (Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin) were offered to the resealed ghosts, the internal SPQ was released. This could be attributed to the formation of toxin-induced ion channels into the ghost membrane that were so large that SPQ could escape through them. This release was actually independent of the anion-exchanging protein, since DIDS had no inhibitory effect on it. Due to their simplicity, and because they do not lyse, erythrocyte ghosts may serve as useful models to study the action of cytolytic pore-forming toxins. To assess the validity of these model membranes we compared results obtained using RBC and resealed erythrocyte ghosts as targets for the toxin, finding complete consistency. Pre-assembled toxin channels could also be studied on the ghosts. Applying different proteolytic enzymes to the external compartment after channel formation, we found that performed E. coli hemolysin pores were at least partially destroyed by enzymatic digestion.     

高原鼢鼠和高原鼠兔红细胞低氧适应特征

为探讨高原鼢鼠对低氧高二氧化碳洞道生境及高原鼠兔对高海拔低氧生境的适应机制,用Sysmex SF-3000血细胞分析仪及聚丙烯酰胺凝胶电泳对两种高原动物的血常规及血红蛋白类型进行分析,后者采用聚丙烯酰胺凝胶电泳法。结果表明,高原鼢鼠和高原鼠兔的红细胞数(RBC)、红细胞压积(HCT)及平均红细胞容积(MCV)组间无显著差异(P>0.05),但高原鼢鼠和高原鼠兔的红细胞数显著高于SD大鼠,红细胞压积及平均红细胞容积均显著低于SD大鼠(P<0.05);高原鼢鼠的血红蛋白浓度(HBC)与SD大鼠无显著差异(P>0.05),但显著高于高原鼠兔的HBC(P<0.05)。高原鼢鼠血红蛋白主要有2种类型,高原鼠兔血红蛋白主要有3种类型,而SD大鼠血红蛋白主要有5种类型。从血红蛋白电泳迁移来看,2种高原动物血红蛋白类型有明显的趋同特征并与SD大鼠具有明显的差异。上述结果提示,长期适应高海拔低氧环境的高原动物的红细胞和血红蛋白表现出趋同进化,同时因生境和习性的差异又表现出各自的特异性。     

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.     

Human erythrocyte membrane acid proteinase (EMAP): sidedness and relation to cathepsin D

An acid proteinase purified from human erythrocyte membranes (Yamamoto, K. & Marchesi, V.T. (1984) Biochem. Biophys. Acta 790, 208-218), now termed "EMAP," was further characterized with respect to its localization and relation to cathepsin D. The membrane-associated form of EMAP was shown to be latent by demonstrating that no activity was detectable in both resealed (right-side-out) ghosts and inside-out vesicles in the absence of detergents. The enzyme associated with the inside-out vesicles was unstable when exposured to acidic pH between 4.0 and 4.5, whereas the enzyme associated with the resealed ghosts was stable in the wide pH range of 3.7 to 9.0. Tryptic digestion produced the loss of activity for the enzyme associated with the inside-out vesicles but not the resealed ghosts. The antibody to rat spleen cathepsin D, which cross-reacted weakly but detectably with EMAP, selectively bound to the inside-out vesicles. These results indicate the location of EMAP on th inner surface of the membranes. Comparison of a number of enzymatic properties of EMAP with rat cathepsin D showed significant differences between these two enzymes. EMAP was less stable in the pH range of 3.5 to 6.0 than cathepsin D. The enzymes were distinguished from each other by differences in their elution profiles on DEAE-Sephacel and chromatofocusing columns and by differences in the extent of inhibition by a few specific inhibitors. Both enzymes revealed significant differences in the amino acid composition and specific activity towards bovine hemoglobin. The immunological relationship between these two enzymes is discussed.     

The interaction of resealed reticulocyte ghosts with Fe3+-transferrin-CO3(2-)

The present studies were designed to investigate the interaction of Fe3+transferrin-CO3(2-) with the transferrin receptors of the resealed reticulocyte ghost and to assess the degree to which the iron release reaction can be reconstituted in resealed ghosts supplemented with entrapped cytoplasmic components. Reticulocyte, but not erythrocyte, ghosts displayed an intact Fe3+transferrin-CO3(2-) binding capability. When ATP, NADH and ferritin were included during the resealing process, some iron release to the ghosts was observed.     

Cytosolic protein concentration is the primary volume signal in dog red cells

It is not known whether the activation of Na/H exchange by shrinkage in dog red cells is due to the packing of cell contents or a change in cell configuration. To make this distinction we prepared resealed ghosts that resembled intact cells in hemoglobin concentration and surface area, but had one-third their volume. A shrinkage-induced, amiloride-sensitive Na flux in the ghosts was activated at a much smaller volume in the ghosts than in the intact cells, but at the same concentration (by weight) of dry solids in both preparations. Na/H exchange in ghosts containing a mixture of 40% albumin and 60% hemoglobin (weight/weight) was activated by osmotic shrinkage at a dry solid concentration similar to that of intact cells or of ghosts containing only hemoglobin. We conclude that the process of Na/H exchange activation by cell shrinkage originates with an increase in the concentration of intracellular protein and not with a change in membrane configuration or tension. The macromolecular crowding that accompanies the reduction in cell volume probably alters the activities of key enzymes that in turn modulate the Na/H exchanger.     

Age‐related changes in mean corpuscular volumes in patients without anaemia: An analysis of large‐volume data from a single institute

Although the mean corpuscular volume (MCV) has been associated with various diseases, these associations in relation to the age‐related trends in MCV remain unclear. Therefore, we used a dataset with over one million values to identify the relationship between ageing and MCV changes. All laboratory data obtained between November 1998 and November 2019 at Chungbuk National University Hospital were retrospectively collected. After excluding cases with missing values for individual complete blood count parameters, outlier MCV values, and ages less than 1 year and more than 88 years, 977,335 MCV values were obtained from 309,393 patients. Principal component analysis of blood components with ages and analysis of the median value changes for each blood component across decade‐wise age groups were conducted to identify relationships between ageing and changes in blood components. The median values of MCV showed gradual increments with age. The linear relationship for patients aged 1–25 years had a larger slope than that for patients aged 26–88 years. For MCV, the equation for patients aged 1–25 years was 0.40*(age) + 81.24 in females and 0.45*(age) + 79.58 in males. The equation for patients aged 26–90 years was 0.04*(age) + 88.97 in females and 0.06*age + 88.30 in males. Among patients aged >40 years, the MCV value was higher in men than in women. Analysis of a large dataset showed that the MCV gradually increased with age and the linear relationship differed between patients aged 1–25 and 26–88 years.     

Effect of the period of resting in elite judo athletes

The purpose of this study was to evaluate the effect of the resting period on hematological and copper-zinc-dependent antioxidant indices in Brazilian elite judo athletes (n=7). Venous blood samples were collected after 24-h and 5-d periods of resting following a competition, with an interval of 30 d between collections. Two months prior to and during the study, each athlete received an individualized adequate diet. Body composition was determined at both study periods. The following were analyzed: in whole blood, hemoglobin, hematocrit, red cell count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, and white cell count; in plasma, zinc, copper, iron, ceruloplasmin, and total iron-binding capacity; in erythrocytes, metallothionein, copper/zinc superoxide dismutase, and osmotic fragility. Dietary intake and body composition did not affect the biochemical measurements. A significant reduction in ceruloplasmin and superoxide dismutase activity was found after 5 d compared to 24 h of resting. A significant correlation between erythrocyte metallothionein and red cell distribution width was observed after 24 h of resting (r=−0.83, p=0.02) whereas positive correlations of metallothionein with hemoglobin, red cell count, and mean corpuscular hemoglobin concentration were observed after 5 d of resting (r≥0.76, p≤0.05). Our results suggest that a longer resting period favors homeostatic adjustments in the erythrocyte population and in the copper/zinc-dependent antioxidant system in elite judo athletes.     

The Role of Reticulocyte Hemoglobin Content for the Diagnosis of Functional Iron Deficiency in Hemodialyzed patients

The effectiveness of reticulocyte hemoglobin content (CHr) had been reported to detect early functional iron deficiency especially among Chronic kidney disease (CKD) patients. CHr is more superior to classic biochemical indices in reflecting transient iron-deficiency status, therefore improving diagnosis and treatment. This study was conducted to determine the sensitivity of CHr in the diagnosis of functional iron deficiency (FID) in hemodialyzed patients. One hundred hemodialyzed patients along with 60 healthy controls were recruited and blood specimens were collected. Venous blood was used for hematological and biochemical investigations collected via 3 ml lavender-top tubes for hematological tests including CBC, blood film, ESR and CHr, and red-top tube for biochemical tests including TIBC, SF and CRP. A statistically significant decrease was noted in CHr values between hemodialysis patients and the control group (24.8 ± 2.0 pg vs. 30.9 ± 1.3 pg, p<0.001). CHr values showed a significant correlations with RBCs, Hb- hemoglobin, Hct- hematocrit level, MCV- mean corpuscular volume, MCH- mean corpuscular hemoglobin, MCHC, RDW- red cell distribution width , SI-Serum Iron, TIBC- Total iron binding capacity and TSAT- Transferrin saturation. The present study showed that CHr in comparison to the conventional hematological and biochemical markers commonly used to diagnose iron deficiency.     

Involvement of spectrin and ATP in infection of resealed erythrocyte ghosts by the human malarial parasite, Plasmodium falciparum

Resealed erythrocyte ghosts were prepared under different experimental conditions and were tested in vitro for susceptibility to infection with the human malarial parasite, Plasmodium falciparum. Resealed ghosts, prepared by dialyzing erythrocytes in narrow membrane tubing against low ionic strength buffer that was supplemented with magnesium ATP, were as susceptible to parasite infection as were normal erythrocytes. There was a direct correlation between intraerythrocytic ATP content and susceptibility to parasite infection. Neither MgCl2 nor sodium ATP could be substituted for magnesium ATP in maintaining high intraerythrocytic ATP concentration. When resealed ghosts were loaded with antispectrin IgG, malaria merozoite invasion was inhibited. At an average intracellular antispectrin IgG concentration of 3.5 micrograms/10(8) cells, there was a 35% inhibition of parasite invasion. This inhibition was due to spectrin crosslinking within the resealed ghosts, since the monovalent, Fab' fragments of antispectrin IgG had no inhibitory effect on invasion. These results indicate that the cytoskeleton plays a role in the complex process of merozoite entry into the host erythrocyte.     

Hematology and serum chemistry in the white-crowned mangabey (Cercocebus torquatus lunulatus) and in the mandrill (Mandrillus sphinx)

Abstract: Reference values of some hematologic and plasma chemical parameters were established in two species of clinically normal Cercopithecidae. The following variables were studied in seven mandrills (Mandrillus sphinx) and nine white-crowned mangabeys (Cercocebus torquatus lunulatus): hematocrit, hemoglobin concentration, erythrocyte and leucocyte counts, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, glucose, urea, uric acid, cholesterol, creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine phosphokinase, lactic dehydrogenase, gamma glutamyl transpeptidase, total serum proteins, albumin, globulins, albumin-globulin ratio, sodium, potassium, calcium, magnesium, total phosphorus, chloride, and serum osmolality. Few differences were observed when compared with human hematological data and with other species of Cercopithecidae Primates.     

Hematological and biochemical reference intervals for wild caught Eurasian otter from Spain

Hematologic and serum chemistry reference intervals were determined from 33 wild caught Eurasian otters (Lutra lutra lutra) between November 1995 and May 1998 during a reintroduction project. Blood was obtained by jugular venipuncture after administration of ketamine and medetomidine. The mean, standard deviation, and range for 19 hematology parameters and 28 serum chemistry values are presented. There were no significant differences between sexes in most analytes. The results are in agreement with those reported previously for Eurasian otters with the exception of higher leukocyte and neutrophil counts, lower eosinophil and lymphocyte counts and higher activities for aspartate aminotransferase and creatine kinase. The Eurasian otters have lower erythrocyte counts but higher mean corpuscular volume and mean corpuscular hemoglobin values than the river otter (Lutra canadensis) in North America.