Young Erythrocytes Research Articles

組換え体ヒトエリスロポエチン投与によるラット赤血球の諸指数，代謝ならびにレオロジー変化 ＩＩ レオロジー的性質

Rheological properties of erythrocytes induced by intravenous administration of recombinant human erythropoietin (rhEPO; 180 or 1800IU/kg daily) to 6 weeks-old rats for 1 or 2 weeks were examined. (1) Increase of blood viscosity was entirely dependent upon the hematocrit. (2) No change of erythrocyte deformability was detected with a high shear rheoscope, in spite of the appearance of large and light (presumably, young) erythrocytes. (3) Velocity of rouleau formation in autologous plasma decreased, probably due to high density of negative charge on the surface of young erythrocytes. In addition, the composition of plasma proteins was not affected by rhEPO-administration. (4) Upon the long-term administration, these rheological findings were not essentially altered.

Erythropoiesis and thrombopoiesis in the head-kidney of the sea bass (Dicentrarchus labrax L.): An ultrastructural study.

The head-kidney of the sea bass is a source of erythropoietic and thrombopoietic cells. No significant lymphopoietic activity is found. Erythropoiesis, thrombopoiesis and granulopoiesis are intermingled, which suggests that only one environmental "niche" exists to modulate the lineage development. No numerous proerythroblasts are present. Erythropoiesis consists of a proerythroblast, basophilic erythroblast, polychromatophilic erythroblast, acidophilic erythroblast, young erythrocyte and old erythrocyte, the latter being nucleated and without nuclear vacuolation. The numbers of the free ribosomes and polyribosomes decrease progressively from proerythroblasts to young erythrocytes, suggesting a high synthetic activity from early on. Immature erythropoietic cells show pits and protrusions correlated to micropinocytotic vesicles, indicating ropheocytosis. The peripheral band of microtubules is the most remarkable cytoplasmic structure in proerythroblasts. Granular cytoplasmic inclusions or lysosomes were not observed in erythropoietic cells. The old erythrocyte reveals an electron-dense homogeneous cytoplasm with occasional mitochondria and a small Golgi apparatus. Thrombopoietic cells comprise both the immature and mature prothrombocyte and adult thrombocyte. Nuclear and cytoplasmic densities increase and the surface connected canalicular system develops during maturation. A marginal band of microtubules is present in the cytoplasm from prothrombocytes to mature thrombocytes. Some pseudopodial processes, dense granules and vesicles, probably indicating passive storage, are also observed.

A study on hemolysis in hemodialysis patients

In order to investigate the pathogenesis of hemolysis in chronic renal failure, red cell life span and hemolysis starting point (HSP) by coil planet centrifuge method were studied in 32 hemodialysis (HD) patients and 16 healthy subjects. Mean red cell life span was 22.0 days before recombinant human erythropoietin (r-HuEPO) therapy, and prolonged up to 28.1 days after r-HuEPO therapy. Mean HSP in HD patients was significantly elevated than healthy subjects. Mean HSP was 106.7 mOsm before r-HuEPO therapy, and improved to 101.0 mOsm after r-HuEPO therapy. HSP improved from 106.8 mOsm to 100.8 following the correction of bicarbonate level with HD. HSP was negatively correlated to bicarbonate level pre- and post-r-HuEPO therapy, but HSP was not correlated to BUN, serum creatinine, hydrogen ion, anion gap and amount of body water removal during HD. When blood PH in 18 HD patients was adjusted by 7% NaHCO3 from 7.21 to 7.40, HSP improved to normal range in all. These data suggested that osmotic fragility was normal in younger erythrocyte in HD patients, and a decrease of plasma bicarbonate level resulted in an increase of hemolysis.

Age-related and phenylhydrazine-induced activation of the membrane-associated cathepsin E in human erythrocytes.

We examined the effects of cell aging and phenylhydrazine-induced oxidant damage on erythrocyte cathepsin E, which is present as a latent, membrane-associated enzyme in normal human erythrocytes. When young erythrocytes isolated from human mature erythrocytes by Percoll density gradient centrifugation were aged in vitro, the membrane-associated cathepsin E was progressively released from the membrane as an active enzyme. During the cell aging up to 100 h, about 40% of the membrane-associated enzyme was activated and solubilized. When phenylhydrazine was incubated with the erythrocytes, it also caused the activation and solubilization of cathepsin E in a dose-dependent and time-dependent manner. Exposure of erythrocytes to 2.5 mM phenylhydrazine for up to 2 h led to about 40% activation of the membrane-associated enzyme. Both aging and phenylhydrazine-treatment were accompanied with an increase in the association of the cytosolic proteins, primarily hemoglobin, with the membrane, which occurred prior to the release of cathepsin E from the membrane. A similar activation for the membrane-associated enzyme was observed with in vitro-aged hemoglobin-free membrane ghosts. Thus, the primary mechanism for activation of cathepsin E in the intact cells seems to be through lesion of the membrane framework that results from increased binding of hemoglobin to the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting employing polyclonal IgG antibodies for human spectrin and band 3 revealed that breakdown of the membrane proteins was enhanced in both aged and phenylhydrazine-treated cells. The relation between the cathepsin E activation and the membrane protein breakdown is discussed.

The fetal blood erythrocyte micronucleus assay: classification of RNA-positive erythrocytes into two age populations by RNA aggregation state

The use of a fluorescent stain containing Hoechst 33258 and pyronin Y in the fetal mouse transplacental micronucleus assay allows classification of erythrocytes into three subpopulations on the basis of RNA staining, and permits micronuclei to be scored in all three subpopulations. The youngest erythrocytes stain uniformly positive for RNA (UEs). In older erythrocytes RNA aggregates to give the cells a stippled appearance (SEs) and ultimately disappears, leaving cells which do not stain positively for RNA. Frequencies of micronucleated UEs and SEs were determined at 30 and 48 h following a single dose of methyl methanesulfonate, benzo[a]pyrene, lasiocarpine, monocrotaline or heliotrine. With each agent and dose tested, the frequency of micronuclei increased first in the younger UEs and later in SEs. The use of the Hoechst/pyronin staining procedure, which permits DNA to be distinguished from RNA, minimizes the potential for mis-scoring RNA artefacts as micronuclei and also increases the efficiency of the assay by permitting two age populations of erythrocytes to be scored in each sample.

Sodium-lithium countertransport activity and its sensitivity to inhibitors with erythrocyte ageing in man

Sodium-lithium countertransport is believed to depend on a specific protein and since such proteins usually decline with erythrocyte age it would be expected to be reduced in older cells. In fact, sodium-lithium countertransport increased with ageing, whereas the rate constant of the sodium pump decreased. The increase in sodium-lithium countertransport with erythrocyte ageing was due to a phloretin insensitive component that was not present in young erythrocytes. Raised sodiumlithium countertransport in patients with essential hypertension was due mainly to an increased phloretin sensitive component but the phloretin insensitive component was also higher in middle aged erythrocytes. Amiloride had no effect upon sodiumlithium countertransport or unidirectional sodium influx in cells of any age. This suggests that sodium-lithium countertransport is not a mode of action of the sodium-proton exchanger in the erythrocyte.

Glucose-6-Phosphate Dehydrogenase Deficiency in Newborn Infants of Eastern Saudi Arabia

ABSTRACT Neonatal screening for glucose-6-phosphate dehydrogenase (G6PD) deficiency was established in a large general hospital in the Eastern Province of Saudi Arabia. During the first 2 years of ...

The in vivo ageing of red cell enzymes: direct evidence of biphasic decay from polycythaemic rabbits with reticulocytosis.

The rate at which the activities of red cell enzymes decay during maturation of the reticulocyte and ageing of the erythrocyte is poorly understood. It has recently been suggested that loss of enzyme activity may be rapid during reticulocyte maturation and occur slowly thereafter. We have now devised a rabbit model in which reticulocytosis is combined with transfusional polycythaemia. This makes it possible to follow the enzymatic activity of a cohort of reticulocytes without the interfering effect of newly formed cells. In this model the reticulocyte count of the experimental animals falls from about 60% to subnormal levels within 4 d. Red cell hexokinase activity declined rapidly with more than half of the activity being lost within 7 d, little change occurring thereafter. Glucose-6-phosphate dehydrogenase activity declined more gradually, but also followed a biphasic course. The fall of pyrimidine 5' nucleotidase activity was the slowest of the three age-dependent enzymes studied. This is consistent with our observations in children with transient erythroblastopenia of childhood, in which this enzyme, alone of the age-dependent enzymes, was found to be decreased in a senescent red cell population. In contrast to the three age-dependent enzymes studied, the activity of lactate dehydrogenase remained unchanged during the course of the experiment. These studies provide direct verification of the suggestion that the decline of red cell enzyme activities may be biphasic. They show, moreover, that enzyme decay is not only rapid in reticulocytes, but also in young erythrocytes.

Training induced effects on blood volume, erythrocyte turnover and haemoglobin oxygen binding properties.

The effect of three weeks ergometer training (Tr) 5 times a week for 45 min at 70% VO2max by 6 subjects on erythrocyte turnover and haemoglobin O2 affinity has been studied. Increased reticulocytosis could be observed from the second day after beginning Tr until a few days after its end, probably caused by increased erythropoietin release by the kidney. Erythrocyte destruction was most pronounced in the first and markedly reduced in the third week of Tr. Elevated glutamate oxalacetate transaminase activity and creatine as well as lowered mean corpuscular haemoglobin indicate a younger erythrocyte population in the first week of recovery. Total blood volume increased during the course of Tr by 700 ml, mainly caused by a raised plasma volume (74%). Red cell volume increased later with maximal values one week after Tr (+280 ml). In this week the standard oxygen dissociation curve was most shifted to the right (P50 increased from 3.77 +/- 0.05 kPa to 3.99 +/- 0.07 kPa) and the Bohr coefficients had their lowest values. Both effects are completely explainable by the haemoglobin O2 binding properties of young erythrocytes. After training, all parameters of physical performance (VO2max, maximal workload, heart rate during rest and exercise) were markedly improved, indicating fast adaptation mechanisms. The increased erythrocyte turnover, including higher erythropoiesis, seems to be one important part of these effects.

Number and distribution density of ABH and MN antigen sites on young and old human erythrocyte surfaces

There were no differences in the number of A and M antigen sites between young and old human erythrocyte surfaces. No essential differences in the number of A 1, N and Vicia graminea N antigen sites could be observed between young and old erythrocytes. The number of B and H antigen sites on cell surface was significantly higher in young erythrocytes than in old ones. The distribution density of A and M antigen sites on young erythrocyte was remarkably higher than that on old ones. Compared with young erythrocytes, significant increases in the distribution density of A 1, B, H, N and Vicia graminea N antigen sites were observed in aged erythrocytes. It is suggested from these and other observations that human erythrocytes aging is accompanied by elimination of a small amount of B and H antigens from cell membranes, while A, A 1, M, N and Vicia graminea N antigens are not released from cell membranes during in vivo aging.

Comparative study on the main membrane-surface sialoglycopeptides released from young and old human erythrocytes with trypsin

1. 1. The amount of sialoglycopeptide (SGP) mixture released from young (Y) erythrocytes by trypsin treatment was significantly larger than that from old (O) ones. 2. 2. The two main membrane-surface sialoglycopeptides (Y-SGP and O-SGP) were isolated in yields of about 45% and 42% from the mixtures by a combination of Sephadex G-50 superfine gel filtration with DEAE-cellulose chromatography, respectively. Molecular weights of 12,000 and 12,100, respectively, were found for the Y-SGP and O-SGP. 3. 3. The Y-SGP showed a greater proportion of sialic acid than the O-SGP. The contents of galactose, N-acetylglucosamine and N-acetylgalactosamine of the Y-SGP were significantly lower than those of the O-SGP. 4. 4. Both the SGPs exhibited the same reactivities against the MN reagents, but the reactivities against the lectins of Limulus polyphemus and Phaseolus vulgaris (E) were stronger in the Y-SGP than in the O-SGP. Neither the Y-SGP nor the O-SGP showed the reactivity to Arachis hypogaea (anti-T) lectin.

Tetrahydrobiopterin deficiency: assay for 6-pyruvoyl-tetrahydropterin synthase activity in erythrocytes, and detection of patients and heterozygous carriers.

6-Pyruvoyl-tetrahydropterin synthase (PTS), a key enzyme in the synthesis of tetrahydrobiopterin in man, is defective in the most frequent variant of tetrahydrobiopterin-deficient hyperphenylalaninaemia (atypical phenylketonuria). An assay for PTS activity in erythrocytes was developed. It is based on the PTS-catalysed formation of tetrahydrobiopterin from dihydroneopterin triphosphate in the presence of magnesium, sepiapterin reductase, NADPH, dihydropteridine reductase, and NADH, and fluorimetric measurement of the product as biopterin by high performance liquid chromatography (HPLC) after oxidation with iodine. The PTS activity was higher in younger erythrocytes, including reticulocytes, than in older ones. Fetal erythrocytes showed approx. four times higher activities than those of adults. Using a more purified human liver sepiapterin reductase fraction which gave a lower yield than a crude preparation, adult controls (n = 8) showed a mean erythrocyte PTS activity of 17.6 (range 11.0-29.5) microU/g Hb. Nine of 11 patients with typical PTS deficiency showed activities between 0% and 8% of the mean of controls, and two of 11 showed 14% and 20%, respectively. The obligate heterozygotes (n = 16) had activities of 19% (range 8%-31%) of the mean of controls, i.e., significantly less than the expected 50%. Four patients with the "peripheral" type of the disease showed 7%-10% of the mean of controls. Thus, the assay did not distinguish between patients and heterozygotes in every family. The assay is well suited to the identification of heterozygotes of PTS deficiency in family studies.

赤血球プロテアーゼと機能制御

To understand the physiological significance of erythrocyte membrane aspartic proteinase (EMAP), its catalytic properties and possible mechanisms for its activation were investigated, and the results are as follows. (1) The enzyme was indistinguishable from cathepsin E but not cathepsin D enzymatically and immunochemically. (2) The enzyme was found at high levels in neutrophils, lymphocytes and platelets as well as erythrocytes. (3) The membrane-bound latent enzyme was activated by various procedures that induce the disturbance of normal erythrocyte membrane organization, e.g., by treatments with phospholipase C and trypsin or simply by heating at 40°C. The activation was accompanied by dissociation of the enzyme from the membrane. (4) When the young erythrocyte obtained by density gradient centrifugation on Percoll gradients were aged in vitro, a decrease in the membrane-bound form of EMAP and an increase in the soluble form were observed in a time-dependent manner. The erythrocyte aging also caused an increase in the membrane-bound form of cytosolic proteins such as hemoglobin.

Red cell age effects on metabolism and oxygen affinity in humans

Hemoglobin-oxygen-binding characteristics and essential influencing factors were investigated in human erythrocytes of different age separated by density gradient centrifugation. The most important age-dependent changes of the cell milieu are losses of K +, organic phosphates and water; the latter also leads to an increased concentration of negative charges on Hb. This augments the Donnan effect, which is only partly compensated for by a decrease of DPG. The oxygen dissociation curve of the oldest fraction (P 50 23.4 mm Hg) is shifted to the left compared to young cells (P 50 29.2 mm Hg), and Hills 'n' is decreased (old cells 2.31, young cells 2.74). The Bohr effect for CO 2 increases in the old population (BC CO 2 at 50% S O 20.63 and 0.24 for old and young erythrocytes, respectively). This effect is less pronounced for the Bohr coefficients for lactic acid (ΔBC Lac 0.09). Most cell age-dependent alterations of Hb-O 2-binding (including BC CO 2) are explainable by opposite alterations of [Hb] and [DPG], causing the change of the ratio [DPG]/[Hb] from 1.3 to 0.7 during the aging process of the erythrocytes. Minor effects may result from aging of the Hb-molecule itself.

Biochemical changes associated with the osmotic fragility of young and mature erythrocytes caused by parathyroid hormone in relation to the uremic syndrome

The effect of parathyroid hormone at concentrations found in uremic patients on erythrocytes (RBC) from newborn and adult rabbits was studied in relation to the fragility pattern in hypotonic salt solutions and the activities of Ca- and Mg-dependent ATPases. Median osmotic fragility of RBC from newborn rabbits was significantly lower than in mature rabbits. Parathyroid hormone (PTH) stimulated to a greater extent the mean osmotic fragility in RBC from newborn rabbits, than in those from adults. Similarly, the hormone stimulated to a much greater extent the Ca-ATPase but not the Mg-ATPase in RBC from the newborn rabbits, in comparison to those from adult rabbits. PTH, which is greatly elevated in the blood of patients with chronic renal failure, may be one cause of the anemia seen in these patients, and its effect, which is mediated by Ca-ATPase activity, is stronger on young RBC. There were significant morphological changes in the young RBC caused by PTH, as seen with scanning electron microscopy.

Effect of long-term supplementation of folate on folate status in plasma and erythrocytes.

Folate nutritional status was estimated by radioassay of folate levels in plasma and erythrocytes during and after a long-term supplementation of folic acid. A 1-mg dose of folic acid per day was administered orally to 6 healthy subjects for 17 weeks. After 4 weeks of supplementation the mean folate concentration in plasma reached 11 ng/ml and remained constant thereafter, but decreased exponentially after stopping the supplementation. However, the folate concentrations in reticulocytes and erythrocytes increased linearly in all subjects during the supplementation. These results suggest that folate-rich, young erythrocytes are mixed at a constant rate with circulating ripe ones, which have a lower folate content, during folate supplementation.

Age-dependent phagocytosis of erythrocytes by the isolated perfused rat liver after galactosamine hepatitis and alpha-naphthylisothiocyanate cholestasis

Isolated rat livers were perfused with an immunoglobulin-free fluid containing homologous rat erythrocytes suspended in an isotonic saline solution. Galactosamine hepatitis and alpha-naphthylisothiocyanate cholestasis were induced as experimental liver diseases. The glutamic oxalacetic transminase (GOT) and guanosine triphosphate (GPT) activities, the potassium level as well as the redox quotients of lactate/pyruvate and β-hydroxybutyrate/acetoacetate were determined as liver function parameters. Results: (1) The same dose of galactosamine led to two different types of reaction. The group suffering more damage (rendering maximum GOT activities) phagocytosed significantly ( p ≤ 0.05) more erythrocytes than the other group. (2) Galactosamine hepatitis significantly slows down the phagocytosis of erythrocytes. The function of the mononuclear phagocytosing cells in the liver is intact. (3) The alpha-naphthylisothiocyanate (ANIT) cholestasis significantly reduces the capacity of the mononuclear phagocytosing cells in the liver. (4) Young erythrocytes were phagocytosed significantly better than old ones in either type of liver damage, in galactosamine hepatitis and in ANIT cholestasis as well as by healthy livers.

Methotrexate pharmacokinetics in age-fractionated erythrocytes

Age fractionation of erythrocytes is useful for further studies of the pharmacokinetics of methotrexate (MTX) in red blood cells. We separated erythrocytes from five blood donors and four patients at different time points after MTX infusions, using discontinuous Percoll gradients consisting of four solutions with a difference of 3% in density among them. The procedure yielded five distinct fractions of erythrocytes of increasing mean cell age as judged by declining reticulocyte enrichment and erythrocyte aspartate aminotransferase activity among the five fractions. MTX concentrations of the erythrocytes were measured at different times in connection with five 24-h MTX infusions (0.7-4 g/m2) on 14 occasions. Two days after completion of MTX infusion, no MTX was detected in the youngest erythrocyte population in two patients. Seven days after the infusion, the highest MTX concentrations were found in the youngest red blood cells. Ten to fourteen days following the MTX treatment, considerably lower MTX concentrations were found in the young red blood cells, and the MTX-containing erythrocytes seemed to have moved down the gradient. Just before the next MTX infusion (after 28 days) no MTX could be detected in the young erythrocytes. The MTX concentrations at that time were highest in the oldest erythrocyte fractions. This study shows more directly that MTX is incorporated in the red cell precursors of the bone marrow. The pharmacokinetics demonstrated correspond to a maturation time of the erythroblasts of about 7 days.

Anomalous Clustering of Underglycosylated Band 3 in Erythrocytes and Their Precursor Cells in Congenital Dyserythropoietic Anemia Type II

Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II

Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.