Erythrocytes Of Different Ages Research Articles

Erythrocyte Senescent Markers by Flow Cytometry

Background: Mature red blood cells lack protein synthesis and are unable to restore inactivated enzymes, damaged cytoskeleton and membrane proteins. An oxidation breakdown of band 3 is probably part of the mechanism leading to the generation of a senescent cell antigen. This specific signal serves for the clearance of RBCs by inducing the binding of autologous IgG and C3, leading to phagocytosis. In addition, phosphatidilserin molecules appear in the outer membrane and the CD47 expression diminishes. Methods: Erythrocytes of different ages from whole blood were studied by flow cytometry analysing light scatter proprieties, binding of autologous IgG, C3 complement deposits, externalization of phosphatidylserine and CD47 expression. Dot-plot analysis based on forward scatter versus side scatter parameters showed two RBCs populations of different sizes and density. RBCs were further incubated with Alexa 488 IgG, APC-anti-C3, PE-annexin-V and PE-CD47. The comparison of the values obtained for the different variables studied in SeRBC and YRBC populations was carried out by the Student t-test for matched samples or by the Wilcoxon test (after verification of the normality assumption). Results: The percentage of IgG and C3 positive cells was significantly higher in senescent red blood cells population. The fraction of annexin-V positive RBCs was also larger in SeRBCs while the CD47 expression was lower in this population. Conclusions: These results indicate that flow cytometry allow differenciation of erythrocytes populations of different ages, turning this tool into an useful alternative option to study erythrocyte aging process. These findings will contribute to a better understanding of the process and mechanisms involved in erythrocyte senescence process.

Comparative Analysis of PKСα and PKCζ Activities in Rat and Lamprey Erythrocytes of Different Ages

The present study aimed to find out a link between ageing of rat and lamprey erythrocytes and activity of two isoforms of protein kinase C (PKC), РKСα and РKСζ. The whole cell population was separated into fractions of different ages in Percoll density gradient. The validity of separation was confirmed by the number of immature erythrocytes, reticulocytes. PKC activity was analyzed in cytosolic and membrane cell fractions. Rat erythrocytes express both PKC isoforms, РKСα and РKСζ, whereas lamprey erythrocytes express only РKСζ. РKСα is identified as a major band at ~ 80 kDa and minor bands at ~ 55–65 kDa; РKСζ is represented by a single band at ~ 80 kDa. In young rat erythrocytes, РKСα is detected mainly in cytosolic fractions, while in membrane fractions its level is by far lower. As cells age, PKCα is translocated from the cytosol to membranes and undergoes proteolytic degradation due to repeated cycles of activation. As a result, in aged erythrocytes relative total PKCα expression (as a sum of expressions in the cytosol and membranes per total protein level) diminishes, indicating a depletion of the PKCα pool and a decline in its functional activity. In both animal species, a highest PKCζ level is observed in the cytosol of young erythrocytes. Erythrocyte ageing is accompanied by a gradual decrease in expression of free cytosolic PKCζ and concurrent increase in the level of its membrane-bound forms. However, in contrast to PKCα, PKCζ is not proteolyzed; its total level in cells and perhaps functional activity do not change throughout the erythrocyte lifespan.

Influence of chlorpromazine on the resistance of erythrocytes of rats of different ages to hypertonic conditions

The osmotic stability of native and modified with chlopromazine (CPR) erythrocytes of 1- and 12-month rats to hypertonic conditions in sucrose solutions and hypertonic shock (4.0 M NaCl) has been studied. It has been shown that 2-min incubation of rat erythrocytes of different ages in hypertonic sucrose media does not reveal any differences in the osmotic stability of these cells. In this case, CPR does not affect cell hemolysis. An increase of the incubation time in hypertonic sucrose solutions to 30 minutes allowed detecting a greater osmotic sensitivity of erythrocytes of 1-month animals to the action. In this case, the protective effect of CPR for older age rat erythrocytes (12 months) has been established. It has been found that in 4.0 M NaCl the hemolysis level of animal erythrocytes of both age groups increases with preliminary incubation (2 min) in a sucrose medium with a concentration of 0.7 M and above. With increasing exposure time (30 min) in sucrose hypertonic solutions, the sensibilization of animal erythrocytes of both age groups to the action of hypertonic shock is also intensified. In this study it has been shown that the influence of CPR on the sensitivity of 1-month-old animal erythrocytes to the transfer in 4.0 M NaCl depends on the tonicity and duration of the cell initial incubation in sucrose solutions. Thus, CPR increases the osmotic resistance of erythrocytes, which were preexposed in sucrose solutions at a concentration of 0.6–0.8 M for 2 and 30 min. Some increase of hemolysis level of these animals erythrocytes modified with CPR in 4.0 M NaCl has been observed after 2 min of incubation in sucrose solutions at a concentration of 0.27–0.5 M. The exclusively protective influence of CPR on 12-month-old animal erythrocytes in conditions of hypertonic shock has been revealed. A quantitative estimation of the efficiency of CPR at hypertonic shock (4.0 M NaCl) of different age animal erythrocytes has been carried out by calculation of the antihemolytic activity value (AG). Short-term incubation (2 min) in a sucrose media does not reveal any differences in the values of AG of CPR for erythrocytes of both age groups. For the cells of young rats, increase of AG of CPR is observed at incubation in sucrose medium to 30 min and for erythrocytes of the older group – to 10 min. With an increase in incubation time of up to 60 min the CPR efficiency in hypertonic saline media is reduced for rat cells in both age groups but in varying degrees.

Glycated Hemoglobin, Plasma Glucose, and Erythrocyte Aging.

The relationship between HbA1c and blood glucose averages has been characterized many times, yet, a unifying, mechanistic description is still lacking. We calculated the level of HbA1c from plasma glucose averages based solely on the in vivo rate of hemoglobin glycation, and the different turnover rates for erythrocytes of different ages. These calculations were then compared to the measured change of HbA1c due to changes in mean blood glucose (MBG), to complex models in the literature, and our own experiments. Analysis of data on erythrocyte ageing patterns revealed that 2 separate RBC turnover mechanisms seem to be present. We calculated the mean red blood cell (RBC) life span within individuals to lie between 60 and 95 days. Comparison of expected HbA1c levels to data taken from continuous glucose monitors and finger-stick MBG yielded good agreement (r = .87, P < .0001). Experiments on the change with time of HbA1c induced by a change of MBG were in excellent agreement with our calculations (r = .98, P < .0001). RBC turnover seems to be dominated by a constant rate of cell loss, and a mechanism that targets cells of a specific age. Average RBC life span is 80 ± 10.9 days. Of HbA1c change toward treatment goal value, 50% is reached in about 30 days. Many factors contribute to the ratio of glycated hemoglobin, yet we can make accurate estimations considering only the in vivo glycation constant, MBG, and the age distribution of erythrocytes.

Effects of amyloid-β peptide Aβ25–35 on glycolytic and antioxidant enzymes in erythrocytes of different ages

Amyloid-β peptide Aβ25–35 was shown to cause lysis of rat erythrocytes of different ages. The toxicity of Aβ25–35 positively correlated with both the erythrocyte age and the peptide concentration. The activity of glycolytic, antioxidant, and Na+/K+-ATPase enzymes decreased with erythrocyte aging in vivo. In vitro Aβ25–35 reduced the activity of hexokinase, phosphofructokinase, pyruvate kinase, glutathione peroxidase, and glutathione transferase and increased Na+/K+-ATPase activity in aged erythrocytes to a greater degree than in young cells.

Effect of cAMP on Peptide Formation in Human Erythrocytes Depending on Cell Age

We found that exogenous and endogenous cAMP induces changes in the spectrum and amount of intraerythrocytic peptides in human erythrocytes of different age. Activation of cAMP formation with epinephrine leads to the appearance of peptides of the other type and to an increase in their total amount in young cells, while blockade of these β-adrenoreceptors with propranolol eliminates these effects. Inhibition of cAMP phosphodiesterase with imidazole in the absence of hormonal signals elevates the content of longer peptides in erythrocytes compared to the effect of exogenous cAMP. The degree of this elevation depends on erythrocyte age.

Phosphotyrosine phosphatases acting on band 3 in human erythrocytes of different age: PTP1B processing during cell ageing

The phosphorylation of tyrosine residues of human red blood cell (RBC) band 3 is regulated in vivo by constitutively active tyrosine-kinases (PTKs) and phosphotyrosine-phosphatases (PTPs), identified so far as, respectively, p72 syk and p56/53 lyn, and PTP1B and SHPTP-2. Tyr-phosphorylation of band 3 increases upon reduction of cell volume as in hypertonic media or during Ca 2+-induced membrane vesiculation. We show here that old RBCs display higher Tyr-phosphorylation levels of band 3 than younger cells under hypertonic conditions, at least in part due to the reduced cell volume of old RBCs, a condition of lowered threshold for activation of volume-sensitive PTKs. We have also analysed the membrane-bound PTP activity and the relative abundance of PTP1B (as the main membrane-associated PTP) in RBCs of different age. Immunodetection of PTP1B in purified ghost membranes revealed that the catalytic, N-terminal domain of the PTP is partially cleaved, in an age-dependent manner, from the membrane-bound domain, and it is lost during the preparation of ghost membranes. This suggests that erythrocytes may undergo in vivo activation of the Ca 2+-dependent calpain system that proteolytically regulates PTP1B activity, as already documented for other cell types. On the other hand, the assay of the PTP activity that remains associated with the membranes of RBCs of different age indicated that the PTP undergoes oxidative inactivation that can be further differentiated into reversible and irreversible components.

Glycosylated hemoglobin in fractions of erythrocytes of different ages.

In 65 children and adolescents with type I diabetes and in 23 healthy controls, glycosylated hemoglobin (HbA1) was measured by ion exchange chromatography in a specimen of whole blood and in three fractions of erythrocytes of different ages. The red blood cells were separated in a density-gradient medium. Three layers were obtained. The top one consisted of younger and lighter cells, while the heaviest and oldest cells were found in the bottom layer. The separation was evaluated by measuring the potassium concentration, which was significantly different in each fraction (p < 0.001). In all healthy controls the lowest HbA1 value was measured in the youngest and the highest in the oldest erythrocytes. In the diabetic patients, however, the HbA1 levels in individual red cell fractions depended on the degree of metabolic control during the life span of the cells. Significant correlation was found between the HbA1 levels in the youngest erythrocytes and metabolic control in the two-week period before investigation (p < 0.001), between the HbA1 content of the medium-aged cell fraction and control two months before assay (p < 0.001), and between HbA1 levels in the oldest erythrocytes and control three and especially four months prior to investigation (p < 0.001). The results of HbA1 determination in the fraction of the youngest cells which show the highest correlation (-0.75) with the glucosuria two weeks prior to assay indicate to be one of possibilities to get information about metabolic control in that period.

Quantitative determination of native proteins in individual human erythrocytes by capillary zone electrophoresis with laser-induced fluorescence detection.

Intracellular fluid within single human erythrocytes is analyzed by capillary electrophoresis with laser-excited native protein fluorescence. Good signal-to-noise is achieved, allowing even minor components to be quantified. Non-Gaussian distributions were found for total protein, fraction carbonic anhydrase, fraction hemoglobin A0, and an unidentified component. Variations among a group of 29 cells for each quantity are as much as 1 order of magnitude, even though erythrocytes are known to be fairly homogeneous in size distribution. Variations in fraction hemoglobin A0 reflect differences in in vitro oxidation rates to methemoglobin. A positive correlation was observed between carbonic anhydrase and hemoglobin A0 for individual cells. This is consistent with the presence of erythrocytes of different ages within the population, with the older cells being less capable of maintaining enzyme activity and preventing oxidative damage.

Evidence for membrane protein oxidation during in vivo aging of human erythrocytes

Oxidative lesions to membrane proteins were studied in human erythrocytes of different age and were evaluated on ghost membrane preparations by assaying thiol and methionine sulphoxide groups, and in situ on intact cells, after treating erythrocytes with the fluorochrome N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM). DACM reacts with thiol groups and the amount of this reagent bound by membrane proteins was quantified after SDS-PAGE separation. Results obtained show that during aging of normal cells the oxidative state of membrane proteins increases: this was better shown by the assay of methionine sulphoxide residues rather than by the thiol titration, when studies were carried out on ghost membranes. After separation of individual membrane proteins by SDS-PAGE, decreased accessibility of DACM to thiol groups of band 3 and of the main proteins of the membrane skeleton was evidence in senescent erythrocytes. These results show that during aging, band 3 and membrane skeleton proteins undergo conformational changes and/or oxidation. Similar results were obtained when thiol distribution was studied in membrane proteins separated by SDS-PAGE in both reducing and non-reducing conditions.

Acylphosphatase levels of human erythrocytes during cell ageing

Human erythrocytes contain an acylphosphatase isoenzyme, whose concentration during cell ageing was determined by an enzyme immunoassay. Erythrocytes were age-fractionated by isopyenic centrifugation in “Percoll” density gradients. Acylphosphatase concentration was found to rise with the increase of cell density. Maximum values were attained in the mature erythrocytes and there was only a slight decrease in the older cells. Acylphosphatase activity in human erythrocytes of different ages followed a similar pattern, a finding which was confirmed for rabbit red cells with different levels of reticulocytes. The most probable mechanism for the increase of acylphosphatase content and activity during red cell maturation appears to be a de novo synthesis of this enzyme during the reticulocyte stage and its storage, virtually unaffected, in the mature erythrocytes. The possible consequences of increased acylphosphatase levels on the metabolic modifications associated with erythrocyte ageing are discusssed.

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.

Effect of surface modification of rat erythrocytes of different ages on their partitioning behavior in charge-sensitive two-polymer aqueous phases

Partitioning differences between cells in two-polymer aqueous phase systems originate from subtle differences between the surface properties of cells. Because of the exponential relation between the parameters affecting the partition ratio ( P) and the P itself, differences in membrane components suspected of affecting the differential partitioning of closely related cell populations cannot be directly established by conventional chemical assay techniques. In order to study the chemical nature of the components responsible for the age-related changes in surface properties of rat red cells we have devised an approach which uses a combination of isotopic labeling of erythrocyte subpopulations of distinct cell age with different enzyme and/or chemical treatments followed by countercurrent distribution in charge-sensitive two-polymer aqueous phase systems. These studies show that: (a) neuraminidase-susceptible sialic acid is not responsible for the cell age-related surface differences detected by partitioning; (b) the component(s) responsible for the cell age-related surface differences can be extracted (from aldehyde-fixed red cells) with ethanol or cleaved with dilute sulfuric acid. Our data are consistent with the hypothesis that ganglioside-linked sialic acid is the chemical moiety responsible for the cell charge-associated surface differences among rat blood cells of different ages.

The effects of pentoxifylline on rat erythrocytes of different age

Age-separated rat erythrocytes were exposed to pentoxifylline, a dimethylxanthine derivative which increases erythrocyte deformability. A comparison of drug-induced effects in young and old erythrocytes yielded age-specific alterations in: (1) accumulation of intracellular Ca 2+; (2) membrane protein prosphorylation; (3) ATP concentrations; and (4) membrane associated protein kinase activity. The effect of Ca 2+ accumulation and membrane protein phosphorylation appears to be biphasic. Low drug concentrations (0.5–2.5 mM) reduced intracellular Ca 2+ and increased membrane protein phosphorylation, whereas higher concentrations (4.0–5.0 mM) increased Ca 2+ levels and reduced membrane protein phosphorylation. Young cells exhibited increased ATP levels over the whole range of pentoxifylline tested; however, older erythrocytes demonstrated higher ATP levels at 5.0 mM drug only. Membrane-associated protein kinase activity was enhanced 10% in young erythrocytes at 1.0 mM pentoxifylline and decreased to 30% of control values at 4.0 and 5.0 mM drug. Protein kinase of old erythrocytes exhibited gradual inbition over the entire drug concentration range. In general, younger erythrocytes appear to be more responsive to pentoxifylline exposure. Based on these studies, it appears that the ageing of the erythrocyte and loss of deformability in vivo may be a consequence of increased Ca 2+ entry into the cell.

The influence of anti-anaemic treatments on the distribution of circulating erythrocytes of different ages during pregnancy.

The influence of anti-anaemic treatments on the distribution of circulating erythrocytes of different ages during pregnancy.

The age-dependent metabolic decline of the red blood cell

The oxidative and non-oxidative utilization of glucose was evaluated in human erythrocytes of different ages, separated by density gradient ultracentrifugation. Young red blood cells are able to metabolize 2.5 times more glucose than old ones; on the other hadn, the amount of glucose utilized via the hexose monophosphate shunt does not show any age dependence. Glucose metabolism evaluated during in vivo ageing of a rabbit red cell population shows results very similar to those obtained for human cells. Metabolic stimulation of glucose utilization by high phosphate in both young and old human red cells increases glucose utilization by 40%. In the same way young and old erythrocytes were able to increase the amount of glucose metabolized via the hexose monophosphate shunt when an oxidative stimulus (methylene blue) was introduced. Human erythrocytes of different age possess similar abilities to transport glucose so that an age-dependent defect in glucose transport can be excluded. The ATP content of human and rabbit red blood cells, as a function of cell age, follows the decrease in glucose metabolized via the Embden-Meyerhof pathway. Reduced glutathione, on the other hand, after a small decrease associated with the “maturation” of reticulocytes into red cells, remains constant like the rate of the hexose monophosphate shunt.

A commentary to the paper entitled: Diameters of erythrocytes of different ages measured by scanning electron microscopy (T. Vömel, D. Platt and W. Strobelt)

A commentary to the paper entitled: Diameters of erythrocytes of different ages measured by scanning electron microscopy (T. Vömel, D. Platt and W. Strobelt)

Riboflavin deficiency in man: effects on haemoglobin and reduced glutathione in erythrocytes of different ages.

1. Erythrocytes (RBC) from control and marginally riboflavin-deficient subjects were fractionated into nine fractions using a discrete density gradient. 2. Glutathione reductase (NAD(P)H: glutathione oxidoreductase; EC 1.6.4.2) activity and aspartate aminotransferase (EC 2.6.1.1) activity (with and without the appropriate co-enzymes) reduced glutathione, methaemoglobin, sulphaemoglobin and oxyhaemoglobin and susceptibility to peroxide were measured in RBC in the different fractions. 3. Glutathione reductase and aspartate aminotransferase activities and concentrations of reduced glutathione and oxyhaemoglobin all declined with age, while methaemogloblin, sulphaemoglobin and susceptibility to peroxide increased with age. 4. The only significant differences noted in the RBC from marginally-riboflavin-deficient subjects by comparison with controls, were lower glutathione reductase activities and higher concentrations of methaemoglobin. 5. The role of riboflavin in those systems controlling RBC integrity is discussed.

Diameters of erythrocytes of different ages measured by scanning electron-microscopy

Contradictory reports exist about the age-dependent changes of erythrocyte diameters. All the investigations were carried out using Coulter Counter, by which the erythrocytes are sucked through a glass capillary. The contact with a glass surface causes the cell to deform instantly. This explains the lack of accuracy of the measurements subsequently taken. We measured the size of red cells of different ages by scanning electron-microscopy, thus avoiding deformations. We found that the average diameter decreases with the age of the red cell. The mean diameter was 7.77 μm in young cells, 7.34 μm in middle-aged and 7.00 μm in old cells.

Bovine J blood-group activity in the lipids of erythrocytes of different age.

J-positive cattle erythrocytes were separated according to age by ultracentrifugation in a discontinuous density gradient of Ficoll-400 (Pharmacia) in isotonic buffer solution. A decrease in phospholipid and in cholesterol content with increasing age was detected. The J activity was found to increase markedly with increasing cell age. This fact supports the view that the in vivo transfer of the J determinant from plasma to erythrocytes is a rather slow process. It is suggested that the increasing J activity of older cells is probably due to a decrease of erythrocyte membrane bound phospholipids which inhibit the transfer of the J determinant to the red cells.