Erythrocytes Of Different Ages Research Articles

Biochemical mechanisms of glucose-6-phosphate dehydrogenase deficiency.

A solid-phase radioimmunoassay for human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) was developed that allowed the specific activity of this enzyme protein to be measured in lysates from whole erythrocyte populations, in lysates from erythrocytes of different ages, and in purified samples. The enzyme was highly purified from erythrocytes of single donors by a simple procedure of affinity chromatography with insolubilized adenosine 2',5'-bisphosphate. These techniques were used in an attempt to elucidate the molecular mechanisms leading to deficiency of glucose-6-phosphate dehydrogenase activity in two genetic variants of the enzyme, i.e., the Mediterranean and the Seattle-like variants. The results indicate that the lowered activity of erythrocytes containing the Mediterranean variant of glucose-6-phosphate dehydrogenase is related to an enhanced rate of degradation of a catalytically defective protein synthesized at a nearly normal rate. Synthesis of a normally functioning protein and an increased breakdown of it are involved in the Seattle-like variant of the enzyme.

Changes in Surface Carbohydrate of Human Erythrocytes Aged in vivo

In addition to the well-characterized changes in enzyme activity (Pennell, 1964) and lipid content (Prankerd, 1958), which accompany aging of erythrocytes in vivo, there is some evidence to suggest that changes in the oligosaccharide chains of the cell surface also occur. Aged cells showed a decreased electrophoretic mobility when compared with a young fraction of the erythrocyte population (Danon et al., 1971). The decreased surface charge was related to a decreased density of staining of the cell membrane of old cells with a positively charged colloidal iron suspension. Since the surface charge of erythrocytes is largely attributable to sialic acid (Eylar et al., 1962) these results suggest that aging of erythrocytes is accompanied by loss of sialic acid. Danon et al. (1971) have proposed that removal of sialic acid is a major factor in the elimination of old erythrocytes from the circulation. Jancik & Schauer (1974) have demonstrated that removal of 60% of the sialic acid from rabbit erythrocytes results in their rapid removal from the circulation. In view of the observations suggesting a possible relationship between the life span of erythrocytes and cell-surface carbohydrate, we have measured the sialic acid content of human erythrocytes of different ages isolated from a single individual and have examined the cells for changes in their surface carbohydrate by examination of their interaction with lectins. Erythrocytes can be fractionated into populations with different specificgravity by discontinuous-gradient ultracentrifugation in iso-osmotic solutions of bovine plasma albumin (Piomelli et al., 1967). It has been demonstrated by labelling studies that the specific gravity of erythrocytes increases progressively with age. Washed erythrocytes from freshly drawn human blood were fractionated on discontinuous density gradients of albumin by a modification of the method of Piomelli et al. (1967). Table 1 shows a typical distribution of cells. That fractions contained cells of different average age was confirmed by assaying the activity of glucose 6-phosphate dehydrogenase (Piomelli et a/., 1968). Activity was determined as the rate of change of absorbance at 340nm per mg of haemoglobin. Haemoglobin was measured by the method of Van Kampen & Zijlstra (1961). The haemoglobin content per cell was constant for cells of different age. N-Acetylneuraminic acid was determined by the method of Aminoff (1961) after release from erythrocytes either by Clostridium perfringens neuraminidase or by acid hydrolysis. The conditions used for acid hydrolysis were standardized to allow for the effect of haemoglobin on the pH of the reaction mixture and protein was precipitated before analysis by addition of 5 % (w/v) phosphotungstic acid. Analysis of sialic acid released from replicate samples of erythrocytes from a single individual gave a value of 348+5 (s.D., for eight determinations) ng of N-acetylneuraminic acid per mg of haemoglobin. The oldest and most dense cell fractions consistently showed a sialic acid content which was lower by approx. 15 % than that of the cells at the top of the gradient. This loss of sialic acid is substantially greater than the total lipid-bound sialic acid of erythrocytes, which amounts to no more than 2.5 % of the total erythrocytesialicacid. Thus, although old cells are known to be deficient in lipid (Prankerd, 1958), this alone cannot account for the diminished content of sialic acid. The conclusion that sialic acid is lost from erythrocyte glycoprotein is supported by the observation of Balduini et al. (1974) that glycopeptides isolated from membranes of aged erythrocytes showed significant decreases in sialic acid when compared with glycopeptides from young cells. The agglutination of erythrocytes of blood group 0 of different age has been examined using wheat-germ agglutinin and a lectin partially purified from the haemolymph of Carcinus. A progressive increase in agglutination with cell age was observed with both lectins. Wheat-germ agglutinin has a specificity directed towards carbohydrate groups

Effect of acetaldehyde and glutaraldehyde fixation on the surface properties of red blood cells as determined by partition in aqueous phases

A correlation between partition of cells in dextran/polyethylene glycol aqueous phase systems and their relative electrophoretic mobilities is often in evidence. Absence of such a correlation is indicative that partition measures surface properties other than charge at the plane of shear. Acetaldehyde- and glutaraldehyde-fixed erythrocytes were partitioned in two-polymer phases and their electrophoretic mobility examined in order to investigate the circumstances under which the above-indicated correlation holds. Aspects of the effect of fixation of cells on their surface properties was thereby obtained. 1. 1. Acetaldehyde-fixed and normal human red blood cells have similar partitions and similar electrophoretic mobilities; glutaraldehyde-fixed red cells display markedly increased partitions and mobilities. 2. 2. Lipid-extracted aldehyde-fixed cells have substantially increased partitions, but unchanged mobilities, when compared with the fixed cells from which they were prepared. This indicates that the removal of lipid exposes a higher density of charge groups deeper in the membrane than is measurable by electrophoresis under the conditions used. 3. 3. The countercurrent distribution obtained with fixed cells depends on the length of time chosen for phase settling. Short times result in a single distribution while longer settling times give rise to a two-peak distribution. The latter phenomenon probably arises from a time-dependent aggregation of the fixed cells in the phases. 4. 4. Electrophoretic examination of the glutaraldehyde-fixed cells from different cavities along the extraction train indicates that the fixation process does not eliminate differences in the relative electrophoretic mobilities of erythrocytes of different ages.

Relationship between age and properties of human and rabbit erythrocyte glucose-6-phosphate dehydrogenase.

The properties of human and rabbit erythrocyte glucose‐6‐phosphate dehydrogenase have been investigated during aging in vivo. The erythrocytes of different age have been prepared by 2 procedures: (a) by inducing a reticulocytosis by phenylhydrazine administration to rabbits and (b) by separating the young and old red cells of man and rabbit according to the different resistence to osmotic lysis.During aging, human and rabbit glucose‐6‐phosphate dehydrogenase shows a decrease of the catalytic activity and some marked modifications of its properties. Thermolability and NADPase‐dependent inactivation increase; the protective effect of NADP and the affinity for both glucose‐6‐phosphate and NADP substrates are decreased; no modifications have been demonstrated as regards pH optimum and electrophoretic patterns in starch gel. It is possible that these modifications begin after the expulsion of ribosome material.According to the literature concerning the behaviour of other enzymes in the physiological state and of glucose‐6‐phosphate dehydrogenase of some pathological conditions, it is possible that the modified properties of the enzyme during aging in vivo can be related to some structural modifications of the protein.

Glutathione synthesis in cell-free preparations from erythrocytes of different ages

Unequivocal synthesis of glutathione has been demonstrated with cell-free preparations derived from red cells. Three different measures of synthesis have been compared using preparations from red cells of different ages. The difference between the results for young and old cells was not statistically significant with any of the three methods. Estimates of synthesis by [ 14C]glycine incorporation and release of in- organic phosphorus from ATP were similar. Both were approximately twice the es- timate of synthesis from direct measurements of the increment of gluthathione. Data on the formation of glutamylcysteine indicated a four fold excess in the production of this precursor. Differences were found in the results for young and old cells but their significance is unknown.

Factors in the partition of red blood cells in aqueous dextran-polyethylene glycol two-phase systems

Abstract The usefulness of countercurrent distribution of cells in aqueous, buffered two-polymer phase systems is illustrated by the segregation of erythrocytes of different ages, the resolution of two populations of reticulocytes, the separability of red blood cells from white blood cells, a study of membrane characteristics of red cells from different species 1,2 . We now report the effects on erythrocyte partition of a number of factors ( e.g. I , pH, temperature, hypertonicity or hypotonicity, ABO blood group, washing of cells and anticoagulant). It is also shown how some of these parameters can be used to obtain desired partitions for (red) cells in a given investigation. In the dextran-polyethylene glycol phase systems that we have investigated, an increase in I (with constant isotonicity) appears not to alter the partition of red cells except with phosphate in which a decrease in the partition coefficient is observed. Increase in pH or temperature causes an increased partition. Different degrees of hypotonicity give similar partitions which are somewhat greater than those found in isotonic phase. Increasing hypertonicity leads to a rapid decrease in the partition coefficients. Human red cells are found to have the same partition independent of the ABO blood group to which they belong. Washing of cells or anticoagulant used (acid-citrate-dextrose, heparin, EDTA) has no effect on their partition. In view of the previously established correlation between electrophoretic mobility of red cells and their partition coefficients 3 we point out, in the course of the discussion, those instances in which the electrophoretic mobility and partition of cells are similarly affected by changing a given parameter, and those instances in which the effects are markedly different. In the latter instances partition of cells in aqueous two-polymer phase systems and electrophoretic mobility appear to measure different membrane characteristics.

Exchange transport of aldoses in human erythrocytes of different ages.

SummaryFractions of erythrocytes differing in age were isolated by centrifugation. The course of the exchange transport of aldoses (galactose‐glucose) was evaluated on the basis of the galactose level reached in the apparent steady state. In the young population the level of galactose, entering the erythrocytes from the medium in exchange for glucose, attained a higher level than in the old erythrocytes. The possible relation between this finding and the structural changes in the erythrocytes membrane during the aging has been pointed out.RésuméDes erythrocytes d'äges différents ont été isolées par centrifugation. Le mode des échanges des aldoses (galactose‐glucose) a été évalué sur la base du taux de galactose obtenu dans un état d'homéostase apparent. Dans les populations érythrocytaires jeunes, le taux de galactose entrant dans les érythrocytes à partir du milieu en échange de glucose atteint un niveau plus élevé que parmi les populations d'érythrocytes ägées. Le rapport possible entre cette découverte et les modifications structurales de la membrane des érythrocytes pendant le vieillissement est discuté.ZusammenfassungMittels Zentrifugation werden Erythrozytenfraktionen mit unterschiedlichem Zellalter gewonnen. Der Verlauf des gegenläufigen Aldosentransports (Galaktose‐Glukose) wurde an Hand des Galaktosespiegels unter steady state‐Bedingungen ermittelt. In jungen Zellpopulationen trat im Austausch gegen Glukose mehr Galaktose aus dem Medium ins Zellinnere ein als bei alten Erythrozyten. Die Beziehungen zwischen diesem Befund und den Altersveränderungen der Zellmembran werden diskutiert.

Exchange Transport of Aldoses in Human Erythrocytes of Different Ages

Exchange Transport of Aldoses in Human Erythrocytes of Different Ages

Separation of Erythrocytes of Different Ages

Abstract Several methods have been evaluated for the separation of red cells of different ages on the basis of density differences. Simple sedimentation and flotation technics have been found to be unsatisfactory. Centrifugation, followed by a modified sampling procedure, has been demonstrated to provide excellent separation of erythrocyte populations of different ages. Standardization of centrifuge conditions and special precautions against redistribution of red-cell layers have contributed toward an improved and reproducible method for the separation of erythrocytes for subsequent study.

A SIMPLE METHOD OF SEPARATING ERYTHROCYTES OF DIFFERENT AGES.

SummaryCentrifugation for two hours at 22,000 G separates young erythrocytes from old ones. The method is useful when transfused cells of groups different from the patient's own are present in his circulation.RésuméDes centrifugations de deux heures à 22 000 G séparent les érythrocytes jeunes des érythrocytes âgés. La méthode est utilisable lorsque des érythrocytes de groupes différents sont transfusés à un patient et sont présents dans sa circulation.ZusammenfassungMittels einer zweistündigen Zentrifugation bei 22 000 G gelingt es, junge von alten Erythrozyten abzutrennen. Diese Méthode ist brauchbar, wenn transfundierte Zellen mit Gruppen, die von denen des Patienten abweichen, in dessen Zirkulation enthalten sind.

A Simple Method of Separating Erythrocytes of Different Ages

A Simple Method of Separating Erythrocytes of Different Ages

Enzymes and radioactivity in erythorcytes of different ages.

FRACTIONATION of erythrocytes of different ages by differential haemolysis is a useful approach to the study of enzymatic aspects of the maturation of the erythrocyte in the peripheral blood. Young red cells are thought to be resistant to osmotic haemolysis and older cells to be very fragile1. Recently Simon and Topper2 have shown by serial osmotic haemolysis that young erythrocytes have both fragile and resistant components. In the present paper the activities of glutamic oxaloacetic transaminase and lactic dehydrogenase have been related to red blood cells of various ages.