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Adult male, juvenile and pregnant rats were exposed to repeated hypoxia 8 h daily for 3-9 days in a low pressure chamber (altitude of 5000 m, pO2 = 11.3 kPa and 6000 m, respectively, pO2 = 9.89 kPa). The density distribution curves of the red blood cells (RBC) and the incorporation of 59Fe into cells of different density were determined. In the ontogenesis the density of RBC increases (median density on the first day of life 1.100 kg/l; on the 21st day of life 1.102 kg/l, adult rats 1.103 kg/l). The density difference between the 1st and 4th quartile in which 50% of the cells range was 0.010 kg/l for newborn and juvenile animals and 0.004 kg/l for adult ones. Exposure to hypoxia led iN the animals of the investigated age groups to a rise of the proporborns, 0.004 for juveniles, and 0.002 kg/l for adult rats. The cumulative distribution curves of 59Fe incorporation and density of RBC can be described by the equation (formula: see text). The value alpha characterizes the relation between density, on the one hand, and the maturity and age of cells, on the other. For control and hypoxic animals the following values for alpha have been calculated: for newborns 0.515 and 0.520, for juveniles 0.829 and 0.596 (p less than or equal to 0.01), and for adult rats 0.875 and 0.823. Juvenile animals from in regard to density similar heterogenic cells as newborns after hypoxia. It may be that ontogenetically older cell forming sites or clones are reactivated.

The separation of cells with different ages from erythrocyte populations of adult rats and young or adult chickens have been achieved by counter-current distribution (CCD). A thin-layer CCD apparatus has been employed. Erythrocytes from blood samples taken at different times after 59Fe i.p. injection were separated by CCD. By compilation in a "composite curve" of the hemoglobin and radioactivity CCD profiles obtained for each erythrocyte population, the distribution of cells according to age can be inferred. Young erythrocytes of rats are located at the right part of the CCD curves, while older cells are distributed towards the left. An opposite distribution has been found for erythrocytes from adult or young chickens. As a first attempt for the application of the CCD procedure to the assay of enzyme activities, it was found a decrease in phytase activity as the age of chicken erythrocytes increases and an increase in phosphoglycerate kinase and phosphofructokinase as the age of rat erythrocytes increases.

The catalytic properties of depeptidyl aminopeptidase and aminopeptidase isolated from human erythrocytes, and the distribution of their activities in different blood cells were determined. Dipeptidyl aminopeptidase was shown to be of the dipeptidyl aminopeptidase III type, activated by Co2+ and inhibited by EDTA, pCMB and partially by DFP and leupeptin. Aminopeptidase preferred Lys-, Phe-, ARg-, and Met-2-naphthylamides as substrates. It was activated by Co2+ and inhibited by EDTA amastatin, bestatin and leupeptin. Dipeptidyl aminopeptidase III activity and aminopeptidase activity on arginine-2-naphthylamide was higher in the population of younger red blood cells. The same activities were also found in thrombocytes, mononuclear and polymorphonuclear leukocytes in concentrations higher than those in erythrocytes.

Mechanisms by which macrophages recognize and remove senescent red blood cells (RBC) were investigated. Present evidence indicates that the antigen binding portion of immunoglobulin G (IgG) autoantibodies in normal serum selectively binds to senescent RBC in situ and initiates their removal by mononuclear phagocytes. IgG autoantibodies eluted from senescent RBC were conjugated to cyanogen bromide activated Sepharose 4B, and used to isolate and purify the IgG binding receptor. Sialoglycoprotein mixtures were processed with the senescent cell IgG affinity column. Gel electrophoresis of the bound material eluted from the column revealed a single band migrating with a molecular weight of approximately equal to 62000. This isolated peptide, but not the sialoglycoprotein mixture from which it was isolated, absorbed the phagocytosis inducing ability of IgG. Thus, the senescent cell antigen to which IgG binds, is a 62000 Dalton glycopeptide.

Membrane glycoprotein structure regulates the fate in the circulation of mammalian erythrocytes: the mechanism of this phenomenon was observed in rabbit and human red cells by in vivo and in vitro experiments. Glycoprotein remodeling can occur as a reversible desialylation process, produced by different events, or as an irreversible process consisting in a loss of sialoglycopeptides. The effect of these two phenomena on the viability of the red cell in the circulation was the subject of our investigation.

Human o-erythrocytes aged in situ do not expose free receptors for anti-T agglutinin of Arachis hypogea. New receptors for concentrated anti-A agglutinin of Helix pomatia are manifested but are lost again in the course of further ageing of the cells in situ. A remasking of exposed receptors for anti-TAH and anti-AHP byautologous, strongly binding globulins is supposed. These globulins could constitute the decisive signal for the autologous phagocytosis of senescent red blood cells.

An integral hypothesis is submitted on the interaction of endogenous and exogenous factors in the mechanism of senescence of erythrocytes and on their selective phagocytosis by autologous macrophages. A method that allows quantitative determination and preparative yield of senescent erythrocytes is proposed.

By means of a simple filtration procedure it is shown that the deformability of red blood cells depends indirectly on their relative ATP-concentration. Losses in deformability were observed even with concentrations of ATP, which occur in the course of normal ageing of red blood cells in situ. If erythrocytes are incubated for longer times, their membrane is alterated to such a degree, that the changes cannot be reversed solely by restitution of ATP. The cells remain rigid.

The separation of red blood cells according to their maturity and age was compared in the isopyknic dextran and albumin density gradients, and the quality of separation was appraised by selected criteria (density distribution curves, accumulation of reticulocytes, activity of G-6-PD). The achieved quality of separation, as measured by the quotient of G-6-PD activity in the cells of lower density/G-6-PD activity in cells of higher density is of equal value in the two media. In the dextran gradient, separation of old cells seems to be accomplished more readily, while albumin affords better separation of young erythrocytes. The reticulocytes are in both media 4-5 mg/ml lighter than the total cell population. When shifting pH from 7.4 to 6.8 both in erythrocytes and reticulocytes the density decreases by 3-6 mg/ml irrespective of the type and pretreatment of the separation medium.

Some results from storage experiments at +4 degrees C with adenine and purine nucleosides (cf. [1]) are compared with incubation experiments at 37 degrees C with guanosine at pH 7.0 and 7.4 and three different levels of inorganic phosphate (iP). At the lower pH and 20 mmol . l-1 iP the synthesis of GTP was extremely rapid; the intracellular concentration reached 0.5 mmol . l-1 within 25 min constituting 1/3 of the purine nucleoside triphosphate pool. Replacement of ATP by an ATP-GTP mixture has metabolic effects on several enzymic steps, e.g., phosphofructose kinase and phosphoglycerate kinase explaining the reported beneficial effect on human erythrocytes during preservation. In order to explore the role of erythrocytes in the physiological transport of purines and purine nucleosides in vitro experiments were designed in which the substances were continuously added to the appropriate concentrations by means of a dialysis tubing. Prior to incubation the erythrocytes were stored with NaF in order to reduce the total adenylate concentration. The rate of synthesis of adenylates was about 0.05 mmol . h-1 (calculated per litre of erythrocytes) both when adenosine and adenine were supplied as source for the adenine moiety of the adenylates. Analyses of purines, nucleosides, glucose, lactate and iP in the efferent dialysis solution and the extra- and intracellular volumes reveal the metabolic processes in the erythrocytes if the transport across the dialysis tubing is accounted for. The low capacity of the erythrocyte adenylate synthesis indicates that only a minor part of the adenine compounds physiologically exported by the liver could be delivered as intraerythrocytic adenylates.

The content and distribution of phospholipids (PL) in red blood cells (RBC) of PK and of G6PD deficiency were found to differ in both disorders. RBC immaturity could not be excluded as reason for PL alterations in PK deficiency. The relative diminution of phosphatidylethanolamine in G6PD deficiency may be connected with the enzyme depletion in this disorder and its influence on the PL of RBC membranes. Moist or all fatty acid deviations seem to be conditioned by RBC immaturity. Nearly all PL fatty acid aberrations in G6PD deficiency RBC were observed also in PK deficiency RBC. Additional deviations of PL fatty acids in PK deficiency RBC may be caused by the stronger immaturity of the cells in this disorder.

An acid-citrate dextrose solution which contains phosphoenolpyruvate and sucrose was used as a preservative and medium for rejuvenating depleted erythrocytes. 2,3-bisphosphoglycerate and ATP in washed erythrocytes, red cell concentrate or whole blood were increased effectively by incubating the cells with the solution at 37 degrees C for 30--60 min. The transport of phosphoenolpyruvate through the erythrocyte membrane was essential to the increase of 2,3-bisphosphoglycerate and ATP. During storage of the cells at 4 degrees C in the presence of phosphoenolpyruvate, no increase of 2,3-bisphosphoglycerate and ATP was observed because no transport of phosphoenolpyruvate into the cells occurred. By an incubation at 37 degrees C for 30 min at the end of storage periods, however, levels of ATP and 2,3-bisphosphoglycerate in the cells were raised.

The still unexplained occurrence of hemolytic anemia in cases of alcoholic liver disease prompted us to study the toxic effect of ethanol on the membrane and metabolic properties of erythrocytes (RBC) in chronic alcoholics (n = 7). Raised susceptibility of RBC to hydrogen peroxide with significantly increased fluidity of membrane lipids were obtained signaling an oxidative metabolic stress. A twofold reduction of ATP and GSH initiated an oxidation of cellular sulfhydryl groups thus impairing enzyme kinetics of pyruvate kinase (PK). Yet heat stability of PK was markedly reduced only in those fractions containing older RBC. Further results indicate that ethanol-induced acquired PK instability may be triggered by a dialyzable and hence non-protein-bound compound, not detectable by experimental procedures owing to its low molecular weight. Thus it is conceivable that the hemolytic implication of ethanol in alcoholic liver disease may be due to abnormal membrane fluidity and metabolic disorder finally involving PK-control mechanism.

Hemolytic sensitivity of various density (and age) fractions of bovine erythrocytes to oxidant stress induced by H2O2, ascorbate and gamma radiation, as well as to prolonged incubation (autohemolysis) and heat treatment was compared. The sensitivity of older red cells was increased for higher but not for lower rates of "oxidative load" nor to autohemolysis and hyperthermic hemolysis. Changes in the sensitivity of senescent erythrocytes to redox agents are mediated, at least partly, by factors other than glutathione concentration which was fairly constant during the intravascular red cell aging.

Acetylcholine esterase activity and sialic acid content were estimated in human erythrocytes separated according to their densities and after fragmentation in vitro by heating or a combined hyperosmolaric-mechanical stress. In vivo aged cells showed a disproportional loss of both membrane constituents. Fragmentation of erythrocytes in vitro resulted in a proportional loss of acetylcholine esterase activity and sialic acid. Young cells are more prone to fragmentation by heat than older ones.

Segregation of integrated proteins, detachment of actin and spectrin from cytoplasmic aspect as well as decomposition of phospholipids may cause the release of vesicles from spicula of banked erythrocytes. The findings provide evidence for a mechanism of plasmalemmal rearrangement, because a clear distinction between the protein composition of the vesicle membrane and of the erythrocyte plasmalemma could be shown. The vesiculation mechanism is discussed.

Any work on red blood cells requires information about the characteristics of a cell population. Using buoyant density separation of red blood cells (RBC) in a dextran medium during a bleeding anemia of rabbits cell-physiological, ribosomal, mitochondrial and cytoplasmic criteria were estimated in order to select parameters useful for the characterization of a given RBC population according to its developmental stages. Useful criteria for an unknown overall RBC population are reticulocyte count and aspartate amino transferase, or glucose-6-phosphate dehydrogenase activity or creatine concentration. Useful additional criteria for reticulocytes are the mean cellular volume and the RNA concentration.

With a simple experimental system the changes of endogenous, antimycin A-suppressed, oligomycin-suppressed and antimycin A-resistant oxygen consumption are studied during the maturation of intact cells of the 6th day of bleeding. All functional characteristics of oxygen consumption decrease during maturation. The rate of decrease is strongly increased by high inorganic phosphate concentrations (125 mM). This effect is most obvious for the oligomycin-suppressed and the endogenous respiration. The degree of uncoupling of non-incubated cells is 14%. During 24 h incubation it rises to 75%. Inorganic phosphate accelerates the increase of uncoupling during maturation. Reticulocytes of the 4th day of bleeding are characterized by a higher respiratory capacity and also by a higher rate of maturation of antimycin A-suppressed and endogenous respiration. The degree of uncoupling does not increase during maturation. This may be attributed to the low lipoxygenase activity of these cells. 25% of the endogenous oxygen consumption of unmatured cells are antimycin A-resistant. This type of respiration declines by 50% in 4 h incubation irrespective of inorganic phosphate concentrations and day of bleeding. In nitrogen all functional characteristics of respiration during the maturation decline more rapidly than in oxygen. The antimycin A-resistant respiration, however decreased more slowly and reached 50% after 12 h. A pH dependence of maturation (maximum at pH 8.4) was found for the endogenous and the antimycin A-suppressed respiration. The degree of uncoupling rises most quickly at pH 7.4. This is possibly related to the pH maximum of lipoxygenase.

Utilizing age-dependent differences in the density of red cells, a mixture of silicone oils with density of 1.0806 g/ml was used for the enrichment of rabbit reticulocytes. During concentration, heavy cells, mainly consisting of erythrocytes, move into the silicone oil, whereas the reticulocytes remain above the oil. Suspensions of apparently intact cells with reticulocyte counts of 87 +/- 7.8% and a recovery rate of about 37% were reproducibly obtained. Neither inhibition of cell respiration nor depletion of adenine nucleotides were observed after this enrichment procedure. A micro-modification in hematocrit capillaries seems to be applicable to obtain rapid information about reticulocyte contents in blood samples without staining and counting of cells.

A sialoglycoprotein with an apparent molecular weight of 210000 appears to serve as a cell-age specific antigen on senescent human red blood cells (RBC). The protein is present both in young and old RBC membranes. The bulk of it copurifies with cytoskeletons. Exposure of this protein as a cell-age specific antigen appears to be due to detachment of a small fraction of this protein from cytoskeletons. The detached fraction is Triton extractable and is predominantly found in Triton extracts from senescent RBC.

The activity of inorganic pyrophosphatase (PPase) of red blood cells (rbc) from newborns is about 40% higher than that of adults. To answer the question whether the higher activity was due to the enhanced reticulocyte count of the newborn's cell population, the rbc of both newborns and adults were separated into different density subpopulations by means of isopycnic density gradient centrifugation in dextran. To judge the quality of separation in the individual age populations of rbc, the activity of glucose-6-phosphate dehydrogenase (G-6-PD) and the reticulocyte count were determined. The activity fall of PPase during maturation and aging of rbc is stronger than that of G-6-PD. The density subpopulations of rbc from newborns and adults with equal reticulocyte counts show the same PPase activities yet different G-6-PD activities. Obviously, the higher PPase activity of a cell population of newborns is due to the higher proportion of reticulocytes. The inorganic PPase is well suited as a guiding criterion to judge the maturity and age of rbc of newborns.