Dense Cell Fractions Research Articles

Peroxiredoxin II is essential for sustaining life span of erythrocytes in mice

AbstractPeroxiredoxins (Prxs) are a family of antioxidant proteins that reduce peroxide levels by using reducing agents such as thioredoxin. These proteins were characterized to have a number of cellular functions, including cell proliferation and differentiation and protection of specific proteins from oxidative damage. However, the physiological roles of the peroxiredoxins have not been determined. To clarify the physiological relevance of this protein type, we generated a mouse model deficient in Prx II, which is abundantly expressed in all types of cells. The Prx II–/– mice were healthy in appearance and fertile. However, they had splenomegaly caused by the congestion of red pulp with hemosiderin accumulation. Heinz bodies were detected in their peripheral blood, and morphologically abnormal cells were elevated in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). The Prx II–/– mice had significantly decreased hematocrit levels, but increased reticulocyte counts and erythropoietin levels, indicative of a compensatory action to maintain hematologic homeostasis in the mice. In addition, a labeling experiment with the thiol-modifying reagent biotinylated iodoacetamide (BIAM) in Prx II–/– mice revealed that a variety of RBC proteins were highly oxidized. Our results suggest that Prx II–/– mice have hemolytic anemia and that Prx II plays a major role in protecting RBCs from oxidative stress in mice.

Type-2 Phosphatidylserine (PS)-Positive Erythrocytes and Their Association with Markers of Hemolysis and Hemostatic Activation in Children with Sickle Cell Disease (SCD)

Introduction: Type-2 phosphatidylserine (PS)-positive red cells are a subpopulation of erythrocytes that are highly positive for PS, contain low levels of fetal hemoglobin, are specific for sickle cell disease (SCD) and have been identified in the dense red cell fraction. Studies have implicated PS-positive red cells in enhancing anemia due to phagocytosis and hemolysis. Shielding of red cell PS by diannexin, a synthetic homodimer of human annexin-V, has been demonstrated to provide protection against hemolysis and prevent activation of prothrombinase.Methods: Using flow cytometry, we measured the levels of type-1 (red cells with low PS positivity) and type-2 PS-positive red cells in 50 children with SCD (31 with HbSS and 19 with HbSC), and assessed their association with various markers of hemolysis and hemostatic activation. Markers of hemolysis evaluated included plasma lactate dehydrogenase (LDH), reticulocyte count, and hemoglobin. Whole blood tissue factor (WBTF), pro-thrombin fragment F1+2, and D-dimer were evaluated as markers of hemostatic activation.Results: We demonstrate that the levels of type-2 PS-positive red cells are significantly increased in HbSS patients (1.37 ± 0.97%, p<0.01) compared to children with HbSC disease (0.32 ± 0.21%) and age- and race-matched controls (0.15 ± 0.15%, n=19). WBTF and D-dimer showed significant associations with both type-1 and -2 red cells with no significant differences in the strength of their association. However, significantly greater correlations were noted between type-2 PS red cells and hemolytic markers compared to those noted with type-1 (Steiger's Z=3.05 to 4.59, p<0.01). In addition our in vitro studies demonstrate increased osmotic fragility of these red cells.Table 1Association of PS-positive RBCs with markers of hemolysis and hemostatic activationBiomarkerType-1 PS-positive RBCsType-2 PS-positive RBCsMarkers of HemolysisLDHr = 0.44, p<0.002r = 0.63, p<0.00001% Reticulocyter = 0.43, p=0.002r = 0.66, p<0.00001Hemoglobinr =-0.35, p=0.014r =-0.63, p<0.00001Markers of Hemostatic ActivationWBTFr = 0.41, p=0.008r = 0.56, p<0.0002F1+2r = 0.26, p=0.07r = 0.31, p<0.03D-dimerr = 0.46, p<0.001r = 0.56, p<0.0005Conclusions: Type-2 PS-positive red cells are elevated in SCD and the number of these cells correlates significantly with both markers of hemolysis and hemostasis. These findings provide a patho-physiologic link between the intravascular hemolytic milieu of SCD and the hemostatic perturbations previously noted in this disease. DisclosuresNo relevant conflicts of interest to declare.

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

In‐depth analysis of cysteine oxidation by the RBC proteome: Advantage of peroxiredoxin II knockout mice

Peroxiredoxin II (Prdx II, a typical 2-Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Mice lacking Prdx II proteins had heinz bodies in their peripheral blood, and morphologically abnormal cells were detected in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). In this study, a labeling experiment with the thiol-modifying reagent biotinylated iodoacetamide (BIAM) in Prdx II-/- mice revealed that a variety of RBC proteins were highly oxidized. To identify oxidation-sensitive proteins in Prdx II-/- mice, we performed RBC comparative proteome analysis in membrane and cytosolic fractions by nano-UPLC-MSE shotgun proteomics. We found oxidation-sensitive 54 proteins from 61 peptides containing cysteine oxidation, and analyzed comparative expression pattern in healthy RBCs of Prdx II+/+ mice, healthy RBCs of Prdx II-/- mice, and abnormal RBCs of Prdx II-/- mice. These proteins belonged to cellular functions related with RBC lifespan maintain, such as cytoskeleton, stress-induced proteins, metabolic enzymes, signal transduction, and transporters. Furthermore, protein networks among identified oxidation-sensitive proteins were analyzed to associate with various diseases. Consequently, we expected that RBC proteome might provide clues to understand redox-imbalanced diseases.

The hydration state of human red blood cells and their susceptibility to invasion by Plasmodium falciparum

AbstractIn most inherited red blood cell (RBC) disorders with high gene frequencies in malaria-endemic regions, the distribution of RBC hydration states is much wider than normal. The relationship between the hydration state of circulating RBCs and protection against severe falciparum malaria remains unexplored. The present investigation was prompted by a casual observation suggesting that falciparum merozoites were unable to invade isotonically dehydrated normal RBCs. We designed an experimental model to induce uniform and stable isotonic volume changes in RBC populations from healthy donors by increasing or decreasing their KCl contents through a reversible K+ permeabilization pulse. Swollen and mildly dehydrated RBCs were able to sustain Plasmodium falciparum cultures with similar efficiency to untreated RBCs. However, parasite invasion and growth were progressively reduced in dehydrated RBCs. In a parallel study, P falciparum invasion was investigated in density-fractionated RBCs from healthy subjects and from individuals with inherited RBC abnormalities affecting primarily hemoglobin (Hb) or the RBC membrane (thalassemias, hereditary ovalocytosis, xerocytosis, Hb CC, and Hb CS). Invasion was invariably reduced in the dense cell fractions in all conditions. These results suggest that the presence of dense RBCs is a protective factor, additional to any other protection mechanism prevailing in each of the different pathologies. (Blood. 2005; 105:4853-4860)

Proteomics of Membrane from Human Sickle and Normal Fractioned Red Cells Identifies Different Expression of Stress-Response Proteins.

Sickle cell disease (SCD) is characterized by the presence of sickle hemoglobin (HbS), which has the unique property of polymerizing when deoxygenated and thereby triggering erythrocyte sickling and dehydration. Repeated HbS polymerization is associated with generation of dense, distorted red cells characterized by an extensive membrane oxidative damage and abnormal activation of membrane cation transport pathways. The recently developed proteomic technologies represent a useful tool for identification of new functional pathways in sickle red cells. We studied the membrane proteoma of sickle red cells from homozygous patients (SS), not carrying alpha-thalassemia, not undergoing hydroxyurea treatment and not transfused at least three months before the study. The buffy-coat was removed and the red cells were separated into two fractions corresponding to the reticulocytes enriched fraction and to the dense red cell fraction (n= 8). Following osmotic lysis, the red cell ghost proteins were separated by one- dimensional or by two-dimensional gel electrophoresis (1-DE, 2-DE). Using the ImageMaster 2D Platinum (5.0 Amersham-Biosciences) software the 2-DE gels from control and sickle cells were compared, allowing us to identify spots differently expressed in sickle cells compared to normal cells. The selected spots were excised and trypsinized before mass spectrometric analysis and compared with the SWISS-PROT and NCBI non-redundant databases. In sickle reticulocytes enriched fraction compared to the control one, we selectively identified the following cystokeleton proteins: four spots for beta actin, two spots for myosin light chain and a train of four proteins identified as protein 4.1, one of which was phosphorylated on Tyr13, a protein similar to flotillin 2, carbonic anhydrase I and the following stress-response proteins: heat shock protein 70 (HSP70) protein 9B, HSPC108 and peroxiredoxin II (PrxII), which was phosphorylated in Thr142. In dense sickle red cell fraction compared to normal one we selectively identified a train of two spots for 4.1R, band 3 and aldolase and two spots identified as HSP70 protein 5.The identified proteins were then confirmed by western-blotting analysis.These data suggest that in sickle red cells HSP70, HSP108 function may be required by the extensive assistance in the refolding of misfolded proteins most likely related to abnormal sickle red cell oxidative environment as also supported by the presence of phosphorylated peroxiredoxin II, which has been recently proposed as regulator of redox-sensitive signaling in other cell types.

How Old Are Dense Red Blood Cells? The Dog's Tale

To the Editor: Decades ago it was observed that young cohorts of radiolabeled human red blood cells (RBC) became progressively enriched in the more dense cell fractions as the cohorts aged.[1][1] It was also shown that the most dense cells were short-lived in circulation after reinfusion into the

Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE).

We describe a duplication of 10 nucleotides (2,455-2,464) in the band 3 gene in a kindred with autosomal dominant hereditary spherocytosis and a partial deficiency of the band 3 protein that is reflected by decreased rate of transmembrane sulfate flux and decreased density of intramembrane particles. The mutant allele potentially encodes an abnormal band 3 protein with a 3.5-kD COOH-terminal truncation; however, we did not detect the mutant protein in the membrane of mature red blood cells. Since the mRNA levels for the mutant and normal alleles are similar and since the band 3 content is the same in the light and dense red cell fractions, we conclude that the mutant band 3 is either not inserted into the plasma membrane or lost from the membrane prior to the release of red blood cells into circulation. We further show that the decrease in band 3 content principally involves the dimeric laterally and rotationally mobile fraction of the band 3 protein, while the laterally immobile and rotationally restricted band 3 fraction is left essentially intact. We propose that the decreased density of intramembrane particles decreases the stability of the membrane lipid bilayer and causes release of lipid microvesicles that leads to surface area deficiency and spherocytosis.

Effects of deoxygenation on active and passive Ca2+ transport and on the cytoplasmic Ca2+ levels of sickle cell anemia red cells.

Elevated [Ca2+]i in deoxygenated sickle cell anemia (SS) red cells (RBCs) could trigger a major dehydration pathway via the Ca(2+)-sensitive K+ channel. But apart from an increase in calcium permeability, the effects of deoxygenation on the Ca2+ metabolism of sickle cells have not been previously documented. With the application of 45Ca(2+)-tracer flux methods and the combined use of the ionophore A23187, Co2+ ions, and intracellular incorporation of the Ca2+ chelator benz-2, in density-fractionated SS RBCs, we show here for the first time that upon deoxygenation, the mean [Ca2+]i level of SS discocytes was significantly increased, two- to threefold, from a normal range of 9.4 to 11.4 nM in the oxygenated cells, to a range of 21.8 to 31.7 nM in the deoxygenated cells, closer to K+ channel activatory levels. Unlike normal RBCs, deoxygenated SS RBCs showed a two- to fourfold increase in pump-leak Ca2+ turnover. Deoxygenation of the SS RBCs reduced their Ca2+ pump Vmax, more so in reticulocyte- and discocyte-rich than in dense cell fractions, and decreased their cytoplasmic Ca2+ buffering. Analysis of these results suggests that both increased Ca2+ influx and reduced Ca2+ pump extrusion contribute to the [Ca2+]i elevation.

Measurement of intracellular Ca 2+ in young and old human erythrocytes using 19F-NMR spectroscopy

Elevated cell calcium has been implicated in functional changes with human erythrocyte aging. However, until recently it has been difficult to measure free ionic intracellular calcium in red cells. We have made use of a fluorinated calcium chelator probe (5,5′-difluoroBAPTA) and fluorine nuclear magnetic resonance ( 19F-NMR) techniques to measure changes of intracellular Ca 2+ concentrations ([Ca 2+] i) with cell aging. We have demonstrated in these studies that human erythrocyte [Ca 2+] i is significantly elevated as a function of in-vivo aging. Young cells, the least dense fraction of density-separated erythrocytes, contained an average of 62 (± 4) nM Ca 2+ (± S.E.), whereas the oldest, most dense cell fraction contained 221 nM Ca 2+ (± 25). Mechanisms by which intracellular [Ca 2+] increases with in-vivo aging are currently under investigation.

Effects of deficiencies of glycophorins C and D on the physical properties of the red cell

Red cells of the rare Leach phenotype lack the membrane glycophorins C and D, and a proportion of the red cells are elliptocytes. Judging from tests on suspensions of red cell ghosts sheared rotationally in an ektacytometer, it has previously been suggested that these membranes are relatively fragile and poorly deformable. We have carried out analyses of individual red cells to investigate possible factors which underlie the physical changes in these glycophorin-deficient cells. Micropipette analysis of the red cell membrane showed that the rigidity and viscosity were normal, both for elliptocytes and discocytes, for three donors deficient in glycophorins C and D. Red cell transit times through 5 microns pores, measured electronically for 2000 individual cells, showed no differences from controls. It was confirmed that the index of deformation obtained using an ektacytometer was reduced, but our results suggest that this arises from shape rather than membrane changes. The elliptocytes were found to have a lower volume and surface area than discocytes from the same donor (measured by micropipette aspiration of single red cells), and were rarely found in less dense red cell fractions. No reticulocytes were found to be elliptical. These data suggest that the elliptocytes are older red cells, and are formed from red cells which are initially released into the circulation with normal shape. Their elongated shape might arise from permanent distortion of the unstable membrane by shear forces in the circulation.

Mechanical properties of sickle cell membranes

The mechanical properties of sickle erythrocyte membranes were evaluated in the ektacytometer. When ghosts from the total red blood cell population were examined, the rigidity of the resealed ghosts and their rate of fragmentation by shear stress (t1/2) were normal. However, fractionation on Stractan density gradients revealed that sickle cells were heterogenous in their membrane mechanical properties. The ghosts from dense cell fractions exhibited both increased rigidity and decreased stability. Presumably, these altered mechanical properties are a reflection of the well-documented biochemical damage found in irreversibly sickle cell membranes. Nevertheless, neither of the alterations in mechanical properties are likely to be significant elements in the hemolysis of sickle cell anemia. Earlier studies of abnormal erythrocytes suggest that increases in membrane rigidity per se do not increase hemolysis, and they are, therefore, unlikely to do so in this case. The stability of membranes from the dense cell fractions was reduced to about two thirds of the control value. Comparison with the results of studies of red blood cell membranes with genetically defective or deficient spectrin suggests that a reduction in t 1/2 of 50% is not associated with significant increases in the rate of hemolysis. Although altered ghost stability and flexibility can be demonstrated in dense sickle cells, these changes in membrane mechanical properties are not likely to be significant factors in the hemolytic process.

Mechanical Properties of Sickle Cell Membranes

The mechanical properties of sickle erythrocyte membranes were evaluated in the ektacytometer. When ghosts from the total red blood cell population were examined, the rigidity of the resealed ghosts and their rate of fragmentation by shear stress (t1/2) were normal. However, fractionation on Stractan density gradients revealed that sickle cells were heterogenous in their membrane mechanical properties. The ghosts from dense cell fractions exhibited both increased rigidity and decreased stability. Presumably, these altered mechanical properties are a reflection of the well-documented biochemical damage found in irreversibly sickle cell membranes. Nevertheless, neither of the alterations in mechanical properties are likely to be significant elements in the hemolysis of sickle cell anemia. Earlier studies of abnormal erythrocytes suggest that increases in membrane rigidity per se do not increase hemolysis, and they are, therefore, unlikely to do so in this case. The stability of membranes from the dense cell fractions was reduced to about two thirds of the control value. Comparison with the results of studies of red blood cell membranes with genetically defective or deficient spectrin suggests that a reduction in t 1/2 of 50% is not associated with significant increases in the rate of hemolysis. Although altered ghost stability and flexibility can be demonstrated in dense sickle cells, these changes in membrane mechanical properties are not likely to be significant factors in the hemolytic process.

Studies on the pathogenesis of the erythrocyte destruction associated with the anemia of inflammatory disease.

Density-separated feline RBC were analyzed for surface immunoglobulin and in vitro susceptibility to macrophage phagocytosis before and after introduction of a sterile abscess. Prior to abscess induction, least dense cells did not have detectable surface immunoglobulin G (IgG) as a direct antiglobulin reaction was negative. Two of four intermediate and three of four most dense cell fractions had detectable surface IgG as indicated by a positive direct antiglobulin reaction. On Days 3 and 5 after induction of sterile abscesses all three RBC fractions were positive and the size of the RBC clumps tended to increase. In vitro macrophage phagocytosis of all three RBC fractions increased significantly after abscess induction. The phagocytosis could be partially blocked by preincubation of macrophages with feline IgG.

Endogenous calcium in sickle cells does not activate polyphosphoinositide phospholipase C.

Sickle-cell-anaemia erythrocytes (SS cells) are known to have a high Ca2+ content (particularly the dense cell fraction) and to take up Ca2+ on deoxygenation. It has been reported that this high Ca2+ was responsible for the activation of the Ca2+-dependent K+ loss, and of the Ca2+-sensitive polyphosphoinositide phospholipase C (PIC) in dense SS cells. We found that, either in the total population of SS cells or in the light or dense fractions, the content of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was not changed, whereas that of phosphatidylinositol 4-phosphate was increased and that of phosphatidic acid (PtdOH) was decreased compared with normal (AA) erythrocytes. Deoxygenation-induced Ca2+ entry into SS cells did not change the concentration or, in 32P-prelabelled cells, the radioactivity of polyphosphoinositides and PtdOH. It also failed to induce the formation of inositol 1,4,5-trisphosphate, the product of PtdIns(4,5)P2 hydrolysis by PIC, which was measured by an original method using ion-pair reverse-phase h.p.l.c. Thus there was no evidence of an endogenous Ca2+ effect on the PIC activity in SS cells, in agreement with the demonstration that the excess Ca2+ in SS cells is compartmentalized into internal vesicles and unavailable as free Ca2+. The 32P incorporation in polyphosphoinositides and PtdOH was markedly higher in SS than in AA cells, but this increase was the same in both dense and light SS cells. The increase in the turnover of these phospholipids in SS cells is consistent either with an activation of the lipid kinases and phosphatases or with perturbation in the metabolic compartmentation of these lipids.

Assessment of the hydration state of sickle cells by phthalate ester density distribution.

Intracellular hemoglobin S (Hb SS) concentration, a function of cell hydration, has a major influence on the rate of Hb SS polymerization and, therefore, cellular sickling. To determine the density distribution of homozygous sickle hemoglobin cells as a function of cell hydration, cells were incubated in autologous plasma buffer mixtures with final osmolalities ranging from 195 to 490 mosm/kg at ambient Po2. The density distribution of the cells was determined by differential flotation on 20 mixtures of di-n-butyl and dimethyl phthalates with specific gravities of 1.062 to 1.142. Mean cell hemoglobin concentration (MCHC) and mean cell volume (MCV) were determined by standard manual procedures. Cell shape was assessed by scanning electron microscopy (SEM), and the axial ratio (L/W) of the elliptical dense cell fraction measured by an image analyzer interfaced with a computer. The density distribution of normal red blood cells lies within a narrow 1.090 to 1.118 gm/ml density band with the middle or transitional 60% (T60) of the cells occupying a density range of 0.0067 +/- 0.0007 gm/ml (+/- SD). The density distribution of sickle cells shows a broader density band of 1.064 to 1.134 gm/ml, and the T60 was 0.0139 +/- 0.0022 gm/ml. The mean T60 did not change with osmotic variation but the mean T60 of Hb SS cells was significantly greater (P less than 0.005). MCHC and 1/MCV varied directly with the median density of the density distribution. By linear regression analysis and Ponder's osmotic equation, it is evident that sickle cells exhibit restricted volume increases in hypotonic media.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium Accumulated by Sickle Cell Anemia Red Cells Does Not Affect Their Potassium (86Rb+) Flux Components

We investigate here the hypothesis that the high Ca content of sickle cell anemia (SS) red cells may produce a sustained activation of the Ca2+-dependent K+ permeability (Gardos effect) and that the particularly high Ca levels in the dense SS cell fraction rich in irreversibly sickled cells (ISCs) might account for the Na pump inhibition observed in these cells. We measured active and passive 86Rb+ influx (as a marker for K+) in density-fractionated SS cells before and after extraction of their excess Ca by exposure to the Ca ionophore (A23187) and ethylene glycol tetra-acetic acid and with or without adenosine triphosphate depletion or addition of quinine. None of these maneuvers revealed any evidence of a Ca2+-dependent K leak in SS discocytes or dense cells. Na pump inhibition in the dense SS cells was associated with normal activation by external K+ and a low Vmax that persisted after Ca extraction from the cells. These results are consistent with our recent findings that the excess Ca in these cells is compartmentalized in intracellular inside-out vesicles and unavailable as free Ca2+ to the inner membrane surface. Although the steady-state free cytoplasmic Ca2+ in oxygenated SS cells must be below the levels needed to activate the K+ channel, possible brief activation of the channels of some SS cells resulting from transient elevations of cell Ca2+ during deoxygenation-induced sickling cannot be excluded. The dense, ISC-rich SS cell fraction showed a Ca2+-independent increase in the ouabain-resistant, nonsaturable component of 86Rb+ influx that, if uncompensated by Na+ gain, could contribute to the dehydration of these cells.

Calcium accumulated by sickle cell anemia red cells does not affect their potassium (86Rb+) flux components

We investigate here the hypothesis that the high Ca content of sickle cell anemia (SS) red cells may produce a sustained activation of the Ca2+-dependent K+ permeability (Gardos effect) and that the particularly high Ca levels in the dense SS cell fraction rich in irreversibly sickled cells (ISCs) might account for the Na pump inhibition observed in these cells. We measured active and passive 86Rb+ influx (as a marker for K+) in density-fractionated SS cells before and after extraction of their excess Ca by exposure to the Ca ionophore (A23187) and ethylene glycol tetra-acetic acid and with or without adenosine triphosphate depletion or addition of quinine. None of these maneuvers revealed any evidence of a Ca2+-dependent K leak in SS discocytes or dense cells. Na pump inhibition in the dense SS cells was associated with normal activation by external K+ and a low Vmax that persisted after Ca extraction from the cells. These results are consistent with our recent findings that the excess Ca in these cells is compartmentalized in intracellular inside-out vesicles and unavailable as free Ca2+ to the inner membrane surface. Although the steady-state free cytoplasmic Ca2+ in oxygenated SS cells must be below the levels needed to activate the K+ channel, possible brief activation of the channels of some SS cells resulting from transient elevations of cell Ca2+ during deoxygenation-induced sickling cannot be excluded. The dense, ISC-rich SS cell fraction showed a Ca2+-independent increase in the ouabain-resistant, nonsaturable component of 86Rb+ influx that, if uncompensated by Na+ gain, could contribute to the dehydration of these cells.

Erythrocyte density distribution in sickle cell anemia.

Density distributions were determined for sickle cell erythrocytes from 27 patients with HbSS genotype using the phthalate ester microcapillary differential flotation method of Danon and Marikovsky. Mean density distribution curves showed HbSS erythrocytes to have trimodal populations with significant increases in both dense and light cell fractions when compared to 20 normal controls of HbAA genotype (p less than 0.05). Irreversibly sickled cell (ISC) and reticulocyte counts were compared with density distributions. Integrated unit areas under the dense cell population curves correlated with ISC percentages, while corresponding unit areas for light populations correlated to a lesser extent with reticulocyte percentages. Mean cell density, D50, varied widely among patients and correlated poorly with the ISC or reticulocyte percentage; however, D50 did correlate with the net change in integrated unit areas. Several patients had repeated density distributions over a 2-year time period. All HbSS patients showed increased but variable dense cell fractions which could not be definitively correlated with the clinical state of the patient. Transfusion reproducibly resulted in a lowering of the dense cell fraction. Erythrocytes from the HbSC patients showed a uniform increase in density and absence of the large dense cell fraction seen in most HbSS patients. This method provides a simple means for quantitation of the light and dense cell fraction in blood of patients with sickling disorders and displays the profile of erythrocyte density heterogeneity for the individual sickle cell patient.

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