Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decreased membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC)§ ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipo-proteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80–85% of abetalipo-proteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity ({ie116-1}) between 5 and 42°C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively, at 10°C; 4.63 vs 4.04 poise at 37°C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Genetic systems of red cell blood groups in goats.

Evidence is presented for six genetic systems of goat red cell blood groups. The A system presently consists of one specificity, two alleles, two phenotypes (A1 and no-A1) and appears to be homologous to the A system of sheep. The B system, like its homologue in sheep, is very complex. Fourteen of 21 specificities detected in the present study, i.e. B2, B3, B4, B5, B7, B8, B9, B10, B11, B14, B15, B16, B17 and B20, belong to the B system which involves a large number of phenogroups (31 different B phenogroups identified in 26 sires). Because of their homology with sheep C and R systems, two other genetic systems of goat blood groups are named C and R respectively. Each of the two goat systems is presently a one blood group specificity, two phenotype (C12 and no-C12; R and no-R detectable on the red cells) two allele system. Two specificities, namely E6 and E18, belong to a genetic system called E in which four phenotypes are under the control of two alleles codominant and one recessive at a single locus. The F system involves but a single pair of alleles and two phenotypes (F19 and no-F19). Because of its low frequency in the goats tested, the X13 specificity remains unassigned.     

Some aspects of zinc transport across eel (Anguilla anguilla) red blood cell membranes.

Zinc movement across eel and human red blood cell membranes was measured by atomic absorption spectrophotometry. It was observed that:

• 1) In human red blood cells zinc uptake is twice as rapid as in fish red blood cells over a temperature range of 10-40°C. The low rate of zinc uptake in eel red blood cell may be simply the side effect of different surface area to volume ratios for the differences in cell size or, it may be due to the low permeability of bicarbonate through the red blood cell membranes.
• 2) Zinc uptake measured in eel and human red blood cells treated and untreated with external trypsin shows different features. The zinc uptake was reduced by about 40% in treated eel red blood cells with respect to the total uptake of untreated red blood cells. Human red blood cells treated and untreated with trypsin do not show any differences in the amount of zinc transported.
• 3) In fish red blood cells, zinc uptake in NANO₃ medium is markedly reduced, compared with that measured in NaCl medium. The [Zn²⁺_i slightly increases in the presence of bicarbonate. In human red blood cells in NANO₃ medium the zinc uptake is strongly reduced and the presence of bicarbonate marginally increases the zinc influx.
• 4) In eel red blood cells there seem to be two independent pathways for zinc uptake: one DIDS-sensitive and the other DIDS-insensitive. DIDS 10 μM inhibits only 64% of the total zinc transported. Iincreasing the DIDS concentration did not give more inhibition. In human red blood cells only one DIDS-sensitive pathway for zinc transported seems to exist, because, 2,5 μM DIDS inhibits 97% of zinc uptake.

     

Comparative studies of the protein composition of red blood cell membranes from eight mammalian species

The polypeptide pattern of red blood cell (RBC) membranes from cow, sheep, horse, rabbit, guinea pig, rat, mouse, analyzed by polyacrylamide gel electrophoresis, was compared to human RBC counterpart. Some qualitative and quantitative differences were noted. Among the high molecular weight components the bands 2.1- 2.3 appeared slightly decreased in rabbit and rat and increased in sheep RBC membranes. Band 3 appeared to have a higher molecular weight in the cow, guinea pig and mouse RBCs, and a lower molecular weight in the sheep RBCs. Band 4.1 from the RBC membranes of cow, sheep, rabbit and guinea pig was splitted into two sub-bands, while band 4.2 overlapped with band 4.1 in horse and guinea pig RBC membranes. There are marked differences in the number and position of bands in the 4.5 region, while band 4.9 is present in higher amounts in horse, rabbit and guinea pig RBC membranes. Band 6 (glyceraldehyde 3-phosphate dehydrogenase) was undetectable in horse, rat and mouse RBC membranes and was decreased in sheep, rabbit and guinea pig. There are also major differences in the region of band 7 and below ("post-7"). Band 8 was undetectable in horse, cow and guinea pig, and was in higher amounts in rat. A band corresponding to a molecular weight of about 22 kD in the "post-8" region was present only in guinea pig RBC membranes.     

Non-invasive in situ simultaneous measurement of multi-parameter mechanical properties of red blood cell membrane

The purpose of this study was to develop a new dynamic image analyzing technique that will give us the ability to measure the viscoelastic parameters of individual living red blood cells non-invasively, in situ and in real time. With this technique, the bending modulus Kc, the shear elasticity μ and their ratio ε were measured under different temperatures, oxygen partial pressures and osmotic pressures. The results not only show the effects of external conditions on mechanical properties of cell membranes including deformability,flexibility, adhesive ability and plasticity, but also demonstrate that the technique can be used to measure cell membrane parameters continuously under several physiological and pathological conditions.     

Disuccinimidyl suberate cross-linked hemoglobin as a novel red blood cell substitute

Disuccinimidyl suberate (DSS) intramolecularly cross-linked hemoglobin (Hb) was developed as a novel red blood cell substitute. A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb. SDS-PAGE was also used as a complement. It was proved that 83.8% of the product was intramolecularly cross-linked Hb with weight-average molecular weights (Mw) of 67.5 kD, 12% was dimeric Hb with Mw of 146.6 kD, and 4.2% was trimeric Hb with Mw of 306.4 kD. The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCl2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the twoα subunits within a Hb molecule, resulting in stabilization of the tetramer structure. However, the cross-linking was not site-specific. The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg, and the Hill coefficient decreased from 2.22 to 1.41. Result of isoelectric focusing showed that the pI of DSS cross-linked Hb was in the range of 4.6-5.2, similar to that of serum albumin. The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs. Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects. The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.     

Disuccinimidyl suberate cross-linked hemoglobin as a novel red blood cell substitute

Disuccinimidyl suberate (DSS) intramolecularly cross-linked hemoglobin (Hb) was developed as a novel red blood cell substitute. A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb. SDS-PAGE was also used as a complement. It was proved that 83.8% of the product was intramolecularly cross-linked Hb with weight-average molecular weights (Mw) of 67.5 kD, 12% was dimeric Hb with Mw of 146.6 kD, and 4.2% was trimeric Hb with Mw of 306.4 kD. The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCI2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the two a subunits within a Hb molecule, resulting in stabilization of the tetramer structure. However, the cross-linking was not site-specific. The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg, and the Hill coefficient decreased from 2.22 to 1.41. Result of isoelectric focusing showed that the pi of DSS cross-linked Hb was in the range of 4.6-5.2, similar to that of serum albumin. The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs. Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects. The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.     

Resistance to the Brownian movement of red blood cells on flat horizontal surfaces

The movements of red blood cells (RBC), suspended in plasma, on plastic, glass, rhodium metal plate, siliconized glass, and siliconized rhodium were recorded on cinéfilm and analyzed. Values for the drag coefficient were calculated, using Einstein's theory of Brownian movement, and compared with the theoretical Stokes' hydrodynamic drag. The difference between the computed and Stokes' values gave the frictional coefficient or resistance resulting from the interaction of the cells, with the test surface. Of the three uncoated test surfaces, plastic was found to have the least interaction with the RBC. The frictional coefficient for plastic was found to be 1.75×10⁻⁷ N s m⁻¹ compared with a value of 2.82×10⁻⁷ N s m⁻¹ for rhodium metal, which had the largest interaction. Upon siliconization of the test surfaces, the interaction decreased by 40%. Reduction in the pH of the suspending plasma increased the interaction between the cells and the uncoated test surfaces, but the pH effect of diminished when the surfaces were siliconized.     

Modulated red blood cell survival by membrane protein clustering

Human and murine blood cells treated with ZnCl₂ and bis(sulfosuccinimidyl)suberate (BS³) (a cross linking agent) undergo band 3 clustering and binding of hemoglobin to red blood cell membrane proteins. These clusters induce autologous IgG binding and complement fixation, thus favouring the phagocytosis of ZnCl₂/BS³ treated cells by macrophages. The extension of red blood cell opsonization can be easily modulated by changing the ZnCl₂ concentration in the 0.1–1.0 mM range thus providing an effective way to affect blood cell recognition by macrophages. In fact, murine erythrocytes treated with increasing ZnCl₂ concentrations have proportionally reduced survivals when reinjected into the animal. Furthermore, the organ sequestration of ZnCl₂/BS³ treated cells strongly resembles the typical distribution of the senescent cells. Since the ZnCl₂/BS³ treatment can also be performed on red blood cells loaded with drugs or other substances, this procedure is an effective drug-targeting system to be used for the delivery of molecules to peritoneal, liver and spleen macrophages.     

Human red blood cell aging: correlative changes in surface charge and cell properties

Red blood cells (RBCs) during microcirculation, aging and storage, lose N-acetylneuraminic acid (NANA) and other biomaterials thereby altering cell structures, some properties and functions. Such cell damage very likely underlies the serious adverse effects of blood transfusion. However, a controversy has remained since 1961-1977 as to whether with aging, the RBCs, suffering loss of NANA, do have a decreased charge density. Any correlation between the changes in the cell properties with cell aging is also not clear. Therefore, to remove the ambiguity and uncertainty, we carried out multiparameteric studies on Percoll fractions of blood of 38 volunteers (lightest-young-Y-RBCs, densest-old-O-RBCs, two middle fractions).We found that there were striking differences between the properties of Y-RBCs and O-RBCs. The ζ-potential of Y-RBCs decreased gradually with aging. Studies in parallel on RBC fractions incubated with both positively charged quantum dots and Sambucus Nigra-fluorescein isothiocyanate (FITC) along with their ζ-potentials provide for the first time direct visual evidence about the lesser amount of charge density and NANA on O-RBCs, and a collinear decrease in their respective ζ-potentials. Close correlation was found between the surface charge on an aging RBC and its structure and functions, from the cell morphology, the membrane deformability to the intracellular Hb structure and oxidation ability. This quantitative approach not only clarifies the picture but also has implications in biology and medicine.     

Association of HLA-DRB1 and DQB1 alleles with red blood cell alloimmunization in Chinese

Few systematic investigations have assessed the correlations between red blood cell (RBC) antibodies and human leukocyte antigen (HLA)-DRB1 alleles in the Chinese population. In this case-control study, we investigated whether specific HLA-DRB1 alleles were associated with RBC alloimmunization by calculating the odds ratios for the frequencies of HLA alleles associated with alloimmunization to different RBC antigens. Three hundred and eight patients harboring RBC alloantibodies were analyzed as the case group, and the frequencies of the HLA-DRB1and HLA-DQB1 alleles in control individuals were analyzed by collecting data from the China Marrow Donor Program (including more than 1.6 million healthy people). HLA alleles were genotyped by single specific primer-polymerase chain reaction. The development of anti-C was associated with DRB1*07, DQB1*06, and DQB1*08; anti-C,e was associated with DRB1*07 and DQB1*06; and anti-E and anti-M were associated with DQB1. Other associations were identified between anti-E and DRB1*09 and between anti-Le^a and DRB1*01. Thus, our findings confirmed that HLA-DRB1 and DQB1 restriction played an important role in the generation of RBC alloantibodies in Chinese individuals.     

Association of HLA-DRB1 and HLA-DQB1 alleles with red blood cell auto-immunization in the Chinese population

Red blood cell (RBC) auto-immunization poses a clinical dilemma, because the formation of RBC autoantibodies in certain patients is a significant causative factor for autoimmune hemolytic anemia. To clarify whether specific HLA-DRB1 and HLA-DQB1 alleles are associated with the response to RBC auto-immunization, we conducted a case-controlled retrospective study. The case group included 117 patients with autoantibodies potentially harmful to RBC after transfusion at the Beijing Red Cross Blood Center, and the controls were 1.6 million healthy subjects whose data are stored in the China Marrow Donor Program database. In the case group, the frequencies of HLA-DRB1 and HLA-DQB1 allele were determined by PCR-rSSO Luminex high-resolution technology. HLA-DRB1*13 allele expression was not detected in the 75 autoantibody positive patients with anti-C3d production (adjusted P=0.0383), whereas the presence of HLA-DRB1*14 allele showed a positive correlation with the autoantibody positive group with anti-C3d production (odds ratio = 2.22, 95% confidence interval = 1.293.84, adjusted P=0.0383). The HLA-DQB1*02 allele was more frequent in patients with both RBC auto-antibodies and alloantibodies. There was no significant association between a positive antiglobulin test result and the presence of HLA-DRB1 and HLA-DQB1 alleles. In summary, we found that HLA-DRB1*13 may serve as a protective factor, whereas HLA-DRB1*14 appears to be a risk factor for the production of autoantibodies positive with anti-C3d in RBC auto-immunization. To our knowledge, this is the first study to propose HLA-DRB1*13 as a protective factor in auto-immunization.     

溶解氧对虹鳟呼吸机能及红细胞特性的影响

目的：初步研究溶解氧对虹鳟呼吸机能及红细胞特性的影响。方法：设四组溶氧水平,测定呼吸频率、血液中PO2、PCO2和pH值,并以RBC、Hb、脆性、膜粘滞性及丙二醛含量等指标观察溶氧对红细胞的影响。结果：溶解氧升高使鱼呼吸频率降低,血液PO2升高（P〈0.05）;高溶氧导致红细胞数、丙二醛含量降低。红细胞膜粘滞系数在试验第10d时,高氧组明显高于其它组,但30d时各溶氧组差异不显著。结论：在一定范围内溶解氧升高可以改善机体组织氧含量,提高呼吸功能,红细胞对溶氧变化有其生理适应性。     

Reconsidering the relation between serum homocysteine and red blood cell distribution width: a cross-sectional study of a large cohort

Purpose: In a recent small sample study, red blood cell distribution width (RDW) was suggested as a predictor of homocysteine levels. The current study was aimed to reexamine this association in a large scale sample.

Methods: A retrospective cross-sectional study of healthy adults, conducted at Rabin Medical Center, during 2000–2014. Data were retrieved from the medical charts and a logistic regression controlling for interfering factors was carried out. Sensitivity analysis was implemented by exclusion of individuals with anaemia.

Results: Five thousand, five hundred fifty-four healthy individuals were included. Mean serum homocysteine level was 10.10 (SD 2.72) μmol/L. 34.4% of the study population had a homocysteine level higher than the upper limit of normal (10.8?μmol/L). Homocysteine showed no association with RDW (OR 1.00; 95% CI 0.97–1.03), but increased with age (OR 1.05; 95% CI 1.04–1.06) and decreased with a rise in haemoglobin (OR 0.77; 95% CI 0.71–0.83), and in the mean corpuscular volume (OR 0.86; 95% CI 0.85–0.88). Exclusion of individuals with anaemia did not reveal an association between homocysteine and RDW but found a somewhat smaller association between haemoglobin and RDW [OR 0.82; 95% CI 0.73–0.91].

Conclusions: In our large scale sample we did not find an association between RDW and serum homocysteine.     

Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells

Cholesterol and phospholipid are the two major lipids of the red cell membrane. Cholesterol is insoluble in water but is solubilized by phospholipids both in membranes and in plasma lipoproteins. Morever, cholesterol exchanges between membranes and lipoproteins. An equilibrium partition is established based on the amount of cholesterol relative to phospholipid (C/PL) in these two compartments. Increases in the C/PL of red cell membranes have been studied under three conditions: First, spontaneous increases in vivo have been observed in the spur red cells of patients with severe liver disease; second, similar red cell changes in vivo have been induced by the administration of cholesterol-enriched diets to rodents and dogs; third, increases in membrane cholesterol have been induced in vitro by enriching the C/PL of the lipoprotein environment with cholesterol-phospholipid dispersions (liposomes) having a C/PL of >1.0. In each case, there is a close relationship between the C/PL of the plasma environment and the C/PL of the red cell membrane. In vivo, the C/PL mole ratio of red cell membranes ranges from a normal value of 0.9–1.0 to values which approach but do not reach 2.0. In vitro, this ratio approaches 3.0. Cholesterol enrichment of red cell membranes directly influences membrane lipid fluidity, as assessed by the rotational diffusion of hydrophobic fluorescent probes such as diphenyl hexatriene (DPH). A close correlation exists between increases in red cell membrane C/PL and decreases in membrane fluidity over the range of membrane C/PL from 1.0 to 2.0; however, little further change in fluidity occurs when membrane C/PL is increased to 2.0–3.0. Cholesterol enrichment of red cell membranes is associated with the transformation of cell contour to one which is redundant and folded, and this is associated with a decrease in red cell filterability in vitro. Circulation in vivo in the presence of the slpeen further modifies cell shape to a spiny, irregular (spur) form, and the survival of cholesterol-rich red cells is decreased in the presence of the spleen. Although active Na-K transport is not influenced by cholesterol enrichment of human red cells, several carrier-mediated transport pathways are inhibited. We have demonstrated this effect for the cotransport of Na + K and similar results have been obtained by others in studies of organic acid transport and the transport of small neutral molecules such as erythritol and glycerol. Thus, red cell membrane C/PL is sensitive to the C/PL of the plasma environment. Increasing membrane C/PL causes a decrease in membrane fluidity, and these changes are associated with a reduction in membrane permeability, a distortion of cell contour and filterability and a shortening of the survival of redcells in vivo.     

Rat red blood cell hexokinase purification,properties and age-dependence

Summary Rat erythrocytes, in contrast to red blood cells from other mammals, have been shown to contain only one hexokinase isozymic form identified as type I by chromatographic and kinetic properties. Rat reticulocytes contain 3.6-times the hexokinase activity found in mature erythrocytes but exactly the same isozyme. By a combination of ion-exchange chromatography, dye-ligand chromatography and high-pressure liquid chromatography the rat erythrocyte hexokinase was purified more than 84 000-fold to a specific activity of 143 units/mg protein and shown to be homogeneous by sodium dodecyl sulfate-gel electrophoresis. The native protein showed a molecular weight of 100 000 by gel-filtration and an apparent molecular weight of 98 000 under denaturating conditions in sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was shown to be 6.3 pH units. This data provides evidence of only one form of hexokinase in the erythrocytes of a mammal.     

Even patients with mild COVID‐19 symptoms after SARS‐CoV‐2 infection show prolonged altered red blood cell morphology and rheological parameters

Infection with the novel severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and the associated coronavirus disease‐19 (COVID‐19) might affect red blood cells (RBC); possibly altering oxygen supply. However, investigations of cell morphology and RBC rheological parameters during a mild disease course are lacking and thus, the aim of the study. Fifty individuals with mild COVID‐19 disease process were tested after the acute phase of SARS‐CoV‐2 infection (37males/13 females), and the data were compared to n = 42 healthy controls (30 males/12 females). Analysis of venous blood samples, taken at rest, revealed a higher percentage of permanently elongated RBC and membrane extensions in COVID‐19 patients. Haematological parameters and haemoglobin concentration, MCH and MCV in particular, were highly altered in COVID‐19. RBC deformability and deformability under an osmotic gradient were significantly reduced in COVID‐19 patients. Higher RBC‐NOS activation was not capable to at least in part counteract these reductions. Impaired RBC deformability might also be related to morphological changes and/or increased oxidative state. RBC aggregation index remained unaffected. However, higher shear rates were necessary to balance the aggregation‐disaggregation in COVID‐19 patients which might be, among others, related to morphological changes. The data suggest prolonged modifications of the RBC system even during a mild COVID‐19 disease course.     

Changes in alanine and glutamine transport during rat red blood cell maturation

Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na⁺-dependent L-alanine transport were:K _m 0.43 and 1.88 mM andV _max 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na⁺-dependent L-glutamine transport in reticulocytes wereK _m 0.51 mM andV _max 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.     

Long ultradian rhythms in red blood cells and ghost suspensions: Possible involvement of cell membrane

Summary Oscillations in glyceraldehyde-3-phosphate dehydrogenase (GAPD) and glucose-6-phosphate dehydrogenase (G6PD) activities were recorded in suspensions of intact human red blood cells (RBCs) exposed to various light regimens. The periods of these oscillations, defined as “long ultradian,” ranged between 13 and 18 h regardless of light regimen. The patterns of enzymatic activities were the same when assayed at each time point, in full hypotonic hemolysates, and membrane-free hemolysates. However, if hemolysates were prepared by sonication the activity pattern did not exhibit significant oscillations and the activity was higher than that recorded in hypotonic hemolysates. The observed rhythms may reflect a time-dependent attachment and detachment of enzyme molecules from cell membrane, suggesting that at the bound state the enzyme molecules are (temporarily) inactive. Oscillations with similar long ultradian periods were also observed in Ca⁺⁺ concentration of suspended RBCs and in the binding of Ca⁺⁺⁴⁵ to human RBC ghosts. Treatment of the RBCs with A₂C or Diamide before the preparation of the ghosts changed or distorted the rhythmic pattern of Ca⁺⁺⁴⁵ binding. These results point to the role of the membrane in processing the long ultradian oscillations. The relation between this type of oscillations to circadian rhythm is discussed.