Erythrocyte aging in neurodegenerative disorders.

In the present paper, we have reviewed the principal studies on red cell membrane abnormalities associated with neurodegenerative disorders. In the literature, two lines of investigation may be recognized: one based on the hypothesis of the presence of an oxidative environment responsible for red cell oxidative damage in Alzheimer's disease (AD), Alzheimer's dementia type (DAT) and Parkinson' disease (PD); the other one based on the identification of structural and/or functional abnormalities in red cell membrane band 3 and/or in red cell membrane lipid composition in "neuroacanthocytosis". In AD, DAT and PD patients, an increased red cell membrane lipid peroxidation suggests an increase red cell oxidative damages and precocious red cell aging. In "neuroacanthocytosis", grouping chorea-acanthocytosis, Mcleod syndrome and abetalipoproteinemia, the red cells are characterized by thorn or spur-like protrusions, known as "acanthocytes". The presence of circulating acanthocytes, characterized by abnormalities in red cell band 3 structure and/or function, is associated with increase levels of anti-band 3 antibodies which are physiologically produced against aged red cells and are known to mediate red cell removal from the peripheral circulation by macrophages. We have reviewed the mechanism(s) of the loss of red cell membrane stability and of the precocious red cell aging in neurodegenerative disorders.     

Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells

To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.     

Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias. I. Increased phosphatidylcholine and reduced sphingomyelin in patients with elevated levels of triacylglycerol-rich lipoproteins.

The composition of red blood cell membrane and plasma phospholipids has been analyzed in patients with hyperlipidemias. In red cells of patients with elevated levels of triacylglycerol-rich lipoproteins, phosphatidylcholine (PC) was raised and sphingomyelin (SM) reduced, resulting in a 20% increase of the membrane PC/SM ratio. In plasma phospholipids of these patients PC and SM levels were also higher and lower, respectively and the plasma PC/SM ratio was elevated by more than 50%. Close positive correlations between plasma and membrane phospholipids were obtained for PC, SM and the PC/SM ratio in normolipidemic and hyperlipidemic donors. Plasmalogen phosphatidylethanolamine (PE), a supposed endogenous protector against lipid oxidation, was reduced by about 20% in red cell membrane lipids in hyperlipidemic patients. Also plasmalogen-PE in plasma tended to be reduced in hyperlipidemic donors. Plasma HDL levels were positively related to the content of plasmalogen PE in the red cell membrane. In conclusion, there are closely related increases in PC/SM ratios in plasma and the red cell membrane in patients with elevated levels of triacylglycerol-rich lipoproteins. It is speculated that decreases in red cell membrane plasmalogen-PE in hyperlipidemic patients could be related to impaired antioxidant protection, possibly as a consequence of reductions in plasma HDL levels.     

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 decrease 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 abetalipoproteinemia 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 abetalipoproteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity (eta) between 5 and 42 degrees 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 degrees C; 4.63 vs 4.04 poise at 37 degrees 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.     

The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that α- and β-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly α-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential β-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-β-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.     

Hydrolysis of phosphatidylserine-exposing red blood cells by secretory phospholipase A2 generates lysophosphatidic acid and results in vascular dysfunction

Secretory phospholipase A(2) (sPLA(2)) type IIa, elevated in inflammation, breaks down membrane phospholipids and generates arachidonic acid. We hypothesized that sPLA(2) will hydrolyze red blood cells that expose phosphatidylserine (PS) and generate lysophosphatidic acid (LPA) from phosphatidic acid that is elevated in PS-exposing red blood cells. In turn, LPA, a powerful lipid mediator, could affect vascular endothelial cell function. Although normal red blood cells were not affected by sPLA(2), at levels of sPLA(2) observed under inflammatory conditions (100 ng/ml) PS-exposing red blood cells hemolyzed and generated LPA (1.2 nM/10(8) RBC). When endothelial cell monolayers were incubated in vitro with LPA, a loss of confluence was noted. Moreover, a dose-dependent increase in hydraulic conductivity was identified in rat mesenteric venules in vivo with 5 microM LPA, and the combination of PS-exposing red blood cells with PLA(2) caused a similar increase in permeability. In the presence of N-palmitoyl L-serine phosphoric acid, a competitive inhibitor for the endothelial LPA receptor, loss of confluence in vitro and the hydraulic permeability caused by 5 microM LPA in vivo were abolished. The present study demonstrates that increased sPLA(2) activity in inflammation in the presence of cells that have lost their membrane phospholipid asymmetry can lead to LPA-mediated endothelial dysfunction and loss of vascular integrity.     

Radiation-induced structural alterations in fish red blood cells

The structural properties of gamma-irradiated fish red blood cells were studied using a spin labelling method. The gradient increase of lipid fluidity with the increasing gamma radiation doses was indicated by methyl 5-doxylpalmitate and methyl 12-doxylstearate spin labels spectra. In turns, the spectra of maleimide spin label (4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl) indicated a modification of the internal proteins and the increased internal viscosity of red blood cells. The results encourage the conclusion that the increase in membrane fluidity may result from theernations in lipid-protein interactions rather than lipid peroxidation.     

Alterations in erythrocyte membrane lipid and fatty acid composition in Chediak-Higashi syndrome

Chediak-Higashi syndrome (CHS) is an autosomal recessive disease characterized by the presence of abnormally large cytoplasmic organelles in all body granule producing cells. The molecular mechanism for this disease is still unknown. Functional disorders in membrane-related processes have been reported. Erythrocyte membranes from four CHS patients and 15 relatives including obligatory heterozygous were studied to examine potential alterations in the lipid and fatty acid profile of erythrocyte membranes associated with this syndrome. Plasma concentrations of cholesterol, triglycerides, phospholipids, and apolipoproteins AI and B100, and the lipid components of very low-, intermediate-, low- and high-density lipoproteins were also determined. CHS erythrocyte membranes were found to be enriched with lipids in relation to protein and to show: (1) an increase in cholesterol and choline-containing phospholipids (sphingomyelin and phosphatidylcholine) that predominate in the outer monolayer, which is higher than the increase in phosphatidylserine and phosphatidylethanolamine, that are chiefly limited to the inner monolayer in normal red blood cells; (2) a relative palmitic acid and saturated fatty acid increase and arachidonic acid and unsaturated fatty acid decrease, this resulting in a lower unsaturation index than controls. Changes in CHS erythrocyte membrane lipids seem to be unrelated to serum lipid disorders as plasma lipid and apolipoprotein concentrations were apparently in the normal range, with the exception of a modest hypertriglyceridemia in patients and relatives and a decreased concentration of HDL cholesterol in patients. These findings indicate that CHS erythrocyte membranes contain an abnormal lipid matrix with which membrane proteins are defectively associated. The anomalous CHS membrane composition can be explained on the postulated effects of the CHS1/Lyst gene.     

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.     

Reduction in the stearic to oleic acid ratio in leukaemic cells--a possible chemical marker of malignancy

Total lipid extracts of peripheral blood cells from patients with chronic leukaemias were analysed for relative values of saturation of the eighteen carbon chain length fatty acids (C 18 FA). The results are expressed as saturation index (C 18 S:C 18 U) of the saturated C 18 FA (stearic acid) over the unsaturated C 18 FA (oleic, linoleic and linolenic acids). The saturation indices of the white blood cells (WBC) and the red blood cells (RBC) in specimens from 14 patients with chronic granulocytic leukaemia (CGL) and 17 patients with chronic lymphocytic leukaemias (CLL) were significantly and consistently lower than control specimens. It is proposed that the relative increase in the unsaturated oleic acid could prove to be a chemical marker of malignancy reflecting a deficient cellular control of the process of stearic acid desaturation. The theoretical implications of the implied increase in membrane fluidity for the cells are discussed.     

Membrane mobility agents: Alteration of human red blood cell membrane properties

Brief incubation of human red cells with the membrane mobility agent, 2-(2-methoxy)-ethoxyethyl 8-(2-n-octylcyclopropyl)-octanoate (A₂C), produces stomatocytes (with invaginations) along with a decrease in osmotic fragility, an altered distribution in a two-phase dextran-polyethylene glycol-water system, and increased permeability to the GSH oxidant, the diazene derivative, 1,2-diazenedicarboxylic acid bis(N′-methylpiperazide). These changes are consistent with what would be expected for agents which increase membrane lipid disorder. Membrane mobility agents provide a very convenient means for altering membrane properties and should be useful for studies on both normal and abnormal red cells.     

Abnormalities of erythrocyte stromal lipids in atresia of the intrahepatic bile ducts

Detailed analysis of plasma and erythrocytes lipid composition of patient with intrahepatic biliary atresia is presented. Abnormalities have been outlined and are characterized as following: (1) an increase of total cholesterol compounds and total phospholipids in serum; (2) an increase of free cholesterol and lecithin up to 50 per cent of total phosphatides in red cells; (3) the fatty-acids pattern isolated from total phospholipids of red cells shows a rise of palmitic and palmitoleic acids and a decrease of stearic and longer-chain fatty acids; (4) in PC and PE of red cells, there is an overall tendency for the degree of unsaturation of long-chain fatty acids to increase. In addition to these lipid changes, it was demonstrated by polyacrylamide-gel electrophoresis that the composition of membrane proteins was normal. It is of particular interest to establish whether these abnormalities are either induced by complex metabolic pathways and exchange processes between the lipids of circulating erythrocytes and the altered lipids of serum environment or are the direct result of modified hepatic cellular or enzymatic function.     

Dehydration rate determines the degree of membrane damage and desiccation tolerance in bryophytes

Desiccation tolerant (DT) organisms are able to withstand an extended loss of body water and rapidly resume metabolism upon rehydration. This ability, however, is strongly dependent on a slow dehydration rate. Fast dehydration affects membrane integrity leading to intracellular solute leakage upon rehydration and thereby impairs metabolism recovery. We test the hypothesis that the increased cell membrane damage and membrane permeability observed under fast dehydration, compared with slow dehydration, is related to an increase in lipid peroxidation. Our results reject this hypothesis because following rehydration lipid peroxidation remains unaltered, a fact that could be due to the high increase of NO upon rehydration. However, in fast‐dried samples we found a strong signal of red autofluorescence upon rehydration, which correlates with an increase in ROS production and with membrane leakage, particularly the case of phenolics. This could be used as a bioindicator of oxidative stress and membrane damage.     

The structural organization of skeletal proteins influences lipid translocation across erythrocyte membrane

In order to define the influence of skeletal protein organization on transmembrane phospholipid movement in erythrocyte membranes, we measured the translocation rate of lysophosphatidylcholine in pathologic red cells. A simple method based on the differential extraction of lysophosphatidylcholine from the red cell membrane by saline and albumin solutions was used to quantitate the translocation rate. Two groups of pathologic red cells were chosen for these studies: red cells with quantitative deficiencies of the skeletal proteins, spectrin and protein 4.1, and sickle erythrocytes in which controlled reorganization of the membrane was induced by hemoglobin polymerization. Marked increase in lipid translocation rate was seen in red cells having quantitative deficiencies of spectrin and protein 4.1. The magnitude of the increase in translocation rate in spectrin-deficient red cells was related to the magnitude of protein deficiency. Translocation rate in sickle erythrocyte membranes increased by 50% upon deoxygenation as a result of sickle hemoglobin polymerization. No increase in translocation rate was seen in normal cells upon deoxygenation. By manipulating the extent of membrane reorganization that occurred following deoxygenation of sickle cells, we have been able to show that skeletal reorganization induced by hemoglobin polymerization and not hemoglobin polymerization per se is responsible for the increase in translocation rate. Together, these findings imply that the structural organization of membrane skeletal proteins plays an important role in regulating the rate of transbilayer movement of lipids across the erythrocyte membrane.     

Impact of exogenous lysolipids on sensitive and multidrug resistant K562 cells: 1H NMR studies

The ability of lysolipids to enter into a membrane bi-layer and disturb the membrane structure was used to study the behavior of K562 erythroleukemic cells, K562 wild type (K562wt) as well as the multidrug resistant cells K562adr. Both types of cells, when analyzed by proton NMR spectroscopy exhibit the high resolution signals assigned to so-called "mobile lipid" signals, which, in most cases, are located outside the lipid bi-layer as lipid droplets. In order to perform these studies, the K562wt and K562adr cells were treated for 48h with lysophosphatidylcholine oleoyl (LPC18), lysophosphatidylcholine palmitoyl (LPC16) and L-alpha-lysophosphatidyslerine (LPS). After evaluating toxicity of lysolipids, proton NMR of whole treated cells was used to analyze the mobile lipid content. Nile red staining and fluorescence microscopy were used to detect the presence of intracellular lipid droplets. Membrane lipid asymmetry perturbation was estimated by annexin V staining with use of flow cytometry. Using fluorescence spectroscopy the functioning of P-glycoprotein (P-gp) responsible for multidrug resistance was also evaluated after the treatment with lysolipids. Lysolipids were found to be more toxic for K562wt than for K562adr cells. LPS and LPC16 produced an increased of a mobile lipid NMR signal and amount of lipid droplets in K562wt cells only. LPC18, with the lowest toxicity, has shown more intense effects on NMR spectra with a large increase of lipid NMR signal without changes in lipid droplet staining. The functioning of the P-gp pump and membrane asymmetry were not modified by any of the lysolipids used.     

Phenylhydrazine-induced changes in erythrocyte membrane surface lipid packing

Phenylhydrazine-induced oxidative damage in red cells results in increased binding of merocyanine 540, a fluorescence probe sensitive to changes in lipid packing. Fluorescence polarization studies with diphenylhexatriene did not reveal major changes in order parameters both in intact red cells and lysates treated with phenylhydrazine. These fluorescence studies indicate that major changes are observed in membrane lipids. Analytical studies of membrane phospholipids revealed a significant decrease in phosphatidylethanolamine. The results of the fluorescence and lipid studies, taken in association with our previously reported findings on spectrin and other cytoskeletal protein degradation in red cells exposed to phenylhydrazine, suggests that degradation of cytoskeleton membrane proteins is also responsible for changes in the lipid bilayer surface of the red cell membrane.     

Vitamin E in young and old human red blood cells

Young and old human red blood cells contain about the same amount of alpha-tocopherol, a compound which has previously been shown to be the major lipid-soluble, chain-breaking antioxidant present in such cells. Since red blood cells lose up to ca. 20% of lipid material from their membrane as they age, the alpha-tocopherol/membrane-lipid ratio actually rises with age rather than declining as might have been expected on the basis of the free radical theory of aging. The alpha-tocopherol/arachidonic acid moiety ratios increase in the order: young red blood cells less than old red blood cells less than plasma, which argues against the suggested membrane stabilizing effect of alpha-tocopherol/arachidonic acid moiety complexes.     

The effect of hypoxia due to iron-deficiency anemia on the physicochemical properties of biological membranes]

The lipid structure and Ca2+ permeability of red blood cell, hepatocyte and cardiomyocyte membranes were determined while investigating the effect of hypoxia caused by iron deficiency anemia upon the structural and functional state of biological membranes. The lipid composition and barrier characteristics of membranes change under conditions of hypoxia caused by experimental iron deficiency anemia. Quantitative changes in the cell membrane lipids may be considered as an important molecular mechanism of Ca2+ transport disorder in membranes, increase of Ca2+ permeability producing its surplus in the cells and subsequent metabolic homeostatic disturbances.     

Comparative study on fluorescent probes distributed in human erythrocytes and platelets

Important cellular functions, such as rheological properties of cells are presumably related to the membrane lipid fluidity which may be approached by the use of fluorescence polarization method. However, biological membranes represent very heterogeneous media and the knowledge of the fluidity of membrane compartments requires the use of different probes. Two fluorescent probes, DPH and its cationic derivative, TMA-DPH, have been employed to probe the lipid fluidity of human platelets and red cell membranes. The results show that the informations given by DPH and TMA-DPH can present important differences, suggesting that DPH and TMA-DPH are localized in different regions of cell membranes. In an attempt to investigate relations between lipid fluidity and rheological properties of red cells, the behavior of probes was studied in a "Couette" viscometer with a device for studying the emissive properties of probes when red cell membranes are under shear conditions.     

Modification of the erythrocyte membrane by a non-specific lipid transfer protein

The non-specific phospholipid transfer protein purified from bovine liver has been used to modify the phospholipid content and phospholipid composition of the membrane of intact human erythrocytes. Apart from an exchange of phosphatidylcholine between the red cell and PC-containing vesicles, the protein appeared to facilitate net transfer of phosphatidylcholine from the donor vesicles to the erythrocyte and sphingomyelin transfer in the opposite direction. Phosphatidylcholine transfer was accompanied by an equivalent transfer (on a molar basis) of cholesterol. An increase in phosphatidylcholine content in the erythrocyte membrane from 90 to 282 nmol per 100 microliters packed cells was observed. Phospholipase C treatment of modified cells showed that all of the phosphatidylcholine which was transferred to the erythrocyte was incorporated in the lipid bilayer. The nonspecific lipid transfer protein used here appeared to be a suitable tool to modify lipid content and composition of the erythrocyte membrane, and possible applications of this approach are discussed.