Erythrocyte Membrane Phospholipids Research Articles

Ethanol-Induced Alterations in Erythrocyte Membrane Phospholipid Composition

Ethanol's effects on erythrocyte membrane lipid composition were examined in male squirrel monkeys divided into three groups receiving three different regimens: Controls were fed a chemically defined liquid diet, and low and high ethanol primates were given diets with vodka substituted isocalorically for 12% and 24% of calories, respectively. After membrane lipid extraction, phospholipid mass, class composition, and fatty acid profiles were measured in each group. Although there were no differences in the total phospholipid mass, the low ethanol primates had significantly elevated phosphatidylethanolamine in their membranes as compared with the other monkeys. Membrane phospholipid fatty acid profiles showed no differences among the three groups. There were also no differences in the animals' plasma liver enzymes. Results of this investigation suggest that, despite the absence of nutritional deficiencies and liver malfunction, low amounts (12%) of dietary ethanol cause elevations in phosphatidylethanolamine that may represent a specific change in the membrane's inner leaflet where this phospholipid is located. These results may have clinical significance because ethanol-induced modifications in membrane lipids may contribute to alterations in fluidity and lead to pathologic changes in function.

Osmotic fragility of sheep erythrocytes and alterations in their membrane constituents in Dictyocaulus filaria infection

Alterations in the sheep erythrocyte membrane constituents during the course of Dictyocaulus filaria infection were studied in 4–6 month old Nali lambs. During the acute course of infection, plasma cholesterol, membrane cholesterol, cholesterol: phospholipid ratio and acetylcholinesterase activity fell significantly when compared with uninfected controls. The onset of the fall in the values of these parameters was observed 1–2 weeks prior to an increase in the osmotic fragility of erythrocytes. The altered values persistently remained at sub-normal levels during the chronic stage of the infection. However, the membrane proteins and phospholipids of sheep erythrocytes remained unaffected during the entire period of study. The clinico-parasitological picture of the disease, as judged by the clinical course of disease, faecal larval output and necropsy worm recovery, was typical of ovine dictyocauliosis.

Effect of Zinc Deficiency and Food Restriction in Rats on Erythrocyte Membrane Zinc, Phospholipid and Protein Content

Zinc deficiency in rats increases the osmotic fragility of erythrocytes, suggesting a structural defect in the plasma membrane. The purpose of this study was to determine the effect of zinc deficiency on membrane components that might be responsible for the increased fragility. Immature male rats were fed for 3 wk a zinc-deficient (0.5 mg Zn/kg) or a control (100 mg Zn/kg) diet either ad libitum or pair-fed. Red blood cell membranes were analyzed for zinc after wet ashing in nitric-perchloric acid. The fatty acid composition of membrane phosphatidylcholine and phosphatidylethanolamine was determined by gas-liquid chromatography, and the protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane zinc concentration was decreased by zinc deficiency, but returned to control levels upon repletion with a zinc-adequate diet for 3 d. The increased osmotic fragility was also restored to the control level during the same repletion period, suggesting that zinc is an important determinant of membrane stability. The phospholipid fatty acid composition was altered by zinc deficiency, but these changes were not rapidly reversed by repletion. The membrane cholesterol:phospholipid ratio was also increased by zinc deficiency. Zinc status had no influence on the membrane protein profiles of the components visualized by staining. Only the concentration of membrane zinc correlated with osmotic fragility during zinc depletion and repletion.

Very Long‐Chain Fatty Acids in Erythrocyte Membrane Phospholipids in Adrenoleukodystrophy

Mass fragmentography was used to analyze the very long-chain fatty acid (VLCFA) composition of erythrocyte membrane phospholipids. The VLCFA content decreased in the order sphingomyelin (SM), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The tetracosanoic acid (C24:0) and hexacosanoic acid (C26:0) content of both SM and PC in patients with adrenoleukodystrophy (ALD) were significantly higher than those in controls. The VLCFA content of PE was too small, in comparison with those of SM, to be accurately determined.

Insulin binding to erythrocytes and fatty acid composition of erythrocyte membrane phospholipids in healthy men

The relationship between specific insulin binding to insulin receptors on erythrocytes and erythrocyte membrane phospholipid fatty acid pattern was evaluated in 11 healthy men. A significant negative correlation between insulin binding and the proportion of w-6 family essential fatty acids, especially linoleic acid ( r = -0.82, p < 0.01) and arachidonic acid ( r = -0.73, p < 0.05) in erythrocyte membrane was found. On the other hand significant positive correlation between insulin binding and the content of nonessential fatty acids ( r = +0.65, p < 0.05) was seen. Data presented support the hypothesis that the fatty acid composition of membrane phospholipids may modify properties of insulin receptors.

Influence of chlorpromazine on the transverse mobility of phospholipids in the human erythrocyte membrane; relation to shape changes

The influence of chlorpromazine (CPZ) on the transverse mobility of spin-labeled phospholipids incorporated into human erythrocytes was investigated by electron spin resonance. The very slow transverse diffusion of phosphatidylcholine, as well as the absence of transverse mobility of sphingomyelin were not modified even by sublytic concentrations ( ≈ 1 mM ) of CPZ. On the other hand, the rapid outside-inside translocation of the aminophospholipids (Seigneuret and Devaux (1984) Proc. Natl. Acad. Sci. USA 81, 3751–3755), was slightly hindered in CPZ containing membranes. If the spin-labeled aminolipids were incorporated in erythrocytes and allowed to flip to the inner monolayer before CPZ addition, a fraction of the spin labels (10–15%) flipped back instantaneously from the inner to the outer leaflet, upon incubation with CPZ. Similar experiments carried out with spin-labeled phosphatidylcholine and spin-labeled sphingomyelin showed that a fraction of the spin-labeled choline derivatives flip instantaneously to the inner leaflet if CPZ was added after the spin labels. Addition of lysophosphatidylcholine had no effect on the spin-labeled phospholipid redistribution nor on their transmembrane mobility. We interpret the immediate effect of CPZ addition as being due to a reorganization of the bilayer accompanying the rapid CPZ membrane penetration, phenomenon which is independent of the CPZ effect on the steady-state activity of the ‘aminophospholipid translocase’, the latter effect being probably a direct CPZ-protein interaction. By comparison of the time course of phosphatidylserine transverse diffusion in control discocyte cells and in CPZ-induced stomatocytes, we infer that the difference in cell shape is not a major factor in the regulation of the active inward transport of aminophospholipids in human erythrocytes.

Odd-numbered long-chain fatty acid contents in erythrocyte membrane phospholipids in patients with an impaired propionate utilization.

Patients with the inherited disorders of propionate and methylmalonate metabolism such as propionic acidaemia (PA; McKusick 23200) and methylmalonic acidurias (MMA; McKusick 25100) accumulate odd-numbered long-chain fatty acids (OLCFA) in lipids of various organs (Hommes et al., 1968; Gompertz, 1970; Gompertz et al., 1970; Kishimoto et al., 1973). The accumulation of OLCFA also occurs in cultured skin fibroblasts of PA and MMA patients (Giudici et al., 1986). This is due to the fact that intracellularly accumulated propionyl-CoA can replace acetyl-CoA as a ‘primer’ for long-chain fatty acid synthesis.

Effects of alpha-tocopherol-nicotinate administration on the microdynamics of phospholipids of erythrocyte membranes in human subjects.

The effects of alpha-tocopherol-nicotinate on the microdynamic parameters, membrane viscosity, and wobbling angle of phospholipid molecules of healthy human erythrocyte membranes were studied with a nanosecond time-resolved fluorometer. The incorporation of alpha-tocopherol was achieved by oral administration of alpha-tocopherol-nicotinate for one month with dosage of 400 mg/day. The increase in alpha-tocopherol concentration in erythrocytes was confirmed in two subjects. The membrane viscosity in seven healthy subjects decreased significantly from 1.015 +/- 0.137 to 0.888 +/- 0.133 mPa.s. But no changes in the wobbling angle of phospholipid molecules were observed.

Influences of Diet and Postnatal Age on the Lipid Composition of Red Blood Cell Membrane in Newborn Infants

The fatty acid and cholesterol contents of tissue membranes are determinants of their stability and functionality. This study was designed to evaluate the influences of diet and postnatal age on the polyunsaturated fatty acid (PUFA) composition of erythrocyte membrane phospholipid fractions and on the red blood cell membrane cholesterol and phosphorus contents in newborn infants during the 1st month of life. A group of infants was fed on human milk and another group on adapted milk formula. Blood samples were obtained at birth, from cord blood, and at 7 and 30 days of age. Long-chain w6 PUFA declined with advancing age in all membrane phosphoglycerides and sphingomyelin (SM) in those infants fed formula. w6 PUFA also decreased in phosphatidylcholine (PC) and phosphatidylserine (PS) in infants fed human milk and were maintained constant in phosphatidylethanolamine (PE) and SM. w3 PUFA were less affected by postnatal age. PE and SM showed significantly higher percentages of w6 and w3 long-chain PUFA in infants fed human milk than in those fed formula. Membrane cholesterol content increased in all infants from birth to 1 month of life but phosphorus levels were unaffected by diet and postnatal age. These results suggest that diets with a low content of long-chain PUFA, such as adapted cow's milk formulas, may induce changes in membrane functionality and that incorporation of PUFA to the diet in amounts similar to those found in human milk should be considered at least in early life.

Erythrocyte membrane microviscosity and phospholipid composition in lead workers.

The microviscosity and fluidity of erythrocyte ghost membranes from lead workers and control subjects was measured by fluorescence polarisation using the fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). Increased lead was associated with a significant decrease in the average microviscosity of resealed and unsealed erythrocyte membranes. Since DPH fluorescence reflects the organisation of lipids in the central core of the membrane, two aspects of phospholipid metabolism were investigated. Phospholipids were extracted from red blood cell ghost membranes and identified by high performance liquid chromatography. The ratio of phosphatidyl choline to phosphatidyl ethanolamine, an established correlate of membrane fluidity, was significantly increased in lead workers. This is attributed to the known increases in red blood cell cholesterol in lead workers and the structural incompatibility of phosphatidyl ethanolamine and cholesterol, which result in a compensatory increase of phosphatidyl choline. Erythrocyte ghost membranes from control subjects were resealed with the intermediates in phospholipid synthesis that increase with a lead inhibited decrease in red blood cell pyrimidine 5'-nucleotidase. Membrane fluidity was not modified by incubation with cytidine triphosphate, uridine triphosphate, cytidine diphosphate choline, or cytidine diphosphate ethanolamine. Alterations in the microviscosity of the lipid regions of the hydrophobic core of the erythrocyte membrane bilayer and in the phospholipid composition of the membrane may be defects which contribute to the clinical and biochemical instability of the red blood cell on exposure to lead.

Effects of dietary nucleotides on the fatty acid composition of erythrocyte membrane lipids in term infants.

Human milk contains relatively high amounts of acid-soluble nucleotides, mainly CMP, AMP, UMP, IMP, and UDP sugars, and lacks orotate. On the contrary, cow's milk and adapted formulas contain high amounts of orotate and very low amounts of CMP and AMP. Nucleotides may be modulators of metabolic functions in gut and liver. To evaluate the possible role of dietary nucleotides in newborn fatty acid metabolism, we studied the polyunsaturated fatty acid (PUFA) composition of erythrocyte membrane phospholipids in 58 term infants at 30 days of age. Twenty of them were fed human milk (HM), 19 an adapted formula (MF), and 19 with the same formula supplemented with nucleotides (NMF) in an amount similar to that present in HM. Relative content of omega 6 and 3 PUFA greater than 18 carbon atoms was significantly reduced especially in phosphatidylethanolamine and phosphatidylserine for infants fed regular MF compared with those fed HM or NMF. Unsaturation index of red blood cell phospholipids showed a similar effect. These results suggest that dietary nucleotides play a role in the in vivo desaturation and elongation of essential fatty acids to long chain PUFA during early life for the human newborn.

Effects of Dietary Nucleotides on the Fatty Acid Composition of Erythrocyte Membrane Lipids in Term Infants

SummaryHuman milk contains relatively high amounts of acid‐soluble nucleotides, mainly CMP, AMP. UMP. IMP, and UDP sugars, and lacks orotate. On the contrary, cow's milk and adapted formulas contain high amounts of orotate and very low amounts of CMP and AMP. Nucleotides may he modulators of metabolic functions in gut and liver. To evaluate the possible role of dietary nucleotides in newborn fatty acid metabolism, we studied the polyunsaturated fatty acid (PUFA) composition of erythrocyte membrane phospholipids in 58 term infants at 30 days of age. Twenty of them were fed human milk (HM). 19 an adapted formula (MF). and 19 with the same formula supplemented with nucleotides (NMF) in an amount similar to that present in HM. Relative content of omega 6 and 3 PUFA &gt;18 carbon atoms was significantly reduced especially in phosphatidylethanol‐amine and phosphatidylserine for infants fed regular MF compared with those fed HM or NMF. Unsaturation index of red blood cell phospholipids showed a similar effect. These results suggest that dietary nucleotides play a role in the in vivo desaturation and elongation of essential fatty acids to long chain PUFA during early life for the human newborn.

Abnormal erythrocyte membrane phospholipid organisation in chronic myeloid leukaemia

The membrane phospholipid organisation in the red cells of humans suffering from chronic myeloid leukaemia has been analysed using the amino-group labelling reagent trinitrobenzenesulphonic acid and the fluid-sensing fluorophore, Merocyanine 540. Unlike the normal human erythrocytes, trinitrobenzenesulphonic acid in intact chronic myeloid leukaemia erythrocytes modified about 30% phosphatidylserine, under controlled conditions. Also, the chronic myeloid laukaemia red cells, but not the normal cells, were found to bind the fluorescent dye Merocyanine 540. These results demonstrate that loss of the transmembrane phospholipid asymmetry in chronic myeloid leukaemia erythrocytes is accompanied by an enhancement in the outer surface fluidity and, therefore, suggest that the red cells membrane phase-state asymmetry originates probably from the asymmetric arrangements of phospholipids across the membrane bilayer.

ラットのバター, マーガリン飼育によるリポ蛋白, 肝細胞膜, 赤血球膜の脂肪酸組成の変化について

It is well recognized that a change of fatty acid composition of plasma lipoproteins and cellular membranes alters membrane fluidity, producing significant influences on the functions of membrane-bound enzymes or lipoprotein receptors. It is important, then, to know whether or to what extent dietary fat manipulation may influence the fatty acid composition of lipoproteins and cellular membranes. We studied fatty acid composition of lipoproteins and membranes of hepatocytes and erythrocytes in rats after feeding of different fats.Wister male rats were divided into two groups which received a diet supplemented with either butter or margarine. After several feeding periods plasma levels of lipids and fatty acid composition of phospholipids in high density lipoprotein (HDL), hepatocyte membranes and erythrocyte membranes were examined. Extraction of total lipids from HDL and hepatocyte membranes was conducted by the method of Folch, and from erythrocytes by the method of Rose. Isolation of total phospholipids was done by thin layer chromatography and fatty acid composition was analyzed by gas liquid chromatography.Plasma levels of triglyceride and phospholipid after two weeks of feeding were significantly higher in the butter-fed rats than in the margarine-fed rats. The difference of cholesterol level did not reach a statistical significance.Fatty acid composition of phospholipids in HDL, hepatocyte and erythrocyte showed a characteristic response for each feeding. The level of linoleic acid in HDL, hepatocyte and erythrocyte was higher in the margarine-fed rats and the level of arachidonic in HDL and hepatocyte was also higher in the margarine-fed rats. The level of oleic acid in HDL and hepatocyte and the level of palmitic acid in hepatocyte were higher in the butter-fed rats. These results show that the fatty acid composition in lipoproteins and cellular membranes responds well to a change of dietary fats.

Red cell and hemorheological changes in multiple sclerosis

Blood rheology in multiple sclerosis (MS) was investigated in 15 subjects with varying degrees of locomotor difficulties who were members of the local MS Society. Control data were obtained from blood samples from 25 male and 25 female normal blood donors. Whole blood viscosity was measured and blood filterability was assessed. Six MS females provided blood samples for scanning electron microscopy. Erythrocyte membrane fatty acids and phospholipids were assayed. Whole blood viscosity in MS females was higher than controls at 3 of 4 shear rates (p less than 0.001) but in MS males blood viscosity was higher only at shear rate of 1.0 s-1 (p less than 0.05). MS erythrocyte filtration rates were significantly lower than controls (p less than 0.001). Leucocyte counts in MS were greater than controls both in males (p less than 0.01) and females (p less than 0.001). MS erythrocyte morphology was greatly different from controls (p less than 0.0001) and erythrocyte membranes contained less sphingomyelin than controls (p less than 0.01) but more phosphatidylinositol plus phosphatidylserine (p less than 0.02). We conclude that, because our findings indicate an identifiable and potentially correctable abnormality, it is possible to envisage an inhibition of the progressive nature of MS, with the hope of a better prognosis for patients.

Effect of high-fat diet on cholesterol distribution in plasma lipoproteins, cholesterol esterifying activity in leucocytes, and erythrocyte membrane components studied: importance of body weight

Effects of isocaloric changes in dietary fat on plasma lipoproteins and lipids and enzymes of erythrocytes and leucocytes were assessed. Subjects with a higher Brocca index showed increase in total and LDL cholesterol, significant reduction in HDL cholesterol, and increased total cholesterol:HDL cholesterol ratio after high-fat diet consumption. Due to high-fat diet feeding, erythrocyte membrane and leucocyte cholesterol and phospholipid contents were increased, cholesterol: phospholipid molar ratio was elevated, and erythrocyte enzymes (G6PD and 6PGD) and leucocyte enzymes (CEH and CES) were elevated. Erythrocyte membrane glycoprotein components showed marked increase, indicating possible alterations of membrane surfaces. The metabolic alterations were reversed slowly after resumption of the normal (low-fat) diet. Body weight plays an important role in the alterations in major lipoprotein cholesterol contents in response to changes in dietary fat composition. Cellular changes indicate alterations in structure and function of blood cells due to high-fat diet feeding.

Modification of phospholipids in erythrocyte membranes by phospholipase D. A fluorescence and ESR spectroscopic study

The conversion of more than 65% of the phospholipids in human erythrocyte membranes to phosphatidyl-methanol and phosphatidic acid by incubation with phospholipase D and methanol increased the dissociation constant of the fluorescence probe ANS compared to untreated membranes, but did not affect the number of binding sites and the limiting fluorescence enhancement at maximal binding ( I max). On the contrary, the cationic fluorescence probe dansylcadaverin showed additional binding sites without a change in K d and an increase of I max upon incubation with phospholipase D treated erythrocyte membranes compared to incubations of membranes with the original phospholipid pattern. The characteristic temperature-dependence of the quenching of the membrane protein fluorescence by a membrane-bound nitroxide-labeled stearic acid was not influenced by the modification of the phospholipids. A slight reduction of the order parameter, S, determined by ESR-spectroscopy with the same nitroxide spin-labeled fatty acid incorporated into modified membranes compared to controls was found at 40°C, but not at 25°C. The results were interpreted as an indication of membrane domains that retained their physical properties and lipid composition during the incubation with phospholipase D.

Overproduction and high‐yield purification of phospholipase A from the outer membrane of Escherichia coli K‐12

The cloned gene for the outer-membrane-bound phospholipase A from Escherichia coli was placed under control of the strong PL promoter of phage lambda. Induction of PL resulted in a 250-fold overexpression up to about 2% total cellular protein. This overproduced enzyme was indistinguishable from the wild-type enzyme. A homogeneous phospholiphase A preparation was obtained in high yield from overproducing bacteria, using the zwitterionic detergent C12-Sulfobetaine and anion-exchange chromatography. Detergent gradients were found to exert great influence on the elution characteristics. Considerations for the choice of optimal detergent gradients are discussed. The purified enzyme migrated as a single 29-kDa band in SDS/polyacrylamide gels, and required Ca(II) for activity. Maximum activity was displayed by enzyme samples taken from solutions with detergent concentrations near the critical micelle concentration. However, upon switching from high to optimal detergent concentration, maximum activity was restored in several hours, probably reflecting a slow conformational transition of the protein. Because the final pure protein was found to hydrolyze phospholipids in the intact erythrocyte membrane, a densely packed bilayer, we assume that this protein is in its biological native state.

Asymmetric lateral mobility of phospholipids in the human erythrocyte membrane.

The fluorescent phospholipid 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminododecanoyl]phosphatidylcholine (NBD-phosphatidylcholine) and the corresponding aminophospholipid derivatives (NBD-phosphatidylethanolamine and NBD-phosphatidylserine) were introduced in the human erythrocyte membrane by a nonspecific phospholipid exchange protein purified from corn. The lateral mobility of the fluorescent phospholipids was measured by using an extension of the classical photobleaching recovery technique that takes advantage of a modulated fringe pattern and provides a high sensitivity. In intact erythrocytes and in ghosts resealed in the presence of ATP, the fluorescence-contrast curves after photobleaching decayed biexponentially corresponding to two lateral diffusion constants. With NBD-phosphatidylcholine, the majority of the signal corresponded to a "slow" component (1.08 X 10(-9) cm2/sec at 20 degrees C), whereas with the amino derivatives the majority of the signal corresponded to a "fast" component (5.14 X 10(-9) cm2/sec at 20 degrees C). If the ghosts were resealed without ATP, the fast component of the aminophospholipids disappeared. We interpret these results as follows: (i) Provided the cells or the ghosts contain ATP, the three fluorescent phospholipids distribute spontaneously between inner and outer leaflets as endogenous phospholipids, namely NBD-phosphatidylcholine is located in the outer leaflet, while both aminophospholipids are preferentially located in the inner leaflet. (ii) The viscosity of the inner leaflet of human erythrocyte membranes is lower than that of the outer leaflet.

T-Butyl hydroperoxide alters fatty acid incorporation into erythrocyte membrane phospholipid

Because the ability of cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be an important determinant of the ability of cells to tolerate oxidative stress, incorporation of exogenous fatty acid into phospholipid by human erythrocytes has been examined following exposure of the cells to t-butyl hydroperoxide. Exposure of human erythrocytes to t-butyl hydroperoxide (0.5–1.0 mM) results in oxidation of glutathione, formation of malonyldialdehyde, and oxidation of hemoglobin to methemoglobin. Under these conditions, incorporation of exogenous [9,10- 3H]oleic acid into phosphatidylethanolamine is enhanced while incorporation of [9,10- 3H]oleic acid into phosphatidylcholine is decreased. These effects of t-butyl hydroperoxide on [9,10- 3H]oleic acid incorporation are not affected by dissipating transmembrane gradients for calcium and potassium. When malonyldialdehyde production is inhibited by addition of ascorbic acid, t-butyl hydroperoxide still decreases [9,10- 3H]oleic acid incorporation into phosphatidylcholine but no stimulation of [9,10- 3H]oleic acid incorporation into phosphatidylethanolamine occurs. In cells pre-treated with NaNO 2 to convert hemoglobin to methemoglobin, t-butyl hydroperoxide reduces [9,10- 3H]oleic acid incorporation into phosphatidylcholine by erythrocytes but does not stimulate [9,10- 3H]oleic acid incorporation into phosphatidylethanolamine. Under these conditions oxidation of erythrocyte glutathione and formation of malonyldialdehyde still occur. These results indicate that membrane phospholipid fatty acid turnover is altered under conditions where peroxidation of membrane phospholipid fatty acids occurs and suggest that the oxidation state of hemoglobin influences this response.