Changes in the lipid composition of erythrocytes during prolonged fasting in lizard (Tropidurus torquatos) and rat (Rattus norvegicus)

During prolonged fasting in lizard and rat, plasma levels of unesterified cholesterol (UC) and phospholipids (TPL) decreased and there were reductions and increases, respectively, in the molar ratios of lecithin (PC) to sphingomyelin (SPH) and UC to TPL. Plasma lecithin: cholesterol acyltransferase (LCATase) activity in lizard and rat plasma was reduced during prolonged fasting. Erythrocyte lipid composition for fasted animals was also characterized by a reduction in the molar ratio PC/SPH and an increase in UC/TPL, and in both species there were positive correlations between these molar ratios in red cells and those in plasma. In both species these were changes in the morphology of the erythrocytes, and those from fasted rats showed alterations in osmotic fragility and permeability which correlated with alterations in lipid composition. These results suggest that changes in plasma lipoprotein lipid composition, linked to reduced LCATase activity, may cause similar alterations in the lipid composition of red cell membranes leading to altered membrane properties.     

The mode of action of some antibiotics on red blood cell membranes

Data are presented on the interaction of gramicidin, primycin and valinomycin with red blood cell membranes and compared with those obtained for artificial lipid bilayer membranes. The channel forming antibiotics gramicidin and primycin show specific kinetic behaviour in living cell membranes. It could be shown that the penetration of these antibiotics into the red blood cell membrane is a cooperative process resulting in the occurrence of aggregates in the lipid lattice of the membrane.     

Protective effect of Montilla-Moriles appellation red wine on oxidative stress induced by streptozotocin in the rat

This study evaluated the protective effect of Montilla-Moriles appellation red wine (Cordoba, Spain) on oxidative stress, course and intensity of symptoms in experimental diabetes induced by the injection of streptozotocin in male Wistar rats. The rats were injected with a single dose of streptozotocin (60 mg/kg i.p.) and given water and red wine separately. After 4 weeks of treatment, blood samples were obtained to determine sugar and fructosamine concentrations in blood plasma, serum insulin concentration, and percentage of glycosylated hemoglobin in blood. The kidney, liver, and pancreas were removed to determine lipid peroxidation levels, reduced glutathione content, and antioxidative enzyme activity. A significant increase of glucose concentration in urine was found in the rats after injecting the streptozotocin. The administration of red wine before streptozotocin elevated reduced glutathione content and antioxidative enzyme activity, while lowering the lipid peroxidation level. Moreover, the red wine induced decreased levels of glycemia, plasma fructosamine and percentage of glycosylated hemoglobin, while increasing levels of insulin. These data suggest that red wine has a protective effect against oxidative stress and diabetes induced by streptozotocin.     

The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability

The bovine seminal plasma protein PDC-109 exerts an essential influence on the sperm cell plasma membrane during capacitation. However, by any mechanism, it has to be ensured that this function of the protein on sperm cells is not initiated too early, that is, upon ejaculation when PDC-109 and sperm cells come into first contact, but rather at later stages of sperm genesis in the female genital tract. To answer the question of whether changes of the bovine sperm lipid composition can modulate the effect of PDC-109 on sperm membranes, we have investigated the influence of PDC-109 on the integrity of (i) differently composed lipid vesicles and of (ii) membranes from human red blood cells and bovine spermatozoa. PDC-109 most effectively disturbed lipid membranes composed of choline-containing phospholipids and in the absence of cholesterol. The impact of the protein on lipid vesicles was attenuated in the presence of cholesterol or of noncholine-containing phospholipids, such as phosphatidylethanolamine or phosphatidylserine. An extraction of cholesterol from lipid or biological membranes using methyl-beta-cyclodextrin caused an increased membrane perturbation by PDC-109. Our results argue for a oppositional effect of PDC-109 during sperm cell genesis. We hypothesize that the lipid composition of ejaculated bull sperm cells allows a binding of PDC-109 without leading to an impairment of the plasma membrane. At later stages of sperm cell genesis upon release of cholesterol from sperm membranes, PDC-109 triggers a destabilization of the cells.     

Prooxidant and antioxidant effects of ascorbate on tBuOOH-induced erythrocyte membrane damage

1. t-Butylhydroperoxide (tBuOOH) a lipoperoxide analog, causes rapid and considerable sulphydryl (SH) oxidation but almost no lipid peroxidation in red blood cell membranes (ghosts) containing no detectable haemoglobin. 2. tBuOOH, in the presence of ascorbate, produces significant lipid peroxidation the level of which is proportional to the ascorbate concentration. The initiation of lipid peroxidation is thought to occur by the reactive tBuO (butoxyl) species via the reductive decomposition of tBuOOH by ascorbate. 3. Ascorbate protects ghost membranes from the tBuOOH-induced SH oxidation in a dose-dependent fashion. 4. There is no parallelism between lipid peroxidation and SH oxidation in these systems. This suggests that the two processes occur independently of each other. 5. These findings indicate that, simultaneously, ascorbate can have both a protective and a prooxidant action in different membrane components under the same oxidative stress.     

Structural-functional status of the cardiomyocyte cell membrane under experimental pathology

The results of research conducted on the cardiomyocytes plasma membranes structural and functional state under the experimental stress and atherosclerosis are displayed in this article. These experimental pathology is determined to be accompanied by some stereotypic quantitative and qualitative modifications occurred in the lipid matrix of the cardiomyocytes plasma membranes--increase of cholesterol content, decrease of phospholipids, accumulation of lisophospholipids and fatty acid. There are demonstrated results that the experimental stress has an atherogenic effect on the plasma membranes of cells by imputting the cholesterol into the membrane even in the intact animals with normal lipid metabolism. All these modifications are also accompanied by the activation of free-radical oxidation. All these changes are capable to lie in the basis of physical and chemical properties mechanism modification of membranes: modification of lipid matrix, change of viscosity, ion-transport properties of cardiomyocytes membranes, oppression of Na+, K(+)-ATPase activity.     

Evaluation of Antioxidants,Protein, and Lipid Oxidation Products in Blood from Sporadic Amiotrophic Lateral Sclerosis Patients

Several parameters indicators of oxidative stress were evaluated in blood from individuals with the sporadic form of amiotrophic lateral sclerosis (SALS) and compared to healthy controls. Plasma levels of 2-thiobarbituric-reactive substances (TBARS), products of lipid peroxidation, were significantly higher (p < 0.03) in the SALS patients compared to controls. The concentration of plasma antioxidants (-tocopherol, -carotene, ubiquinol-10 and glutathione) and the activity of red blood cell CuZn superoxide dismutase were not significantly different between the groups. The ratio TBARS/-tocopherol was 47% higher in the SALS individuals than in controls. Protein thiols and protein-associated carbonyls in red blood cell membranes and supernates were similar for both groups. A positive correlation (r² = 0.91) was found between the concentration of protein-associated carbonyls in red blood cells and the onset of clinical symptoms. These findings are in agreement with several reports showing higher levels of oxidative damage to cell components in ALS.     

Lipid peroxidation and antioxidant systems in the blood of young rats subjected to chronic fluoride toxicity

Wistar albino rats were exposed to 30 or 100 ppm fluoride in drinking water during their fetal, weanling and post-weaning stages of life up to puberty. Extent of lipid peroxidation and response of the antioxidant systems in red blood cells and plasma to prolonged fluoride exposure were assessed in these rats in comparison to the control rats fed with permissible level (0.5 ppm) of fluoride. Rats treated with 100 ppm fluoride showed enhanced lipid peroxidation as evidenced by elevated malondialdehyde (MDA) levels in red blood cells but, 30 ppm fluoride did not cause any appreciable change in RBC MDA level. 30 ppm fluoride-intake resulted in increased levels of total and reduced glutathione in red blood cells and ascorbic acid in plasma while 100 ppm fluoride resulted in decreases in these levels. The activity of RBC glutathione peroxidase was elevated in both the fluoride-treated groups, more pronounced increase was seen with 100 ppm. Reduced to total glutathione ratio in RBC and uric acid levels in plasma decreased in both the groups. RBC superoxide dismutase activity decreased significantly on high-fluoride treatment. These results suggest that long-term high-fluoride intake at the early developing stages of life enhances oxidative stress in the blood, thereby disturbing the antioxidant defense of rats. Increased oxidative stress could be one of the mediating factors in the pathogenesis of toxic manifestations of fluoride.     

Separation and Partial Characterization Of Rat Liver Cell Membrane Components

The objectives of this research were to separate and partially characterize the components of rat liver cell plasma membranes. The plasma membranes of liver cells obtained from normal Wistar male rats were isolated using density gradients. The purified membrane was then solubilized in phenol-urea-acetic acid and gel electrophoresis was performed on the solubilized membrane components. Samples of the eluate collected following preparative gel electrophoresis were phosphorus positive-ninhydrin positive; phosphorus negative-ninhydrin positive; phosphorus po6itive-ninhydrin negative; and phosphorus negative-ninhydrin negative. The amino acid analyses showed that the proteins of the separated components were different and that no one component contained more than 6% of the total membrane protein. Liver cell plasma membranes contain components that differ from those of red blood cell plasma membranes. These components can be further divided into subgroups of proteins, i.e., phospholipoproteins and protein alone. The concept of a group of proteins composing the membrane is extended to one of various subgroups on the basis of their different affinity for lipid.     

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.     

Correlation between immuno-gold labels and activities of the cytochrome-c oxidase (aa3-type) in membranes of salt stressed cyanobactria

Abstract Anacystis nidulans ( Synechococcus PCC6301) and Synechocystis PCC6803 were grown photoautotrophically in a turbido-statically operated chemostat at a constant cell concentration of 2.0±0.3 μ l packed cell mass per ml in the presence of elevated NaCl concentrations up to 0.5 M ('salt stress'). The impact of salt stress on ccytochrome- c oxidase (EC 1.9.3.1) was` studied on isolated and purified membranes, and by immuno-gold labeling of thin-sectioned whole cells ATPase activities of membranes isolated and separated from cells under varying salt stress were also measured. Anacystis and Synechocystis adapted to the presence of 0.5 M NaCl in the medium with lag phases of 2 days and 2 hours, respectively. Both isolated plasma and thylakoid membranes from salt adapted Synechocystis displayed 5- to 8-times enhanced cytcytochrome- c oxidase activities while in Anacystis the effect was restricted to the plasma membrane. In either case less than proportionately increased counts of immuno-gold labeled cytochrome- c oxidase molecules in the respective membranes were obtained, the additional increment being attributed to the increased lipid content of the membranes from salt-adapted cells, leading to increased specific activities of the enzyme compared to control cells. ATPase activity of plasma membranes from Synechocystis was far more increased than of those from Anacystis while in thylakoid membranes the differentiating effect was less pronounced. Our results are discussed in terms of distinct strategies for salt adaptation in the two cyanobacterial species whereby in Anacystis the plasma membrane-bound respiratory chain and in Synechocystis the plasma membrane-bound ATPase(s) play the major role for plasma membrane energization which, in turn, is necessary for the active exclusion of sodium from the cell interior.     

Increased oxidative stress and decreased activities of Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase in the red blood cells of the hibernating black bear

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.     

The proteomics of sickle cell disease: profiling of erythrocyte membrane proteins by 2D-DIGE and tandem mass spectrometry

Quantitative changes in the red blood cell membrane proteome in sickle cell disease were analyzed using the two-dimensional fluorescence difference gel electrophoresis 2D-DIGE technique. From over 500 analyzed two-dimensional gel spots, we found 49 protein gel spots whose content in sickle cell membranes were changed by at least 2.5-fold as compared to control cells. In 38 cases we observed an increase and in 11 cases a decrease in content in the sickle cell membranes. The proteins of interest were identified by in-gel tryptic digestion followed by liquid chromatography in line with tandem mass spectrometry. From 38 analyzed gel spots, we identified 44 protein forms representing different modifications of 22 original protein sequences. The majority of the identified proteins fall into small groups of related proteins of the following five categories: actin accessory proteins--four proteins, components of lipid rafts--two proteins, scavengers of oxygen radicals--two proteins, protein repair participants--six proteins, and protein turnover components--three proteins. The number of proteins whose content in sickle RBC membrane is decreased is noticeably smaller, and most are either components of lipid rafts or actin accessory proteins. Elevated content of protein repair participants as well as oxygen radical scavengers may reflect the increased oxidative stress observed in sickle cells.     

Effect of Ascorbate on Red Blood Cell Lipid Peroxidation

The present study investigates the effect of ascorbate on red cell lipid peroxidation. At a concentration between 0.2 mmol-20 mmol/l ascorbic acid reduces hydrogen peroxide-induced red blood cell lipid peroxidation resulting in a marked decrease in ethane and pentane production as well as in haemolysis. Ascorbic acid also shows an antioxidant effect on chelated iron-catalyzed hydrogen peroxide-induced peroxidation of erythrocyte membranes. At a concentration of 10 mmol/l ascorbic acid totally inhibits oxidative break-down of polyunsaturated fatty acids by radicals originating from hydrogen peroxide.

Our results indicate that ascorbate at the chosen concentration has an antioxidant effect on red blood cell lipid peroxidation.     

Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes)

Vesicles are released during the in vitro culture of sheep reticulocytes which can be harvested by centrifugation at 100,000 X g for 90 min. These vesicles contain a number of activities, characteristic of the reticulocyte plasma membrane, which are known to diminish or disappear upon reticulocyte maturation. The activities include acetylcholinesterase, cytochalasin B binding (glucose transporter) nucleoside binding (i.e. nucleoside transporter), Na+-independent amino acid transport, and the transferrin receptor. Enzymes of cytosolic origin are not detectable or are present at low activity in the vesicles. Cultures of whole blood, mature red cells, or white cells do not yield comparable levels of these activities, supporting the conclusion that the activities arise from the reticulocytes. In addition, the lipid composition of the vesicles shows the high sphingomyelin content characteristic of sheep red cell plasma membranes, but not white cell or platelet membranes, also consistent with the conclusion that the vesicles are of reticulocyte origin. It is suggested that vesicle externalization may be a mechanism for shedding of specific membrane functions which are known to diminish during maturation of reticulocytes to erythrocytes.     

Induction of lipid peroxidation in the erythrocytes during cholesterol oxidation catalysed by cholesterol oxidase

Using the chemiluminescence technique to assay the activity of cholesterol oxidase it has been shown that enzymic oxidation of cholesterol to cholest-4-en-3-one red cell membranes is accompanied by accumulation of lipid peroxidation products--malonyl dialdehyde (MDA). The amount of MDA formed was dependent on the amount of cholesterol oxidized. The free radical scavenger 4-methyl-2,6-ditretbutylphenol, the transition metal chelator EDTA and catalase inhibited lipid peroxidation in red blood cells. The participation of OH radicals in the initiation of lipid peroxidation in red cell membranes in the course of cholesterol oxidation is discussed.     

Effect of the endothelial surface layer on transmission of fluid shear stress to endothelial cells

Responses of vascular endothelial cells to mechanical shear stresses resulting from blood flow are involved in regulation of blood flow, in structural adaptation of vessels, and in vascular disease. Interior surfaces of blood vessels are lined with a layer of bound or adsorbed macromolecules, known as the endothelial surface layer (ESL). In vivo investigations have shown that this layer has a width of order 1 microm, that it substantially impedes plasma flow, and that it excludes flowing red blood cells. Here, the effect of the ESL on transmission of shear stress to endothelial cells is examined using a theoretical model. The layer is assumed to consist of a matrix of molecular chains extending from the surface, held in tension by a slight increase in colloid osmotic pressure relative to that in free-flowing plasma. It is shown that, under physiological conditions, shear stress is transmitted to the endothelial surface almost entirely by the matrix, and fluid shear stresses on endothelial cell membranes are very small. Rapid fluctuations in shear stress are strongly attenuated by the layer. The ESL may therefore play an important role in sensing of shear stress by endothelial cells.     

Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form HII-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Galphaq/11 protein and hence, signal transduction.     

Isolation of plasma membranes from the bovine retinal pigment epithelium

Retinal pigment epithelium plasma membranes have been isolated by differential and density gradient centrifugation of glass-bead-bound, collagenase-treated cells. Electron microscopic evidence indicates that the glass-bead-bound cells were devoid of red blood cells, rod outer segments and other ocular cell contaminants. The plasma membranes were recovered in 4–6 μg/eye yields and purified 10-fold by 5′-nucleotidase and alkaline phosphodiesterase 1, and 6.5-fold by (Na⁺ + K⁺)-ATPase. Plasma membrane purity as measured by covalent labeling of the epithelial cell plasma membrane proteins with p-(diazonium) benzene[³²S]sulfonic acid was 8–19-fold. In purified plasma membranes contamination by mitochondria was undetectable and lysosomal contamination reduced 100-fold, while endoplasmic reticulum was 2-fold enriched. SDS-polyacrylamide gel electrophoresis of the plasma membrane proteins revealed 23–26 major bands by Coomassie blue staining and 12–16 major bands by radioactive labeling. The plasma membranes exhibited a 3-fold lower concentration of docosahexaenoic acid, a 3-fold higher cholesterol/phosphate ratio, and were 10-fold enriched in cholesterol per μg protein when compared to the whole cell fraction. Retinal epithelial plasma membranes contain an average of 1 mol cholesterol per mol of lipid phosphorus, a high palmitic acid concentration (39 mol%) and a low concentration of docosahexaenoic acid (2 mol%). The lipid profile of the retinal pigment epithelial plasma membranes indicates that they are typical of plasma membranes from many other cell types and that they appear to be less fluid than total rod outer segment membranes.     

Biochemical and immunological changes on oral glutamate feeding in male albino rats

 High altitude stress leads to lipid peroxidation and free radical formation which results in cell membrane damage in organs and tissues, and associated mountain diseases. This paper discusses the changes in biochemical parameters and antibody response on feeding glutamate to male albino Sprague Dawley rats under hypoxic stress. Exposure of rats to simulated hypoxia at 7576 m, for 6 h daily for 5 consecutive days, in an animal decompression chamber at 32±2° C resulted in an increase in plasma malondialdehyde level with a concomitant decrease in blood glutathione (reduced) level. Supplementation of glutamate orally at an optimal dose (27 mg/kg body weight) in male albino rats under hypoxia enhanced glutathione level and decreased malondialdehyde concentration significantly. Glutamate feeding improved total plasma protein and glucose levels under hypoxia. The activities of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) and the urea level remained elevated on glutamate supplementation under hypoxia. Glutamate supplementation increased the humoral response against sheep red blood cells (antibody titre). These results indicate a possible utility of glutamate in the amelioration of hypoxia-induced oxidative stress. Received: 23 March 1998 / Accepted: 19 October 1998