Alterations in the physicochemical properties of renal cortical membranes in rats with experimental cirrhosis of the liver.

Changes in the major component of renal cortical membranes as well as membrane fluidity and Na+, K+, ATPase activity have been studied in membranes from the renal cortex of rats with experimental liver cirrhosis, which show renal sodium and water retention, and in normal animals. Rats with cirrhosis of the liver show a decrease in cholesterol, phospholipid and protein content, without changes in cholesterol/phospholipid molar ratio. In addition there is a small decrease in 14:0 and 18:2 and an increase in 20:4 content, without differences in unsaturation degree. Membrane fluidity was decreased in renal membranes from cirrhotic rats when compared with normal ones. Na+, K+, ATPase activity was higher in cirrhotic than in normal renal membranes could be related with the changes in renal water and electrolyte changes shown by cirrhotic rats.     

Differential response of chick liver and brain membranes to short ethanol treatment

The effect of 60 hr ethanol ingestion on lipid composition of liver and brain membranes from 2-day-old chicks was investigated. Analysis of hepatic membrane cholesterol shows that ethanol induced a slight increase in microsomes exclusively due to free cholesterol while mitochondria was not affected. In brain, both fractions showed a clear increase in their cholesterol content, while a high decrease was observed in myelin. Free cholesterol was also the main responsible for the changes found in brain. The ethanol-treated animals showed an alteration in their phospholipid composition exclusively in brain microsomes and myelin. Despite all these changes, the values of cholesterol/phospholipid molar ratio in both liver and brain membranes remained unaltered after short ethanol treatment. Our results indicate that neonatal chick brain membranes appears to be especially sensitive to the presence of ethanol.     

The effect of clofibrate on the phospholipid composition of the peroxisomal membranes in mouse liver

Membranes were prepared from peroxisomes which had been isolated from the livers of normal mice and from mice treated with clofibrate (a hypolipidemic drug and peroxisome proliferator). Phospholipid analysis of these membranes revealed that clofibrate treatment resulted in a decrease in the membrane content of phosphatidylcholine, the most abundant phospholipid, and a concomitant increase in the amount of lysophosphatidylcholine, this latter component reaching a level of almost 6% of the total membrane phospholipid. The concentrations of other phospholipids in these membranes were not significantly altered. The parallel analysis of microsomal membranes demonstrated an analogous increase in the level of lysophosphatidylcholine following clofibrate treatment. In control experiments with microsomal membranes employing quinacrine, an inhibitor of phospholipase A2, the increased lysophosphatidylcholine concentration was still observed in clofibrate-treated animals. As well, a decrease in the proportion of microsomal phosphatidylcholine with clofibrate treatment was seen when quinacrine was used. Fatty acid analysis of the phosphatidylcholines from peroxisomal membranes showed some minor changes, including an increase in one component tentatively identified as docosahexaenoic acid, in clofibrate-treated animals. Overall, these data demonstrate that clofibrate causes a marked perturbation of the phospholipid composition of peroxisomal membranes, and are interpreted as indicating that the main site of action of the drug is the deacylation-reacylation cycle between phosphatidylcholine and lysophosphatidylcholine.     

Changes in the phospholipid and fatty acid composition in normal erythrocytes from sheep of different ages. Aminophospholipid organization in the membrane bilayer

Development and aging processes in mammals are associated with changes in several physiological parameters. The aim of the present study was to investigate the changes in erythrocyte lipid composition during sheep development. In all the age groups studied, cholesterol/phospholipid ratios remained constant, at close to unity, while phospholipid patterns (sphingomyelin: 45-51%, phosphatidylethanolamine: 26-33%, phosphatidylserine: 13-19% and phosphatidylcholine: less than 2%) changed during development, with a statistically significant decrease (P less than 0.01) in phosphatidylserine and an increase in sphingomyelin content. These data suggest an increase in the rigidity of the erythrocyte lipid bilayer in adult sheep when compared with 1-month-old animals due to a decrease in the phosphatidylserine/sphingomyelin ratio. Fatty acid profiles consistently showed 5 main acids: oleic (52-54%), stearic (17-18%), linoleic (9-15%), palmitic (8.5-11%) and arachidonic acid (2-3%), mainly with significant variations (P less than 0.01) in palmitic and linoleic acid contents, respectively reaching the highest and lowest percentages in the youngest sheep. However, the developmental process seems to have no influence on the aminophospholipid topology of erythrocytes. This study suggests that the animals' developmental process has a marked effect on the lipid composition of erythrocyte membranes, which could affect cell functions.     

Biological properties of the mechanism of "marine" phospholipids containing omega-3 fatty acids

The Laboratory mainly deals with the development of biologic technologies for producing physiologically active lipid-protein-nature compounds from marine organisms containing omega-3 fatty acids (that are membrane components) with further evaluation of their action both in the normal state and in some simulated pathologic states as well as with creation of new preparations for application in medicine and agriculture on their basis. As a result of the experiments performed, a technology for producing two biologic preparations, namely, surface active compounds (phospholipids) and a nucleopeptid-lipid complex, exhibiting a specific action, was developed. The phospholipid complex, being surface-active in composition, was characterized as a complex possessing some surfactant-type properties and displaying an antioxidant and a membrane-stabilizing effect. On the basis of the complex including marine phospholipids with omega-3 fatty acids, the preparation "Phylomek", which is the concentrate of essential marine phospholipids, and the preparation "Morephyl", which is a marine agent with surfactant-type effect, intended for animals and poultry, were created. The ingredients of the nucleopeptid-lipid complex were identified, and its effect on the increase of testosterone levels in the blood of old and sick animals was determined. A stimulating agent of genital hormones secretion was recommended for application in the geriatrics in the case of genital function disturbance and presenilation. A fraction similar in chemical content and specific activity, found in the velvet antlers, was used as the base of the biologic preparation "Pantheron". The natural complexes of marine phospholipids were shown to be able to change the composition of lipids of membranes, but the intensity of these changes differed in cells differing in function. In the study of the biochemical mechanism of correction of disturbances in the cell membranes under an oxidative stress, the interrelation between the composition of lipids of membranes, their oxidation, and the content of natural antioxidants was determined. The reparative effect of marine phospholipids on the cell membranes under progressing pathology, caused by the restoration of the composition of phospholipids, by increases in the activities of antioxidant enzymes (SOD, catalase), and by a decrease of the accumulation of LPO products, was established. Under interaction of marine phospholipids and alpha-tocopherol, synergism was noticed, the antioxidant potential of the investigated substances and their membrane-stabilizing effect increased. The phospholipids with various residues of PUFA in the molecule were found to affect the inhibition and oxidation processes, as well as the modelling of lipid membranes. This is especially true for arachidonic and docosahexaenoic acids, the ratio of which changes under the oxidative stress. At administration of phospholipids omega-3, their ratio decreases due a decrease in the level of unetherificated PUFA. The main changes of the PUFA were found to occur in phosphatidylethanolamine isolated from microsomes. The particular role of phosphatidylethanolamine and arachidonic acid in the reparation of membranes under the action of phospholipids PUFA omega-3 and alpha-tocopherol was noted.     

Relationship between structural and ionic changes in cardiosclerosis

The parallelism between hypoxic changes in the myocardium revealed by Lie's technique and by fluorescence microscopy and the ionic changes (Na+, K+, Ca2+) was studied in 51 autopsy cases. The Lie technique was positive in 52.6 per cent of the cases of cardiosclerosis with myocardial infarction and in 15 per cent of the cases of cardiosclerosis without infarction, while fluorescent areas were found in all cases of infarction and in 35 per cent of cardiosclerosis cases without infarction. Na+ and K+ were decreased and Ca2+ ions were increased in both ventricles and in both sexes, both in infarctions and in cardiosclerosis cases without infarction. Ionic changes are more probably related to the intensity of cardiosclerosis rather than to age or sex.     

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl2 from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which incrased in phosphatidylethanolamine content from 39% (low Na+) to 48% (high Na+) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca2+-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na+-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca2+ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.     

Change of synaptic membrane lipid composition and fluidity by chronic administration of lithium

The effect of chronic administration of lithium salts on the lipid composition and physical properties of the synaptosomal plasma membrane was examined in rat brain. The effect of lithium treatment has been studied on the fluorescence polarization of synaptosomal plasma membrane and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from lithium-treated animals. Altered DPH polarization was due to a decrease in the order parameter of the probe. The lithium-treatment also changed the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS), a probe that binds to the polar head group of the phospholipids and to proteins on the membrane surface. Synaptic plasma membranes from treated rats presented no significant changes on the cholesterol-to-phospholipid ratio, although the phospholipid class distribution was altered and the membrane phospholipid unsaturation increased. In summary, the neural plasma membranes became disorder after chronic lithium administration at therapeutic levels. This structural change may be due to changes in plasma membrane phospholipid distribution and to the degree of unsaturation of phospholipid fatty acids.     

Structural and functional changes in erythrocyte membranes in experimental atherosclerosis

Structural and functional characteristics of erythrocyte membranes were studied in rabbits with experimental atherosclerosis. In animals with single lipid spots in the aorta, a significant rise of the plasma cholesterol level was associated with the increased cholesterol/phospholipid (CS/PL) ratio and diminished activity of erythrocyte membrane Na+, K+-ATPase. EPMR spin probe data point to changes in structural membrane characteristics--an increase in order parameter for fatty acid chains of lipids and expansion of the temperature interval of the transition phase in the membranes. In rabbits with total aorta injury, a further increase both in the plasma cholesterol concentration and in the CS/PL ratio as well as in structural changes in erythrocyte membranes does not lead to another decrease in the enzymatic activity. In aorta homogenates of the experimental animals, the activity of Na+, K+-ATPase correlated with that in the erythrocyte membrane. This suggests the existence of similar chemical and structural changes in aorta cell membranes. The data may provide an indirect evidence in favour of the hypothesis of the involvement of smooth muscle cells and membrane enzymatic activity alterations in atherosclerosis.     

Structural state of rat liver mitochondrial and microsomal membranes in aging and after treatment with thyroxine

Rotation mobility of stearic acid spin labelling in mitochondria and microsomes of 24 months old rats is shown to decrease in comparison with that of 1 month rats. This decrease correlates with an increase in the free cholesterol content in membranes. The phospholipid composition is practically unchanged. With age the osmoresistance of organelles decreases and the cryoresistance increases, supporting indirectly the existence of structural changes in membranes with ageing. The most pronounced activation of NADPH-cytochrome C reductase in the microsomal membrane after the treatment with triton X-100 is observed in case of old animals. The thyroxine treatment of old rats causes a "rejuvenative" effect on the structural state of mitochondrial and microsomal membranes, evidencing for the important role of hormonal regulation in mechanisms of their age rearrangement.     

Some studies on lipid peroxidation in monomolecular and bimolecular lipid films

Summary Hydrogen peroxide generated from dissolved oxygen through the alloxandialuric acid cycle affected both the permeability and the stability of lipid bilayer membranes. The permeability of the artificial membranes varied directly with the hydrogen peroxide concentration. Membrane stability varied inversely with the hydrogen peroxide concentration. Bilayers formed from solutions containing both phospholipid and the antioxidant vitamin E were less permeable and more stable in the presence of hydrogen peroxide than bilayers generated from solutions containing phospholipid alone. Peroxidation of phospholipid monolayers caused first an expansion of the films presumably through the introduction of peroxide groups. Further oxidation of phospholipid monolayers led to contraction of the films presumably through the formation of water-soluble products. The results of the monolayer studies and a consideration of the possible kinetics for the peroxidation reaction sequence have been used to explain the changes in the permeability and the stability of lipid bilayer membranes. Our data suggest that oxidation of lipid in biological membranes may first increase membrane permeability and then decrease membrane stability.     

Changes in the microviscosity of lipid bilayer membranes of various malignant cells and tumor transplantability

It is shown that cholesterol incorporation into the membranes of Zajdel hepatoma cells, lymphoblast leukemia cells L1210 and into those of ovary tumour causes an increase in the membrane phospholipid bilayer microviscosity measured by pyrene as fluorescent probe. The increase in the membrane lipid microviscosity resulted in a decrease in the activity of Na,K-ATPase and 5-nucleotidase of the tumour cells. After the injection of tumour cells with an increase of cholesterol/phospholipid ratio we observed an increase of the life-span of experimental animals as compared to the control groups.     

Reparation of hepatocyte mitochondrial membranes using phosphatidylcholine liposomes]

The influence of multibilayer phosphatidylcholine liposomes on the properties of rat hepatocyte mitochondria membranes damages caused by hepatotropic toxin--CCl4 was investigated. Alterations of the membrane structure were estimated by the decrease in phospholipid/protein ratio /by 33%/ and via changes of quantitative and qualitative composition of phospholipid components. The possibility of reparation of the hepatocytes mitochondria damaged membrane by egg yolk phosphatidylcholine liposomes is shown. Phosphatidylcholine reparative effect on the damaged membranes is revealed in quantitative phospholipid fractions redistribution.     

Changes in the phospholipid composition of mitochondria during heat incubation

Incubation of liver mitochondria at 37 degrees C causes changes in the phospholipid composition, such as the decrease in the levels of major phospholipids (e.g. phosphatidylcholine, phosphatidylethanolamine, cardiolipin) and their lysoderivatives as well as an increase in the levels of phosphatidic and lysophosphatidic acids. Similar changes in the phospholipid composition are observed upon heat incubation of mitochondrial fragments ("ghosts", inner and outer mitochondrial membranes). Ca2+ accelerate the heat-induced changes in the phospholipid levels resulting from heat incubation, whereas EGTA, in contrast, decelerates them. The role of an endogenous system of lipolytic enzymes in the observed conversions of mitochondrial phospholipids is discussed.     

Modulation of the lipid composition of boar sperm plasma membranes during an acrosome reaction in vitro

An in vitro acrosome-like reaction was induced in spermatozoa from the boar cauda epididymis by incubation in Tyrode's solution containing 1 mg/ml fatty acid-free bovine serum albumin. Plasma membranes were isolated from the spermatozoa at different times during the incubation and analyzed for their lipid composition. The total lipid, phospholipid, and glycolipid content of the membranes did not change during the acrosome-like reaction, whereas the amount of diacylglycerols and free fatty acids increased. Within the phospholipid class, a decrease of the inositol phospholipid and and sphingomyelin content was observed, whereas the other phospholipids of the plasma membranes did not decrease significantly after 2 h of incubation. Changes in the sterol composition of the membranes were also observed. The onset of the lipid changes was correlated with the uptake of extracellular calcium by the spermatozoa. These results for the lipid changes in isolated sperm plasma membranes during an in vitro acrosome reaction provide the first direct evidence that a modulation of the plasma membrane lipid composition is involved in an acrosome-like reaction of mammalian spermatozoa.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction

After force-feeding a protein-free diet to male rats for 5-7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.     

Induction of Ca-independent PLA(2) and conservation of plasmalogen polyunsaturated fatty acids in diabetic heart

Diabetes-induced changes in phospholipase A(2) (PLA(2)) activity have been measured in several tissues but are undefined in diabetic myocardium. We measured ventricular PLA(2) activity in control, streptozotocin-induced diabetic, and insulin-treated diabetic rats and characterized myocardial phospholipids to determine whether diabetes altered myocardial phospholipid metabolism. Increased membrane-associated Ca(2+)-independent PLA(2) (iPLA(2)) activity was observed in diabetes that was selective for arachidonylated phospholipids. Increased iPLA(2) activity was accompanied by an increase in choline lysophospholipids. Diabetes was associated with marked alterations in the phospholipid composition of the myocardium, characterized by decreases in esterified arachidonic and docosahexaenoic acids and increases in linoleic acid. The decrease in polyunsaturated fatty acids was confined to diacylphospholipids, whereas the relative amount of these fatty acids in plasmalogens was increased. Diabetes-induced changes in PLA(2) activity, lysophospholipid production, and alterations in phospholipid composition were all reversed by insulin treatment of diabetic animals. Diabetes-induced changes in membrane phospholipid content and phospholipid hydrolysis may contribute to some of the alterations in myocardial function that are observed in diabetic patients.     

Protective effect of blackcurrant on liver cell membrane of rats intoxicated with ethanol

Chronic ethanol intoxication oxidative stress participates in the development of many diseases. Nutrition and the interaction of food nutrients with ethanol metabolism may modulate alcohol toxicity. One such compound is blackcurrant, which also has antioxidant abilities. We investigated the effect of blackcurrant as an antioxidant on the composition and electrical charge of liver cell membranes in ethanol-intoxicated rats. Qualitative and quantitative phospholipid composition and the presence of integral membrane proteins were determined by high-performance liquid chromatography. Electrophoresis was used to determine the surface charge density of the rat liver cell membranes. Ethanol intoxication is characterized by changes in cell metabolism that alter the structure and function of cell membrane components. Ethanol increased phospholipid levels and altered the level of integral proteins as determined by decreased phenylalanine, cysteine, and lysine. Ethanol significantly enhanced changes in the surface charge density of the liver cell membranes. Administration of blackcurrant to rats intoxicated with ethanol significantly protected lipids and proteins against oxidative modifications. It is possible that the beneficial effect of blackcurrant is connected with its abilities to scavenge free radicals and to chelate metal ions.     

Creatine phosphokinase activity in myocardial sarcoplasmic reticulum membranes in experimental coronary insufficiency

Experiments on rats with acute coronary blood flow restriction (intravenous injection of vasopressin in a dose of 0.5 units/kg) have shown a decrease in the activity of creatine phosphokinase (CPK) and activation of lipid peroxidation in sarcoplasmic reticulum (SPR) membranes. These changes were attended by structural abnormalities in the membrane apparatus of the SPR, primarily on the part of the lipid composition. Pretreatment (1 h before vasopressin) of the animals with the antioxidant AV-156Na averted the development in the SPR membranes of the changes described. This evidences that the rate of peroxide reactions in the myocardium might determine in many respects the metabolic and structural supply of the cell apparatus.     

The effect of choline deficiency on the outer membranes of rat liver mitochondria

The outer membranes of mitochondria prepared from the liver of rats kept 12 days on a choline-deficient diet were analyzed for changes in phospholipid and protein content. The total amount of phospholipid in the outer membranes was not affected by the deficiency. There was, however, a significant decrease in the amount of phosphatidylcholine and an increase in phosphatidylethanolamine. The alterations in the membrane phospholipids were reflected in a reduction in the fluorescence of the membrane probe, 8-anilino-1-naphthalene sulfonate. Choline deficiency also affected the protein composition of the outer membranes as judged by electrophoretic analysis; however, the activity of several enzymes which serve as markers for the outer membrane was not affected by the deficiency.