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.     

Effect of "marine" phospholipids omega-3 fatty acids on the composition of fatty acids of rat liver microsomal membrane under oxidative stress

As a result of experiments conducted the marine phospholipids preparation enriched by omega-3 fatty acids was defined to modify fatty acids content due to changes of fatty acids level change in the neutral lipids and phospholipids fractions. As well it was identified, that at the oxidative stress induced by administration of CCl4 the growth of arachidonic and docozahexaenoic acids in the neutral lipids fractions was observed if compare with the norm. At the same time, the presented fatty acids in the phospholipids fractions remained unchanged. At oxidative stress the phospholipids fraction reacts to levels of arachidonic and docozahexaenoic acids just only as a result of administrating phospholipids with omega-3 fatty acids. The most attractive is the change of correlation C20:4/C22:6--increasing at administration of CCl4 and decreasing both at phospolipids and vitamin E injection. Thus, at the oxidative stress the first reacting ones are the fatty acids of neutral lipids microsomal membranes.     

Effect of phospholipids containing omega-3 fatty acids on structural changes of microsomal lipids in cell membranes of functionally different cells

As a result of the experimental researches conducted it has been shown that administration of some normal animal marine phospholipids (PL) including in their structure omega-3 polyunsaturated fatty acids (PUFA) provides for quantitative changes of individual PL, fatty acids (FA) content and quantity in general and individual PL of liver, heart, brain and gonads microsomes. While estimating general microsomal PL fraction FA content under the action of PL omega-3 PUFA FA concentration change, unsaturation index (omega 6/omega 3) and relation of arachidonic acid to docosahexenic (AA/DHA) decrease have been identified. The decrease of AA/DHA relationship occurs due to AA and DHA quantitative changes. In the case of AA increase in some tissues there is observed the decrease of docosapentaenic acid and increase of DHA and eucosapentaenic (EPA) acidds. As a result of studying FA content in the individual PL composition it has been identified that certain PL classes characteristic for some tissues respond by changes of some certain FA. The relationship omega 6/omega 3 has been shown as decreasing in phosphatidilcholine (PC) all tissues microsomes (liver, gonads, heart, brain), in phosphatidilethanolamine (PEA) of liver and cardiac microsomes, in phosphatidilserine (PS) this relationship relationship decreases in the liver, brain and heart, for phosphatidilinositole (PI) the changes take place in liver, gonads, brain. Simultaneously, the decrease of AA/DHA relationship in the individual PL decrease of AA and increase of EPA and DHA depend on the tested tissues. The marine phospholipids might be supposed to render their effect on AA metabolism resulting in AA/DHA relationship in PEA and PS relationship displays itself as specific and depends on the tissues functions. The preference of PEA and PS use by certain tissues microsomes could be explained by their membrane protective capability.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : II. Lipids

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ~20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.     

Influence of dietary fat on the lipid composition of rat brain synaptosomal and microsomal membranes.

The modulation of rat brain microsomal and synaptosomal membrane lipid by diet fat was examined. Brain synaptosomal and microsomal membrane composition was compared for rats fed on diets containing either soya-bean oil (SBO), SBO plus choline, SBO lecithin, sunflower oil (SFO), chow or low-erucic acid rape-seed oil (LER) for 24 days. Cholesterol and phosphatidylcholine levels in both membranes were altered by diet. Diet fat also affected the microsomal content of sphingomyelin. Change in membrane phosphatidylcholine level was related to the relative balance of omega-6, omega-3 and monounsaturated fatty acids within the diets fed. The highest phosphatidylcholine levels appeared in membranes of animals fed on SBO lecithin and the lowest in those fed on LER. Microsomal membrane cholesterol and sphingomyelin content increased by feeding on SBO lecithin. In both synaptosomal and microsomal membranes a highly significant correlation was observed between membrane phosphatidylcholine and cholesterol content. The fatty acyl composition of phospholipids from both membranes also altered with diet and age. Alteration in fatty acid composition was observed in response to dietary levels of omega-6, omega-3 and monounsaturated fatty acids, but the unsaturation index of each phospholipid remained constant for all diet treatments. These changes in lipid composition suggest that dietary fat may be a significant modulator in vivo of the physicobiochemical properties of brain synaptosomal and microsomal membranes.     

Regulation of intracellular lipid metabolism by the preparation of omega-3 phospholipids from marine organisms in deficiency of essential fatty acids in rats

It has been established that a deficit of essential fatty acids (EFA) in the animal organism induces specific modifications of composition of fatty acid (FA) of general phospholipids and plasmalogenic P1 in microsomal tissue membranes with various functions and affects the activity of phospholipase A2. It has been shown that arachidonic (AA), docosapentaenoic (DPA) and docosahexaenoic (DHA) acids in the composition of general phospholipids - phosphatidylcholine (PC), phosphatidylethanolamine (PEA) and plasmalogens PC and PEA react to EFA deficit in the organism. Quantitative redistribution of AA, DPA, DHA of FA in general phospholipids and plasmalogenic microsomal membranes depending on their functions was found under EFA deficit in the organism. Deficit of DHA and plasmalogenic phospholipids evidences that the status of cell plasmalogens affects the level of PUFA at EFA deficit in the organism. AA and DHA can be a selective target for plasmalogens. The drug of omega-3 phospholipids, considerable amount of DHA and eicosapentaenoic (EPA)FA being present in their structure, increases the amount of plasmalogens and decreases the amount of AA in the brain, heart and reproductive organs. It was also found that EFA deficit in the organism favours the increase of lisoPEA, lisoPC, free FA (FFA) connected with the increase of activity of endogenic phospholippase A2 and plasmalogen-selective phospholipase A2. The omega-3 phospholipase from marine organisms at EFA defecit decreases the amount of FFA, lisophospholipids and activity of phospholipase A2 in the microsomas of the studied tissues. The drug of omega-3 phospholipids normalizes the state and functions of the brain, liver, and heart tissues, reproductive organs against a background of EFA defecit and regulates the synthesis of biologocically active metabolites of AA in the organism.     

Omega-3 and omega-6 fatty acid concentrations in red blood cell membranes relate to schizotypal traits in healthy adults

Reduced omega-3 and omega-6 fatty acids in red blood cell (RBC) membranes are often found in patients with schizophrenia. Here we investigated whether membrane concentrations of these fatty acids might vary as a function of schizotypal traits in non-psychotic individuals. Twenty-five healthy adults completed the O-LIFE schizotypal trait inventory and fatty acid composition of their venous blood samples was analysed via gas-liquid chromatography. Correlations between schizotypy measures and RBC fatty acids were examined and comparisons made between groups high and low on fatty acid measures and schizotypy scores. The omega-6 fatty acids arachidonic, adrenic and docosapentaenoic acid were directly related to positive schizotypal trait measures, as were most omega-3 fatty acids, but none were related to a negative, withdrawn form of schizotypy. Our findings of high RBC concentrations of omega-3 and omega-6 fatty acids in healthy adults with positive schizotypal traits clearly contrast with the low levels often found in schizophrenia, but are quite consistent with evidence that omega-3 fatty acids (notably EPA) can be useful in the treatment of schizophrenic illness.     

Age-dependent modifications in membrane lipids: lipid composition, fluidity and palmitoyl-CoA desaturase in Tetrahymena membranes

The membrane lipid composition of Tetrahymena pyriformis NT-I was observed to change in a manner markedly dependent on the progress of culture age. The pellicular, mitochondrial and microsomal membranes were isolated from cell harvested at various growth phases (I, early exponential; II, mid-exponential; III, late exponential; IV, early stationary; V, late stationary) and their lipid composition was analyzed by thin-layer and gas-liquid chromatography. Although the phospholipid composition varied somewhat among membrane fractions, the most general age-dependent alteration was a considerable decrease in the content of phosphatidylethanolamine accompanied by a small increase in phosphatidylcholine. The 2-aminoethylphosphonolipid, enriched in the surface membrane pellicle, did not undergo a consistent change. As for fatty acid composition the most notable variation occurred in unsaturated fatty acids; a great increase in oleic and linoleic acids and a compensatory decrease in palmitoleic acid. This resulted in an augmented unsaturation of the overall phospholipid fatty acid profile of the aged membranes. The age-associated drastic decline in the palmitoleic acid content in membrane phospholipids could be accounted for by the markedly lowered activity of palmitoyl-CoA desaturase. The microsomes from the early exponential phase cells possess a 4-fold higher activity of the desaturase as compared to that of the late stationary phase microsomes. The decreased desaturase activity associated with the culture age was also reflected in the corresponding decrease in the conversion rate of [14C]palmitate to [14C]palmitoleate in cells labelled in vivo. The ESR spectra of the spin-labeled phospholipids extracted from the pellicular and microsomal membranes have led to the suggestion that these types of membrane would become more fluid with the age of growth.     

Lipid composition and protein profiles of outer and inner membranes from pig heart mitochondria. Comparison with microsomes.

1. Mitochondria, inner and outer mitochondrial membranes and microsomes were isolated and purified from pig heart. Their lipid composition and protein components were studied. 2. The fatty acid distribution in the main phospholipids seemed specific rather of a given phospholipid and not of one type of membrane. 3. Inner mitochondrial membranes were characterized by a high content in cardiolipin and a very low level of triglycerides together with a high degree of unsaturation and C18 acids. Gel electrophoresis revealed 13 different polypeptide subunits of which 5 were major ranging in molecular weights from 10000 to 215000. 4. In outer mitochondrial membranes, total lipid, phosphatidylcholine, phosphatidylinositol, plasmologen and triglyceride contents were much higher than in inner membranes. Fatty acids of phospholipids were mostly saturated and the polypeptide pattern showed 12 components, of which 4 were major of mol. wt 75000, 60000, 20000 and below 10000. 5. Compared to outer membrane, microsomes exhibited a much higher cholesterol content and markedly different protein profiles. They contained significant amounts of cardiolipin and phosphatidylserine, this latter phospholipid being exclusively located in microsomes. However odd similarities were observed in some lipid components of microsomes and inner mitochondrial membranes, but fatty acids were more saturated in microsomes and electrophoretic profiles of protein components appeared very different and revealed components of high mol. wt.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.     

Morphological and Physical Analysis of Natural Phospholipids-Based Biomembranes

Background

Liposomes are currently an important part of biological, pharmaceutical, medical and nutritional research, as they are considered to be among the most effective carriers for the introduction of various types of bioactive agents into target cells.

Scope of Review

In this work, we study the lipid organization and mechanical properties of biomembranes made of marine and plant phospholipids. Membranes based on phospholipids extracted from rapeseed and salmon are studied in the form of liposome and as supported lipid bilayer. Dioleylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) are used as references to determine the lipid organization of marine and plant phospholipid based membranes. Atomic force microscopy (AFM) imaging and force spectroscopy measurements are performed to investigate the membranes'' topography at the micrometer scale and to determine their mechanical properties.

Major Conclusions

The mechanical properties of the membranes are correlated to the fatty acid composition, the morphology, the electrophoretic mobility and the membrane fluidity. Thus, soft and homogeneous mechanical properties are evidenced for salmon phospholipids membrane containing various polyunsaturated fatty acids. Besides, phase segregation in rapeseed membrane and more important mechanical properties were emphasized for this type of membranes by contrast to the marine phospholipids based membranes.

General Significance

This paper provides new information on the nanomechanical and morphological properties of membrane in form of liposome by AFM. The originality of this work is to characterize the physico-chemical properties of the nanoliposome from the natural sources containing various fatty acids and polar head.     

Polyunsaturated fats,membrane lipids and animal longevity

Fatty acids are essential for life because they are essential components of cellular membranes. Lower animals can synthesize all four classes of fatty acids from non-lipid sources, but both omega-6 and omega-3 cannot be synthesized de novo by ‘higher’ animals and are therefore essential components of their diet. The relationship between normal variation in diet fatty acid composition and membrane fatty acid composition is little investigated. Studies in the rat show that, with respect to the general classes of fatty acids (saturated, monounsaturated and polyunsaturated) membrane fatty acid composition is homeostatically regulated despite diet variation. This is not the case for fatty acid composition of storage lipids, which responds to diet variation. Polyunsaturated fatty acids are important determinants of physical and chemical properties of membranes. They are the substrates for lipid peroxidation and it is possible to calculate a peroxidation index (PI) for a particular membrane composition. Membrane PI appears to be homeostatically regulated with respect to diet PI. Membrane fatty acid composition varies among species and membrane PI is inversely correlated to longevity in mammals, birds, bivalve molluscs, honeybees and the nematode Caenorhabditis elegans.     

Changes in mitochondrial and microsomal lipid peroxidation and fatty acid profiles in adrenal glands, testes, and livers from alpha-tocopherol-deficient rats

Studies were done to evaluate the effects of alpha-tocopherol deficiency in rats on the fatty acid composition and sensitivity to lipid peroxidation (LP) of mitochondria and microsomes from adrenal glands, testes, and livers. In control (alpha-tocopherol-sufficient) animals, adrenal concentrations of alpha-tocopherol were approximately 10 times greater than those in livers and testes. Dietary deficiency of alpha-tocopherol for 8 weeks decreased adrenal and hepatic concentrations by 80-90% and testicular concentrations by approximately 60-70%. Incubation of testicular or hepatic mitochondria and microsomes from control rats with FeSO(4) (1.0 mM) caused a time-dependent stimulation of LP as indicated by the formation of thiobarbituric acid reactive substances (TBARS); the rate of TBARS production increased in preparations from alpha-tocopherol-deficient animals. TBARS formation was not demonstrable in adrenal mitochondria or microsomes from alpha-tocopherol sufficient rats, but reached high levels in alpha-tocopherol-deficient preparations. The fatty acid composition of mitochondria and microsomes was tissue-dependent. In particular, arachidonic acid comprised approximately 40% of the total fatty acids in adrenal membranes, but only 20-25% in testes and livers. alpha-Tocopherol deficiency increased oleic acid concentrations in adrenal and hepatic mitochondria and microsomes but not in testes. In all three tissues, linoleic acid concentrations decreased by approximately 50%, but arachidonic acid levels were unaffected by alpha-tocopherol deficiency. The results indicate a close relationship between tissue sensitivity to LP in vitro and alpha-tocopherol concentrations. Nonetheless, any oxidative stress in vivo caused by alpha-tocopherol deficiency seems to spare arachidonic acid in mitochondria and microsomes but decreases linoleic acid concentrations. It is possible that because of the important physiological functions of arachidonic acid, metabolic adaptations serve to maintain membrane content during periods of oxidative stress.     

Evidence for a mechanism by which omega-3 polyunsaturated lipids may affect membrane protein function

We have calculated the lateral pressure profile from well-converged, experimentally validated, molecular dynamics simulations of hydrated lipid bilayer membranes containing highly polyunsaturated fatty acids. The three simulations, each 30 ns in length, contain omega-3 fatty acids, omega-6 fatty acids, and a mixture of omega-3 fatty acids and cholesterol and were continued from previously published simulations that demonstrated excellent agreement with a wide variety of experimental measurements. We find that the distribution of lateral stress within the hydrophobic core of the membrane is sensitively dependent on the degree of chain unsaturation and on the presence of cholesterol. Replacing omega-3 fatty acids with omega-6 chains, or incorporating cholesterol into the membrane, shifts the repulsive lateral chain pressure away from the lipid/water interface toward the bilayer interior. This may support a previously proposed mechanism by which lipid composition may affect conformational equilibrium for integral membrane proteins.     

Modulation of enzymatic activities by n-3 polyunsaturated fatty acids to support cardiovascular health

Epidemiological evidence from Greenland Eskimos and Japanese fishing villages suggests that eating fish oil and marine animals can prevent coronary heart disease. Dietary studies from various laboratories have similarly indicated that regular fish oil intake affects several humoral and cellular factors involved in atherogenesis and may prevent atherosclerosis, arrhythmia, thrombosis, cardiac hypertrophy and sudden cardiac death. The beneficial effects of fish oil are attributed to their n-3 polyunsaturated fatty acid (PUFA; also known as omega-3 fatty acids) content, particularly eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3). Dietary supplementation of DHA and EPA influences the fatty acid composition of plasma phospholipids that, in turn, may affect cardiac cell functions in vivo. Recent studies have demonstrated that long-chain omega-3 fatty acids may exert beneficial effects by affecting a wide variety of cellular signaling mechanisms. Pathways involved in calcium homeostasis in the heart may be of particular importance. L-type calcium channels, the Na+-Ca2+ exchanger and mobilization of calcium from intracellular stores are the most obvious key signaling pathways affecting the cardiovascular system; however, recent studies now suggest that other signaling pathways involving activation of phospholipases, synthesis of eicosanoids, regulation of receptor-associated enzymes and protein kinases also play very important roles in mediating n-3 PUFA effects on cardiovascular health. This review is therefore focused on the molecular targets and signaling pathways that are regulated by n-3 PUFAs in relation to their cardioprotective effects.     

Lipid composition of liver microsomes in rats fed a high monounsaturated fatty acid diet

The fatty acid and cholesterol contents of tissue membranes are the determinants of membrane stability and functionality. This study was designed to evaluate the influence of a high monounsaturated fatty acid diet on the fatty acid composition of rat liver microsomes and on their cholesterol and lipid phosphorus content. Weanling animals were fed for 5 weeks with high fat diets containing olive oil or corn oil. Saturated fatty acids were increased and oleic acid decreased in microsomal total phospholipids and in the three major phosphoglycerides, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), of rats fed corn oil as compared to the olive oil group. The percentage of linoleic acid was higher in the corn oil group, but only for total phospholipids and PC. Linoleic and alpha-linolenic metabolites were significantly increased in total phospholipids of olive oil-fed animals with respect to those fed corn oil. These changes were responsible for the low unsaturation index found in microsomal phospholipids of the corn oil group. The diet did not affect the microsome cholesterol or the lipid phosphorus content. These results show that, in olive oil-fed rats, the cholesterol content and the degree of unsaturation of liver microsomes was similar to that observed in weanling animals; this probably suggests an adequate maintenance of functionality of membranes in olive oil-fed animals.     

The effect of alpha-tocopherol on the lipid peroxidation of mitochondria and microsomes obtained from rat liver and testis.

The effect of intraperitoneal administration of alpha-tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the alpha-tocopherol treated group were not observed. The light emission was significantly higher in the control than in the alpha-tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the alpha-tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with alpha-tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbate-Fe2+ lipid peroxidation. The protector effect observed by alpha-tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

Lipid diet and enterocyte microsomal membrane fluidity in rats

Rats were fed on diets more or less enriched with n-3 and n-6 unsaturated fatty acids, before removal of the small intestine. The global protein, cholesterol and phospholipid contents of enterocyte microsomes were measured. Fatty acids of the total lipid extracts were determined. Acyl coenzyme A: cholesterol acyl transferase (ACAT) was chosen as the enzyme whose activity reflects metabolic changes induced by lipid diets. Fluorescence measurements using diphenylhexatriene as the membrane probe were performed. As dietary fat may change the fatty acid composition of membranes, the order parameter S calculated from fluorescence measurements was studied with regard to dietary fatty acid composition. The S values, distributed over a large range, were not different between rat groups. They were positively correlated with the ratios of cholesterol and proteins to phospholipids and the molar percentage of saturated fatty acids. ACAT activity was negatively correlated with S. Variations in S values among rats, whatever the diet, could in part be attributed to individual factors.     

Regional differences in lipid composition and incorporation of saturated and unsaturated fatty acids into microsomal membranes of rat small intestine

Incorporation of [1-14C]palmitic (16:0) and [1-14C]linoleic (18:2 omega 6) acids into microsomal membranes of proximal (jejunum) and distal (ileum) regions of rat small intestine was investigated, and the lipid composition, including fatty acid profiles of membrane phospholipids, was determined. Jejunal microsomes contained significantly higher amounts of total phospholipids, phosphatidylcholine, and phosphatidylinositol, and lower amounts of cholesterol and sphingomyelin when compared with ileal microsomes. Jejunal microsomal phospholipids contained higher levels of stearic (18:0), 18:2 omega 6, and eicosapentaenoic (20:5 omega 3) acids followed by reduced levels of oleic (18:1 omega 9), arachidonic (20:4 omega 6), and docosahexaenoic (22:6 omega 3) acids when compared with those from the ileum, except for phosphatidylinositol where no significant difference between 20:4 omega 6 content of each site was observed. In both jejunal and ileal microsomes, incorporation of [1-14C]18:2 omega 6 was significantly higher than that of [1-14C]16:0. Incorporation of both [1-14C]16:0 and [1-14C]18:2 omega 6 was significantly higher in jejunal microsomal lipid fractions (phospholipids, diacylglycerols, triacylglycerols) when compared with the ileal microsomal fraction. These data suggest that (1) jejunal and ileal microsomal membranes differ from each other in terms of lipid composition and lipid synthesis, (2) site variations in the specificity of acyltransferases for different fatty acids exist, and (3) higher delta 9-, delta 6-, delta 5-, and delta 4-desaturase activities exist in ileal compared with jejunal enterocytes.     

Thermally induced heterogeneity in microsomal membranes of fatty acid-supplemented Tetrahymena: lipid composition, fluidity and enzyme activity

Thermally induced phase separation was observed to occur in microsomal membranes of the ciliate Tetrahymena pyriformis, using the technique of freeze-fracture electron microscopy. In the present study, we attempted to fractionate the phase-separated membranes which were produced by chilling cells by sucrose density gradient centrifugation. When Tetrahymena was grown in the presence of palmitic acid, cells rapidly incorporated the fatty acid into their phospholipids. The resulting endoplasmic reticulum containing a high level of palmitic acid was more susceptible to thermotropic phase separation. Despite the profound alterations in the fatty acid composition, the cells retained normal growth rate, appearance and cell motility. Smooth microsomes isolated from palmitic acid-supplemented Tetrahymena cells were sonicated and then fractionated into three major subfractions. Fraction-I with lower buoyant density was rich in phospholipids and saturated fatty acids, while Fraction-III with higher density was rather rich in proteins and contained more unsaturated fatty acids in the phospholipids. A significant change was also observed in the polar head composition of phospholipids in these fractions. ESR analysis demonstrated that the extracted lipids from Fraction-III were more fluid than those from Fraction-I. In addition, the motion of the spin probe in the native membranes was more restricted than in extracted lipids. These results indicate that the lipid phase separation causes "squeezing out" of the membrane proteins from the less fluid to the fluid areas. Furthermore, we examined the temperature dependence of the activities of glucose-6-phosphatase and palmitoyl CoA desaturase.