Modification Of Lipid Composition Research Articles

Molecular breeding strategies for the modification of lipid composition

Lipids are key components of all living cells. Acyl lipids and sterols provide the matrix of the biological membranes that both define the boundaries of cells and organelles, and act as sites for the trafficking of molecules within and into/out of cells. Lipids are also important metabolic intermediates and the most efficient form of energy storage that is available to a cell. It is the latter, energy-storing function that is of most relevance to this review. Storage lipids are accumulated in abundance in many of our most important crops, including maize, soybean, rapeseed, and oil palm, giving rise to a commercial sector valued at over $50 billion/year. Because the storage lipids of the major global oil crops have a relatively restricted composition, there is great interest in using all available breeding technologies, whether traditional or modern, to enhance the variation in lipid quality in existing crops and/or to domesticate new crops that already accumulate useful novel lipids. Over the past few decades, there has been a great deal of effort to manipulate fatty acid composition in order to produce novel lipids, especially for industrial applications. However, these attempts, many based on genetic engineering, have met with only limited commercial success to date. More recently, there has been a resurgence of interest in the modification of both acyl and non-acyl lipids to enhance the nutritional quality of plant oils. In this review, we will examine the background to plant lipid modification and some of the latest developments, with a particular focus on edible oils.

Polyunsaturated eicosapentaenoic acid changes lipid composition in lipid rafts

Polyunsaturated fatty acids (PUFAs) modulate immune responses particularly by affecting T cell function and are applied clinically as adjuvant immunosuppressants in the treatment of various inflammatory diseases. However, the molecular mechanisms of PUFA-induced immunosuppressive effects are not yet elucidated. Membrane lipid rafts are functional plasma membrane microdomains characterized by a unique lipid environment. Since lipid interactions are crucial for the formation of lipid rafts, the immunomodulatory effects of PUFAs may be due to changes of fatty acid composition in lipid rafts. We investigated the effects of eicosapentaenoic acid (EPA, 20:5 n - 3) supplementation on modulating lipid composition and fatty acyl substitution in their cytoplasmic and exoplasmic lipid leaflet in lipid rafts. The human Jurkat E6-1 T cells were cultured in EPA-supplemented medium and the cells treated with stearic acid served as a control. Lipid rafts were isolated by discontinuous sucrose density gradient ultracentrifugation. The lipids in raft and soluble fractions from EPA-treated and control T cells were extracted and separated by gas chromatography. Raft phospholipids were analyzed by mass spectrometry. Our results showed that EPA treatment could alter lipid composition resulting in a considerable increase of unsaturated fatty acyl chains in lipid rafts from EPA-treated T cells compared with control cells. Effective incorporation of EPA to rafts was not only in the exoplasmic but also in the cytoplasmic membrane lipid leaflet. EPA treatment altered the lipid environment in lipid rafts. EPA presented an inhibiting effect on Jurkat T cells proliferation and inhibited IL-2Ralpha expression on the surface of T cells. Our data provided evidence for an important modification in lipid composition of membrane lipid rafts and T cell function by EPA supplementation.

Dietary seed oil rich in conjugated linolenic acid from bitter melon inhibits azoxymethane‐induced rat colon carcinogenesis through elevation of colonic PPARγ expression and alteration of lipid composition

Our previous short-term experiment demonstrated that seed oil from bitter melon (Momordica charantia) (BMO), which is rich in cis(c)9, trans(t)11, t13-conjugated linolenic acid (CLN), inhibited the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF). In our study, the possible inhibitory effect of dietary administration of BMO on the development of colonic neoplasms was investigated using an animal colon carcinogenesis model initiated with a colon carcinogen AOM. Male F344 rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for 2 weeks to induce colon neoplasms. They also received diets containing 0.01%, 0.1% or 1% BMO for 32 weeks, starting 1 week before the first dosing of AOM. At the termination of the study (32 weeks), AOM induced 83% incidence (15/18 rats) of colonic adenocarcinoma. Dietary supplementation with 0.01% and 0.1% BMO caused significant reduction in the incidence (47% inhibition by 0.01% BMO, p<0.02; 40% inhibition by 0.1% BMO, p<0.05; and 17% inhibition by 1% BMO) and the multiplicity (64% inhibition by 0.01% BMO, p<0.005; 58% inhibition by 0.1% BMO, p<0.02; and 48% inhibition by 1% BMO, p<0.05) of colonic adenocarcinoma, though a clear dose response was not observed. Such inhibition was associated with the increased content of CLA (c9,t11-18:2) in the lipid composition in colonic mucosa and liver. Also, BMO administration in diet enhanced expression of peroxisome proliferator-activated receptor (PPAR) gamma protein in the nonlesional colonic mucosa. These findings suggest that BMO rich in CLN can suppress AOM-induced colon carcinogenesis and the inhibition might be caused, in part, by modification of lipid composition in the colon and liver and/or increased expression of PPARgamma protein level in the colon mucosa.

Oral intake of shark liver oil modifies lipid composition and improves motility and velocity of boar sperm

The natural ether-lipids 1- O-alkylglycerols (alkyl-Gro) from shark liver oil improve boar sperm motility and fertility in vitro. We examined the effects of oral shark liver oil on motility and velocity parameters of sperm together with modifications of lipid composition. Eleven boars were used as control and 11 were fed with 40 g/day for 28 days and sperm was collected on Days 0, 14 and 28 in control and treated groups. After 28 days treatment, sperm motility was improved by 2.9% as well as velocity parameters (curvilinear velocity +10.75%, progressive velocity +18.8% and average path velocity +13.5%) and sperm lipid composition was modified as follows: alkyl-Gro with saturated chains were increased (C16:0 +40.1%, C18:0 +87.2%) while alkyl-Gro with unsaturated chains remained absent, as in the control group, despite the prominence of C18:1 and C16:1 in shark liver oil. The treatment also resulted in an overall increase in the proportion of n−3 and n−6 polyunsaturated fatty acids in sperm lipids with a prominent increase of docosahexaenoic acid over time (18.9±1.34% at Day 0 to 25.7±1.11% at Day 28) and compared to control (25.7±1.11% for treated versus 16.1±0.81% for control at Day 28, respectively). These data demonstrate the influence of lipid intake on boar sperm composition and functions and suggest that oral intake of shark liver oil might improve reproduction.

Linoleate supplementation in steers modifies lipid composition of plasma lipoproteins but does not alter their fluidity.

The health value for man of lipids in bovine muscles can be improved by the addition of PUFA to the animals' diets, but such treatments can modify fluidity of plasma lipoproteins and therefore their metabolic functions. The aim of the present study was to analyse whether changes in chemical composition of lipoproteins in steers fed sunflower oil-rich diets altered lipoprotein fluidity, measured by fluorescence polarization and electron spin resonance. LDL, light HDL and heavy HDL fractions were isolated by ultracentrifugation from plasma of eighteen crossbred Charolais x Salers steers. For a period of 70 d, animals were given a control diet (C, n 6) consisting of hay (540 g/kg) and concentrate mixture (460 g/kg) or the same basal diet supplemented with sunflower oil rich in n-6 PUFA (40 g/kg diet DM), given either as crushed seeds (S, n 6) or as a free oil infused directly into the duodenum (O, n 6), thus avoiding ruminal hydrogenation of PUFA. We have shown that in bovine animals: (1) fluidity measurements by fluorescence polarization must be made at the bovine physiological temperature (38.5 degrees C); (2) heavy HDL always appear as the less fluid lipoparticles; (3) electron spin resonance, which does not depend on lipoparticle size, is more appropriate to compare the fluidity of LDL with that of light HDL. The values for lipoprotein fluidity measured by both methods indicated that linoleate-rich diets did not have any effect when compared with diet C; however, chemical variables support a fluidification of lipoparticles, since in steers given the diet O, n-6 PUFA concentrations increased in polar (x1.8) and neutral (x1.6) lipids in lipoparticles (P=0.0001). The phospholipid:protein ratio increased in light (+20 %, P=0.019) and heavy (+23 %, P=0.06) HDL and especially in LDL (+46 %, P=0.0001); the total cholesterol:phospholipid ratio decreased in the three lipoprotein classes (-15 to -30 %, NS). Diet S led to similar but less pronounced effects. We concluded that linoleate-rich diets modified the chemical composition of plasma lipoproteins in steers, but did not alter their fluidity; this probably occurred as a result of 'homeoviscous adaptation', which ensured their functional capacity.

Modification of lipid composition and oxidation in porcine muscle and muscle microsomes as affected by dietary supplementation of n-3 with either n-9 or n-6 fatty acids and α-tocopheryl acetate

The effect of different oil combinations: olive oil (20 g/kg), sunflower oil (20 g/kg), linseed oil (5 g/kg) and either olive (15 g/kg) or sunflower oil (15 g/kg) and α-tocopheryl acetate supplementation (200 mg/kg feed) on fatty acid composition and lipid oxidation of pig muscle and muscle microsomes was investigated. The proportions of α-linolenic acid ( P<0.001), eicosapentaenoic acid ( P<0.001) and docosahexaenoic acid ( P<0.05) in neutral lipids were significantly greater when linseed oil was added to diets. However, the proportion of docosahexaenoic acid in polar lipids was not significantly affected by the addition of linseed oil to the diets. Adding n-3 fatty acids in the diet produced a higher ( P<0.05) linoleic acid proportion in muscle neutral lipids, particularly in those groups fed n-9 MUFA. Pigs fed n-9 and n-3 enriched diets had the closest n-6:n-3 ratio to human dietary recommendations. The addition of linseed oil produced a significantly ( P<0.05) lower proportion of oleic acid (C18:1n-9) in polar lipids of muscles from pigs fed olive and linseed oils (OL + LIN), but not in those fed sunflower and linseed when compared with groups fed diets not enriched with linseed oil. Supplementation with 200 mg α-tocopheryl acetate/kg feed also led to higher ( P<0.05) oleic acid concentrations in polar lipids. Muscle from the pigs fed sunflower and linseed oils had significantly ( P<0.05) higher Thiobarbituric acid Reactive Substances (TBARS) values throughout the study compared to those fed olive and linseed oils. α-Tocopheryl acetate supplementation consistently improved lipid stability. No interactions between α-tocopherol and oils were observed on lipid oxidation. Iron-induced peroxidation showed a similar trend to that observed for TBARS development. Iron-induced peroxidation of muscle microsomes showed a similar trend to oxidation in muscle. No interactions were detected between different oils in muscle microsomes. The partial replacement of n-9 MUFA (as olive oil) by n-3 PUFA in diet, results in a small increase in muscle and membrane lipid oxidation and could be an interesting alternative in pig diet formulation.

Eicosapentaenoic acid regulates scallop (Placopecten magellanicus) membrane fluidity in response to cold.

The lipid core of a biological membrane requires a certain degree of structural rigidity, but it must also be sufficiently fluid to permit lateral movement of the constituent lipids and embedded proteins. Ectotherms can counteract the ordering effects of reduced temperature by changing the structure of their membranes, a process known as homeoviscous adaptation (1). Although the content of unsaturated fatty acids in the membranes of ectothermic animals is generally known to increase in response to cold (2), no clear and direct relationship between unsaturated fatty acids and membrane fluidity has been established in marine organisms. For example, phospholipid molecular species containing docosahexaenoic acid (22:6 3) are believed to be important in controlling finfish membrane fluidity (3–6), but a direct correlation between 22:6 3 and membrane fluidity has not been found (4, 5, 7, 8). In contrast, we show here a simple but very strong relationship between fluidity and a single polyunsaturated fatty acid, eicosapentaenoic acid (20:5 3), in gill membranes from a marine bivalve mollusc, the sea scallop Placopecten magellanicus. Phospholipids are the main structural elements of biological membranes, and their physical characteristics are key determinants of membrane structure and function. Many vital cell activities that depend on the optimal functioning of membranes are therefore sensitive to the chemistry of the membrane lipids (9) and to environmental conditions, such as temperature and pressure, that perturb the phase behavior and dynamics of lipids in membranes (10). Under extreme or variable conditions, organisms can exploit the tremendous chemical diversity among membrane lipids to defend the physical properties of the membrane (10). Thus in ectotherms, where changes in temperature cause important membrane perturbations, the usual adaptive response includes a modification of lipid composition (11). Sessile animals living in Newfoundland waters must maintain membrane structure and function in the face of extreme cold in deep waters (as low as 1.4°C) or seasonally highly variable conditions in surface waters (as much as 22°C in 6 months) (12). In the present study, we exposed sea scallops to a 10°C decrease in temperature for up to 3 weeks and then examined the relationship between the fatty acid composition of branchial phospholipids and membrane fluidity. Vesicles were prepared from the gills of scallops acclimated to temperatures of 15 and 5°C. After three weeks of thermal acclimation, the structural order of the phospholipids was measured by electron spin resonance (ESR) spectroscopy at five temperatures (0–20°C) that span the physiological range of Placopecten magellanicus (Fig. 1). The vesicles prepared from gills of 5°C-acclimated scallops were significantly (ANCOVA, P 0.03) less ordered than vesicles from 15°C-acclimated scallops. Temperature acclimation had shifted the order parameter curve 1–2°C toward lower assay temperatures, giving a homeoviscous efficacy (13) of 14%. Such a partial adjustment towards an ideal or complete homeoviscous response has also been found in crabs (14) and crayfish (15). In these invertebrates, the costs of perfect compensation may be too high, or the benefits too low. On the other hand, the ESR measurements in this study were made with the spin probe 5-doxyl stearic acid, reflecting the homeoviscous response in the outer region of the purified lipid bilayer. It is possible that the response deeper in the bilayer, in the actual region of alkenyl chain unsaturation, would have been greater (16). Received 30 November 2001; accepted 22 March 2002. 1 Present address: Department of Physiology and Experimental Medicine, The George Washington University Medical Center, Ross Hall Room 402, 2300 Eye Street, NW, Washington, DC 20037. 2 To whom correspondence should be addressed. E-mail: cparrish@ mun.ca Reference: Biol. Bull. 202: 201–203. (June 2002)

Effect of glycation of high density lipoproteins on their physicochemical properties and on paraoxonase activity.

We investigated the effect of incubation of high density lipoprotein (HDL) under hyperglycaemic conditions on lipid composition, physicochemical properties and activity of paraoxonase (PON), a calcium-dependent enzyme associated with HDL that contributes to the antiatherogenicity of this lipoprotein. HDL incubated for three days with various glucose concentrations (0-100 mM) had significant increases in thiobarbituric acid-reactive substances (TBARS) and conjugated dienes with respect to control HDL. These results suggest that lipid peroxidation accompanies HDL glycation in vitro. The susceptibility to lipid peroxidation was higher in HDL isolated from subjects with low HDL-paraxonase activity with respect to subjects with higher HDL-PON activity. The lipid compositional changes were associated with modifications of apoprotein conformation as shown by the red-shifted position of the maximum emission of tryptophan in treated HDL. The decrease in the Gp (generalized polarization) value and the red-shifted position of the maximum emission of Laurdan incorporated in treated HDL demonstrate modifications of order and polarity with respect to control HDL. The negative correlation established between the Gp value and TBARS demonstrates that the modifications in molecular order are likely related to the increase in lipid peroxidation products. The activity of paraoxonase was significantly decreased in HDL incubated at 37 degrees C; a greater decrease occurred in the presence of 50 mM and 100 mM glucose. This study demonstrates modifications of lipid composition, apoprotein conformation and physicochemical properties of HDL incubated in the presence of glucose. These modifications affect the activity of HDL-associated paraoxonase. The physicochemical properties of lipoproteins play a regulatory role in lipoprotein function. The modification of order and polarity of glycated HDL and the alterations in paraoxonase activity could potentially contribute to the accelerated atherosclerosis in diabetic patients.

Changes in membrane lipid and free fatty acid composition during low temperature preconditioning against SO 2 injury in coleus

The effects of a low temperature (13 °C) treatment known to provide protection against sulphur dioxide (SO 2) injury were assessed on leaf lipid composition in two cultivars of Coleus blumei Benth, found previously to differ in sensitivity to SO 2 and other environmental stresses. After 5 days growth at 13 °C, there were significant differences in membrane lipid fatty acid composition as well as in free fatty acid (FFA) levels between SO 2-sensitive ‘Buckley Supreme’ (‘BS’) and SO 2-insensitive ‘Marty’ (‘M’). Molecular species of chloroplast galactolipids in ‘M’ contained increased levels of linolenic acid (18:3). In the leaf FFA pools, the saturated components, palmitic (16:0) and stearic (18:0) acids, were predominant at 20 °C. After temperature hardening at 13 °C, the total amount of FFAs decreased in ‘M’ but increased in ‘BS.’ These modifications in lipid composition suggest an additional mechanism for cultivar differences in tolerance to SO 2 and other stressors in coleus.

Changed cellular membrane lipid composition and lipid peroxidation of kidney in rats with chronic fluorosis

An animal model of chronic fluorosis was produced by subjecting Wistar rats to high doses of fluoride in drinking water for a prolonged period. Phospholipid and neutral lipid contents in rat kidney were then analyzed by high-performance liquid chromatography (HPLC), and fatty acid compositions from individual phospholipids were measured by gas chromatography. Lipid peroxidation was detected by the thiobarbituric-acid-reactive substance assay. Results showed that the total phospholipid content significantly decreased in the kidney of the rats treated with high doses of fluoride and the main species influenced were phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Decreased proportions of polyunsaturated fatty acids were observed in PE and PC in kidney of fluoride-treated animals compared to controls. No changes could be detected in the amounts of cholesterol and dolichol in kidneys between the rats treated with fluoride and controls. A significant decrease of ubiquinone in rat kidney was observed in the groups treated with excessive fluoride. High levels of lipid peroxidation were detected in kidney of the rats with fluorosis. It is plausible that the specific modification of lipid composition results from lipid peroxidation. The oxidative stress and modification of cellular membrane lipids may be involved in the pathogenesis of chronic fluorosis and provide a possible explanation for the gross system damage observed in the body, especially in soft tissues and organs.

Intracellular distribution and mobilization of unesterified cholesterol in adipocytes: triglyceride droplets are surrounded by cholesterol-rich ER-like surface layer structures.

In addition to their central role in triglyceride storage, fat cells are a primary depot of unesterified cholesterol (FC) in the body. In comparison, peripheral cells contain very little FC. This difference in adipocytes versus peripheral tissues is inconsistent with the current theory of cholesterol homeostasis. Attempting to resolve this discrepancy, we examined intracellular storage sites of FC in murine 3T3-F442A adipocytes. Using the cholesterol-binding antibiotic, filipin, in combination with high resolution fluorescence microscopy, intense fluorescent staining characteristically decorated the periphery of triglyceride droplets (TGD) as well as the plasma membrane (PM) of fat cells. Filipin-staining was not visible inside the lipid droplets. Purification of TGD by subcellular fractionation demonstrated that the rise in total FC content of adipocytes upon differentiation was attributable to an increase in TGD-FC, which contributed up to one third of the total cellular FC. The protein component of purified TGD from cultured adipocytes as well as from murine adipocytes obtained from fresh tissues contained the lumenal endoplasmic reticulum (ER) immunoglobulin binding protein (BiP) and the integral ER membrane protein calnexin. Efflux experiments using the extracellular FC acceptors (&bgr;)-cyclodextrin or apolipoprotein A-I demonstrated that TGD-associated FC was releasable from TGD. Whereas FC efflux from adipocytes was unaffected in the presence of brefeldin A or monensin, the secretion of a control protein, lipoprotein lipase, was effectively reduced. In summary, our findings identify the TGD surface layer as primary intracellular storage site for FC within adipocytes. We suggest that the structural role of ER-resident proteins in this adipocyte TGD envelope has been previously neglected. Our findings support the suggestion that an ER-like structure, albeit of modified lipid composition, constitutes the lipid droplets' surface layer. Finally, the efflux process of FC from adipocytes upon extracellular stimulation with (beta)-cyclodextrin provides evidence for an energy-dependent intracellular trafficking route between the TGD-FC pool and the PM-FC sites which is distinct from the secretory pathway of proteins.

Growth and lipid composition of scallop juveniles, Placopecten magellanicus , fed the flagellate Isochrysis galbana with varying lipid composition and the diatom Chaetoceros muelleri

The effect of feeding the flagellate Isochrysis galbana (Parke; clone T-Iso) of modified lipid composition on the growth and lipid composition of juvenile scallops [Placopecten magellanicus (Gmelin)] was investigated in the spring of 1993. I. galbana grown in 85-liter cage culture turbidostats under conditions of nitrogen limitation had a significantly higher total lipid content than when grown under nutrient-replete conditions. This was due mainly to a doubling in the amount of less unsaturated triacylglycerol in the cells. The concentrations of methyl and ethyl ketones were also greater in nitrogen-limited cells. Diets of nitrogen-limited I. galbana and nutrient-replete I. galbana grown in continuous and semi-continuous cultures were compared. Scallop juveniles were batch fed daily, and measurements of ingestion were determined. Samples of juveniles were removed periodically for determination of organic weight. The juveniles did not grow when fed nitrogen-limited or nutrient-replete I. galbana alone; however, when each diet was supplemented with 20% of the diatom Chaetoceros muelleri (Lemm.), there was a significant increase in growth in the juveniles receiving the nitrogen-limited I. galbana compared with juveniles on other diets. In comparison with I. galbana, C. muelleri provided a rich source of carbohydrates and the essential fatty acid 20:4ω6. This study shows the importance of providing optimal dietary levels of ω3 and ω6 polyunsaturated fatty acids, as well as less unsaturated fatty acids and carbohydrates.

Salt Tolerance and Crop Potential of Halophytes

Although they represent only 2% of terrestrial plant species, halophytes are present in about half the higher plant families and represent a wide diversity of plant forms. Despite their polyphyletic origins, halophytes appear to have evolved the same basic method of osmotic adjustment: accumulation of inorganic salts, mainly NaCl, in the vacuole and accumulation of organic solutes in the cytoplasm. Differences between halophyte and gly-cophyte ion transport systems are becoming apparent. The pathways by which Na+ and Cl− enters halophyte cells are not well understood but may involve ion channels and pinocytosis, in addition to Na+ and Cl− transporters. Na+ uptake into vacuoles requires Na+/H+ antiporters in the tonoplast and H+ ATPases and perhaps PPi ases to provide the proton motive force. Tonoplast antiporters are constitutive in halophytes, whereas they must be activated by NaCl in salt-tolerant glycophytes, and they may be absent from salt-sensitive glycophytes. Halophyte vacuoles may have a modified lipid composition to prevent leakage of Na+ back to the cytoplasm.Becuase of their diversity, halophytes have been regarded as a rich source of potential new crops. Halophytes have been tested as vegetable, forage, and oilseed crops in agronomic field trials. The most productive species yield 10 to 20 ton/ha of biomass on seawater irrigation, equivalent to conventional crops. The oilseed halophyte, Sali-cornia bigelovii, yields 2 t/ha of seed containing 28% oil and 31% protein, similar to soybean yield and seed quality. Halophytes grown on seawater require a leaching fraction to control soil salts, but at lower salinities they outperform conventional crops in yield and water use efficiency. Halophyte forage and seed products can replace conventional ingredients in animal feeding systems, with some restrictions on their use due to high salt content and antinutritional compounds present in some species. Halophytes have applications in recycling saline agricultural wastewater and reclaiming salt-affected soil in arid-zone irrigation districts.

Age-related changes in phospholipid fatty acid composition and monoaminergic neurotransmission in the hippocampus of rats fed a balanced or an n-3 polyunsaturated fatty acid-deficient diet

The influence of aging (2, 6, 12, and 24 months) on hippocampal lipid composition and neurochemical markers (endogenous noradrenaline, serotonin levels, monoamine oxidase (MOA) activities) was studied in rats fed a control or an n-3 polyunsaturated fatty acid (PUFA)-deficient diet. The n-3 PUFA deficiency reduced the 22:6 (n-3) level, compensated by the increase in 22:5 (n-6). However, the difference in 22:6 (n-3) content between control and deficient rats was less between 2 and 12 months and then became stable. There was an overall age-induced decrease in the major phospholipid classes phosphatidylethanolamine (PE), and phosphatidylcholine (PC) whereas the minor classes, phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM), were greatly increased, regardless of diet. The n-3 PUFA deficiency induced a reduction in the PS level, concomitant with a higher level in MAO-B activity as compared to control rats at the age of 24 months. The age-related evolution of the MAO-B activity was parallel with that of noradrenaline levels in both dietary groups. The noradrenaline and serotonin levels were modified according to age but without effect of the n-3 PUFA deficiency. Results showed that the hippocampus sustained specific age-induced modifications in lipid composition and neurotransmission factors, often with a transition period between 6 and 12 months.

Altered lipid composition and enzyme activities of plasma membranes from Trypanosoma (Schizotrypanum) Cruzi epimastigotes grown in the presence of sterol biosynthesis inhibitors

The accepted mechanism for the antiproliferative effects of sterol biosynthesis inhibitors (SBI) against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, is the depletion of specific parasite sterols that are essential growth factors and cannot be replaced by cholesterol, the main sterol present in the vertebrate host. However, the precise metabolic roles of these specific parasite steroles are unknown. We approached this problem by subjecting T. cruzi epimastigotes to two types of SBI, inhibitors of sterol C-14 demethylase and Δ 24(25) methyl transferase, and investigating the modification of lipid composition and enzyme activities in the plasma membranes of the parasite. We found in purified plasma membrane from SBI-treated cells that, together with the expected changes in the sterol composition, there was also an inversion of the phosphatidylcholine (PC) to phosphatidylethanolamine (PE) ratio and a large increase in the content of saturated fatty acids esterified to phospholipids. The modification of the phospholipid headgroup composition correlated with a 70% reduction in the specific activity of the membrane-bound PC-PE- N-methyl transferase SBI-treated cells; it was shown that this inhibition was not due to a direct effect of the drug on the enzyme. Finally, the specific activity of the Mg 2+-dependent, vanadate-sensitive ATPase present in the membranes was also inhibited by ca. 50% in SBI-treated cells. The results suggest that one of the primary effects of the depletion of endogenous sterols induced by SBI in T. cruzi is a modification of the cellular phospholipid composition as a consequence of a reduced activity of PE-PC- N-methyl transferase and probably of the acyl δ 9 and Δ 9 desaturases; this, in turn, could affect the activity of other enzymatic and transport proteins.

Lipid Peroxidation and Modification of Lipid Composition in an Endothelial Cell Model of Ischemia and Reperfusion

Among the changes that accompany the development of ischemia are alterations in the composition and turnover of membrane phospholipids. To study these effects, a cell culture model was developed to facilitate accurate measurements of lipids over varying intervals of ischemia and reperfusion (I/R). In order to mimic ischemia, rabbit aortic endothelial cells were grown to confluency on collagen coated beads and the bead cultures allowed to settle to the bottom of a conical test tube or spectrofluorometric cuvette. The cell-coated beads were then resuspended in media to simulate the process of reperfusion. Survival after ischemia/reperfusion, was determined by measurements of cellular replating efficiency, and found to decrease after periods longer than three hours of ischemia (followed by 24 h of reperfusion). Plating efficiencies were reduced to nearly 50% after 5 h of ischemia followed by reperfusion. Release of LDH inversely correlated with cell survival, and lactate production, ATP levels, and extracellular H2O2 concentration were all affected by the duration of ischemia. These changes could be directly related to rates of cellular oxygen consumption which decreased by 50% after 5 h of ischemia, while the percentage of oxygen consumption not be inhibitable by cyanide, increased. Release of esterified fatty acids, which was partly inhibited by the phospholipase A2 inhibitor, mepacrine, was stimulated by increasing periods of ischemia while the incorporation of free fatty acids into phospholipids was inhibited. The incorporation of arachidonic acid was inhibited to a lesser degree than that of oleic or linoleic acids with a resulting change in phospholipid fatty acyl composition favoring greater proportions of unsaturated fatty acids. In some experiments, the effects of vitamin E or ascorbic acid administered prior to ischemia were studied. The degree of fatty acid unsaturation, fatty acid incorporation into phospholipids, and release from phospholipids into the free fatty acid pool during ischemia/reperfusion were not affected by prior administration of vitamin E or ascorbic acid. However, the extent of lipid peroxidation during ischemia was inhibited by 100 mM ascorbic acid when present during the ischemia/reperfusion period, but not by vitamin E administered for 24 h prior to ischemia. Ascorbic acid treatment, but not vitamin E, also enabled cells to recover substantial amounts of the ATP lost following prolonged ischemia. The ATP recovery corresponded to an increased cell survival and decreased lipid peroxidation. Progressive intervals of ischemia followed by reperfusion result in compromised cell respiratory activity and decreased ATP production, and decreased phospholipid acylation leading to net hydrolysis. The associated changes in phospholipid composition, and specifically increased unsaturation appear to favor peroxidation of membrane phospholipids.

Influence of a subinhibitory dose of antifungal fatty acids from Sporothrix flocculosa on cellular lipid composition in fungi.

Antifungal fatty acids produced by the biocontrol fungus Sporothrix flocculosa were studied on the basis of their effect on growth and cellular lipid composition of three fungi, Cladosporium cucumerinum, Fusarium oxysporum, and S. flocculosa, whose growth was decreased by 51, 33, and 5%, respectively, when exposed to 0.4 mg fatty acid per ml. The sensitivity to fatty acid antibiotics from S. flocculosa was related to a high degree of unsaturation of phospholipid fatty acids and a low proportion of sterols. The major responses of sensitive fungi to sublethal doses of antifungal fatty acids from liquid culture of S. flocculosa were: (i) a decrease in total lipid; (ii) an increase in the degree of fatty acid unsaturation (18:1 > 18:2 > 18:3); (iii) an increase in free fatty acids and phosphatidic acid and a decrease in total phospholipids; and (iv) an increase in sterol/phospholipid ratio. These modifications in lipid composition led to an increase in membrane fluidity in sensitive fungi as demonstrated by assessment of fluoresence anisotropy using liposomes and 1,6-diphenyl-1,3,5-hexatriene probe. This alteration in the physical state of lipids appears to be responsible for the previously demonstrated alteration of membrane structure and function in fungi confronted to S. flocculosa.

The Effects of Cadmium on the Fluidity and H +-ATPase Activity of Plasma Membrane from Sunflower and Wheat Roots

Summary The effects of cadmium on the H + -ATPase activity and on the membrane fluidity were studied in plasma membrane prepared from roots of sunflower and wheat. Plasma membrane purified from the microsomal fraction by phase partitioning showed a decreased ATPase activity by in vivo Cd 2+ treatment (30 % and 90 % decrease in wheat and sunflower, respectively) and the in vitro application caused an inhibition, as well. The ordering state of the membrane at the C-5 and C-16 levels of the fatty acid chains and at the membrane surface was determined by electron spin resonance (ESR) and by fluorescence spectroscopy, respectively. The presence of Cd 2+ increased the ordering of the plasma membrane in both in vivo treatments and in vitro experiments. Under Cd 2+ treatment, a more rigid structure of the PM was found at different depths in the membrane; changes were larger in sunflower than in wheat. The alterations can be the results of direct effects on the membrane constituents (proteins, lipids) or of the modification in lipid composition. Preliminary studies suggest that there are changes caused by Cd 2+ in the fatty acid composition of the phospholipid fraction in PM, which were different in the case of wheat and sunflower.

Peroxidation reactions in plant membranes: effects of free fatty acids.

Free fatty acids accumulate in plant membranes after exposure of plants to environmental stress, such as freezing and desiccation. Fatty acid accumulation has been linked to various biophysical changes and to the occurrence of lipid peroxidation, but the relationships appear complex and inconsistent. The interactions between oxygen free radicals, free fatty acids and lipid peroxidation in plant membranes were examined further by studying peroxidation reactions in a model membrane system composed of a complex mixture of plant phospholipids, including various free fatty acids. Multilamellar liposomes were treated with oxygen free radicals generated from iron ascorbate. Increased concentrations of free palmitic acid up to 10 mol% (fatty acid/phospholipid) reduced the production of aldehydes detected by the thiobarbituric acid assay, but enhanced the production of fluorescent products. By contrast, increased concentrations of free linolenic acid increased aldehyde production and reduced the formation of fluorescent products. The two free fatty acids both enhanced the susceptibility of phospholipids to degradation as shown by the reduced recovery of esterified polyunsaturated fatty acids (linoleic and linolenic). The free radical reactions with or without free fatty acid additions catalyzed the selective degradation of phospholipids in the order phosphatidylethanolamine > phosphatidylcholine > phosphatidylinositol > phosphatidylglycerol. Selective degradation of phospholipids is often observed after periods of environmental stress or during senescence of plants, and has been cited as evidence for the involvement of phospholipases in these degenerative processes. The results indicate that selectivity is not a criterion for eliminating the involvement of oxygen free radicals in these degenerative processes. Furthermore, the results suggest that modifications of lipid composition during a plant's acclimation to adverse environments may determine the types of free radical reactions that occur due to stress.

Diet-induced structural and functional modifications in the pig liver endoplasmic reticulum membrane: effect of polyunsaturated fatty acid deficiency

Weaned 39-day-old female piglets were fed diets containing either corn-soybean oil (control) or hydrogenated coconut oil (essential fatty acid (EFA) deficient) for 12 weeks. EFA deficiency produced increased cholesterol (Chol) and decreased phospholipid (PL) contents relative to protein, and thereafter an enhanced Chol/PL molar ratio in the liver endoplasmic reticulum (E.R.) membrane. The primary changes included modified PL distribution, with significant decrease in phosphatidylcholine (PC) and increase in phosphatidylinositol (PI) and phosphatidylserine (PS) levels. Furthermore, fatty acid profiles of the four main PL classes were altered by EFA deficiency. In particular, linoleic, arachidonic, and docosapentaenoic acid levels were largely reduced, whereas palmitoleic and oleic acid levels were increased. The biosynthesis of 5, 8, 11-eicosatrienoic acid (from oleic acid) was strongly stimulated in EFA-deficient pig liver E.R. membranes. The lower polyunsaturated fatty acid level of these latter induced a decreased peroxidability as measured by thiobarbituric assay. Moreover, the modified lipid composition due to EFA deficiency was followed by a decrease in membrane fluidity and an alteration in the activity of several membrane proteins. Liver E.R. membrane from EFA-deficient piglets exhibited a decreased Ca −+ uptake, although passive Ca ++ efflux was unaffected. NADH-Cyt.b 5 and NADH-Cyt.b 5 reductase activity were enhanced, whereas NADPH-Cyt. P450 electron transferring system and some of the liver detoxifying enzyme activities were significantly depressed.