Bicuculline induces free fatty acid release from phospholipids in Neuro-2A cells in culture

This study characterizes free fatty acid release in a neuroblastoma cell line (Neuro-2A), a potential model system for the study of factors that control phospholipase A₂ in neurons. Two compounds, bicuculline (an antagonist at -aminobutyric acid receptors), and A23187 (a Ca²⁺ ionophore), were examined. The release of endogenous fatty acids and the turnover of radiolabeled arachidonic and docosahexaenoic acids were measured. The cells actively incorporated radiolabeled fatty acids into various glycerolipid pools. Both endogenous fatty acids and radiolabeled fatty acids were released from glycerolipids in a time-dependent manner. Phosphatidylcholine was a major source of released fatty acids. Release of free fatty acids was markedly stimulated by both bicuculline and A23187. We conclude that the Neuro-2A cell contain phospholipase activity that is sensitive to Ca²⁺ ionophore and bicuculline, and may provide a good system for further studies on the regulation of phospholipase A₂ in neurons.Abbteviations 160 palmitic acid - 180 stearic acid - 181 oleic acid - 182 linoleic acid - 183 linolenic acid - 204 arachidonic acid - 226 docosahexaenoic acid - DG diacylglycerol - FAME fatty acid methyl ester - FFA free fatty acid - GABA -aminobutyric acid - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - TG triacylglycerol     

Fatty acid composition of total lipids and phospholipids of membrane preparations of transport ATPases

The fatty acid composition of total lipids and phospholipids of duck salt gland Na,K-ATPase (outer plasma membrane) and of rabbit skeletal muscle Ca-ATPase (intracellular membrane) was investigated. The bulk of Na,K-ATPase fatty acids is represented by palmitic (16:0), oleic (18:1), stearic (18:0) and arachidonic (20:4) acids. The duck salt gland is characterized by rather a high content of unsaturated fatty acids, especially of arachidonic acid. The unsaturation index of total-lipid fatty acids increases during purification of these preparations in the following order: homogenate greater than microsomal fraction greater than purified enzyme. The fatty acid composition of Na,K-ATPase total lipids and phospholipids reveals certain differences. Phospholipids contain more stearic and liholeic (18:2) acids than total lipids, but the level of arachidonic acid in them is twice as low. Besides, phospholipids were found to contain polyunsaturated docosohexaenic (22:6) acid. The bulk of fatty acids of rabbit skeletal muscle Ca-ATPase total lipids and phospholipids is represented by 16:0, 18:0, 18:1 and 18:2 acids. The content of polyunsaturated fatty acids in this preparation is much lower than in duck salt gland Na,K-ATPase. The fatty acid composition of total lipids and phospholipids in rabbit skeletal muscle Ca-ATPase differ insignificantly. The differences in the fatty acid composition of membrane preparations under study is conditioned mainly by the fractional composition of their lipids.     

Dietary n-6 PUFA deprivation for 15 weeks reduces arachidonic acid concentrations while increasing n-3 PUFA concentrations in organs of post-weaning male rats

Few studies have examined effects of feeding animals a diet deficient in n-6 polyunsaturated fatty acids (PUFAs) but with an adequate amount of n-3 PUFAs. To do this, we fed post-weaning male rats a control n-6 and n-3 PUFA adequate diet and an n-6 deficient diet for 15 weeks, and measured stable lipid and fatty acid concentrations in different organs. The deficient diet contained nutritionally essential linoleic acid (LA,18:2n-6) as 2.3% of total fatty acids (10% of the recommended minimum LA requirement for rodents) but no arachidonic acid (AA, 20:4n-6), and an adequate amount (4.8% of total fatty acids) of α-linolenic acid (18:3n-3). The deficient compared with adequate diet did not significantly affect body weight, but decreased testis weight by 10%. AA concentration was decreased significantly in serum (− 86%), brain (− 27%), liver (− 68%), heart (− 39%), testis (− 25%), and epididymal adipose tissue (− 77%). Eicosapentaenoic (20:5n-3) and docosahexaenoic acid (22:6n-3) concentrations were increased in all but adipose tissue, and the total monounsaturated fatty acid concentration was increased in all organs. The concentration of 20:3n-9, a marker of LA deficiency, was increased by the deficient diet, and serum concentrations of triacylglycerol, total cholesterol and total phospholipid were reduced. In summary, 15 weeks of dietary n-6 PUFA deficiency with n-3 PUFA adequacy significantly reduced n-6 PUFA concentrations in different organs of male rats, while increasing n-3 PUFA and monounsaturated fatty acid concentrations. This rat model could be used to study metabolic, functional and behavioral effects of dietary n-6 PUFA deficiency.     

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A2-VIA (iPLA2β)-knockout mice

Calcium-independent phospholipase A₂ group VIA (iPLA₂β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA₂β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA₂β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA₂β^+/+) and knockout (iPLA₂β^−/−) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA₂, cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA₂β^+/+ mice, iPLA₂β^−/− mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA₂β^−/− mice, brain levels of iPLA₂β mRNA, protein, and activity were decreased, as was the iPLA₂γ (Group VIB PLA₂) mRNA level, while levels of secretory sPLA₂-V mRNA, protein, and activity and cytosolic cPLA₂-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA₂β deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.     

Secretory phospholipase A2-alpha from Arabidopsis thaliana: functional parameters and substrate preference

The secretory phospholipase A2-alpha from Arabidopsis thaliana (AtsPLA2-alpha), being one of the first plant sPLA2s obtained in purified state, has been characterised with respect to substrate preference and optimum conditions of catalysis. The optima of pH, temperature, and calcium concentration were similar to the parameters of secretory PLA2s from animals. However, substrate preferences markedly differed. In contrast to pancreatic PLA2s, AtsPLA2-alpha preferred zwitterionic phospholipids, and showed lower activity toward anionic phospholipids. In substrates with two identical fatty acid chains, AtsPLA2-alpha showed optimum activity toward phospholipids with decanoyl groups. In substrates with palmitoyl groups in sn-1 position, acyl chains with higher degree of unsaturation in sn-2 position were preferred, excluding arachidonic acid, showing the evolutionary adaptation of the enzyme to substrate composition in plants. Km values for short chain phospholipids were comparable to sPLA2s from animals, whereas k cat values were much smaller and interfacial activation was less important.     

Fatty acid oxidation and myocardial phospholipase A2 activity

Summary Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A₂ activity (PLA₂) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca²⁺-dependent PLA₂ was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA, activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC₅₀ for arachidonic acid was approx 65 M. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37°C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA₂ inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA₂s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury.     

Bile formation and hepatic plasma membrane composition in guinea-pigs and rats

We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl⁻, phosphate and Ca²⁺ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na⁺,K⁺ ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs. Total fatty acid analysis of the membrane revealed that palmitic acid (C16:0), stearic acid (C18:0) and linoleic acid (C18:2, n-6) were the predominant fatty acids in guinea-pigs, while palmitic acid (C16:0), stearic acid (C18:0) and arachidonic acid (C20:4, n-6) were the most important in rats. Thus, high bile flow rate and BAIF in the guinea-pig may be attributed to the low bile acid concentration (below the critical micellar concentration), secretion of hypercholeretic bile acids (e.g. ketolithocholate) and high bicarbonate output.     

Degumming of vegetable oils by a novel phospholipase B from Pseudomonas fluorescens BIT-18

Pseudomonas fluorescens BIT-18 was isolated from soil near a vegetable oil factory and shown to produce a B-type phospholipase. The enzyme was partially purified by ammonium sulfate precipitation. Gas chromatography demonstrated that the enzyme preparation hydrolyzed both the 1- and 2-ester bonds of phosphatidylcholine. When degumming of soybean, rapeseed, and peanut oil was performed with this enzyme preparation, oils with phosphorous contents lower than 5 mg/kg were obtained after 5 h of enzyme treatment at 40 °C. The enzyme preparation did not show lipase activity, thus free fatty acids were only generated from the phospholipids. Therefore, this novel phospholipase B is potentially useful for the refining of high-quality oils with attractive yields.     

Regulation of FFA by the acyltransferase pathway in focal cerebral ischemia-reperfusion

Cerebral insult is associated with a rapid increase in free fatty acids (FFA) and arachidonic acid release has been linked to the increase in eicosanoid biosynthesis. In transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, there is an inverse relationship between the increase in FFA and the decrease in ATP, both during the ischemia period and at later time periods after reperfusion. In this study, the focal cerebral ischemia model was used to examine incorporation of [¹⁴C]arachidonic acid into the glycerolipids in rat MCA cortex at different reperfusion times after a 60 min ischemia. The label was injected intracerebrally into left and right MCA cortex 1 hr prior to decapitation. Labeled arachidonic acid was incorporated into phosphatidylcholine, phosphatidylethanolamine and neutral glycerides. With increasing time (4–16 hr) after a 60 min ischemia, an inhibition of labeled arachidonate uptake could be found in the right ischemic MCA cortex, whereas the distribution of radioactivity among the major phospholipids was not altered. When compared to labeled PC, there was a 3–4 fold increase in incorporation of label into phosphatidic acid and triacylglycerols (TG) in the right MCA cortex, suggesting of an increase in de novo biosynthesis of TG. In an in vitro assay system, synaptosomal membranes isolated from MCA cortex 8 and 16 hr after a 60 min ischemia showed a significant decrease in arachidonoyl transfer to lysophospholipids, due mainly to a decrease in lysophospholipid:acylCoA acyltransferase activity. Assay of phospholipase A₂ activity with both syaptosomes and cytosol, however, did not show differences between left and right MCA cortex or with time after reperfusion. These results suggest that besides ATP availability, the decrease in acyltransferase activity may also contribute to the increase in FFA in cerebral ischemia-reperfusion.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PEpl ethanolamine plasmalogen - PI phosphatidylinositol - PS phosphatidylserine - poly-PI polyphosphoinsoitide - DG diacylglycerol - TG triacylglycerol - FFA free fatty acids - PUFA polyunsaturated fatty acids - MCA middle cerebral artery - CCAs common carotid arteries - HPTLC high performance thin layer chromatography - GLC gas-liquid chromatography - PLA₂ phospholipase A₂ Special issue dedicated to Dr. Leon S. Wolfe.     

Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts

Although the activation of phospholipase A₂ (PLA₂) in ras-transformed cells has been well documented, the mechanisms underlying this activation are poorly understood. In this study we tried to elucidate whether the membrane phospholipid composition and physical state influence the activity of membrane-associated PLA₂ in ras-transformed fibroblasts. For this purpose membranes from non-transfected and ras-transfected NIH 3T3 fibroblasts were enriched with different phospholipids by the aid of partially purified lipid transfer protein. The results showed that of all tested phospholipids only phosphatidylcholine (PC) increased PLA₂ activity in the control cells, whereas in their transformed counterparts both PC and phosphatidic acid (PA) induced such effect. Further we investigated whether the activatory effect was due only to the polar head of these phospholipids, or if it was also related to their acyl chain composition. The results demonstrated that the arachidonic acid-containing PC and PA molecules induced a more pronounced increase of membrane-associated PLA₂ activity in ras-transformed cells compared to the corresponding palmitatestearate- or oleate- containing molecular species. However, we did not observe any specific effect of the phospholipid fatty acid composition in non-transformed NIH 3T3 fibroblasts. In ras-transformed cells incubated with increasing concentrations of arachidonic acid, PLA₂ activity was altered in parallel with the changes of the cellular content of this fatty acid. The role of phosphatidic and arachidonic acids as specific activators of PLA₂ in ras-transformed cells is discussed with respect to their possible role in the signal transduction pathways as well as in the processes of malignant transformation of cells.     

The influence of ionic detergents on the phospholipid fatty acid compositions of Porphyridium purpureum

The phospholipid fatty acid composition of Porphyridium purpureum on a solid medium was studied in the presence of sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB). The most common fatty acids in phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) were palmitic (16:0), stearic (18: 0), linoleic (18:2ω 6), arachidonic (20:4ω 6) and eicosapentaenoic (20:5ω 3) acids, 20:4ω 6 being very abundant. In phosphatidyl glycerol (PG) the most common acids were 16:0, trans-hexadecenoic acid (tr 16:1ω 13), oleic acid (18:1) and 20:4ω 6. Both detergents increased the saturation grade of PC and PE by decreasing the relative amount of the polyunsaturated acids, especially 20:4ω 6. A corresponding increase in the amounts of saturated acids was observed in PC and PE. The changes in PG fatty acid composition were not very significant: a slight increase was observed in the amounts of 16:0 and tr 16:1ω 13 , with a corresponding decrease in the amounts of 20:4ω 6 and 20:5ω 3. Both detergents decreased the PC/PE and the (PC + PE)/PG ratios very markedly, most probably as a result of increases in the amounts of PE and PG. In the presence of CTAB the cells seemed to contain much more phospholipids than in the presence of SDS, perhaps as a result of the mucilage-precipitating effect of CTAB. The significance of the findings is discussed.     

Incorporation of exogenous fatty acids into phospholipids by cultured hamster fibroblasts. Effect of SV40 transformation

In situ incorporation of two saturated (palmitic, 16:0; stearic, 18:0) and three unsaturated fatty acids (oleic, 18:1; linoleic, 18:2; arachidonic, 20:4) into the four major phospholipids, sphingomyelin, PC, PI and PE, was followed. Transformed cells incorporated unsaturated fatty acids more rapidly, whereas no significant differences were found concerning saturated fatty acids. In vitro determination of phospholipid acylation showed that incorporation of coenzyme A-activated forms of two saturated fatty acids (16:0 and 18:0) and one unsaturated fatty acid (18:1) into phospholipids was increased in transformed cells. Comparison of results obtained in situ and in vitro strongly suggests that incorporation of fatty acids into phospholipids in cultured cells is not limited by acyltransferase activities.     

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE₂, PGD₂, and PGF_2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A₂ (PLA₂) isozymes revealed that cytosolic PLA₂α, but not calcium-independent PLA₂s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.     

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.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.     

GABA, progesterone and zona pellucida activation of PLA2 and regulation by MEK-ERK1/2 during acrosomal exocytosis in guinea pig spermatozoa

We investigated whether GABA activates phospholipase A2 (PLA2) during acrosomal exocytosis, and if the MEK-ERK1/2 pathway modulates PLA2 activation initiated by GABA, progesterone or zona pellucida (ZP). In guinea pig spermatozoa prelabelled with [14C]arachidonic acid or [14C]choline chloride, GABA stimulated a decrease in phosphatidylcholine (PC), and release of arachidonic acid and lysoPC, during exocytosis. These lipid changes are indicative of PLA2 activation and appear essential for exocytosis since inclusion of aristolochic acid (a PLA2 inhibitor) abrogated them, along with exocytosis. GABA activation of PLA2 seems to be mediated, at least in part, by diacylglycerol (DAG) and protein kinase C since inclusion of the DAG kinase inhibitor R59022 enhanced PLA2 activity and exocytosis stimulated by GABA, whereas exposure to staurosporine decreased both. GABA-, progesterone- and ZP-induced release of arachidonic acid and exocytosis were prevented by U0126 and PD98059 (MEK inhibitors). Taken together, our results suggest that PLA2 plays a fundamental role in agonist-stimulated exocytosis and that MEK-ERK1/2 are involved in PLA2 regulation during this process.     

Effects of postnatal age and diet on the fatty acid composition of plasma lipid fractions in preterm infants

Diet and postnatal age effect the fatty acid composition of plasma and tissue lipids. This work was designed as a transversal study to evaluate the changes in the fatty acid composition of plasma phospholipids, cholesteryl esters, triglycerides and free fatty acids in preterm infants (28-35 weeks gestational age), fed human milk (HM) and milk formula (MF) from birth to 1 month of life. Sixteen blood samples were obtained from cord, and 19 at 6-8 h after birth, 14 at 1 week and 9 at 4 weeks from HM-fed infants and 18 at 1 week and 14 at 4 weeks from MF-fed ones. Groups had similar mean birth weight, gestational age and sex ratio. The MF provided 69 kcal/dl and contained 16% of linoleic acid and 1.3% of alpha-linolenic acid on the total fat. Plasma lipid fractions were extracted and separated by thin-layer chromatography and fatty acid methyl esters were quantitated by gas liquid chromatography. In plasma phospholipids, linoleic acid (18:2 omega 6) continuously increased from birth to 1 month of age, but no changes were seen as related to type of diet; polyunsaturated fatty acids greater than 18 carbon atoms of both the omega 6 and omega 3 series (PUFA omega 6 greater than 18 C and omega 3 greater than 18 C) dropped from birth to 1 week and continued to decrease in MF-fed infants until 1 month; eicosatrienoic (20:3 omega 6), arachidonic (20:4 omega 6) and docosahexaenoic (22:6 omega 3) were the fatty acids implicated. In cholesteryl esters palmitoleic (16:1 omega 7) and oleic (18:1 omega 9) acids decreased from birth to 1 month and linoleic acid increased and arachidonic acid dropped, especially in MF fed infants. In triglycerides, palmitic, palmitoleic and stearic acid (18:0) decreased during the first month of life; oleic acid remained constant and linoleic acid increased in all infants, but arachidonic acid decreased only in those fed formula. Free fatty acids showed a similar behavior in fatty acids and in plasma triglycerides. Preterm neonates seem to have special requirements of long-chain PUFA and adapted MF should contain these fatty acids in similar amounts to those of HM to allow the maintenance of an adequate tissue structure and physiology.     

Inhibitory effect of n-3 fish oil fatty acids on cardiac Na+/Ca2+ exchange currents in HEK293t cells

Abnormal activity of the cardiac Na+/Ca2+ exchanger (NCX1) can affect intracellular Ca2+ homeostasis and cause arrhythmias. The n-3 polyunsaturated fatty acids (PUFAs), however, may prevent arrhythmias. To test the effect of PUFAs on the cardiac NCX1 current (I(NCX1)), the canine NCX1 cDNA was expressed in human embryonic kidney (HEK293t) cells. The average density of I(NCX1) was 10.9+/-2.6 pA/pF (n=44) in NCX1-transfected cells and eicosapentaenoic acid (EPA, C20:5n-3) significantly inhibited I(NCX1) The suppression of I(NCX1) by EPA was concentration-dependent with an IC50 of 0.82+/-0.27 microM. EPA had a similar effect on outward or inward I(NCX1). Docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6) also significantly inhibited I(NCX1), whereas the saturated fatty acid, stearic acid (SA, C18:0), did not. Our data demonstrate that the n-3 PUFAs significantly suppress cardiac I(NCX1), which is probably one of their protective effects against lethal arrhythmias.     

Use of phospholipase A to compare phospholipid organization in synaptic membranes,myelin, and liposomes

Summary The pattern of fatty acid release from rat synaptic membranes in the presence of phospholipase A₂ (Vipera russelli) was compared to that from liposomes comprised of phospholipids. Phospholipase A₂ more readily attacked myelin and synaptic membranes than liposomes prepared from total phospholipids derived from myelin. Although hydrolysis of liposomal phospholipids occurred in the absence of added calcium, the presence of 2mm CaCl₂ or 2% bovine serum albumin significantly enhanced the phospholipase attack of liposomes, but not synaptic membranes or myelin. Phospholipase exhibited a marked preference for phospholipids containing docosahexaenoic acid (226) in the synaptic membranes, while with liposomes the pattern of released fatty acid reflected the fatty acid composition in the two-position of the phospholipids. Although either calcium or albumin markedly increased the phospholipase hydrolysis of liposomes, neither affected the hydrolysis of synaptic membranes or the pattern of fatty acid release from liposomes. It was concluded that the nonlipid constituents, particularly the proteins, of biomembranes were responsible for the organization of the phospholipids and accounted for the observed differences between liposomes and synaptic membranes with respect to enzymic accessibility.     

Transient Formation of Superoxide Radicals in Polyunsaturated Fatty Acid-Induced Brain Swelling

The involvement of superoxide free radicals and lipid peroxidation in brain swelling induced by free fatty acids has been studied in brain slices and homogenates. The polyunsaturated fatty acids linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), and docosahexaenoic acid (22:6) caused brain swelling concomitant with increases in superoxide and membrane lipid peroxidation. Palmitic acid (16:0) and oleic acid (18:1) had no such effect. Furthermore, superoxide formation was stimulated by NADPH and scavenged by the addition of exogenous superoxide dismutase in cortical slice homogenates. These in vitro data support the hypothesis that both superoxide radicals and lipid peroxidation are involved in the mechanism of polyunsaturated fatty acid-induced brain edema.