Phospholipid fatty acid composition affects enzymatic antioxidant defenses in cultured Swiss 3T3 fibroblasts

The aim of this work was to study the adaptation of enzymatic antioxidant cell defense to the nature of the membrane polyunsaturated fatty acids (PUFA). 3T3 Swiss fibroblasts were grown for 5 days in a medium supplemented with 50 microM linoleic acid (LA) or eicosapentaenoic acid (EPA) and compared to control cells (C). The phospholipid fatty acid content was evaluated: LA were enriched in n-6 PUFA (27.8%) in comparison to C (6.7%) or EPA (5.6%); EPA were enriched in n-3 PUFA (26.2%) in comparison to LA (4.4%) or C (4.6%). The fatty acid double bond index (DBI) increased from C to LA and EPA. The activities of the three key enzymatic antioxidant defenses, SOD, GPx and GST, increased with the degree of unsaturation of the phospholipid fatty acids. In the cells with fatty acids that are very sensitive to oxidative stress, the higher activities of SOD and GPx might act to limit the initiation of lipid peroxidation and the higher activities of GST and GPx to decrease the toxic effects of the various species produced from lipid degradation.     

Potential use of cytokine therapy in poultry

The time course of incorporation of [14C]arachidonic acid and [3H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [14C]arachidonic acid and [3H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Fatty acyl desaturation in isolated hepatocytes from Atlantic salmon (Salmo salar): stimulation by dietary borage oil containing gamma-linolenic acid

The effects of different dietary oils on the fatty acid compositions of liver phospholipids and the desaturation and elongation or [1-14C]18:3n-3 and [1-14C]18:2n-6 were investigated in isolated hepatocytes from Atlantic salmon. Atlantic salmon smolts were fed diets containing either a standard fish oil (FO) as a control diet, a 1:1 blend of Southern Hemisphere marine oil and tuna orbital oil (MO/TO), sunflower oil (SO), borage oil (BO), or olive oil (OO) for 12 wk. The SO and BO diets significantly increased the percentages of 18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, and total n-6 polyunsaturated fatty acids (PUFA) in salmon liver lipids in comparison with the FO diet. The BO diet also increased the percentage of 20:4n-6. Both the SO and BO diets significantly reduced the percentages of all n-3 PUFA in comparison with the FO diet. The OO diet significantly increased the percentages of 18:1n-3, 18:2n-6, total monoenes, and total n-6 PUFA in liver lipids compared to the FO diet, and the percentages of all n-3 PUFA were significantly reduced. With [1-14C]18:3n-3, the recovery of radioactivity in the products of delta 6 desaturation was significantly greater in the hepatocytes from salmon fed SO, BO, and OO in comparison with the FO diet. The BO diet also increased the recovery of radioactivity in the products of delta 5 desaturation. Only the BO diet significantly affected the desaturation of [1-14C]18:2n-6, increasing recovery of radioactivity in both delta 6- and delta 5-desaturation products. In conclusion, dietary BO, enriched in gamma-linolenic acid (18:3n-6), significantly increased the proportions of both 20:3n-6 and 20:4n-6 in salmon liver phospholipids and also significantly increased the desaturation of both 18:2n-6 and 18:3n-3 in salmon hepatocytes. The possible relationships between dietary fatty acid composition, tissue phospholipid fatty acid composition, and desaturation/elongation activities are discussed.     

Incorporation of n-3 fatty acids into plasma lipid fractions, and erythrocyte membranes and platelets during dietary supplementation with fish, fish oil, and docosahexaenoic acid-rich oil among healthy young men

The effects of n-3 fatty acid supplementation in the form of fresh fish, fish oil, and docosahexaenoic acid (DHA) oil on the fatty acid composition of plasma lipid fractions, and platelets and erythrocyte membranes of young healthy male students were examined. Altogether 59 subjects (aged 19-32 yr, body mass index 16.8-31.3 kg/m2) were randomized into the following diet groups: (i) control group; (ii) fish diet group eating fish meals five times per week [0.38 +/- 0.04 g elcosapentaenoic acid (EPA) and 0.67 +/- 0.09 g DHA per day]; (iii) DHA oil group taking algae-derived DHA oil capsules (1.68 g/d DHA in triglyceride form); and (iv) fish oil group (1.33 g EPA and 0.95 g DHA/d as free fatty acids) for 14 wk. The fatty acid composition of plasma lipids, platelets, and erythrocyte membranes was analyzed by gas chromatography. The subjects kept 4-d food records four times during the study to estimate the intake of nutrients. In the fish diet, in DHA oil, and in fish oil groups, the amounts of n-3 fatty acids increased and those of n-6 fatty acids decreased significantly in plasma lipid fractions and in platelets and erythrocyte membranes. A positive relationship was shown between the total n-3 polyunsaturated fatty acids (PUFA) and EPA and DHA intake and the increase in total n-3 PUFA and EPA and DHA in all lipid fractions analyzed. DHA was preferentially incorporated into phospholipid (PL) and triglyceride (TG) and there was very little uptake in cholesterol ester (CE), while EPA was preferentially incorporated into PL. and CE. The proportion of EPA in plasma lipids and platelets and erythrocyte membranes increased also by DHA supplementation, and the proportion of linoleic acid increased in platelets and erythrocyte membranes in the DHA oil group as well. These results suggest retroconversion of DHA to EPA and that DHA also interferes with linoleic acid metabolism.     

Spontaneous transfer of monoacyl amphiphiles between lipid and protein surfaces

The kinetics of transfer of natural and fluorescent nonesterified fatty acids (NEFA) and lysolecithins (lysoPC) from phospholipid and protein surfaces were measured. The kinetics of transfer of 12-(1-pyrenyl)dodecanoic acid, from liquid crystalline and gel phase single unilamellar phospholipid vesicles, very low, low, and high density lipoproteins, human serum albumin, and rat liver fatty acid-binding protein, were first-order and characterized by similar rate constants. The halftimes (t1/2) of NEFA transfer from lipids and proteins were dependent on the acyl chain structure according to log t1/2 = -0.62n + 0.59m + 12.0, where n and m, respectively, are the numbers of carbon atoms and double bonds. The structure of the donor surface had a measurable but smaller effect on transfer rates. The kinetics of NEFA and lysoPC transfer are slow relative to the lipolytic processes that liberate them. Therefore, one would predict a transient accumulation of NEFA and lysoPC during lipolysis and an attendant modulation of many metabolic processes within living cells and within the plasma compartment of blood. These data will be useful in the refinement of current models of membrane and lipoprotein function and in the selection of fluorescent NEFA analogs for studying transport in living cells.     

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.     

Biosynthesis of the unsaturated 14-carbon fatty acids found on the N termini of photoreceptor-specific proteins

In the vertebrate retina, a number of proteins involved in signal transduction are known to be N-terminal acylated with the unusual 14 carbon fatty acids 14:1n-9 and 14:2n-6. We have explored possible pathways for producing these fatty acids in the frog retina by incubation in vitro with candidate precursor fatty acids bearing radiolabels, including [3H]14:0, [3H]18:1n-9, [3H]18:2n-6, and [3H]18:3n-3. Rod outer segments were prepared from the radiolabeled retinas for analysis of protein-linked fatty acids, and total lipids were extracted from the remaining retinal pellet. Following saponification of extracted lipids, fatty acid phenacyl esters were prepared and analyzed by high pressure liquid chromatography (HPLC) with detection by continuous scintillation counting. Transducin, whose alpha-subunit (Gt alpha) is known to bear N-terminal acyl chains, was extracted from the rod outer segments and subjected to SDS-polyacrylamide gel electrophoresis and fluorography to detect radiolabeled proteins. Gt alpha was also subjected to methanolysis, and the resulting fatty acyl methyl esters were analyzed by HPLC. The identities of HPLC peaks coinciding with unsaturated species of both phenacyl esters and methyl esters were confirmed by reanalyzing them after catalytic hydrogenation. The results showed that 14:1n-9 can be derived in the retina from 18:1n-9 and 14:2n-6 from 18:2n-6, most likely by two rounds of beta-oxidation, but that neither is produced in detectable amounts from 14:0. Retroconversion of unsaturated 18 carbon fatty acids to the corresponding 14 carbon species showed specificity, in that 18:3n-3 was not converted to 14 carbon fatty acids in detectable amounts. Myristic acid (14:0), 14:1n-9, and 14:2n-6 were all incorporated into Gt alpha. A much less efficient incorporation of 18:1n-9 into Gt alpha was also observed, but no radiolabeling of Gt alpha was observed in retinas incubated with 18:3n-3. Thus, retroconversion by limited beta-oxidation of longer chain unsaturated fatty acids appears to be the most likely metabolic source of the unusual fatty acids found on the N termini of signal transducing proteins in the retina.     

Effects of feeding medium-chain triacylglycerols on maternal lipid metabolism and pup growth in lactating rats

To study the effects of medium-chain triacylglycerols (MCT) on maternal lipid metabolism and pup growth, MCT (200 g/kg) were incorporated into a commercial chow diet and fed to lactating rats for 8-10 d. The results were compared with similar diets containing sunflower oil (polyunsaturated fatty acids; PUFA), tristearin (saturated fatty acid) or triolein (monounsaturated fatty acid). There was decreased food and energy intake with the MCT diet and this was accompanied by decreased (35%) pup growth. All the high-fat diets inhibited lipogenesis in vivo in the lactating mammary gland, the order of effectiveness being PUFA > triolein > tristearin > MCT. Only the MCT diet increased the rate of hepatic lipogenesis (180%). Experiments feeding an MCT meal containing [1-14C]octanoate indicated that very little (3-4%) of the C was present in mammary gland lipid, unlike the findings with [1-14C]triolein meal (40%). The major portion (65%) of the absorbed [1-14C]octanoate was oxidized to 14CO2. There was no evidence for adaptation of the mammary gland to increased dietary lipid uptake on the triolein or MCT diets. It is concluded that the decreased pup growth on the MCT diet is due in part to the decreased energy intake and to the inability of dietary medium-chain fatty acids to provide substrates for milk lipid synthesis.     

Abnormality in fatty acid composition of gastric mucosal phospholipids in patients with liver cirrhosis and its correction with a polyunsaturated fatty acid-enriched soft oil capsule

The abnormal composition of the fatty acids in gastric mucosal phospholipids was examined in 11 patients with non-alcoholic liver cirrhosis accompanied by gastritis and in 10 healthy subjects without liver disease and gastritis. The cases positive for serum anti-Helicobacter pylori immunoglobulin G antibody were excluded. The arachidonic acid (AA, C20:4n-6) contents of the two main components of phospholipid, the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fractions, in gastric biopsy specimens were significantly lower in the cirrhosis group, although PC and PE contents in gastric biopsy specimens were not altered. In the cirrhosis group, AA contents of the PC fractions in gastric biopsy specimens were significantly correlated with AA contents of plasma PC fractions and serum albumin levels. Soft oil capsules containing polyunsaturated fatty acid (PUFA) such as AA, eicosapentanoic acid (EPA, C20:5n-3) and docosahexanoic acid (DHA, C20:6n-3) were administered after each meal daily for 4 weeks to six cirrhotic patients and four control subjects, who were randomly selected from the patients and control subjects. After administration of PUFA-enriched oil capsules, gastric mucosal AA contents of PC and PE fractions were significantly improved almost to the control levels. In three cirrhotic patients with severe or mild gastritis whose gastric mucosal lesions were endoscopically shown to have responded to PUFA-enriched oil capsules, AA contents of plasma PC and PE fractions before administration were much lower than in the remaining three cirrhotics that did not respond to the PUFA-enriched oil capsules, but significantly improved to the control levels after administration of oil capsules. The results demonstrate that administration of PUFA-enriched soft oil capsules increased AA contents of the phospholipids in gastric mucosa and thus improved gastric mucosal lesions endoscopically in cirrhotic patients with gastritis.     

Intestinal absorption of polyunsaturated phosphatidylcholine in the rat

The mechanism of intestinal absorption of polyunsaturated phosphatidylcholine in an oil medium was studied with 1,2-di-[9,10,12,13-3H4]linoleoyl-sn-glycero-3-phospho-[N-14CH3]-choline, 1-[1-14C]linoleoyl-2-[9,10,12,13-3H4]-linoleoyl- and 1-[9,10,12,13-3H4]linoleoyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine, especially with regard to the stability of the ester bonds in position 1 and 2 of the phospholipid molecule. The absorption rate, as measured by the disappearance from the gastro-intestinal tract, was comparatively rapid in the first 6 - 8 h, but then became considerably slower. After 24 h more than 90% of the applied radioactivity was absorbed from the intestinal tract. Respiratory 14CO2 from the degradation of the unsaturated acyl moiety in position 2 is produced much more rapidly than that from the acyl group attached to the 1-position of the glycerophosphocholine backbone. Analyses of the liver phosphatidylcholine by specific enzymatic hydrolysis with phospholipase A2, 6 h after the application, showed that in the isolated PC 9 times more labelled fatty acids from the original 1-position were present than from the corresponding 2-position. In rats with lymph fistula it was shown that more than 90% of the acyl moieties of the administered 1,2-di-[9,10,12,-13-3H4]linoleoyl-[N-14CH3] glycerophosphocholine was transported by the chylomicrons. About one half of the 14C choline radioactivity of the glycerophosphocholine backbone was found in the chylomicrons and the other half in the liver. The 3H radioactivity distribution in the chylomicrons amounted to 25% in the phosphatidylcholine fraction and 75% in the neutral lipids. Positional specific analyses of the phosphatidylcholine present in chylomicrons confirmed the fact that the 1-position remained practically intact while the 2-position underwent considerable exchange with unlabelled fatty acids. Analysis of the liver of the animals with lymph fistula indicated that it was practically free of the 3H radioactivity derived from the acyl moieties but contained a high percentage of the 14C radioactivity of the choline group. The methyl groups of choline were oxidized only to a very small extent. These results demonstrate that during the absorption process, about one half of the absorbed polyunsaturated phosphatidylcholine is hydrolyzed to 1-acyl-lysophosphatidylcholine and reacylated again to phosphatidylcholine upon entering the mucosa cell, while the other half is completely hydrolyzed to free fatty acids and glycerophosphocholine or its hydrolysis products. The fatty acids released are utilized for the reassembly of triacylglycerides and phosphatidylcholine found in the chylomicrons.     

Effect of long chain polyunsaturated fatty acids in the sn-2 position of phosphatidylcholine on the interaction with recombinant high density lipoprotein apolipoprotein A-I

The effects of polyunsaturated fatty acids (PUFA) on the structure of recombinant high density lipoprotein (rHDL) was investigated using homogeneous particles containing phosphatidylcholine (PC), [3H]cholesterol, and apolipoprotein A-I (apoA-I). The PC component of the rHDL contained sn -1 16:0 and sn -2 18:1 (POPC), 18:2 (PLPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC). The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.57-1.70 m) compared to those containing POPC (2.83 m). Intrinsic apoA-I tryptophan fluorescence emission intensity and lifetimes were decreased for rHDL containing long chain PUFA compared to POPC and PLPC rHDL. Monoclonal antibody binding studies demonstrated that apoA-I had decreased immunoreactivity with monoclonal antibodies spanning amino acid residues 115-147 in rHDL containing long chain PUFA. PC lipid fluidity, measured as diphenylhexatriene (DPH) fluorescence polarization, was increased in PUFA rHDL compared to POPC rHDL. There also was a strong correlation between the number of sn -2 double bonds in rHDL and DPH fluorescence lifetime (r 2 = 0. 89). LCAT reactivity of the homogeneous size rHDL was ordered POPC = PLPC>PAPC> PEPC>PDPC. We conclude that rHDL with long chain PUFA in the sn -2 position of PC contain apoA-I that is less stable and in a different conformation than that in POPC rHDL and have a fatty acyl region that is more fluid and hydrated. The weaker interaction of apoA-I with PC containing PUFA may lead to hypercatabolism of apoA-I in plasma explaining, in part, the decreased plasma HDL and apoA-I concentrations seen with PUFA diets.     

Polyunsaturated fatty acids exert antiarrhythmic actions as free acids rather than in phospholipids

Previous studies have shown that exogenous free n-3 polyunsaturated fatty acids (PUFA) can prevent tachyarrhythmias caused by specific agents in isolated cardiac myocytes. However, the question as to whether incorporation of the n-3 PUFA into membrane phospholipids has the same immediate protective effects remained to be answered. To answer this question, we increased the content of n-3 PUFA in the phospholipids of cultured neonatal rat myocytes by growing them 2-3 d in a culture to which eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in 15 microM concentration was added. Analysis of the fatty acid composition of membrane phospholipids revealed a significantly higher level of EPA and DHA (from 0.2 to 7.6% and from 1.2 to 6.5%) in cells supplemented with EPA or DHA, respectively. The responses of the myocytes grown in normal media or in media enriched with the PUFA to arrhythmogenic agents were examined after free fatty acids were removed from the medium and the cells. The arrhythmogenic agents used were the beta-adrenergic agonist isoproterenol or an elevated extracellular concentration of calcium. The results showed that there was no significant difference in the induction of tachyarrhythmias by isoproterenol or by elevated [Ca2+]o in cells grown in media enriched with PUFA, as compared with cells grown in normal media in the absence of the free PUFA. Under the conditions of this study, only the unesterified PUFA were able to protect the cardiomyocytes against induced arrhythmias. There was no antiarrhythmic effect due to an increased fraction of EPA or DHA in membrane phospholipids.     

Fatty acid composition of human brain phospholipids during normal development

The fatty acid composition of phosphatidylethanolamine (PE), ethanolamine plasmalogens (EPs), phosphatidylserine (PS), phosphatidylcholine (PC), and sphingomyelin was studied in 22 human forebrains, ranging in age from 26 prenatal weeks to 8 postnatal years. Phospholipids were separated by two-dimensional TLC, and the fatty acid methyl esters studied by capillary column GLC. Docosahexaenoic acid (22:6n-3) increased with age in PE and PC, whereas arachidonic acid (20:4n-6) remained quite constant. In EP, 22:6n-3 increased less markedly than 20:4n-6, adrenic (22:4n-6) and oleic (18:1n-9) acids being the predominant fatty acids during postnatal age. In PS, 18:1n-9 increased dramatically throughout development, and 20:4n-6 and 22:4n-6 increased only until approximately 6 months of age. Although 22:6n-3 kept quite constant during development in PS, its percentage decreased due to the accretion of other polyunsaturated fatty acids (PUFAs). As a characteristic myelin lipid, sphingomyelin was mainly constituted by very long chain saturated and monounsaturated fatty acids. Among them, nervonic acid (24:1n-9) was the major very long chain fatty acid in Sp, followed by 24:0, 26:1n-9, and 26:0, and its accretion after birth was dramatic. As myelination advanced, 18:1n-9 increased markedly in all four glycerophospholipids, predominating in EP, PS, and PC. In contrast, 22:6n-3 was the most important PUFA in PE in the mature forebrain.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [14C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [14C] 18:1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (< or = 1.0 per cent; v/v) had no effect. At concentrations greater than 1.5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [3H]choline or [14C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (> or = 1.5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40-50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [14C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18:1 and PL-base, the ratios of incorporation (base/18:1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [14C]ethanolamine into [14C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Binocular processes in vernier acuity

The contribution of synthesis and dietary sequestration to the high arachidonate content of the lone star tick, Amblyomma americanum, salivary glands was investigated by assessing the salivary metabolites of various radiolabeled fatty acid substrates administered to partially fed females. A portion of each of the fatty acids studied was incorporated into the fatty acid moiety of the phospholipid fraction. [14C]acetate was metabolized only into myristic, palmitic, palmitoleic, steric, and oleic acids. [3H]oleic acid, [14C]linoleic acid, [14C]gamma-linolenic acid and [14C]eicosatrienoic acids were incorporated into salivary gland phospholipids but underwent little change including elongation and/or desaturation to arachidonate. Ingested [3H]arachidonic acid was readily taken up by the salivary gland and distributed among the lipid classes in a pattern markedly different from that of the other fatty acids tested. We conclude that ticks are unable to synthesize arachidonic acid for incorporation into the salivary glands, but rather sequester it from the host bloodmeal.     

Incorporation of exogenous fatty acids into molecular species of rat hepatocyte phosphatidylcholine

Freshly isolated rat hepatocytes were incubated for 20 and 60 min with [U-14C]glycerol and unlabeled palmitic (16:0), oleic (18:1), or arachidonic (20:4) acid, added as albumin complex in 10% ethanol. Each fatty acid increased glycerol incorporation into total lipids by a factor of 8-10 over control, whereas ethanol alone (final concentration 100 mM) yielded a threefold increase of glycerol uptake. Glycerol incorporation stopped after 20 min and cellular acyl turnover continued in the absence of useable labeled substrate. In each case, radioactivity recovered in hepatocyte lipids was present primarily in triacylglycerol (37-64%), phosphatidylcholine (22-37%), and phosphatidylethanolamine (10-22%). Separation by high-performance liquid chromatography of the diacylglycerol dinitrobenzoates derived from phosphatidylcholine showed that the molecular species had drastically different labeling patterns in the presence of the exogenous fatty acids, whereas the pattern obtained in the presence of ethanol alone was virtually the same as that for the control incubations. The labeling patterns indicated that exogenous fatty acids, including arachidonic acid, were incorporated into phosphatidylcholine primarily by the de novo pathway yielding highly labeled species with the exogenous fatty acid esterified at both the sn-1 and sn-2 positions of glycerol. After 20 min incubation with arachidonic acid, the 20:4-20:4 phosphatidylcholine contained about one-half of the [U-14C]glycerol label recovered in this lipid class. The data also showed that newly synthesized molecular species were extensively remodeled within 1 h.     

Influence of nicotine and cotinine on retinal phospholipase A2 and its significance to macular function

The macula is a constituent of the sensory retina that is necessary for sharp contrast and color vision. A significant relationship has been found between tobacco smoking and age-related macular degeneration. Opsin, rhodopsin and phospholipase A2 (PLA2) are located in excitable membranes of retina which are enriched with polyunsaturated fatty acids (PUFA). A question may arise as to whether nicotine and its major metabolite cotinine influence PLA2 so that arachidonic acid (AA) and proinflammatory prostaglandins (PG) are produced in the retina. Therefore, the effects of nicotine and cotinine on the retinal PLA2 were studied. PLA2 activity of rat retinal sonicates was assayed using 1-palmitoyl-2[1-14C]arachidonyl-Phosphatidylethanolamine (PE, 2.2 nmol) as a substrate in Tris buffer (10 mM, pH 7.4) at 37 degrees C with and without nicotine or cotinine in the assay medium. These studies gave the following results: (1) Rat retinal PLA2 activity was 4.2+/-0.8 pmol PE hydrolyzed/100 ng protein/hr. (2) Nicotine in low concentrations (1-150 nM) activated PLA2 (EC50 5 nM). (3) Cotinine also activated PLA2 (EC50 300 nM). (4) Only high concentrations of nicotine (> 1.0 microM) and cotinine (> 25 microM) exhibit inhibition of PLA2. (5) All three known PLA2 inhibitors, mepacrine, 4-bromophenacyl bromide and bromoacetylcholine bromide (IC50: 0.5mM, 88 microM, 30 mM, respectively) inhibited retinal PLA2 activity. These observations suggest that polyunsaturated fatty acids are cleaved, and arachidonic acid, the precursor for prostaglandins and related pro-inflammatory mediators, is liberated by nicotine and cotinine. Oxidative stress (reduced levels of antioxidants), vascular insufficiency, as well as activation of PLA2 by nicotine and cotinine may contribute for retinal degeneration in smokers during aging.     

Study of mechanisms involved in the collision-induced dissociation of carboxylate anions from glycerophospholipids using negative ion electrospray tandem quadrupole mass spectrometry

The collision-induced dissociation of the carboxylate anions from human blood phosphatdilycholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidic acid (PA) containing the C18:0 (sn-1) and C20:4 (sn-2) fatty acyl residues was studied using normal phase liquid chromatography coupled with negative ion electrospray tandem mass spectrometry. The product ion peak area ratio of C18:0 to C20:4 was calculated for each phospholipid species and was found to increase with increasing collision energy for all classes. For the phospholipids with a net neutral charge (PE, PC) there was a preferential loss of the sn-2 carboxylate anion (C20:4) at low collision energy, while at higher energy there was a preferential loss of the sn-1 carboxylate anion (C18:0). For the phospholipids with a net negative charge (PI, PA, PS) the intensity of the sn-1 carboxylate anion peak was equal to or higher than the sn-2 carboxylate anion peak at the energies measured. At a given collision energy the product ion peak area ratio decreased in the order PA > or = PS > PI. Studying PS and PE species at different collision energies, it was found that for both classes the increase in the abundance ratio with increasing collision energy was largely dependent on the chain length and degree of unsaturation of the sn-2 acyl chain.     

Serum phospholipid fatty acid composition and habitual intake of marine foods registered by a semi-quantitative food frequency questionnaire

OBJECTIVE: To examine the relation between consumption of fish and fish products registered by a comprehensive food frequency questionnaire and the composition of fatty acids in serum phospholipids. DESIGN: Cross-section study. SETTING: Cardiovascular screening centre in Trondheim, Mid-Norway. SUBJECTS: Of 256 eligible women 242 agreed to participate in the present study. Altogether 234 middle-aged women (91.4%) completed the questionnaire and gave a valid blood sample. RESULTS: Total frequency consumption of fish for dinner showed only weak association with serum phospholipid fatty acid composition. In separate analyses of lean and fatty fish, consumption of fatty fish was negatively associated with n-6 and positively associated with n-3 fatty acids in serum phospholipids, while no significant associations were found for lean fish consumption. Cod liver oil consumption was strongly related to the phospholipid fatty acid composition. The associations improved moderately when adding portion size information. Spearman's correlation coefficient between dietary intake of eicosapentaenoic acid (EPA) and serum phospholipid EPA was 0.58, and Spearman's correlation coefficient between intake of docosahexaenoic acid (DHA) and serum phospholipid DHA was 0.53. CONCLUSIONS: This study suggests that in populations with a high consumption of fish and cod liver oil, habitual intake can be reflected in serum phospholipids. However, as the fat content of fish is highly variable, separate registration of lean and fatty fish consumption is needed.     

Ratios of carbon isotopes in microbial lipids as an indicator of substrate usage

The occurrence and abundance of microbial fatty acids have been used for the identification of microorganisms in microbial communities. However, these fatty acids can also be used as indicators of substrate usage. For this, a systematic investigation of the discrimination of the stable carbon isotopes by different microorganisms is necessary. We grew 11 strains representing major bacterial and fungal species with four different isotopically defined carbon sources and determined the isotope ratios of fatty acids of different lipid fractions. A comparison of the differences of delta13C values of palmitic acid (C16:0) with the delta13C values of the substrates revealed that the isotope ratio is independent of the growth stage and that most microorganisms showed enrichment of C16:0 with 13C when growing on glycerol. With the exception of Burkholderia gladioli, all microorganism showed depletion of 13C in C16:0 while incorporating the carbons of glucose, and most of them were enriched with 13C from mannose, with the exception of Pseudomonas fluorescens and the Zygomycotina. Usually, the glycolipid fractions are depleted in 13C compared to the phospholipid fractions. The delta13C pattern was not uniform within the different fatty acids of a given microbial species. Generally, tetradecanoic acid (C14:0) was depleted of 13C compared to palmitic acid (C16:0) while octadecanoic acid (C18:0) was enriched. These results are important for the calibration of a new method in which delta13C values of fatty acids from the environment delineate the use of bacterial substrates in an ecosystem.