Sn-2 Fatty Acid Research Articles

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.

Novel lipids in Myxococcus xanthus and their role in chemotaxis

Organisms that colonize solid surfaces, like Myxococcus xanthus, use novel signalling systems to organize multicellular behaviour. Phosphatidylethanolamine (PE) containing the fatty acid 16:1omega5 (Delta11) elicits a chemotactic response. The phenomenon was examined by observing the effects of PE species with varying fatty acid pairings. Wild-type M. xanthus contains 17 different PE species under vegetative conditions and 19 at the midpoint of development; 13 of the 17 have an unsaturated fatty acid at the sn-1 position, a novelty among Proteobacteria. Myxococcus xanthus has two glycerol-3-phosphate acyltransferase (PlsB) homologues which add the sn-1 fatty acid. Each produces PE with 16:1 at the sn-1 position and supports growth and fruiting body development. Deletion of plsB1 (MXAN3288) results in more dramatic changes in PE species distribution than deletion of plsB2 (MXAN1675). PlsB2 has a putative N-terminal eukaryotic fatty acid reductase domain and may support both ether lipid synthesis and PE synthesis. Disruption of a single sn-2 acyltransferase homologue (PlsC, of which M. xanthus contains five) results in minor changes in membrane PE. Derivatization of purified PE extracts with dimethyldisulfide was used to determine the position of the double bonds in unsaturated fatty acids. The results suggest that Delta5 and Delta11 desaturases may create the double bonds after synthesis of the fatty acid. Phosphatidylethanolamine enriched for 16:1 at the sn-1 position stimulates chemotaxis more strongly than PE with 16:1 enriched at the sn-2 position. It appears that the deployment of a rare fatty acid (16:1omega5) at an unusual position (sn-1) has facilitated the evolution of a novel cell signal.

A Role for the Pregnane X Receptor in High-Density Lipoprotein Metabolism

High-density lipoprotein (HDL) cholesterol is widely recognized as the good plasma cholesterol for coronary heart disease and is believed to be responsible for cholesterol transport from peripheral tissues back to the liver, the central clearing house for cholesterol in the body. Interestingly, the liver is also a major site for HDL production, although the process of particle assembly is believed to occur intravascularly.1–4 ApoA-I is secreted by the liver in lipid-poor form and then interacts with ATP-binding cassette protein A1 (ABCA1) in liver and peripheral tissues to acquire phospholipids and cholesterol, forming a discoidal HDL intermediate sometimes referred to as pre–β-HDL. These particles are excellent substrates for the liver-derived plasma enzyme lecithin:cholesterol acyltransferase. Using the sn-2 fatty acids of the particle-associated phosphatidylcholine, lecithin:cholesterol acyltransferase catalyzes the esterification of cholesterol, which is partially derived from peripheral tissues. This reaction produces the cholesteryl esters (CE) that form the neutral lipid core of the mature spherical HDL particles. In addition to whole HDL particle uptake by the liver, the CE of mature HDL can also be removed by the liver through a process of selective CE uptake via scavenger receptor-class B type I (SR-BI). Both uptake mechanisms result in a directional flux of cholesterol from the plasma compartment to …

Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine

We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.

Roles of C-Terminal Processing, and Involvement in Transacylation Reaction of Human Group IVC Phospholipase A2 (cPLA2γ)

The phospholipase A2s (PLA2s) are a diverse group of enzymes that hydrolyze the sn-2 fatty acid from phospholipids and play a role in a wide range of physiological functions. A 61-kDa calcium-independent PLA2, termed cPLA2gamma, was identified as an ortholog of cPLA2alpha with approximately 30% overall sequence identity. cPLA2gamma contains a potential prenylation motif at its C terminus, and is known to have PLA2 and lysophospholipase activities, but its physiological roles have not been clarified. In the present study, we expressed various forms of recombinant cPLA2gamma, including non-prenylated and non-cleaved forms, in order to investigate the effects of C-terminal processing. We examined the expression of the wild type and non-prenylated (SCLA) forms of cPLA2gamma, and found that the SCLA form was expressed normally and retained almost full activity. Expression of the prenylated and non-cleaved form of cPLA2gamma using yeast mutants lacking prenyl protein proteases AFC1 (a-factor-converting enzyme) and RCE1 (Ras-converting enzyme) revealed decreased expression in the mutant strain compared to that in the wild type yeast, suggesting that complete C-terminal processing is important for the functional expression of cPLA2gamma. In addition, cPLA2gamma was found to have coenzyme A (CoA)-independent transacylation and lysophospholipid (LPL) dismutase (LPLase/transacylase) activities, suggesting that it may be involved in fatty acid remodeling of phospholipids and the clearance of toxic lysophospholipids in cells.

Effect of Supplementation with Docosahexaenoic Acid Ethyl Ester and sn-2 Docosahexaenyl Monoacylglyceride on Plasma and Erythrocyte Fatty Acids in Rats

Background/Aims: Docosahexaenoic acid (C22:6, DHA) is an omega-3 fatty acid required for the normal development of the mammalian nervous and visual system. DHA is provided by the mother during pregnancy and lactating period. Mother’s DHA supplementation during pregnancy, and even before pregnancy, has been suggested. DHA can be provided by marine oils, egg’s yolk phospholipids, single cell algae oils, the pure fatty acid, or by the ethyl ester derivative (DHA-EE). Another way to provide DHA can be by sn-2 docosahexaenyl monoacylglyceride (DHA-MG), obtained by the treatment of fish oil with stereospecific lipases. sn-2 Fatty acid monoacylglycerides can be more easily absorbed at the intestine than other fatty acid derivatives. Methods: Female rats fed with a synthetic, which provided essentially no DHA, received a 40-day supplementation of either DHA-EE or DHA-MG. Plasma and erythrocyte fatty acid composition were assessed by gas chromatography at day 0 and 40 of supplementation. Results: DHA-EE increased plasma and erythrocyte DHA by 15 and 11.9%, respectively, with no modification of arachidonic acid (AA) content. DHA-MG supplementation increased plasma and erythrocyte DHA by 24 and 23.8%, respectively, but reduced AA by 5.5 and 3%, respectively. Conclusions: We conclude that in the rat, DHA-MG supplementation allows a higher plasma and erythrocyte DHA content than DHA-EE with minor modification of AA content.

Studies on the endogenous phospholipids of mammalian kidney and their in vitro hydrolysis by endogenous phospholipases: a thin layer chromatographic and densitometric study.

The phosphoglycerides profile of six species of mammalian kidney (guinea pig, pig, cat, dog, mouse and rat) and their in vitro response to the endogenous phospholipases were determined by TLC technology in conjunction with densitometric measurements. Changes in their phospholipids profile subsequent to in vitro incubation of whole tissue homogenate of these kidneys for 60 min, at pH 7.4, 38 degrees C, and prior to phospholipids extraction have shown that the deacylation of the endogenous cardiolipin (CL) is the most prevalent lipolytic event of all mammalian kidneys studied. Concurrent with the deacylation of CL, there was also formation of monolysocardiolipin (MLCL) and a reduction in CL level. To a much lesser extent, lyso alkenyl phosphatidyl ethanolamine (LPE) was also produced concomitant with a decrease of the endogenous alkenyl phosphatidyl ethanolamine (PE) level. The deacylation of PE plasmalogen to its lyso form confirms the action of endogenous PLA(2) releasing sn-2 fatty acids.

The determination of the asymmetrical stereochemical distribution of fatty acids in triacylglycerols

The fatty acid distribution in triacylglycerols (TAGs) is a factor that contributes to the intrinsic properties of oils from different species variants. Many hypotheses have been proposed to explain the specific distribution of fatty acids in the different naturally occurring oils. Currently, the 1,3-random-2-random theory is more or less accepted, but it has been widely shown that most vegetable oils do not behave randomly in the sn-1 and sn-3 stereochemical positions. For this reason, complex methodologies have been developed to analyze the fatty acid composition of the three stereochemical positions in TAGs. In this article, we propose that by calculating the asymmetric α coefficient, the stereochemical asymmetry of fatty acids in TAG molecular species can be defined. This coefficient reflects the relative content of fatty acids at the sn-1 and sn-3 positions and may overcome the problems found mainly with complex sn-1, sn-2, and sn-3 stereochemical analysis of fatty acids in TAG. The α coefficient is calculated from the fatty acid, sn-2 fatty acid, and TAG composition of the oil. Indeed, through this coefficient, it has been possible to show that, despite having the same overall content, the stearic acid distribution in the sn-1 and sn-3 positions is not random in some oils.

Catalytic features, regulation and function of myocardial phospholipase A2.

Phospholipase A2 (PLA2) catalyzes the hydrolysis of sn-2 fatty acids from membrane phospholipids resulting in the production of several biologically active phospholipid metabolites such as lysophospholipids, arachidonic acid, eicosanoids and platelet-activating factor. The majority of myocardial PLA2 activity is membrane-associated and does not require Ca2+ for activity (iPLA2). Myocardial iPLA2 demonstrates unique characteristics when compared to other PLA2 isoforms described previously, including a selectivity for plasmalogen phospholipids and resistance to inhibition by methyl arachidonyl fluorophosphonate. Activation of myocardial iPLA2 results in the production of lysoplasmenylcholine and arachidonic acid, both of which can change the electrophysiologic properties of the myocardium. Arachidonic acid can modulate ion channel activity via protein kinase C activation and has been demonstrated to decrease gap junctional conductance. Lysoplasmenylcholine directly produces action potential derangements and alters calcium cycling in cardiac myocytes. Thus, inhibition of iPLA2 activity to block production of phospholipid metabolites that mediate pathologic changes in the myocardium would be of considerable benefit. However, there are situations where inhibition of PLA2 activity would be detrimental to the myocardium, in particular if iPLA2 acts as a phospholipid repair enzyme following oxidative damage. Although little is known regarding the function of cPLA2 or sPLA2 in the myocardium, it is possible that they may be important for signal transduction or may modulate the activity of iPLA2.

Identification by electrospray tandem mass spectrometry of spin-trapped free radicals from oxidized 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine.

GPC radical species formed during oxidation of a glycerophosphocholine (16:0/18:1) under the Fenton reaction conditions were detected using a spin trap, 5,5-dimethyl-1-pyrrolidine N-oxide (DMPO). The stable spin-trapped radical adducts were identified by mass spectrometry (MS) using electrospray (ES) as ionization method and characterized by tandem mass spectrometry (MS/MS). Radical adducts of oxidized free sn-2 fatty acid and of oxidized intact GPC, containing one, two and three additional oxygen atoms, were assigned. DMPO adducts of oxidized intact GPC were observed as singly and doubly charged ions in ES-MS, while adducts of oxidized free fatty acids were observed as singly charged ions. Oxidized free sn-2 fatty acids and intact GPC-DMPO adducts correspond to carbon- and oxygen-centered radicals that were identified by MS/MS as alkyl, hydroxy-alkyl, alkoxyl, hydroxy-alkoxyl, peroxyl and hydroperoxide-alkoxyl spin adducts. The DMPO molecule was attached predominantly at C(9) of the oleic chain. The fragmentation pathway of spin adducts with two DMPO molecules strongly suggests the presence of species that were simultaneously carbon- and oxygen-centered radicals. Several fragments identified are consistent with the presence of isomeric structures contributing to the same ions.

Substrate preference in phosphatidylserine biosynthesis for docosahexaenoic acid containing species.

Neuronal membranes contain high levels of phosphatidylserine (PS) and docosahexaenoic acid (22:6n-3, DHA). In this study, substrate preference in PS synthesis was determined to gain insight on the biochemical basis for concentrating PS in neuronal membranes where 22:6n-3 is highly enriched. We first established an in vitro assay method using unilamellar vesicles (LUV) of deuterium-labeled substrates and reversed-phase HPLC/electrospray ionization (ESI) mass spectrometry. The PS production by the incubation of deuterium-labeled substrate and microsomal fractions was monitored. We found that tissue-specific substrate preference exists in PS synthesis. Microsomes from the cerebral cortex synthesized PS from 18:0,22:6-PC most favorably among the PC substrates tested, followed by 18:0,22:5-PC, resulting in the PC substrate preference in the order of 18:0,22:6 > 18:0,22:5 > 18:0,20:4 = 18:0,18:1. Liver microsomes also preferred 18:0,22:6-PC as the substrate in PS synthesis but did not use 18:0,22:5-PC favorably. The 18:0,22:5-PC species was converted to PS at the similar extent as 18:0,20:4- or 18:0,18:1-PC species in the liver. Both brain and liver microsomes showed a preference for 18:0 over 16:0 as the sn-1 fatty acid. From these data it was deduced that preferential conversion of 18:0,22:6-PC to the corresponding PS species is at least partly responsible for concentrating PS in neuronal tissues where 22:6n-3 is particularly abundant. The distinctive preference for 18:0,22:5-PS observed with brain microsomes may help to maintain PS at a high level in the brain when 22:6n-3 is replaced by 22:5n-3 as in the case of n-3 fatty acid deficiency.

Assaying phospholipase A2 activity.

Mammalian cells contain many structurally and functionally diverse phospholipases A2 (PLA2) that catalyze the hydrolysis of sn-2 fatty acid from membrane phospholipid. Assays are described for measuring the activity of Group IVA cytosolic PLA2alpha(cPLAalpha) and for secreted PLA2s (sPLA2) that are suitable for purified enzymes and for measuring activity in crude cell lysates and culture medium. The assay for cPLA2alpha involves measuring the calcium-dependent release of radiolabeled sn-2 arachidonic acid from small unilamellar vesicles of phosphatidylcholine. Methods are described for distinguishing cPLA2alpha activity in cell lysates from other PLA2s. sPLA2 activity is monitored using a fluorimetric assay that measures the continuous calcium-dependent formation of albumin-bound pyrene fatty acid from the sn-2 position of phosphatidylglycerol.

Age-related changes of the endogenous cardiolipin and plasmalogens of guinea pig kidney and their in vitro hydrolysis by endogenous phospholipases: a thin layer chromatographic analysis in conjunction with densitometric measurement.

The phosphoglycerides profile of guinea pig kidney, fetal, young adult, and aged, and their in vitro response to the endogenous lipolytic enzymes, mainly in the phospholipase group were determined by TLC technology in conjunction with densitometric measurement. Changes in phosphoglycerides profile subsequent to in vitro incubation of these tissues at pH 7.4, and 38 degrees C for 45 min and prior to phospholipid extraction has provided evidence relating to their respective lipolytic enzymes capabilities and age. These changes are mainly related to endogenous cardiolipin (CL), alkenyl phospholipids (phosphatidyl ethanolamine and phosphatidyl choline) and their endogenous deacylation to their respective lyso derivatives monolysocardiolipin (MLCL), lyso alkenyl phosphatidyl ethanolamine (LPE), and lyso alkenyl phosphatidyl choline (LPC) by endogenous phospholipases. The hydrolysis of the plasmalogen confirms the action of endogenous PLA(2) on sn-2 fatty acids of these compounds.

Structural analysis of phosphatidylcholines by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The utility of post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was investigated for the structural analysis of phosphatidylcholine (PC). PC did not produce detectable negative molecular ion from MALDI, but positive ions were observed as both [PC+H] + and [PC+Na] +. The PSD spectra of the protonated PC species contained only one fragment corresponding to the head group ( m/ z 184), while the sodiated precursors produced many fragment ions, including those derived from the loss of fatty acids. The loss of fatty acid from the C-1 position ( sn-1) of the glycerol backbone was favored over the loss of fatty acid from the C-2 position ( sn-2). Ions emanating from the fragmentation of the head group (phosphocholine) included [PC+Na-59] +, [PC+Na-183] + and [PC+Na-205] +, which corresponded to the loss of trimethylamine (TMA), non-sodiated choline phosphate and sodiated choline phosphate, respectively. Other fragments reflecting the structure of the head group were observed at m/ z 183, 146 and 86. The difference in the fragmentation patterns for the PSD of [PC+Na] + compared to [PC+H] + is attributed to difference in the binding of Na + and H +. While the proton binds to a negatively charged oxygen of the phosphate group, the sodium ion can be associated with several regions of the PC molecule. Hence, in the sodiated PC, intermolecular interaction of the negatively charged oxygen of the phosphate group, along with sodium association at multiple sites, can lead to a complex and characteristic ion fragmentation pattern. The preferential loss of sn-1 fatty acid group could be explained by the formation of an energetically favorable six-member ring intermediate, as apposed to the five-member ring intermediate formed prior to the loss of sn-2 fatty acid group.

Biological functions of group X secretory PLA2.

Secretory phospholipase A2s (sPLA2s) are a diverse family of low molecular mass enzymes that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. In addition to the classical types of group IB and IIA (sPLA2-IB and IIA), recent advances in molecular biology have led to the identification of novel types of sPLA2s, including group IID and IIE that we have cloned [1]. Among the nine types of human sPLA2s, group X sPLA2 (sPLA2-X) has a structural similarity with the classical type of sPLA2-IB and IIA [2]. We have shown that sPLA2-X has a potent hydrolyzing activity toward phosphatidylcholine (PC) and elicits more potent production of unsaturated fatty acids and lysophosphatidylcholine (lysoPC) in various inflammatory cells and colon cancer cells compared with sPLA2-IB and IIA [3,4]. The released arachidonic acid by sPLA2-X was efficiently converted to prostaglandins (PGs) by the action of endogenous cyclooxygenase (COX). The expression of sPLA2-X was detected in human lung epithelial cells and mouse splenic macrophages, and greatly augmented in human colon adenocarcinoma tissues in concert with enhanced expression of COX-2 [4]. These findings suggest that sPLA2-X plays a critical role in the lipid mediator productions under various inflammatory states and in the COX-2- dependent PGE2 biosynthesis during colon tumorigenesis. Recently, we have shown that sPLA2-X is a high-affinity ligand for mouse PLA2 receptor (PLA2R) and identified a soluble PLA2R in mouse plasma [5]. Using PLA2 receptor-deficient mice, we have shown that the membrane and soluble form of the receptor are involved in the endogenous inhibitory mechanisms against the strong PC-hydrolizing activity of sPLA2-X in mice [5].

Positional distribution of fatty acids on the glycerol backbone during the biosynthesis of glycerolipids in Ectocarpus fasciculatus

The biosynthesis of glycolipids in E. fasciculatus was studied by 14C label and chase. The fatty acids in sulphoquinovosyl diacylglycerol (SQDG) were almost 16-carbon and 18-carbon ones. In addition to the two fatty acids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained 8.5 mol% and 31.0 mol% of eicosapentaenoic acid (20: 5), respectively, and this fatty acid was usually distributed in the sn-1 position of the glycerol backbone. When plants were incubated with [2−14C] acetate, differences existed in the positional distribution of the labeled fatty acids in sn-1 and sn-2 among the three glycerolipids. In SQDG., 14C-labeled fatty acids were distributed uniformly in the sn-1 and sn-2 positions. In DGDG., 14C-labeled fatty acids were mainly distributed in the sn-2 position. In MGDG., the radioactivity of fatty acids in sn-1 position was far greater than that in sn-2 position after a 30 min pulse label, and the difference in radioactivity between the two positions decreased rapidly. The above results indicated that differences in the positional distribution of 14C-labeled fatty acids between sn-1 and sn-2 positions might be related to 20: 5 and the biosynthesis of DGDG. Our results also suggested that E. fasciculatus had the same DGDG biosynthetic pathway as that in higher plants and galactosyl transferase was selective for MGDG.

Identification of alterations in gene expression in rat recipients with long-term–surviving cardiac grafts

Various human tissues and cells express phospholipase A1 member A (PLA1A), including the liver, lung, prostate gland, and immune cells. The enzyme belongs to the pancreatic lipase family. PLA1A specifically hydrolyzes sn-1 fatty acid of phosphatidylserine (PS) or 1-acyl-lysophosphatidylserine (1-acyl-lysoPS). PS externalized by activated cells or apoptotic cells or extracellular vesicles is a potential source of substrate for the production of unsaturated lysoPS species by PLA1A. Maturation and functions of many immune cells, such as T cells, dendritic cells, macrophages, and mast cells, can be regulated by PLA1A and lysoPS. Several lysoPS receptors, including GPR34, GPR174 and P2Y10, have been identified. High serum levels and high PLA1A expression are associated with autoimmune disorders such as Graves' disease and systemic lupus erythematosus. Increased expression of PLA1A is associated with metastatic melanomas. PLA1A may contribute to cardiometabolic disorders through mediating cholesterol transportation and producing lysoPS. Furthermore, PLA1A is necessary for hepatitis C virus assembly and can play a role in the antivirus innate immune response. This review summarizes recent findings on PLA1A expression, lysoPS and lysoPS receptors in autoimmune disorders, cancers, cardiometabolic disorders, antivirus immune responses, as well as regulations of immune cells.

Comparative composition of total and sn-2 fatty acids in bovine and ovine milk fat

Similar diets were fed to cattle and sheep to compare the total and the sn-2 fatty acid (FA) composition of milk fat. The study confirmed that substrate availability in the bovine and ovine mam- mary gland is a determining factor in the sn-2 FA composition. The acyltransferase that esterifies the FA at the sn-2 position is specific for FA of chain length C14 to C18 and has a reduced affinity for short chain FA in cattle and sheep. fatty acid / sn-2 / triglyceride composition / lysophosphatidic acid acyltransferase

Epoxyisoprostane and Epoxycyclopentenone Phospholipids Regulate Monocyte Chemotactic Protein-1 and Interleukin-8 Synthesis: FORMATION OF THESE OXIDIZED PHOSPHOLIPIDS IN RESPONSE TO INTERLEUKIN-1β

Monocyte recruitment to the vessel wall, mediated by monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8), plays an important role in atherogenesis. We have shown previously that minimally oxidized low density lipoprotein, oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), activates endothelial cells to produce MCP-1 and IL-8. By using liquid chromatography/mass spectrometry methods coupled with bioassay, we report a family of epoxyisoprostane (PEIPC) and epoxycyclopentenone (PECPC) phospholipids that are the components of Ox-PAPC responsible for the majority of this activity. Ox-PAPC contains five chromatographically distinguishable active PEIPC components (m/z 825.5) and four PECPC components (m/z 810.5). All nine components induced endothelial cell synthesis of IL-8 and MCP-1 in a dose-dependent fashion between 0.1 and 5 microm concentrations. The five PEIPC components had identical functional groups and all underwent dehydration to produce m/z 810.5. We present evidence that these phospholipids are regioisomers with epoxide groups at the 5,6-, 8,9-, 11,12-, or 14,15-positions of the sn-2 fatty acid and their epoxide groups is important for biological activity. We have shown previously that peroxisome proliferator-activated receptor alpha is involved in MCP-1 synthesis in response to Ox-PAPC. We now show that PEIPC and PECPC isomers are potent activators of peroxisome proliferator-activated receptor alpha. PEIPC and PECPC isomers are strongly recognized by specific circulating murine natural autoantibodies (EO6) and accumulate in cells treated with IL-1beta. These studies demonstrate that PEIPC and PECPC isomers are potent activators of endothelial cells increasing synthesis of IL-8 and MCP-1. Their accumulation in cells exposed to cytokines and in atherosclerotic lesions suggests that these lipids may play a role in a number of chronic disease processes.

Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.