Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry

Triacylglycerols (TAGs) are neutral lipids present in all mammalian cells as energy reserves, and diacylglycerols (DAGs) are present as intermediates in phospholipid biosynthesis and as signaling molecules. The molecular species of TAGs and DAGs present in mammalian cells are quite complex, and previous investigations revealed multiple isobaric species having molecular weights at virtually every even mass between 600 and 900 Da, making it difficult to assess changes of individual molecular species after cell activation. A method has been developed, using tandem MS and neutral loss scanning, to quantitatively analyze changes in those glyceryl ester molecular species containing identical fatty acyl groups. This was carried out by neutral loss scanning of 18 common fatty acyl groups where the neutral loss corresponded to the free carboxylic acid plus NH(3). Deuterium-labeled internal standards were used to normalize the signal for each nominal [M+NH(4)](+) ion undergoing this neutral loss reaction. This method was applied in studies of TAGs in RAW 264.7 cells treated with the toll-like receptor 4 ligand Kdo(2)-lipid A. A 50:1-TAG containing 18:1 was found to increase significantly over a 24-h time course after Kdo(2)-lipid A exposure, whereas an isobaric 50:1-TAG containing 16:1 did not change relative to controls.     

Lipid profiling reveals arachidonate deficiency in RAW264.7 cells: Structural and functional implications

Glycerophospholipids containing arachidonic acid (20:4) serve as the precursors for an array of biologically active lipid mediators, most of which are produced by macrophages. We have applied mass spectrometry-based lipid profiling technology to evaluate the glycerophospholipid structure and composition of two macrophage populations, resident peritoneal macrophages and RAW264.7 cells, with regard to their potential for 20:4-based lipid mediator biosynthesis. Fatty acid analysis indicated that RAW264.7 cells were deficient in 20:4 (10 +/- 1 mol %) compared to peritoneal macrophages (26 +/- 1 mol %). Mass spectrometry of total glycerophospholipids demonstrated a marked difference in the distribution of lipid species, including reduced levels of 20:4-containing lipids, in RAW264.7 cells compared to peritoneal macrophages. Enrichment of RAW264.7 cells with 20:4 increased the fatty acid to 20 +/- 1 mol %. However, the distribution of the incorporated 20:4 remained different from that of peritoneal macrophages. RAW264.7 cells pretreated with granulocyte-macrophage colony stimulating factor followed by lipopolysaccharide and interferon-gamma mobilized similar quantities of 20:4 and produced similar amounts of prostaglandins as peritoneal macrophages treated with LPS alone. LPS treatment resulted in detectable changes in specific 20:4-containing glycerophospholipids in peritoneal cells, but not in RAW264.7 cells. 20:4-enriched RAW264.7 cells lost 88% of the incorporated fatty acid during the LPS incubation without additional prostaglandin synthesis. These results illustrate that large differences in glycerophospholipid composition may exist, even in closely related cell populations, and demonstrate the importance of interpreting the potential for lipid-mediator biosynthesis in the context of overall glycerophospholipid composition.     

Glycerolipid and cholesterol ester analyses in biological samples by mass spectrometry

Neutral lipids are a diverse family of hydrophobic biomolecules that have important roles in cellular biochemistry of all living species but have in common the property of charge neutrality. A large component of neutral lipids is the glycerolipids composed of triacylglycerols, diacylglycerols, and monoacylglycerols that can serve as cellular energy stores as well as signaling molecules. Another abundant lipid class in many cells is the cholesterol esters that are on one hand sterols and the other fatty acyl lipids, but in either case are neutral lipids involved in cholesterol homeostasis and transport in the blood. The analysis of these molecules in the context of lipidomics remains challenging because of their charge neutrality and the complex mixtures of molecular species present in cells. Various techniques have been used to ionize these neutral lipids prior to mass spectrometric analysis including electron ionization, atmospheric chemical ionization, electrospray ionization and matrix assisted laser desorption/ionization. Various approaches to deal with the complex mixture of molecular species have been developed including shotgun lipidomics and chromatographic-based separations such as gas chromatography, reversed phase liquid chromatography, and normal phase liquid chromatography. Several applications of these approaches are discussed. .     

Reconsideration of hydrophobic lipid distributions in lipoprotein particles

Lipoprotein particles are commonly known as micellar aggregates with hydrophobic lipids located within the core and amphipathic molecules in the surface. Using a new structural model for optimizing the distribution of hydrophobic lipids, namely triglyceride (TG) and cholesterol ester (CE) molecules, we reveal that particle size-dependent proportion of these 'core lipids' may locate in the surface of lipoprotein particles. The composition of the particles also strongly influences the actual molecular content of the surface. For example, in high-density lipoprotein (HDL) particles the percentage of CEs of all surface lipids is between 13% and 27% due to the high tendency of CEs to locate in the surface and the high concentration of CEs in the particles. Conversely, although the percentage of TG molecules in the surface of HDL particles is also high, approximately 60% as for CE, the percentage of TGs of all surface lipids is low, only up to 5%, because HDL particles have a low-TG concentration. These structural models provide an intuitive and coherent structural rationale for various metabolic cascades in lipoprotein metabolism with the catalytic enzyme action and molecular binding for transport proteins taking place at the surface of the particles.     

Simultaneous determination of molecular species of monoacylglycerols,diacylglycerols and triacylglycerols in human very-low-density lipoproteins by reversed-phase liquid chromatography

The aim of the present study was to investigate the applicability of a previously developed method for the analysis of triacylglycerol molecular species to the simultaneous determination of triacylglycerols, diacylglycerols and monoacylglycerols of human very-low-density lipoproteins (VLDL). Ten elderly women were recruited for the study. Blood was obtained in fasting conditions and VLDL were isolated by ultracentrifugation. Neutral lipids were separated by solid-phase extraction and were subsequently injected on a reversed-phase HPLC system, with an elution system composed of acetone in acetonitrile. The method allowed the separation of four monoacylglycerols, 18 diacylglycerols and 24 triacylglycerols, including the resolution of positional isomers of diacylglycerols. Monoacylglycerols were composed of oleic, linoleic, palmitic and stearic acids. The major diacylglycerols were 1,2-dilinoleoyl-glycerol and 1,3-dilinoleoyl-glycerol (14.24+/-1.02 and 17.93+/-1.42%, respectively). The main triacylglycerols quantified were dioleoyl-stearoyl-glycerol (OOS), oleoyl-dipalmitoyl-glycerol (OPP), trilinoleoyl-glycerol (LLL) and linoleoyl-distearoyl-glycerol (LSS), accounting for 11.25+/-2.15, 10.14+/-2.05, 9.35+/-2.30 and 8.56+/-1.56%, respectively. An inverse relationship between polarity and fatty acid disappearance from triacylglycerols (r(2)=0.82, P<0.05) and from diacylglycerols (r(2)=0.93, P<0.01) was discovered. In conclusion, the method allowed, for the first time, the easy, rapid and simultaneous determination in a single chromatogram of triacylglycerol, diacylglycerol and monoacylglycerol molecular species of human VLDL by reversed-phase HPLC.     

Cobalt carbonyl complexes as probes for alkyne-tagged lipids

Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a “tag” that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV_349nm. The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity “pull-down” strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection.     

Electrospray MS/MS reveals extensive and nonspecific oxidation of cholesterol esters in human peripheral vascular lesions

Although LDL is rendered proatherogenic by various experimental treatments (e.g., acetylation), the exact structural changes that drive LDL transformation in vivo remain enigmatic. Among the many hypothesized targets of oxidative modification are cholesterol esters (CE). This family of neutral lipids, which carries a highly unsaturated pool of fatty acyl groups, is the main component of both LDL particles and atherosclerotic plaques. Tandem mass spectrometry (MS/MS) was employed to reveal abundant and diverse oxidized CEs (oxCE), including novel oxidation products, within human peripheral vascular lesions. These oxCE species composed up to 40% of the total CE pool, with cholesteryl linoleate being oxidized to the greatest extent. Imaging mass spectrometry studies showed that oxCE was entirely confined within the plaque, along with unmodified CE and triacylglyceride (TAG). Interestingly, we found no evidence for TAG oxidation, although polyunsaturated species were abundant. Enzymatic oxidation of cholesteryl linoleate by 15-lipoxygenase (15-LO), an enzyme often invoked in CE oxidation, initially results in a regio- and stereospecific product. Analysis of intact cholesteryl hydroxyoctadecadienoate isomers in human atheromata revealed no regio- or stereospecificity, indicating 15-LO was either not a major source of oxCE or nonenzymatic processes had eroded any product specificity.     

Measurement of lysophospholipid acyltransferase activities using substrate competition

Lysophospholipid acyltransferases (LPATs) incorporate fatty acyl chains into phospholipids via a CoA-dependent mechanism and are important in remodeling phospholipids to generate the molecular species of phospholipids found in cells. These enzymes use one lysophospholipid and one acyl-CoA ester as substrates. Traditional enzyme activity assays engage a single substrate pair, whereas in vivo multiple molecular species exist. We describe here an alternative biochemical assay that provides a mixture of substrates presented to the microsomal extracts. Microsomal preparations from RAW 264.7 cells were used to compare traditional LPAT assays with data obtained using a dual substrate choice assay using six different lysophospholipids and eight different acyl-CoA esters. The complex mixture of newly synthesized phospholipid products was analyzed using LC-MS/MS. Both types of assays provided similar results, but the dual choice assay provided information about multiple fatty acyl chain incorporation into various phospholipid classes in a single reaction. Engineered suppression of LPCAT3 activity in RAW 264.7 cells was easily detected by the dual choice method. These findings demonstrate that this assay is both specific and sensitive and that it provides much richer biochemical detail than traditional assays.     

Molecular motions and thermotropic phase behavior of triacylglycerols and cholesteryl esters in herpesvirus-infected arterial smooth muscle cells: a deuterium nuclear magnetic resonance study.

The physical state of lipids in arterial smooth muscle cells (SMC) may contribute to lipid accumulation following injury. We have previously demonstrated that herpes simplex virus (HSV) infection alters the physical state of the neutral lipid accumulating in arterial SMC, as determined by differential scanning calorimetry (Biochem. J. 268 (1990) 693-697). To more precisely determine the molecular packing of neutral lipids in HSV-infected cells, the influence of HSV-infection on the thermotropic and phase-behavior of the lipids in intact arterial smooth muscle cells and in cell-free lipid extracts was evaluated using [2H]-NMR, employing U-[2H]-oleic acid incorporated into cells. Inspection of the [2H]-line-widths indicate that the lipid of HSV-infected cells exhibited more restricted motion or a greater chemical shift dispersity than lipids from uninfected cells, as evidenced by significant broadening of the -CD = CD- signals at 25 degrees C and 45 degrees C. Fatty acid compositional analysis of the neutral lipids of control and HSV-infected cells following C18:1 supplementation (an amount added similar to the NMR experiments) reveals that: (1) there is approximately 55-fold more triacylglycerols (TG) than cholesteryl esters (CE) in control cells and 40-fold more TG than CE in the HSV-infected cells; (2) HSV infection significantly increases the C18:1 content of CE, and C18:3 and C20:4 in TG; and (3) HSV-infection does not alter the ratio of TG to CE. These data support the hypothesis that the greater restriction of the neutral lipids in HSV-infected cells may be due to the rigidifying effects of C18:1 on lipid mobility. Thus, alterations in the physical state of neutral lipids in HSV-infected cells may lead to reduced CE hydrolysis which, in turn, may contribute to or exacerbate lipid accumulation.     

Daxx通过上调caveolin-1的表达介导Ox-LDL诱导巨噬细胞胆固醇蓄积和凋亡

为探讨Daxx对氧化型低密度脂蛋白(oxidized low-density lipoprotein,Ox-LDL)诱导巨噬细胞胆固醇蓄积和凋亡的介导作用及其可能的分子机制,用高效液相色谱法检测细胞内胆固醇含量,油红O染色观察细胞内脂滴的形成情况,流式细胞术和吖啶橙/溴化乙锭(AO/EB)染色法研究Ox-LDL对细胞凋亡的影响,Real time RT-PCR检测细胞内Daxx mRNA的表达水平,Western blot检测caveolin-1蛋白的表达,用特异性siRNA沉默Daxx在RAW264.7 细胞中的表达．Ox-LDL上调Daxx mRNA和caveolin-1的表达、增加细胞内胆固醇含量、促使RAW264.7细胞凋亡,用特异性siRNA干扰Daxx在RAW264.7细胞中的表达能降低caveolin-1的表达、减少细胞内胆固醇含量、以及抑制细胞凋亡．上述结果表明,Daxx对Ox-LDL诱导RAW264.7巨噬细胞胆固醇蓄积和凋亡具有介导作用,这一作用可能与Daxx上调caveolin -1的表达有关．     

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape^TM from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.     

Cytosolic delivery of macromolecules: I. Synthesis and characterization of pH-sensitive acyloxyalkylimidazoles

A series of 1-(acyloxyalkyl)imidazoles (AAI) were synthesized by nucleophilic substitution of chloroalkyl esters of fatty acids with imidazole. The former was prepared from fatty acid chloride and an aldehyde. When incorporated into liposomes, these lipids show an apparent pK(a) value ranging from 5.12 for 1-(palmitoyloxymethyl)imidazole (PMI) to 5.29 for 1-[(alpha-myristoyloxy)ethyl]imidazole (alpha-MEI) as determined by a fluorescence assay. When the imidazole moiety was protonated, the lipids were surface-active, as demonstrated by hemolytic activity towards red blood cells. As expected, AAI were hydrolyzed in serum as well as in cell homogenate. They were significantly less toxic than biochemically stable N-dodecylimidazole (NDI) towards Chinese hamster ovary (CHO) and RAW 264.7 (RAW) cells as determined by MTT assay. When fed to RAW cells, fluorescein-labeled oligonucleotides encapsulated in liposomes containing 20 mol% 1-(stearoyloxymethyl)imidazole (SMI) resulted in punctate as well as partially diffuse fluorescence. In a functional assay involving down-regulation of luciferase in CV-1 cells, neutral liposomes containing imidazole lipids showed suboptimal delivery of antisense phosphorothioate oligomers. Taken together, the results suggest that AAI are of potential use in developing nontoxic, pH-sensitive liposomes. However, these liposomal formulations need to be optimized to achieve higher concentrations of pH-sensitive detergents within the endosome to facilitate efficient cytosolic release of liposome-entrapped contents.     

Changes in lipids containing long- and very long-chain polyunsaturated fatty acids in cryptorchid rat testes

The aim of the present study was to examine the effects of experimental cryptorchidism on rat testicular phospholipids and neutral lipids that contain long-chain (C(18)-C(22)) and very long-chain (VLC) (C(24)-C(32)) polyunsaturated fatty acids (PUFA). The weight of the cryptorchid testis was nearly half that of the contralateral control at postsurgical Days 7-10 owing to the depletion of germ cells. Concomitantly, the amounts of major glycerophospholipids (GPL) and sphingomyelin (SM) per testis decreased. Both these lipids lost their characteristic long-chain and very long-chain PUFA, notably 22:5n-6 and 28:4n-6, respectively, which suggests that these species are linked to the membranes of germ cells. In contrast, the amounts and concentrations of triglycerides (TG; triacylglycerols and 1-O-alkyl-2,3-diacylglycerols) and cholesterol esters (CE) increased several fold in the surviving cells (mainly Sertoli cells) in the cryptorchid testis. All these neutral lipids, but especially CE, accumulated large amounts of the major PUFA of the testis, 22:5n-6, as well as pentaenes with longer carbon chains (i.e., 24:5n-6 in TG and 28:5n-6 in CE). This accretion suggests that neutral lipids may store preformed PUFA coming from dying germ cell GPL and also VLCPUFA no longer needed as a source of PUFA destined to assemble new germ cell GPL. The lipid adjustments observed in cryptorchidism suggest a possible role for Sertoli cell CE in the turnover and conservation of PUFA within seminiferous tubules.     

A potent sphingomyelinase inhibitor from Cordyceps mycelia contributes its cytoprotective effect against oxidative stress in macrophages

A novel water-soluble polysaccharide fraction, CME-1, with a molecular mass of 27.6 kDa and containing mannose and galactose in a respective ratio of 4:6, was prepared from Cordyceps sinensis mycelia and identified by NMR and GC-MS. In the current study, we examined whether CME-1 has anti-inflammatory effects in RAW264.7 cells. The ability of CME-1 to inhibit H(2)O(2)-induced cell death in RAW264.7 cells was assessed by using an MTT assay and annexin V/propidium iodide double staining; we found that CME-1 protected cells against H(2)O(2)-induced injury. H(2)O(2)-induced intracellular oxidative stress and mitochondrial membrane depolarization were also diminished with CME-1 treatment. We evaluated the hydroxyl radical scavenging ability of CME-1 by using the DMPO-electron spin resonance technique, which indicated that CME-1 acts as an intracellular antioxidant in a concentration-dependent manner through a mechanism other than its scavenging activity. Activities of both neutral and acid sphingomyelinases (SMases) were assessed in vitro, and results showed that the CME-1 inhibited activities of both neutral and acid SMases in a concentration-dependent manner. CME-1 reduced H(2)O(2) treatment-elevated C16- and C18-ceramide levels measured by LC/MS/MS in RAW264.7 cells. Results suggest that CME-1 protects RAW264.7 cells against oxidative stress through inhibition of SMase activity and reduction of C16- and C18-ceramide levels.     

干酪乳杆菌LC2W细胞壁组分对RAW264．7巨噬细胞吞噬活性的影响

目的探讨干酪乳杆菌LC2W细胞壁组分体外对小鼠巨噬细胞功能的影响。方法以培养液单纯培养小鼠巨噬细胞系RAW264．7细胞作为对照,研究干酪乳杆菌LC2W细胞壁主要组分磷壁酸和肽聚糖对RAW264．7细胞乳酸脱氢酶（LDH）活性、吞噬中性红和致病菌能力的影响。结果不同浓度磷壁酸和肽聚糖对小鼠巨噬细胞RAW264．7细胞LDH活性、吞噬中性红能力有明显增强作用,并呈一定的剂量效应。在相同质量浓度时,2种细胞壁组分刺激RAW264．7细胞吞噬中性红能力差异无显著性,但磷壁酸对巨噬细胞RAW264．7细胞内LDH活性的增强作用高于肽聚糖。在受到浓度为50μg/ml的磷壁酸和肽聚糖刺激后,磷壁酸和肽聚糖均能显著增强RAW264．7对致病性大肠埃希菌和肠炎沙门菌的吞噬作用（P〈0．01）。经过刺激的巨噬细胞与致病菌共孵育1h后,其吞噬能力达到最大值。结论干酪乳杆菌LC2W细胞壁主要组分磷壁酸和肽聚糖可以增强小鼠巨噬细胞RAW264．7细胞内LDH活性及吞噬能力,并具有剂量效应。     

普伐他汀对泡沫细胞内胆固醇酯的影响及与小凹蛋白-1的关系

本文旨在观察普伐他汀对鼠源巨噬细胞性泡沫细胞内胆固醇酯含量的影响,探讨此作用与小凹蛋白一l的关系。采用体外培养的鼠源性巨噬细胞株作为研究对象,加入氧化低密度脂蛋白（oxidized low density lipoprotein,OX-LDL）使其形成泡沫细胞,运用高效液相色谱测定细胞内胆固醇酯的改变,同时运用Western blot检测细胞中小凹蛋白-1的表达,并观察普伐他汀对细胞内胆固醇酯和小凹蛋白-1影响的量效和时效关系。结果显示：普伐他汀可明显降低泡沫细胞内的胆固醇酯与总胆固醇的比值,且在一定范围内呈剂量依赖性和时间依赖性。在泡沫细胞中加入普伐他汀后能够促进小凹蛋白-1的表达,呈剂量依赖性和时间依赖性。上述结果提示普伐他汀通过降低细胞内胆固醇酯的含量,减轻细胞泡沫化程度。普伐他汀的这一作用可能与促进小凹蛋白-1表达上调有关。     

Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry

Fatty acyl-CoAs participate in numerous cellular processes. This article describes a method for the quantitation of subpicomole amounts of long-chain and very-long-chain fatty acyl-CoAs by reverse-phase LC combined with electrospray ionization tandem mass spectrometry in positive ion mode with odd-chain-length fatty acyl-CoAs as internal standards. This method is applicable to a wide range of species [at least myristoyl- (C14:0-) to cerotoyl- (C26:0-) CoA] in modest numbers of cells in culture ( approximately 10(6)-10(7)), with analyses of RAW264.7 cells and MCF7 cells given as examples. Analysis of these cells revealed large differences in fatty acyl-CoA amounts (12 +/- 1.0 pmol/10(6) RAW264.7 cells vs. 80.4 +/- 6.1 pmol/10(6) MCF7 cells) and subspecies distribution. Very-long-chain fatty acyl-CoAs with alkyl chain lengths > C20 constitute <10% of the total fatty acyl-CoAs of RAW264.7 cells versus >50% for MCF7 cells, which somewhat astonishingly contain approximately as much C24:0- and C26:0-CoAs as C16:0- and C18:0-CoAs and essentially equal amounts of C26:1- and C18:1-CoAs. This simple and robust method should facilitate the inclusion of this family of compounds in "lipidomics" and "metabolomics" studies.     

Evidence for altered positional specificity of LCAT in vivo: studies with docosahexaenoic acid feeding in humans

The percentage of saturated cholesteryl esters (CEs) synthesized by human LCAT is several times higher than expected from the sn-2 acyl composition of plasma phosphatidylcholine (PC), whereas the synthesis of 20:4 CE and 22:6 CE is much lower than expected. To explain these discrepancies, we proposed that LCAT transfers some saturated fatty acids from the sn-1 position of PC species that contain 20:4 or 22:6 at sn-2. The present studies provide in vivo evidence for this hypothesis. We determined the composition and synthesis of CE species in plasma of volunteers before and after a 6 week dietary supplementation with docosahexaenoic acid (22:6; DHA). In addition to an increase in the DHA content of all plasma lipids, there was a significant (+12%; P <0.005) increase of 16:0 CE, although there was no increase in 16:0 at sn-2 of PC. The increase of DHA in CE was much lower than its increase at sn-2 of PC. Ex vivo synthesis of CE species in plasma showed a significant (+24%; P <0.005) increase in the synthesis of 16:0 CE after DHA supplementation, which correlated positively with the increase of 22:6, but not of 16:0, at sn-2 of PC. These results show that the positional specificity of human LCAT is altered when the concentration of 16:0-22:6 PC is increased by DHA supplementation.