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1.  Eicosanoid Profiling in an Orthotopic Model of Lung Cancer Progression by Mass Spectrometry Demonstrates Selective Production of Leukotrienes by Inflammatory Cells of the Microenvironment 
PLoS ONE  2013;8(11):e79633.
Eicosanoids are bioactive lipid mediators derived from arachidonic acid1 (AA), which is released by cytosolic phospholipase A2 (cPLA2). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA2 knockout vs wild-type mice, we demonstrated that prostaglandins (PGE2, PGD2 and PGF2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB4, LTC4, LTD4, LTE4) production required cPLA2 expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.
PMCID: PMC3823604  PMID: 24244531
2.  Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation[S] 
Journal of Lipid Research  2013;54(11):3085-3097.
Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.
PMCID: PMC3793613  PMID: 23883581
Oxidized phospholipids; atherosclerosis; PGE2/D2-PEs
3.  Peroxide Bond Driven Dissociation of Hydroperoxy-Cholesterol Esters Following Collision Induced Dissociation 
The oxidative modification of polyunsaturated fatty acids which occurs through enzymatic and non-enzymatic processes is typically initiated by the attachment of molecular oxygen to an unsaturated fatty acyl chain forming a lipid hydroperoxide (LOOH). Enzymatic pathways are critical for cellular homeostasis but aberrant lipid peroxidation has been implicated in important pathologies. Analysis of primary oxidation products such as hydroperoxides has proven challenging for a variety of reasons. While negative ion electrospray ionization has been used for the specific detection of some LOOH species, hydroperoxide dehydration in the ion source has been a significant drawback. Here we describe positive ion electrospray ionization of ammoniated 13-hydroperoxy-9Z,11E-octadecadienoyl cholesterol and 9-hydroperoxy-10E,12Z-octadecadienoyl cholesterol, [M + NH4]+, following normal phase high-pressure liquid-chromatography. Dehydration in the ion source was not prevalent and the ammoniated molecular ion was the major species observed. Collisionally induced dissociation of the two positional isomers yielded unique product ion spectra resulting from carbon-carbon cleavages along their acyl chains. Further investigation of this behavior revealed that complex collision induced dissociations were initiated by scission of the hydroperoxide bond that drove subsequent acyl chain cleavages. Interestingly some of the product ions retained the ammonium nitrogen through the formation of covalent carbon-nitrogen or oxygen-nitrogen bonds. These studies were carried out using hydroperoxy-octadecadienoate cholesteryl esters as model compounds, however the observed mechanisms of [LOOH+NH4]+ ionization and dissociation are likely applicable to the analysis of other lipid hydroperoxides and may serve as the basis for selective LOOH detection as well as aid in the identification of unknown lipid hydroperoxides.
PMCID: PMC3565457  PMID: 21472521
4.  Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses 
Cell  2012;151(1):138-152.
Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses observed in macrophage foam cells, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism and suppression of inflammatory response genes. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, pro-inflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.
PMCID: PMC3464914  PMID: 23021221
5.  Working towards an exegesis for lipids in biology 
Nature chemical biology  2009;5(9):602-606.
As a field, lipidomics is in its infancy, yet it has already begun to influence lipid biochemistry in myriad ways. As with other omic technologies, the field is driven by advances in analytical chemistry, particularly by mass spectrometry. At the heart of a renaissance in lipid biochemistry, systems biology is being used to define the cellular lipome, build a comprehensive picture of metabolic interconnections, discover new molecular species and determine how lipids modulate biological functions.
PMCID: PMC3785062  PMID: 19690530
6.  Mammalian Fatty Acid Synthase Activity From Crude Tissue Lysates Tracing [13C]-Substrates Using GC Mass Spectrometry 
Analytical biochemistry  2012;428(2):158-166.
Fatty Acid Synthase (FASN, FAS; EC is the sole mammalian enzyme to synthesize fatty acids de novo from acetyl and malonyl coenzyme A esters. A new method is described that directly quantifies uniformly labeled [13C]16-palmitate by tracing [13C]2-acetyl-CoA and [13C]3-malonyl-CoA using an in vitro FASN assay. This method used GC-MS to detect [13C]16-palmitate carboxylate anions (m/z 271) of pentafluorobenzyl derivatives and was highly sensitive at femtomole quantities. Uniformly incorporated [13C]16-palmitate was the primary product of both recombinant and crude tissue lysate FASN. Quantification of FASN protein within crude tissue lysates assured equal FASN amounts, preserved steady state kinetics, and enabled calculation of FASN specific activity. FASN activity determined by [13C]16-palmitate synthesis was consistent with values obtained from NADPH oxidation assays. Analysis of FASN activity from tissue extracts was not hampered by contaminating enzymes or pre-existing fatty acids. Crude mammary gland and liver lysates had significantly different activities at 82 and 65 nmoles minute−1 mg−1 respectively, suggesting tissue specific activity levels differ in a manner unrelated to FASN amount. GC-MS quantification of [13C]16-palmitate synthesis permits sensitive evaluation of FASN activity from tissues of varied physiologic states, and of purified FASN activity in the presence of modifying proteins, enzymes, or drugs.
PMCID: PMC3415257  PMID: 22728958
Fatty Acid Synthase Activity; GC-Mass Spectrometry; Mammary Gland/Liver Lysates; [13C]-substrate incorporation; [13C]16-Palmitic Acid
7.  A Complex LuxR-LuxI Type Quorum Sensing Network in a Roseobacterial Marine Sponge Symbiont Activates Flagellar Motility and Inhibits Biofilm Formation 
Molecular microbiology  2012;85(5):916-933.
Bacteria isolated from marine sponges, including the Silicibacter-Ruegeria (SR) subgroup of the Roseobacter clade, produce N-acylhomoserine lactone (AHL) quorum sensing signal molecules. This study is the first detailed analysis of AHL quorum sensing in sponge-associated bacteria, specifically Ruegeria sp. KLH11, from the sponge Mycale laxissima. Two pairs of luxR and luxI homologues and one solo luxI homologue were identified and designated ssaRI, ssbRI, and sscI (sponge-associated symbiont locus A, B, and C, luxRI or luxI homologue). SsaI produced predominantly long-chain 3-oxo-AHLs and both SsbI and SscI specified 3-OH-AHLs. Addition of exogenous AHLs to KLH11 increased the expression of ssaI but not ssaR, ssbI or ssbR, and genetic analyses revealed a complex interconnected arrangement between SsaRI and SsbRI systems. Interestingly, flagellar motility was abolished in the ssaI and ssaR mutants, with the flagellar biosynthesis genes under strict SsaRI control, and active motility only at high culture density. Conversely, ssaI and ssaR mutants formed more robust biofilms than wild type KLH11. AHLs and transcript of the ssaI gene were detected in M. laxissima extracts suggesting that AHL signaling contributes to the decision between motility and sessility and that it also may facilitate acclimation to different environments including the sponge host.
PMCID: PMC3429658  PMID: 22742196
quorum sensing; acyl-homoserine lactones; LuxI-LuxR type regulation; sponge symbionts; motility; biofilm
8.  MALDI Imaging of Lipids after Matrix Sublimation/Deposition 
Biochimica et biophysica acta  2011;1811(11):970-975.
Mass spectrometric techniques have been developed to record mass spectra of biomolecules including lipids as they naturally exist within tissues and thereby permit the generation of images displaying the distribution of specific lipids in tissues, organs, and intact animals. These techniques are based on matrix-assisted laser desorption/ionization (MALDI) that requires matrix application onto the tissue surface prior to analysis. One technique of application that has recently shown significant advantages for lipid analysis is sublimation of matrix followed by vapor deposition directly onto the tissue. Explanations for enhanced sensitivity realized by sublimation/deposition related to sample temperature after a laser pulse and matrix crystal size are presented. Specific examples of sublimation/deposition in lipid imaging of various organs including brain, ocular tissue, and kidney are presented.
PMCID: PMC3202086  PMID: 21571091
imaging mass spectrometry; sublimation/deposition; MALDI; phosphatidylcholine; sphingolipids; brain; kidney; retina; spinal cord; human lens; glycerophospholipids
9.  Glycerolipid and Cholesterol Ester Analyses in Biological Samples by Mass Spectrometry 
Biochimica et biophysica acta  2011;1811(11):776-783.
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 are 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.
PMCID: PMC3205286  PMID: 21757029
glycerolipids; electrospray ionization; electron ionization; neutral lipid; atmospheric pressure ionization; mass spectrometry; matrix assisted laser desorption/ionization; normal phase; reversed phase; triacylglycerols; diacylglycerols; cholesterol esters
11.  Cytosolic Phospholipase A2α and Eicosanoids Regulate Expression of Genes in Macrophages Involved in Host Defense and Inflammation 
PLoS ONE  2013;8(7):e69002.
The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α+/+ than cPLA2α-/- macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α+/+ than cPLA2α-/- macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α+/+ and cPLA2α-/- macrophages (3 h) was compared by microarray. cPLA2α+/+ macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α-/- macrophages (≥2-fold, p<0.05). Several pro-inflammatory genes were expressed at lower levels (Tnfα, Cx3cl1, Cd40, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna, Ifnγ, several IFNγ-inducible GTPases). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2α+/+ macrophages. Representative genes expressed lower in cPLA2α+/+ macrophages (Tnfα, Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2α, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation.
PMCID: PMC3742295  PMID: 23950842
12.  Analysis of Diacylglycerol Molecular Species in Cellular Lipid Extracts by Normal-Phase LC-Electrospray Mass Spectrometry 
The quantitative determination of 48 molecular species of regioisomeric diacylglycerols has been made in a single analysis of an extract of bone marrow derived macrophages. The analytical procedure involves solvent extraction of neutral lipids, including diacylglycerols, derivatization of free hydroxyl moieties as 2,4-difluorophenyl urethane, and analysis by normal phase liquid chromatography-tandem mass spectrometry. The derivatization step not only prevents fatty acyl group migration, thus allowing determination of both 1,2- and 1,3-diacylglycerols, but also yields species that are sensitively and uniquely determined by constant neutral loss mass spectrometry. The method also detected monoacylglycerols, which were characterized by unique retention time and collisional spectra, and were present in mouse bone marrow derived macrophage extracts.
PMCID: PMC3158596  PMID: 21860599
diacylglycerol; normal phase; electrospray; diflurophenyl urethane derivative; neutral loss; lipid; monoacylglycerols; diacylglycerols
13.  Joint Tissues Amplify Inflammation and Alter Their Invasive Behavior via Leukotriene B4 in Experimental Inflammatory Arthritis 
Mechanisms by which mesenchymal-derived tissue lineages participate in amplifying and perpetuating synovial inflammation in arthritis have been relatively underinvestigated and are therefore poorly understood. Elucidating these processes is likely to provide new insights into the pathogenesis of multiple diseases. Leukotriene B4 (LTB4) is a potent proinflammatory lipid mediator that initiates and amplifies synovial inflammation in the K/BxN model of arthritis. We sought to elucidate mechanisms by which mesenchymal-derived fibroblast-like synoviocytes (FLSs) perpetuate synovial inflammation. We focused on the abilities of FLSs to contribute to LTB4 synthesis and to respond to LTB4 within the joint. Using a series of bone marrow chimeras generated from 5-lipoxygenase–/– and leukotriene A4 (LTA4) hydrolase–/– mice, we demonstrate that FLSs generate sufficient levels of LTB4 production through transcellular metabolism in K/BxN serum-induced arthritis to drive inflammatory arthritis. FLSs—which comprise the predominant lineage populating the synovial lining—are competent to metabolize exogenous LTA4 into LTB4 ex vivo. Stimulation of FLSs with TNF increased their capacity to generate LTB4 3-fold without inducing the expression of LTA4 hydrolase protein. Moreover, LTB4 (acting via LTB4 receptor 1) was found to modulate the migratory and invasive activity of FLSs in vitro and also promote joint erosion by pannus tissue in vivo. Our results identify novel roles for FLSs and LTB4 in joints, placing LTB4 regulation of FLS biology at the center of a previously unrecognized amplification loop for synovial inflammation and tissue pathology.
PMCID: PMC3690310  PMID: 20876351
14.  Mass Spectrometric Analysis of Long-Chain Lipids 
Mass Spectrometry Reviews  2010;30(4):579-599.
PMCID: PMC3117083  PMID: 21656842
15.  Quantitative assays for new families of esterified oxylipins generated by immune cells 
Nature protocols  2010;5(12):1919-1931.
Phospholipid-esterified oxylipins are newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several, in particular hydro(pero)xyeicosatetraenoic acids (H(p)ETEs), hydroxyoctadecadienoic acids (HODEs), hydroxydocosahexaenoic acids (HDOHEs) and keto-eicosatetraenoic acids (KETEs), attached to either phosphatidylethanolamine (PE) or phosphatidylcholine (PC). Lipids are isolated from cells or tissue using a liquid phase organic extraction, then analyzed by HPLC-tandem mass spectrometry (LC/MS/MS) in multiple reaction monitoring (MRM) mode. The protocol can simultaneously monitor up to 23 different species. Generation of standards takes 2 days approximately. Following this, extraction of 30 samples takes approximately 3 hrs, with LC/MS/MS run time of 50 min per sample.
PMCID: PMC3678246  PMID: 21127486
Lipid biochemistry; eicosanoid; phospholipid; mass spectrometry; macrophage; neutrophil; platelet
16.  Dietary linoleate preserves cardiolipin and attenuates mitochondrial dysfunction in the failing rat heart 
Cardiovascular Research  2012;94(3):460-468.
Cardiolipin (CL) is a tetra-acyl phospholipid that provides structural and functional support to several proteins in the inner mitochondrial membrane. The majority of CL in the healthy mammalian heart contains four linoleic acid acyl chains (L4CL). A selective loss of L4CL is associated with mitochondrial dysfunction and heart failure in humans and animal models. We examined whether supplementing the diet with linoleic acid would preserve cardiac L4CL and attenuate mitochondrial dysfunction and contractile failure in rats with hypertensive heart failure.
Methods and results
Male spontaneously hypertensive heart failure rats (21 months of age) were administered diets supplemented with high-linoleate safflower oil (HLSO) or lard (10% w/w; 28% kilocalorie fat) or without supplemental fat (control) for 4 weeks. HLSO preserved L4CL and total CL to 90% of non-failing levels (vs. 61–75% in control and lard groups), and attenuated 17–22% decreases in state 3 mitochondrial respiration observed in the control and lard groups (P < 0.05). Left ventricular fractional shortening was significantly higher in HLSO vs. control (33 ± 2 vs. 29 ± 2%, P < 0.05), while plasma insulin levels were lower (5.4 ± 1.1 vs. 9.1 ± 2.3 ng/mL; P < 0.05), with no significant effect of lard supplementation. HLSO also increased serum concentrations of several eicosanoid species compared with control and lard diets, but had no effect on plasma glucose or blood pressure.
Moderate consumption of HLSO preserves CL and mitochondrial function in the failing heart and may be a useful adjuvant therapy for this condition.
PMCID: PMC3353802  PMID: 22411972
Heart failure; Polyunsaturated fatty acids; Cardiolipin; Mitochondria; Hypertension
Shock (Augusta, Ga.)  2012;37(6):599-604.
The cellular and biochemical mechanisms leading to acute lung injury and subsequent multiple organ failure are only partially understood. In order to study the potential role of eicosanoids, particularly leukotrienes, as possible mediators of acute lung injury, we used a murine experimental model of acute lung injury induced by hemorrhagic shock after blood removal via cardiac puncture. Neutrophil sequestration as shown by immunofluorescence, and protein leakage into the alveolar space, were measured as markers of injury. We used liquid chromatography coupled to tandem mass spectrometry to unequivocally identify several eicosanoids in the bronchoalveolar lavage fluid of experimental animals. MK886, a specific inhibitor of the 5-lipoxygenase pathway, as well as transgenic mice deficient in 5-lipoxygenase, were used to determine the role of this enzymatic pathway in this model. Leukotriene B4 and leukotriene C4 were consistently elevated in shock-treated mice compared to sham-treated mice. MK886 attenuated neutrophil infiltration and protein extravasation induced by hemorrhagic shock. 5-lipoxygenase-deficient mice showed reduced neutrophil infiltration and protein extravasation after shock treatment, indicating greatly reduced lung injury. These results support the hypothesis that 5-lipoxygenase, most likely through the generation of leukotrienes, plays an important role in the pathogenesis of acute lung injury induced by hemorrhagic shock in mice. This pathway could represent a new target for pharmacological intervention to reduce lung damage following severe primary injury.
PMCID: PMC3357208  PMID: 22392149
multiple organ failure; eicosanoids; leukotrienes; mass spectrometry; inflammation; pharmacological inhibition; transgenic mice
18.  The Relationship between MALDI IMS Intensity and Measured Quantity of Selected Phospholipids in Rat Brain Sections 
Analytical chemistry  2010;82(20):8476-8484.
MALDI IMS positive ion images of rat brain show a regional distribution of phosphocholine species that is striking in the apparent distinctiveness, and reproducibility of such depictions. The interpretation of these images, specifically the relationship between MALDI IMS ion intensity and the amount of the phosphocholine (PC) species in the tissue is complicated by numerous factors such as ion suppression, ion molecule chemistry, and effects of tissue structure. This study was designed to test the hypothesis that the intensity of PC molecular species does relate to the quantity of molecules in a tissue sample. A set of comparison studies for a limited but representative selection of cell-derived PC molecular species, was carried out using LC/MS/MS to measure the amounts of these species in brain tissue extracts. There was good correlation between the MALDI IMS ion abundance of PC molecular species and the relative abundance of corresponding PC molecular species in microdissected regions analyzed by LC/MS/MS.
PMCID: PMC3025046  PMID: 20853893
MALDI imaging; MALDI IMS; phospholipids; rat brain; phosphocholine; mass spectrometry; electrospray ionization
Journal of Neurochemistry  2012;120(6):974-984.
The repellent semaphorin 3A (Sema3A) causes growth cone turning or collapse by triggering cytoskeletal rearrangements and detachment of adhesion sites. Growth cone detachment is dependent on eicosanoid activation of protein kinase C epsilon (PKCε), but the characterization of the phospholipase A2 (PLA2) that releases arachidonic acid (AA) for eicosanoid synthesis has remained elusive. Here we show in rat dorsal root ganglion neurons that Sema3A stimulates PLA2 activity, that Sema3A-induced growth cone turning and collapse are dependent on the release of AA, and that the primary PLA2 involved is the Group IV α isoform (GIVA). Silencing GIVA expression renders growth cones resistant to Sema3A-induced collapse, and GIVA inhibition reverses Sema3A-induced repulsion into attraction. These studies identify a novel, early step in Sema3A-signaling and a PLA2 necessary for growth cone repulsion and collapse.
PMCID: PMC3296835  PMID: 22220903
Axonal growth cone; phospholipase A2; growth cone repulsion; signaling
20.  Characterization of Oxidized Phosphatidylethanolamine Derived from RAW 264.7 Cells using 4-(Dimethylamino)benzoic Acid Derivatives 
Recently, a derivative of phosphatidylethanolamine (PE), namely the 4-(dimethylamino)benzoic acid derivative has been developed with various isotope labeled variants that provided a universal precursor ion scan for diacyl, ether, and plasmalogen PE lipids that can not be accomplished otherwise. This derivative was further investigated as a means to facilitate characterization of various oxidized phosphatidylethanolamine lipids by collision activation. Phospholipids derived from RAW 264.7 cells were treated with a free radical generating system to generate a complex mixture of oxidized and nonoxidized lipids that were separated by reversed phase HPLC and detected using a precursors of m/z 191 scan for the d0-DMABA labeled control sample and a precursors of m/z 197 scan for the d6-DMABA labeled oxidized sample. Collisional activation of the corresponding [M-H]− ions permitted the identification of several chained shortened ω-aldehydes, as well as direct oxygen addition products including isoprostane PE and monohydroxy PE oxidized phospholipids that were not easily detected without the use of the DMABA derivatives. The stable isotope labeled derivatives permitted assessment of relative quantitative changes in oxidized lipids based upon ion abundance.
PMCID: PMC3086548  PMID: 20530831
electrospray; tandem mass spectrometry; lipid oxidation; derivatization
21.  Low Concentration Ozone Reacts with Plasmalogen Glycerophosphoethanolamine Lipids in Lung Surfactant 
Chemical research in toxicology  2010;23(1):108-117.
Ozone is a common environmental toxicant to which individuals are exposed to on a daily basis. While biochemical endpoints such as increased mortality, decrements in pulmonary function and initiation of inflammatory processes are known, little is actually understood regarding the chemical mechanisms underlying changes in pulmonary health, especially for low concentrations of ozone. This study was undertaken to investigate ozone induced oxidation of endogenous lipids that are potentially exposed to environmental ozone within lung, specifically focusing on plasmalogen glycerophospholipids present in pulmonary surfactant. Sensitive liquid chromatography-mass spectrometry methods were developed to follow oxidation of diacyl and plasmalogen phosphatidylethanolamine (PE) phospholipids, and to identify and quantitate products generated by ozonolysis. Using a unilamellar vesicle system containing a 1:1 molar mixture of 1-O-octadec-1’-enyl-2-octadecenoyl-PE and 1,2-dihexadecanoyl-PC, these studies revealed the vinyl ether bond of plasmalogens was oxidized preferentially at low concentrations of ozone (100 ppb), when compared to olefinic bond oxidation on ω-9 of the fatty acyl chain in the same phospholipids. Major phospholipid products generated were identified as 1-formyl-2-octadecenoyl-PE and 1-hydroxy-2-octadecenoyl-PE. Heptadecanal and heptadecanoic acid production was also quantitated using gas chromatography-mass spectrometry and production was consistent with oxidation of the vinyl ether, at low concentrations of ozone. Analysis of murine lung surfactant from C57Bl/6 mice revealed several plasmalogen PE lipid species, encompassing ~38% of total PE species. Upon exposure of ozone (0 ppb, 100 ppb) to murine surfactant, plasmalogen PE molecular species preferentially reacted, as compared to diacyl PE molecular species. Lysophospholipids, pentadecanal, and nonanal were found to be the primary products of surfactant ozone oxidation.
PMCID: PMC2807911  PMID: 19916514
Mass Spectrometry; Plasmalogen; Phosphatidylethanolamine; Ozone; Lipid; Surfactant; sn-1 lyso PE
22.  Effects of Acrolein on Leukotriene Biosynthesis in Human Neutrophils 
Chemical research in toxicology  2008;21(12):2424-2432.
Acrolein is a toxic, highly reactive α,β-unsaturated aldehyde that is present in high concentrations in cigarette smoke. In the current study, the effect of acrolein on eicosanoid synthesis in stimulated human neutrophils was examined. Eicosanoid synthesis in neutrophils was initiated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) and 5-LO products in addition to small amounts of COX products were detected using LC/MS/MS. A dose-dependent decrease in the formation of 5-LO products was observed in GM-CSF/fMLP stimulated neutrophils when acrolein (0-50 μM) was present with almost complete inhibition at ≥25 μM acrolein. The production of COX products was not affected by acrolein in these cells. The effect of acrolein was examined on key parts of the eicosanoid pathway, such as arachidonic acid release, intracellular calcium ion concentration, and adenosine production. In addition, the direct effect of acrolein on 5-LO enzymatic activity was probed using a recombinant enzyme. Some of these factors were affected by acrolein, but did not completely explain the almost complete inhibition of 5-LO product formation in GM-CSF/fMLP treated cells with acrolein. In addition, the effect of acrolein on different stimuli that initiate the 5-LO pathway (platelet-activating factor (PAF)/fMLP, GM-CSF/PAF, opsonized zymosan, and A23187) was examined. Acrolein had no significant effect on the leukotriene production in neutrophils stimulated with PAF/fMLP, GM-CSF/PAF, or OPZ. Additionally, 50% inhibition of the 5-LO pathway was observed in A23187 stimulated neutrophils. Our results suggest that acrolein has a profound effect on the 5-LO pathway in neutrophils, which may have implications in disease states, such as COPD and other pulmonary disease where both activated neutrophils and acrolein are present.
PMCID: PMC2772067  PMID: 19548361
23.  Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging 
Common organic MALDI matrices, 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and alpha-cyano-4-hydroxy-cinnamic acid were found to undergo sublimation without decomposition under conditions of reduced pressure and elevated temperature. This solid to vapor phase transition was exploited to apply MALDI matrix onto tissue samples over a broad surface in a solvent-free application for mass spectrometric imaging. Sublimation of matrix produced an even layer of small crystals across the sample plate. The deposition was readily controlled with time, temperature and pressure settings and was highly reproducible from one sample to the next. Mass spectrometric images acquired from phospholipid standards robotically spotted onto a MALDI plate yielded a more intense, even signal with fewer sodium adducts when matrix was applied by sublimation relative to samples where matrix was deposited by an electrospray technique. MALDI matrix could be readily applied to tissue sections on glass slides and stainless steel MALDI plate inserts as long as good thermal contact was made with the condenser of the sublimation device. Sections of mouse brain were coated with matrix applied by sublimation, and were imaged using a Q-q-TOF mass spectrometer to yield mass spectral images of very high quality. Image quality is likely enhanced by several features of this technique including the microcrystalline morphology of the deposited matrix, increased purity of deposited matrix, and evenness of deposition. This inexpensive method was reproducible, and eliminated the potential for spreading of analytes due to solvent deposition during matrix application.
PMCID: PMC2042488  PMID: 17659880
24.  Detection of the Abundance of Diacylglycerol and Triacylglycerol Molecular Species in Cells Using Neutral Loss Mass Spectrometry 
Analytical biochemistry  2007;366(1):59-70.
Triacylglycerols are neutral lipids present in all mammalian cells as energy reserves and diacylglycerols as intermediates in phospholipid biosynthesis and as signaling molecules. The molecular species of triacylglycerols and diacylglycerols present in mammalian cells are quite complex and previous investigations revealed multiple isobaric species having molecular weights at virtually every even mass between 600-900 daltons, making it difficult to assess changes of individual molecular species after cell activation. A method has been developed using tandem mass spectrometry 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 NH3. Deuterium labeled internal standards were used to normalize the signal for each nominal [M+NH4]+ ion undergoing this neutral loss reaction. This method was applied in studies of triacylglycerols in RAW 264.7 cells treated with the toll-like receptor 4 ligand Kdo2-lipid A. A 50:1-TAG containing 18:1 was found to increase significantly over a 24 hr time course after Kdo2-lipid A exposure whereas an isobaric 50:1-TAG containing 16:1 did not change relative to controls.
PMCID: PMC2034497  PMID: 17442253
triacylglycerols; diacylglycerols; mass spectrometry; neutral loss; electrospray ionization; MS3; Kdo2-lipid A; RAW 264.7 molecular species
25.  cPLA2 Is Protective Against COX Inhibitor–Induced Intestinal Damage 
Toxicological Sciences  2010;117(1):122-132.
Cytosolic phospholipase A2 (cPLA2) is the rate-limiting enzyme responsible for the generation of prostaglandins (PGs), which are bioactive lipids that play critical roles in maintaining gastrointestinal (GI) homeostasis. There has been a long-standing association between administration of cyclooxygenase (COX) inhibitors and GI toxicity. GI injury is thought to be induced by suppressed production of GI-protective PGs as well as direct injury to enterocytes. The present study sought to determine how pan-suppression of PG production via a genetic deletion of cPLA2 impacts the susceptibility to COX inhibitor–induced GI injury. A panel of COX inhibitors including celecoxib, rofecoxib, sulindac, and aspirin were administered via diet to cPLA2− / − and cPLA2+ / + littermates. Administration of celecoxib, rofecoxib, and sulindac, but not aspirin, resulted in acute lethality (within 2 weeks) in cPLA2− / − mice, but not in wild-type littermates. Histomorphological analysis revealed severe GI damage following celecoxib exposure associated with acute bacteremia and sepsis. Intestinal PG levels were reduced equivalently in both genotypes following celecoxib exposure, indicating that PG production was not likely responsible for the differential sensitivity. Gene expression profiling in the small intestines of mice identified drug-related changes among a panel of genes including those involved in mitochondrial function in cPLA2− / − mice. Further analysis of enterocytic mitochondria showed abnormal morphology as well as impaired ATP production in the intestines from celecoxib-exposed cPLA2− / − mice. Our data demonstrate that cPLA2 appears to be an important component in conferring protection against COX inhibitor–induced enteropathy, which may be mediated through affects on enterocytic mitochondria.
PMCID: PMC2923287  PMID: 20562220
cytosolic phospholipase A2; COX inhibitor; mitochondria; intestine

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