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.

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.

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.

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.

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.

Summary

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.

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.

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.

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.

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.

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.

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.

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.

Supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil may prevent development of heart failure through alterations in cardiac phospholipids that favorably impact inflammation and energy metabolism. A high-fat diet may block these effects in chronically stressed myocardium. Pathological left ventricle (LV) hypertrophy was generated by subjecting rats to pressure overload by constriction of the abdominal aorta. Animals were fed: (1) standard diet (10% of energy from fat), (2) standard diet with EPA+DHA (2.3% of energy intake as EPA+DHA), (3) high fat (60% fat); or (4) high fat with EPA+DHA. Pressure overload increased LV mass by ≈40% in both standard and high-fat diets without fish oil. Supplementation with fish oil increased their incorporation into cardiac phospholipids, and decreased the proinflammatory fatty acid arachidonic acid and urine thromboxane B2 with both the standard and high-fat diet. Linoleic acid and tetralinoloyl cardiolipin (an essential mitochondrial phospholipid) were decreased with pressure overload on standard diet, which was prevented by fish oil. Animals fed high-fat diet had decreased linoleic acid and tetralinoloyl cardiolipin regardless of fish oil supplemention. Fish oil limited LV hypertrophy on the standard diet, and prevented upregulation of fetal genes associated with heart failure (myosin heavy chain-β and atrial natriuetic factor). These beneficial effects of fish oil were absent in animals on the high-fat diet. In conclusion, whereas treatment with EPA+DHA prevented tetralinoloyl cardiolipin depletion, LV hypertrophy, and abnormal genes expression with pressure overload, these effects were absent with a high-fat diet.

The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication [Thomas, Mitchell, Blanksby in the Journal of the American Chemical Society, 2006, 128, 58−59] we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including, acidic and neutral glycerophospholipids, sphingomyelins and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges or the adduct ion (e.g., [M-H]−, [M-2H]2-, [M+H]+, [M+Na]+, [M+NH4]+). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio. The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used - in conjunction with conventional tandem mass spectral analysis - for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1) and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.

Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A2α (cPLA2α) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA2α in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF–primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the β-glucan receptor dectin-1 was increased in GM-CSF–primed macrophages, and AA release from GM-CSF–primed dectin-1−/− alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal–regulated kinases and phosphorylation of cPLA2α on Ser-505 that occurred in GM-CSF–primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF–primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF–primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans–stimulated increase in TNF-α production that occurred in GM-CSF–primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA2α in GM-CSF–primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF–dependent prostanoid production.

Consumption of ω-3 fatty acids from fish oil, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases risk for heart failure and attenuates pathologic cardiac remodeling in response to pressure overload. Dietary supplementation with EPA+DHA may also impact cardiac mitochondrial function and energetics through alteration of membrane phospholipids. We assessed the role of EPA+DHA supplementation on left ventricular (LV) function, cardiac mitochondrial membrane phospholipid composition, respiration, and sensitivity to mitochondrial permeability transition pore (MPTP) opening in normal and infarcted myocardium. Rats were subjected to sham surgery or myocardial infarction by coronary artery ligation (n=10–14), and fed a standard diet, or supplemented with EPA+DHA (2.3% of energy intake) for 12 weeks. EPA+DHA altered fatty acid composition of total mitochondrial phospholipids and cardiolipin by reducing arachidonic acid content and increasing DHA incorporation. EPA+DHA significantly increased calcium uptake capacity in both subsarcolemmal and intrafibrillar mitochondria from sham rats. This treatment effect persisted with the addition of cyclosporin A, and was not accompanied by changes in mitochondrial respiration or coupling, or cyclophilin D protein expression. Myocardial infarction resulted in heart failure as evidenced by LV dilation and contractile dysfunction. Infarcted LV myocardium had decreased mitochondrial protein yield and activity of mitochondrial marker enzymes, however respiratory function of isolated mitochondria was normal. EPA+DHA had no effect on LV function, mitochondrial respiration, or MPTP opening in rats with heart failure. In conclusion, dietary supplementation with EPA+DHA altered mitochondrial membrane phospholipid fatty acid composition in normal and infarcted hearts, but delayed MPTP opening only in normal hearts.

Abstract

The leukotrienes belong to a family of biologically active lipids derived from arachidonate that are often involved in inflammatory responses. In the central nervous system, a group of leukotrienes, known as the cysteinyl leukotrienes, is generated in brain tissue in response to a variety of acute brain injuries. Although the exact clinical significance of this excess production remains unclear, the cysteinyl leukotrienes may contribute to injury-related disruption of the brain-blood barrier and exacerbate secondary injury processes. In the present study, the formation and role of cysteinyl leukotrienes was explored in the fluid percussion injury model of traumatic brain injury in rats. The results showed that levels of the cysteinyl leukotrienes were elevated after fluid percussion injury with a maximal formation 1 hour after the injury. Neutrophils contributed to cysteinyl leukotriene formation in the injured brain hemisphere, potentially through a transcellular biosynthetic mechanism. Furthermore, pharmacological reduction of cysteinyl leukotriene formation after the injury, using MK-886, resulted in reduction of brain lesion volumes, suggesting that the cysteinyl leukotrienes play an important role in traumatic brain injury.

The mechanism of ozone-induced lung cell injury is poorly understood. One hypothesis is that ozone induces lipid peroxidation and that these peroxidased lipids produce oxidative stress and DNA damage. Oxysterols are lipid peroxide formed by the direct effect of ozone on pulmonary surfactant and cell membranes. We studied the effects of ozone and the oxysterol 5β,6β-epoxycholesterol (β-epoxide) and its metabolite cholestan-6-oxo-3,5-diol (6-oxo-3,5-diol) on human alveolar epithelial type I-like cells (ATI-like cells) and type II cells (ATII cells). Ozone and oxysterols induced apoptosis and cytotoxicity in ATI-like cells. They also generated reactive oxygen species and DNA damage. Ozone and β-epoxide were strong inducers of nuclear factor erythroid 2-related factor 2 (Nrf2), heat shock protein 70 (Hsp70) and Fos-related antigen 1 (Fra1) protein expressions. Furthermore, we found higher sensitivity of ATI-like cells than ATII cells exposed to ozone or treated with β-epoxide or 6-oxo-3,5-diol. In general the response to the cholesterol epoxides was similar to the effect of ozone. The importance of understanding the response of human ATI-like cells and ATII cells to oxysterols may be useful for further studies, because these compounds may represent useful biomarkers in other diseases.

Elevated glucose levels result in enhanced generation of proinflammatory leukotrienes by mouse bone marrow cells and by retinal glial and microvascular endothelial cells. Leukotrienes may contribute to chronic inflammation in diabetic retinopathy.

Purpose.

Evidence suggests that capillary degeneration in early diabetic retinopathy results from chronic inflammation, and leukotrienes have been implicated in this process. The authors investigated the cellular sources of leukotriene biosynthesis in diabetic retinas and the effects of hyperglycemia on leukotriene production.

Methods.

Retinas and bone marrow cells were collected from diabetic and nondiabetic mice. Mouse retinal glial cells and retinal endothelial cells (mRECs) were cultured under nondiabetic and diabetic conditions. Production of leukotriene metabolites was assessed by mass spectrometry, and Western blot analysis was used to quantitate the expression of enzymes and receptors involved in leukotriene synthesis and signaling.

Results.

Bone marrow cells from nondiabetic mice expressed 5-lipoxygenase, the enzyme required for the initiation of leukotriene synthesis, and produced leukotriene B4 (LTB4) when stimulated with the calcium ionophore A23187. Notably, LTB4 synthesis was increased threefold over normal (P < 0.03) in bone marrow cells from diabetic mice. In contrast, retinas from nondiabetic or diabetic mice produced neither leukotrienes nor 5-lipoxygenase mRNA. Despite an inability to initiate leukotriene biosynthesis, the addition of exogenous leukotriene A4 (LTA4; the precursor of LTB4) to retinas resulted in robust production of LTB4. Similarly, retinal glial cells synthesized LTB4 from LTA4, whereas mRECs produced both LTB4 and the cysteinyl leukotrienes. Culturing the retinal cells in high-glucose concentrations enhanced leukotriene synthesis and selectively increased expression of the LTB4 receptor BLT1. Antagonism of the BLT1 receptor inhibited LTB4-induced mREC cell death.

Conclusions.

Transcellular delivery of LTA4 from marrow-derived cells to retinal cells results in the generation of LTB4 and the death of endothelial cells and, thus, might contribute to chronic inflammation and retinopathy in diabetes.

A set of four (d0, d4, d6, and d10) deuterium enriched 4-(dimethylamino)benzoic acid (DMABA) N-hydroxysuccinimide (NHS) ester reagents was developed that react with the primary amine group of glycerophosphoethanolamine (PE) lipids to create derivatives where all subclasses of DMABA labeled PE are detected by a common precursor ion scan. The positive ion collision induced dissociation data from (d0, d4, d6, and d10)-DMABA labeled PE standards indicated that a precursor ion scan of m/z 191.1, 195.1, 197.1, and 201.1 could be used to selectively detect (d0, d4, d6, and d10)-DMABA modified PE, respectively, in a complex biological mixture. The PE lipids from a time course (0, 30, 60, and 300 min) of AAPH treatment of liposomes made of RAW 264.7 cell phospholipids were each labeled with the d0-, d4-, d10-, and d6-DMABA NHS ester reagents, respectively. The DMABA derivatives revealed loss of endogenous PE lipids and an increase in oxidized PE lipid throughout the time course of AAPH treatment. These DMABA NHS ester reagents provide a universal scan for diacyl, ether, and plasmalogen PE lipids that can not be readily observed otherwise, enable differential labeling, and provide an internal standard for each PE lipid.

Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A2 (cPLA2α), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the β-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88−/− but not Dectin-1−/− macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA2α on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA2α phosphorylation, and COX2 expression in response to particulate β-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA2α. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both β-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA2α and eicosanoid production.