Phospholipid Ozonation Products Activate the 5‑Lipoxygenase Pathway in Macrophages

Ozone is a highly reactive environmental toxicant that can react with the double bonds of lipids in pulmonary surfactant. This study was undertaken to investigate the proinflammatory properties of the major lipid-ozone product in pulmonary surfactant, 1-palmitoyl-2-(9′-oxo-nonanoyl)-glycerophosphocholine (16:0/9al-PC), with respect to eicosanoid production. A dose-dependent increase in the formation of 5-lipoxygenase (5-LO) products was observed in murine resident peritoneal macrophages (RPM) and alveolar macrophages (AM) upon treatment with 16:0/9al-PC. In contrast, the production of cyclooxygenase (COX) derived eicosanoids did not change from basal levels in the presence of 16:0/9al-PC. When 16:0/9al-PC and the TLR2 ligand, zymosan, were added to RPM or AM, an enhancement of 5-LO product formation along with a concomitant decrease in COX product formation was observed. Neither intracellular calcium levels nor arachidonic acid release was influenced by the addition of 16:0/9al-PC to RPM. Results from mitogen-activated protein kinase (MAPK) inhibitor studies and direct measurement of phosphorylation of MAPKs revealed that 16:0/9al-PC activates the p38 MAPK pathway in RPM, which results in the activation of 5-LO. Our results indicate that 16:0/9al-PC has a profound effect on the eicosanoid pathway, which may have implications in inflammatory pulmonary disease states where eicosanoids have been shown to play a role

Determination of Double Bond Positions in Polyunsaturated Fatty Acids Using the Photochemical Paternò-Büchi Reaction with Acetone and Tandem Mass Spectrometry

The positions of double bonds along the carbon chain of methylene interrupted polyunsaturated fatty acids are unique identifiers of specific fatty acids derived from biochemical reactions that occur in cells. It is possible to obtain direct structural information as to these double bond positions using tandem mass spectrometry after collisional activation of the carboxylate anions of an acetone adduct at each of the double bond positions formed by the photochemical Paternò-Büchi reaction with acetone. This reaction can be carried out by exposing a small portion of an inline fused silica capillary to UV photons from a mercury vapor lamp as the sample is infused into the electrospray ion source of a mass spectrometer. Collisional activation of [M – H]⁻ yields a series of reverse Paternò-Büchi reaction product ions that essentially are derived from cleavage of the original carbon–carbon double bonds that yield an isopropenyl carboxylate anion corresponding to each double bond location. Aldehydic reverse Paternò-Büchi product ions are much less abundant as the carbon chain length and number of double bonds increase. The use of a mixture of D₀/D₆-acetone facilitates identification of these double bonds indicating product ions as shown for arachidonic acid. If oxygen is present in the solvent stream undergoing UV photoactivation, ozone cleavage ions are also observed without prior collisional activation. This reaction was used to determine the double bond positions in a 20:3 fatty acid that accumulated in phospholipids of RAW 264.7 cells cultured for 3 days

Increased ω6-Containing Phospholipids and Primary ω6 Oxidation Products in the Brain Tissue of Rats on an ω3-Deficient Diet

<div><p>Polyunsaturated fatty acyl (PUFA) chains in both the ω3 and ω6 series are essential for normal animal brain development, and cannot be interconverted to compensate for a dietary deficiency of one or the other. Paradoxically, a dietary ω3-PUFA deficiency leads to the accumulation of docosapentaenoate (DPA, 22:5ω6), an ω6-PUFA chain that is normally scarce in the brain. We applied a high-precision LC/MS method to characterize the distribution of DPA chains across phospholipid headgroup classes, the fatty acyl chains with which they were paired, and the extent to which they were oxidatively damaged in the cortical brain of rats on an ω3-deficient diet. Results indicate that dietary ω3-PUFA deficiency markedly increased the concentrations of phospholipids with DPA chains across all headgroup subclasses, including plasmalogen species. The concentrations of phospholipids containing docosahexaenoate chains (22:6ω3) decreased 20–25%, while the concentrations of phospholipids containing arachidonate chains (20:4ω6) did not change significantly. Although DPA chains are more saturated than DHA chains, a larger fraction of DPA chains were monohydroxylated, particularly among diacyl-phosphatidylethanolamines and plasmalogen phosphatidylethanolamines, suggesting that they were disproportionately subjected to oxidative stress. Differences in the pathological significance of ω3 and ω6 oxidation products suggest that greater oxidative damage among the ω6 PUFAs that increase in response to dietary ω3 deficiency may have pathological significance in Alzheimer’s disease.</p></div

ARA, DPA, and DHA content in the brain tissue of animals fed diets with adequate or deficient amounts of ω3-PUFAs.

<p>Fractions correspond to the five normal phase LC fractions described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.ref018" target="_blank">18</a>]. GC/MS determinations were made as described in methods. LC/MS/MS results are the sum of all data in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.g001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.g003" target="_blank">3</a>. Uncertainties for the five fractions are standard deviations of 6 measurements. Uncertainties for the fraction totals were calculated according to the variance sum law. All units are nmoles/gm tissue wet weight.</p

The concentrations of ARA-containing phospholipid species in the parietal cortex of animals fed a diet with adequate amounts of ω3 PUFAs (black bars) and a diet deficient in ω3 PUFAs (gray bars) in nmoles / gm tissue.

<p>Each result is the mean of 6 different brain extracts, and the error bars represent standard deviations. The absence of a bar for an sn1 chain indicates that the corresponding MRM transition was monitored, but no signal detected. There were no statistically significant differences between groups. See text for information about the nature of quantitative uncertainty in these data.</p

The ratio of +O addition (oxidation) for selected PE species in the parietal cortex of animals fed the diet deficient in ω3 PUFAs, relative to the diet with adequate amounts of ω3 PUFAs.

<p>The three panels represent PE species containing either oxidized (a) ARA, (b) DPA, or (c) DHA. Thin blue bars in each panel represent results for the <i>sn</i>-1 chains indicated, which correspond to the 6 most abundant PE species in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.g001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.g003" target="_blank">3</a>. The wide red cross-hatched bar in each panel is the average ratio for all 6 species in aggregate, with a standard error of the mean indicated. By 2-tailed T test of unpaired samples with unequal variances, the ratio for 22:5(+O)-PE species in aggregate (1.63) differs from the ratio for 20:4(+O)-PE species (1.16) at P ≤ 0.05, and from the ratio for 22:6(+O)-PE species (1.01) at P ≤ 0.01.</p

Identification of 18:0/22:5(OH)-PE by LC/MS/MS.

<p>(a) Single reaction monitoring chromatogram of the 808.8 → 345.2 transition, with solvent flow reduced to 850 μl/min. 18:0/22:5(+O)-PE eluted 0.3 min later than 18:0/22:5-PE, consistent with the slightly greater polarity of the oxidized species. (b) Product ions produced by the <i>m/z</i> 808.8 parent ion at 12.6 min. Due to the low concentrations of the parent species (a maximum of only 400 transitions/sec observed in panel a), this spectrum was collected with the Q1 resolution set to “unit”, while the Q3 resolution was set to “open”. The position of the oxygen on the 22:5 chain is unknown, and possibly variable. The identity of the <i>m/z</i> 672.9 and 749.5 product ions is not known.</p

Mass Spectrometric Collisional Activation and Product Ion Mobility of Human Serum Neutral Lipid Extracts

A novel method for lipid analysis called CTS (collisional activation and traveling wave mass spectrometry), involving tandem mass spectrometry of all precursor ions with ion mobility determinations of all product ions, was applied to a sample of human serum. The resulting four-dimensional data set (precursor ion, product ion, ion mobility value, and intensity) was found to be useful for characterization of lipids as classes as well as for identification of specific species. Utilization of ion mobility measurements of the product ions is a novel approach for lipid analysis. The trends and patterns of product mobility values when visually displayed yield information on lipid classes and specific species independent of mass determination. Collection of a comprehensive set of data that incorporates all precursor–product relationships, combined with ion mobility measurements of all products, enables data analysis where different molecular properties can be juxtaposed and analyzed to assist with class and species identification. Overall, CTS is a powerful, specific, and comprehensive method for lipid analysis

Tandem Mass Spectrometry in Combination with Product Ion Mobility for the Identification of Phospholipids

Concerted tandem and traveling wave ion mobility mass spectrometry (CTS analysis) is a unique method that results in a four-dimensional data set including nominal precursor ion mass, product ion mobility, accurate mass of product ion, and ion abundance. This nontargeted lipidomics CTS approach was applied in both positive- and negative-ion mode to phospholipids present in human serum, and the data set was used to evaluate the value of product ion mobility in identifying lipids in a complex mixture. It was determined that the combination of diagnostic product ions and unique collisional cross-section values of product ions is a powerful tool in the structural identification of lipids in a complex biological sample

Multiple eicosanoids are increased in tumor bearing mice.

<p>LLC-Luc cells or matrigel (control) were injected into left lung lobes of WT C57Bl/6 mice and tumor-bearing and control lungs were harvested 2 or 3 weeks after injection. Eicosanoid levels were analyzed by LC/MS/MS and normalized to the protein content of the sample. Levels on graphs are expressed as fold over control. A. Early eicosanoids – induced >3 fold at 2 weeks, and further increasing at 3 weeks. B. Late eicosanoids – not upregulated (<2-fold) at 2 weeks, but increased at 3 weeks. C. Unchanged eicosanoids.</p