Linoleic Acid Metabolites Research Articles

Exploring the heat-induced structural changes of β-lactoglobulin -linoleic acid complex by fluorescence spectroscopy and molecular modeling techniques.

Linoleic acid (LA) is the precursor of bioactive oxidized linoleic acid metabolites and arachidonic acid, therefore is essential for human growth and plays an important role in good health in general. Because of the low water solubility and sensitivity to oxidation, new ways of LA delivery without compromising the sensory attributes of the enriched products are to be identified. The major whey protein, β-lactoglobulin (β-Lg), is a natural carrier for hydrophobic molecules. The thermal induced changes of the β-Lg-LA complex were investigated in the temperature range from 25 to 85°C using fluorescence spectroscopy techniques in combination with molecular modeling study and the results were compared with those obtained for β-Lg. Experimental results indicated that, regardless of LA binding, the polypeptide chain rearrangements at temperatures higher than 75°C lead to higher exposure of hydrophobic residues causing the increase of fluorescence intensity. Phase diagram indicated an all or none transition between two conformations. The LA surface involved in the interaction with β-Lg was about 497Ǻ(2), indicating a good affinity between those two components even at high temperatures. Results obtained in this study provide important details about heat-induced changes in the conformation of β-Lg-LA complex. The thermal treatment at high temperature does not affect the LA binding and carrier functions of β-Lg.

Chronic‐Binge Alcohol‐Induced Hepatic Injury and Inflammation Were Ameliorated in Mice Deficient for Transient Receptor Potential Vanilloid 1 Gene

IntroductionExperimental alcohol‐induced liver injury is exacerbated by a high polyunsaturated fat diet rich in linoleic acid (LA). The aim of the study was to evaluate the role of bioactive oxidized LA metabolites (OXLAMs) and their signaling through the Transient Receptor Potential Vanilloid 1 (TRPV1) in an experimental animal model of alcoholic liver disease (ALD).MethodsC57BL/6 wild type (WT) and Trpv1 knock out mice were fed a diet containing 5% ethanol for 10 days, followed by a single ethanol gavage. Liver steatosis, injury and inflammation were assessed. In vitro studies using HepG2 cells were performed to evaluate OXLAM/TRPV1 signaling.ResultsChronic‐binge alcohol administration to WT animals resulted in a marked increase in plasma OXLAM levels, specifically 9‐ and 13‐HODEs, in parallel with the up‐regulation of hepatic Trpv1. These effects were associated with hepatic steatosis, inflammation and injury. Genetic depletion of Trpv1 did not blunt hepatic steatosis caused by EtOH, but ameliorated hepatic injury and prevented the increase in pro‐inflammatory cytokine and chemokine expression, including Tnf‐α, IL‐6, Mip‐2, Mcp1, and Pai‐1. Exposure of HepG2 cells to 9‐ and 13‐HODEs resulted in activation of TRPV1 signal transduction with the increased intracellular Ca2+ levels, suggesting that OXLAM/TRPV1/Ca2+ signaling may be a relevant pathway contributing to ALD.ConclusionsTRPV1‐OXLAM interactions appear to play a significant role in ethanol‐induced hepatic inflammation/injury, further supporting an important role for dietary lipids in ALD.

10-oxo-12(Z)-octadecenoic acid, a linoleic acid metabolite produced by gut lactic acid bacteria, potently activates PPARγ and stimulates adipogenesis

Our previous study has shown that gut lactic acid bacteria generate various kinds of fatty acids from polyunsaturated fatty acids such as linoleic acid (LA). In this study, we investigated the effects of LA and LA-derived fatty acids on the activation of peroxisome proliferator-activated receptors (PPARs) which regulate whole-body energy metabolism. None of the fatty acids activated PPARδ, whereas almost all activated PPARα in luciferase assays. Two fatty acids potently activated PPARγ, a master regulator of adipocyte differentiation, with 10-oxo-12(Z)-octadecenoic acid (KetoA) having the most potency. In 3T3-L1 cells, KetoA induced adipocyte differentiation via the activation of PPARγ, and increased adiponectin production and insulin-stimulated glucose uptake. These findings suggest that fatty acids, including KetoA, generated in gut by lactic acid bacteria may be involved in the regulation of host energy metabolism.

A Gut Microbial Metabolite of Linoleic Acid, 10-Hydroxy-cis-12-octadecenoic Acid, Ameliorates Intestinal Epithelial Barrier Impairment Partially via GPR40-MEK-ERK Pathway

Gut microbial metabolites of polyunsaturated fatty acids have attracted much attention because of their various physiological properties. Dysfunction of tight junction (TJ) in the intestine contributes to the pathogenesis of many disorders such as inflammatory bowel disease. We evaluated the effects of five novel gut microbial metabolites on tumor necrosis factor (TNF)-α-induced barrier impairment in Caco-2 cells and dextran sulfate sodium-induced colitis in mice. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a gut microbial metabolite of linoleic acid, suppressed TNF-α and dextran sulfate sodium-induced changes in the expression of TJ-related molecules, occludin, zonula occludens-1, and myosin light chain kinase. HYA also suppressed the expression of TNF receptor 2 (TNFR2) mRNA and protein expression in Caco-2 cells and colonic tissue. In addition, HYA suppressed the protein expression of TNFR2 in murine intestinal epithelial cells. Furthermore, HYA significantly up-regulated G protein-coupled receptor (GPR) 40 expression in Caco-2 cells. It also induced [Ca(2+)]i responses in HEK293 cells expressing human GPR40 with higher sensitivity than linoleic acid, its metabolic precursor. The barrier-recovering effects of HYA were abrogated by a GPR40 antagonist and MEK inhibitor in Caco-2 cells. Conversely, 10-hydroxyoctadacanoic acid, which is a gut microbial metabolite of oleic acid and lacks a carbon-carbon double bond at Δ12 position, did not show these TJ-restoring activities and down-regulated GPR40 expression. Therefore, HYA modulates TNFR2 expression, at least partially, via the GPR40-MEK-ERK pathway and may be useful in the treatment of TJ-related disorders such as inflammatory bowel disease.

Simvastatin and a Plant Galactolipid Protect Animals from Septic Shock by Regulating Oxylipin Mediator Dynamics through the MAPK-cPLA2 Signaling Pathway

Sepsis remains a major medical issue despite decades of research. Identification of important inflammatory cascades and key molecular mediators are crucial for developing intervention and prevention strategies. In this study, we conducted a comparative oxylipin metabolomics study to gain a comprehensive picture of lipid mediator dynamics during the initial hyperinflammatory phase of sepsis, and demonstrated, in parallel, the efficacy of simvastatin and plant galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG) in the homeostatic regulation of the oxylipin metabolome using a lipopolysaccharide (LPS)-induced sepsis C57BL/6J mouse model. LPS increased the systemic and organ levels of proinflammatory metabolites of linoleic acid including leukotoxin diols (9-,10-DHOME, 12-,13-DHOME) and octadecadienoic acids (9-HODE and 13-HODE) and arachidonic acid-derived prostanoid, PGE2, and hydroxyeicosatetraenoic acids (8-, 12- and 15-HETE). Treatment with either compound decreased the levels of proinflammatory metabolites and elevated proresolution lipoxin A4, 5(6)-EET, 11(12)-EET and 15-deoxy-PGJ2. dLGG and simvastatin ameliorated the effects of LPS-induced mitogen-activated protein kinase (MAPK)-dependent activation of cPLA2, cyclooxygenase-2, lipoxygenase, cytochrome P450 and/or epoxide hydrolase lowered systemic TNF-α and IL-6 levels and aminotransferase activities and decreased organ-specific infiltration of inflammatory leukocytes and macrophages, and septic shock-induced multiple organ damage. Furthermore, both dLGG and simvastatin increased the survival rates in the cecal ligation and puncture (CLP) sepsis model. This study provides new insights into the role of oxylipins in sepsis pathogenesis and highlights the potential of simvastatin and dLGG in sepsis therapy and prevention.

Transient Receptor Potential Vanilloid 1 Gene Deficiency Ameliorates Hepatic Injury in a Mouse Model of Chronic Binge Alcohol-Induced Alcoholic Liver Disease

Experimental alcohol-induced liver injury is exacerbated by a high polyunsaturated fat diet rich in linoleic acid. We postulated that bioactive oxidized linoleic acid metabolites (OXLAMs) play a critical role in the development/progression of alcohol-mediated hepatic inflammation and injury. OXLAMs are endogenous ligands for transient receptor potential vanilloid 1 (TRPV1). Herein, we evaluated the role of signaling through TRPV1 in an experimental animal model of alcoholic liver disease (ALD). Chronic binge alcohol administration increased plasma OXLAM levels, specifically 9- and 13-hydroxy-octadecadienoic acids. This effect was associated with up-regulation of hepatic TRPV1. Exposure of hepatocytes to these OXLAMs invitro resulted in activation of TRPV1 signal transduction with increased intracellular Ca(2+) levels. Genetic depletion of TRPV1 did not blunt hepatic steatosis caused by ethanol, but prevented hepatic injury. TRPV1 deficiency protected from hepatocyte death and prevented the increase in proinflammatory cytokine and chemokine expression, including tumor necrosis factor-α, IL-6, macrophage inflammatory protein-2, and monocyte chemotactic protein 1. TRPV1 depletion markedly blunted ethanol-mediated induction of plasminogen activator inhibitor-1, an important alcohol-induced hepatic inflammation mediator, via fibrin accumulation. This study indicates, for the first time, that TRPV1 receptor pathway may be involved in hepatic inflammatory response in an experimental animal model of ALD. TRPV1-OXLAM interactions appear to play a significant role in hepatic inflammation/injury, further supporting an important role for dietary lipids in ALD.

Immunomodulatory activity of a gut microbial metabolite of dietary linoleic acid, 10-hydroxy-cis-12-octadecenoic acid, associated with improved antioxidant/detoxifying defences

Various hydroxy-, oxo-, and conjugated fatty acids are generated by gut microbes throughout the metabolism of dietary PUFAs. Specifically, 10-Hydroxy-cis-12-(HYA), 10-oxo-cis12-(KetoA) and 10-oxo-cis9,trans11-octadecenoic acid (KetoC) are intermediates free fatty acids (iFFA) produced during the isomerization of dietary linoleic acid to cis9,trans11 (c9,t11) isomer of conjugated linoleic acid (CLA). Herein, immunomodulatory and antioxidant activity of selected iFFA was studied in resting (iDCs), LPS-matured DCs (mDCs) and in murine enterocyte cells (MODE-K). Phenotypic maturation of iDCs was not influenced by iFFA, but the release of pro-inflammatory molecules from mDCs was reduced following HYA pre-treatment. HYA ability to improve antioxidant/detoxifying defenses associated with decreased expression of maturation markers in mDCs and in MODE-K cells. The pro-oxidant activity, via NADPH oxidase activation, was responsible for the pro-inflammatory effect of 10-hydroxy-octadecanoic acid. Presented data suggest that the immunomodulatory ability of HYA is determined, at least in part, by its ability to improve antioxidant/detoxifying defenses.

Effect of conjugated linoleic acid mixture supplemented daily after carcinogen application on linoleic and arachidonic acid metabolites in rat serum and induced tumours

Conjugated linoleic acid (CLA) is thought to exert anticarcinogenic, antiatherogenic, anti-inflammatory and weight loss effects. The impact on eicosanoid biosynthesis may be one of the mechanisms of its action. The aim of this study was to establish whether CLA mixture supplemented daily after administration of carcinogen (7, 12-dimethylbenz[a]anthracene, DMBA) influenced the concentration of linoleic and arachidonic acid metabolites: 13- or 9-hydroxyoctadecadienoic acids (13-, 9-HODE) and 15-, 12- or 5-hydroxyeicosatetraenoic acids (15-, 12- or 5-HETE) and prostaglandin E2 (PGE2) in rat serum and DMBA-induced tumours. The correlations between polyunsaturated fatty acids (PUFA) and HETE and HODE contents in serum were also investigated.Female Sprague–Dawley rats divided into three groups according to the diet (1% Bio-C.L.A., 2% Bio-C.L.A. and plant oil in the control group) were used in the study. On the 50th day of life some of the animals in every dietary group were administered DMBA to induce tumours. Since that day, the rats were fed one of the above-mentioned diets. After 15weeks the animals were sacrificed and blood and tumours were collected. HETE and HODE were extracted using a solid-phase extraction (SPE) method on C18 columns and analysed with LC-MS/MS.The results of our study showed that CLA daily supplementation after carcinogen administration influence LA and AA metabolite levels in serum and tumours. However, the ratios of eicosanoids having opposite effects (e.g. 12-HETE/15-HETE), not concentrations of particular compounds, appear to be better indicators of pathological processes.

Placental infection by Salmonella enterica typhimurium in a murine model: mechanisms of pathogenesis and role of inflammatory cell death

Placental infection by Salmonella enterica typhimurium in a murine model: mechanisms of pathogenesis and role of inflammatory cell death

Metabolomics approach to assessing plasma 13- and 9-hydroxy-octadecadienoic acid and linoleic acid metabolite responses to 75-km cycling.

Bioactive oxidized linoleic acid metabolites (OXLAMs) include 13- and 9-hydroxy-octadecadienoic acid (13-HODE + 9-HODE) and have been linked to oxidative stress, inflammation, and numerous pathological and physiological states. The purpose of this study was to measure changes in plasma 13-HODE + 9-HODE following a 75-km cycling bout and identify potential linkages to linoleate metabolism and established biomarkers of oxidative stress (F2-isoprostanes) and inflammation (cytokines) using a metabolomics approach. Trained male cyclists (N = 19, age 38.0 ± 1.6 yr, wattsmax 304 ± 10.5) engaged in a 75-km cycling time trial on their own bicycles using electromagnetically braked cycling ergometers (2.71 ± 0.07 h). Blood samples were collected preexercise, immediately post-, 1.5 h post-, and 21 h postexercise, and analyzed for plasma cytokines (IL-6, IL-8, IL-10, tumor necrosis factor-α, monocyte chemoattractant protein-1, granulocyte colony-stimulating factor), F2-isoprostanes, and shifts in metabolites using global metabolomics procedures with gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS). 13-HODE + 9-HODE increased 3.1-fold and 1.7-fold immediately post- and 1.5 h postexercise (both P < 0.001) and returned to preexercise levels by 21-h postexercise. Post-75-km cycling plasma levels of 13-HODE + 9-HODE were not significantly correlated with increases in plasma cytokines but were positively correlated with postexercise F2-isoprostanes (r = 0.75, P < 0.001), linoleate (r = 0.54, P = 0.016), arachidate (r = 0.77, P < 0.001), 12,13-dihydroxy-9Z-octadecenoate (12,13-DiHOME) (r = 0.60, P = 0.006), dihomo-linolenate (r = 0.57, P = 0.011), and adrenate (r = 0.56, P = 0.013). These findings indicate that prolonged and intensive exercise caused a transient, 3.1-fold increase in the stable linoleic acid oxidation product 13-HODE + 9-HODE and was related to increases in F2-isoprostanes, linoleate, and fatty acids in the linoleate conversion pathway. These data support the use of 13-HODE + 9-HODE as an oxidative stress biomarker in acute exercise investigations.

Oxidized metabolites of linoleic acid mediated hepatotoxicity as a potential mechanism of dietary unsaturated fat and alcohol induced liver injury: in vivo and in vitro studies (959.20)

Introduction: Dietary fat and alcohol both play an important role in the development and progression of alcoholic liver disease. The aim of the present study was to evaluate the role of linoleic acid (LA, a major unsaturated fatty acid in the Western diet), and its bioactive oxidized metabolites (OXLAMs) in alcohol‐induced liver injury. Materials and Methods: In‐vivo study: C57BL/6 mice were fed unsaturated (USF, corn oil enriched) fat diet containing 5% ethanol for 10 days plus a single binge ethanol administration. Liver injury and steatosis as well as 12/15 lipoxygenase and plasma OXLAM levels were evaluated. In‐vitro study: HepG2 cells were exposed to LA and multiple OXLAMs. Seahorse and Cellomics analysis were performed to evaluate effects of treatments on mitochondria function, ER stress, and cell survival. Results: Significant liver injury and steatosis associated with elevated plasma OXLAM levels and hepatic 12/15 lipoxygenase, an enzyme involved in OXLAM production, were observed in response to USF+EtOH. A dose‐dependent decrease in mitochondria oxygen consumption was observed in HepG2 cells in response to multiple OXLAMs. 9‐ and 13‐ HODEs increased cell membrane permeability and ER stress in a dose‐dependent manner, although cell survival was not affected. Conclusion: OXLAM mediated mitochondria dysfunction and ER stress may be a potential mechanism underlying USF+EtOH mediated liver injury.Grant Funding Source: Supported by NIH, DOD and Veterans Administration

Mass spectrometry-based metabolomic profiling identifies alterations in salivary redox status and fatty acid metabolism in response to inflammation and oxidative stress in periodontal disease

Periodontal diseases represent the most common chronic inflammatory diseases in humans and a major cause of tooth loss. Combining mass spectrometry-based ionomics and targeted lipidomics on fatty acid metabolites, we identified significant alterations in redox status and fatty acid metabolism in saliva in response to chronic inflammation and oxidative stress in periodontal disease in a cohort of nonsmoker subjects with chronic periodontitis. For the first time, ionomic profiling of around 30 ions in saliva revealed significantly decreased levels of redox-active metal ions including Mn, Cu, and Zn in the periodontal group, which is consistent with decreased levels of superoxide dismutases in saliva and serum. A targeted lipidomic approach was employed to monitor the major metabolites of arachidonic acid and linoleic acid in saliva. We observed increased levels of cyclooxygenase products including PGE2, PGD2, and PGF2α and TXB2, but decreased level of PGI2 in the periodontal group. A unique pattern of the lipoxygenase products of arachidonic acid and linoleic acid was observed with increased level of 5-HETE but decreased levels of 13-HODE and 9-HODE. Levels of salivary F2-isoprostanes, free radical lipid peroxidation products, and a gold standard for oxidative stress in vivo were also significantly elevated. Taking these data together, our study using multiple powerful omics techniques demonstrates that local redox alteration contributes significantly to periodontitis through the modulation of fatty acid metabolism in response to inflammation and oxidative stress. This study highlights the importance of redox status in periodontitis and provides a rationale for preventing periodontal disease by dietary interventions aiming to restore redox balance.

리놀레산 대사체들의 글루쿠론산 결합화합물 합성

Linoleic acid and its metabolites have various medicinal effects with carboxylic acid functional group. General carboxylic acid compounds are discovered as glucuronide metabolites by UGT glucuronosyl transferase at liver. Consequently, glucuronides of linoleic acid metabolites are expected as potent conjugated metabolite. A previous study reported two epoxide metabolites and two dihydroxy metabolites of linoleic acid. There are prepared their glucuronic acid conjugated compounds as potent linoleic acid metabolites.

The Effect of Parenteral Lipid Emulsions on Pulmonary Hemodynamics and Eicosanoid Metabolites in Preterm Infants

Soy-based intravenous fat emulsion (IVFE) is known to cause a rise in pulmonary artery pressure in the preterm infant, thought to be mediated through eicosanoid metabolites of linoleic acid. We compared the effect of soy-based IVFE and an olive-oil-based IVFE containing less than half the content of linoleic acid on pulmonary artery pressure and eicosanoid metabolites in preterm infants receiving parenteral nutrition. In this pilot study at a regional neonatal intensive care unit (ICU), infants received either a soy-based or olive-oil-based IVFE as part of an otherwise identical feeding protocol. Pulmonary artery pressure and urinary thromboxane B2 and prostaglandin F1 alpha were measured at baseline and maximum lipid infusion. There was a greater fall in pulmonary artery pressure in the olive-oil-based IVFE group compared with the soy-based IVFE group. A decrease in urine thromboxane/prostaglandin F1 alpha ratio was seen only in the olive-oil-based IVFE group. In the parenterally fed preterm infant, an olive-oil-based IVFE may have a beneficial effect on pulmonary artery pressure when compared with soy-based IVFE. Effects on pulmonary vascular tone are likely to be mediated through alterations in eicosanoid metabolism. A randomized trial is warranted to compare the effects of different lipid emulsions.

Oxidized metabolites of linoleic acid as biomarkers of liver injury in nonalcoholic steatohepatitis

The rising prevalence of obesity in the last few decades has been accompanied by an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD encompasses a spectrum of liver pathology from isolated hepatic steatosis to steatohepatitis and advanced fibrosis. Dietary habits characterized by consumption of high-caloric, lipid-rich diets play a major role in the development of NAFLD. Recent studies have uncovered the importance of certain components of the diet. In this review, we will focus on the growing evidence for a central role of n-6 polyunsaturated fatty acids. We will discuss novel findings linking oxidative stress and increased production of reactive oxygen species in the liver to oxidation of n-6 polyunsaturated fatty acids and production of specific lipid oxidation metabolites. In particular, we will highlight the potential role of these metabolites as noninvasive markers to diagnose and monitor the extent of liver damage in patients with NAFLD.

Linoleic acid: Between doubts and certainties

Linoleic acid is the most abundant polyunsaturated fatty acid in human nutrition and represents about 14 g per day in the US diet. Following the discovery of its essential functions in animals and humans in the early 1920's, studies are currently questioning the real requirement of linoleic acid. It seems now overestimated and creates controversy: how much linoleic acid should be consumed in a healthy diet? Beyond the necessity to redefine the dietary requirement of linoleic acid, many questions concerning the consequences of its excessive consumption on human health arise. Linoleic acid is a direct precursor of the bioactive oxidized linoleic acid metabolites. It is also a precursor of arachidonic acid, which produces pro-inflammatory eicosanoids and endocannabinoids. A majority of the studies on linoleic acid and its derivatives show a direct/indirect link with inflammation and metabolic diseases. Many authors claim that a high linoleic acid intake may promote inflammation in humans. This review tries to (i) highlight the importance of reconsidering the actual requirement of linoleic acid (ii) point out the lack of knowledge between dietary levels of linoleic acid and the molecular mechanisms explaining its physiological roles (iii) demonstrate the relevance of carrying out further human studies on the single variable linoleic acid.

Role of endogenous TRPV1 agonists in a postburn pain model of partial-thickness injury

Oxidized linoleic acid metabolites (OLAMs) are a class of endogenous transient receptor potential vanilloid 1 (TRPV1) channel agonists released on exposure of tissue to transient noxious temperatures. These lipid compounds also contribute to inflammatory and heat allodynia. Because persistent pain after a burn injury represents a significant clinical challenge for treatment, we developed an in vivo rat model of partial-thickness cutaneous thermal injury and examined whether TRPV1 and specific OLAM metabolites play a role in mediating postburn pain injury. This peripheral model of burn injury had marked thermal allodynia peaking at 24h after thermal injury, with allodynia being maintained for up to 7d. Immunohistochemical characterization of tissue taken from injury sites revealed an increase in leukocyte/macrophage infiltration that was colocalized with TRPV1-positive fibers. Using this peripheral thermal injury model, we found that pharmacological blockade of peripheral TRPV1 receptors reduced thermal allodynia by about 98%. Moreover, there was a significant increase in OLAM levels compared to naive controls in hind paw skin biopsies. Additional studies of the metabolism of [C14]-linoleic acid in skin biopsies revealed the role of the cytochrome P450 (CYP) system in mediating the metabolism of linoleic acid after thermal injury. Finally, we demonstrated that direct inhibition of OLAMs using OLAM antibodies and indirect inhibition using the CYP inhibitor ketoconazole significantly reduced postburn thermal allodynia. Collectively, these findings point to a novel role of the OLAMs and CYP-related enzymes in generating postburn allodynia via activation of peripheral TRPV1.

Oxidized linoleic acid metabolite–cytochrome P450 system (OLAM-CYP) is active in biopsy samples from patients with inflammatory dental pain

Endogenous TRPV1 agonists such as oxidized linoleic acid metabolites (OLAMs) and the enzymes releasing them [eg, cytochrome P450 (CYP)] are up-regulated after inflammation in the rat. However, it is not known whether such agonists are elevated in human inflammatory pain conditions. Because TRPV1 is expressed in human dental pulp nociceptors, we hypothesized that OLAM-CYP machinery is active in this tissue type and is increased under painful inflammatory conditions such as irreversible pulpitis (IP). The aim of this study was to compare CYP expression and linoleic acid (LA) metabolism in normal vs inflamed human dental pulp. Our data showed that exogenous LA metabolism was significantly increased in IP tissues compared to normal tissues and that pretreatment with a CYP inhibitor, ketoconazole, significantly inhibited LA metabolism. Additionally, extracts obtained from LA-treated inflamed tissues evoked significant inward currents in trigeminal ganglia neurons and were blocked by pretreatment with the TRPV1 antagonist IRTX. Moreover, extracts obtained from ketoconazole-pretreated inflamed tissues significantly reduced inward currents in trigeminal ganglia neurons. These data suggest that LA metabolites produced in human inflamed tissues act as TRPV1 agonists and that the metabolite production can be targeted by CYP inhibition. In addition, immunohistochemical analysis of 2 CYP isoforms, CYP2J and CYP3A1, were shown to be predominately expressed in immune cells infiltrating the inflamed dental pulp, emphasizing the paracrine role of CYP enzymes in OLAM regulation. Collectively, our data indicate that the machinery responsible for OLAM production is up-regulated during inflammation and can be targeted to develop potential analgesics for inflammatory-induced dental pain.

Oxidized fatty acid analysis by charge-switch derivatization, selected reaction monitoring, and accurate mass quantitation

A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r2>0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA2γ–/– mouse liver identifying the role of iPLA2γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems.

Cyclopentanoids from Cyclopentadiene: Synthesis of (-)-Methyl jasmonate and (+)-12-Oxophytodienoic acid

Linoleic acid metabolites, (-)-methyl jasmonate and (+)-12-oxophytodienoic acid ((+)-12-oxo-PDA), were prepared from the same precursor (1,2-trans, 1,3-cis, 2'Z)-2-(pent-2'-enyl)-cyclopent-4-en-1,3-diol, which was obtained by regioselective pent-2-enylation of cyclopentadiene and following photooxidation to cis-1,3-diol. A methoxycarbonylmethyl substituent was introduced to the cyclopentane ring via alkylation of the pi-allyl palladium intermediate derived from (1R,2S,3S,2'Z)-3-acetoxy-2-(pent-2'-enyl)cyclopent-4-ene-1-ol with dimethyl malonate for (-)-methyl jasmonate. The alpha-chain was introduced to the cyclopentane ring via the S(N)2 type nucleophilic substitution of (1S,2R,3R,2'Z)-3-acetoxy-2-(pent-2'-enyl)cyclopent-4-ene-1-ol with a dialkylcuprate for (+)-12-oxo-PDA.