Oxidized Linoleic Acid Metabolites Research Articles

Linoleic Acid: A Narrative Review of the Effects of Increased Intake in the Standard American Diet and Associations with Chronic Disease.

The intake of linoleic acid (LA) has increased dramatically in the standard American diet. LA is generally promoted as supporting human health, but there exists controversy regarding whether the amount of LA currently consumed in the standard American diet supports human health. The goal of this narrative review is to explore the mechanisms that underlie the hypothesis that excessive LA intake may harm human health. While LA is considered to be an essential fatty acid and support health when consumed in modest amounts, an excessive intake of LA leads to the formation of oxidized linoleic acid metabolites (OXLAMs), impairments in mitochondrial function through suboptimal cardiolipin composition, and likely contributes to many chronic diseases that became an epidemic in the 20th century, and whose prevalence continues to increase. The standard American diet comprises 14 to 25 times more omega-6 fatty acids than omega-3 fatty acids, with the majority of omega-6 intake coming from LA. As LA consumption increases, the potential for OXLAM formation also increases. OXLAMs have been associated with various illnesses, including cardiovascular disease, cancer, and Alzheimer's disease, among others. Lowering dietary LA intake can help reduce the production and accumulation of OXLAMs implicated in chronic diseases. While there are other problematic components in the standard American diet, the half-life of LA is approximately two years, which means the damage can be far more persistent than other dietary factors, and the impact of reducing excessive LA intake takes time. Therefore, additional research-evaluating approaches to reduce OXLAM formation and cardiolipin derangements following LA consumption are warranted.

Biochemical and behavioral effects of decreasing dietary linoleic acid and increasing eicosapentaenoic acid and docosahexaenoic acid in a rat chronic monoarthrits model

Clinical studies have demonstrated that decreasing linoleic acid (LA) while increasing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in diets evokes an analgesic effect in headache sufferers. We utilized a rat chronic monoarthritis model to determine if these analgesic effects can be reproduced in rats and to and further probe potential analgesic mechanisms. We fed 8 rats a control diet (with fatty acid levels similar to standard US diets) and 8 rats a low LA diet with added EPA and DHA (H3L6 diet) and after 10 weeks, performed a unilateral intraarticular injection of Complete's Freund Adjuvant (CFA). We evaluated thermal and mechanical sensitivity as well as hind paw weight bearing prior to and at 4 and 20 days post CFA injection. At 28 days post CFA injection rats were euthanized and tissue collected. H3L6 diet fed rats had higher concentrations of EPA and DHA, as well as higher concentrations of oxidized lipids derived from these fatty acids, in hind paw and plasma, compared to control fed rats. LA and oxidized LA metabolites were lower in the plasma and hind paw of H3L6 compared to control fed rats. Diet did not affect thermal or mechanical sensitivity, nor did it affect hind paw weight bearing. In conclusion, the H3L6 diet evoked biochemical changes in rats but did not impact pain related behavioral measures in this chronic monoarthritis model.

Feeding mice a diet high in oxidized linoleic acid metabolites does not alter liver oxylipin concentrations

The oxidation of dietary linoleic acid (LA) produces oxidized LA metabolites (OXLAMs) known to regulate multiple signaling pathways in vivo. Recently, we reported that feeding OXLAMs to mice resulted in liver inflammation and apoptosis. However, it is not known whether this is due to a direct effect of OXLAMs accumulating in the liver, or to their degradation into bioactive shorter chain molecules (e.g. aldehydes) that can provoke inflammation and related cascades. To address this question, mice were fed a low or high LA diet low in OXLAMs, or a low LA diet supplemented with OXLAMs from heated corn oil (high OXLAM diet). Unesterified oxidized fatty acids (i.e. oxylipins), including OXLAMs, were measured in liver after 8 weeks of dietary intervention using ultra-high pressure liquid chromatography coupled to tandem mass-spectrometry. The high OXLAM diet did not alter liver oxylipin concentrations compared to the low LA diet low in OXLAMs. Significant increases in several omega-6 derived oxylipins and reductions in omega-3 derived oxylipins were observed in the high LA dietary group compared to the low LA group. Our findings suggest that dietary OXLAMs do not accumulate in liver, and likely exert pro-inflammatory and pro-apoptotic effects via downstream secondary metabolites.

A Low ω-6 to ω-3 PUFA Ratio (n–6:n–3 PUFA) Diet to Treat Fatty Liver Disease in Obese Youth

ABSTRACTBackgroundRecent literature suggests that the Western diet's imbalance between high ω-6 (n–6) and low ω-3 (n–3) PUFA intake contributes to fatty liver disease in obese youth.ObjectivesWe tested whether 12 wk of a low n–6:n–3 PUFA ratio (4:1) normocaloric diet mitigates fatty liver and whether the patatin-like containing domain phospholipase 3 (PNPLA3) rs738409 variant affects the response.MethodsIn a single-arm unblinded study, obese youth 9–19 y of age with nonalcoholic fatty liver disease were treated with a normocaloric low n–6:n–3 PUFA ratio diet for 12 wk. The primary outcome was change in hepatic fat fraction (HFF%), measured by abdominal MRI. Metabolic parameters included alanine aminotransferase (ALT), lipids, measures of insulin sensitivity, and plasma oxidized linoleic acid metabolites (OXLAMs). Outcomes were also analyzed by PNPLA3 rs738409 genotype. Wilcoxon's signed rank test, the Mann–Whitney U test, and covariance pattern modeling were used.ResultsTwenty obese adolescents (median age: 13.3 y; IQR: 10.5–16.4 y) were enrolled and 17 completed the study. After 12 wk of dietary intervention, HFF% decreased by 25.8% (P = 0.009) despite stable weight. We observed a 34.4% reduction in ALT (P = 0.001), 21.9% reduction in triglycerides (P = 0.046), 3.28% reduction in LDL cholesterol (P = 0.071), and a 26.3% improvement in whole body insulin sensitivity (P = 0.032). The OXLAMs 9-hydroxy-octadecandienoic acid (9-HODE) (P = 0.011), 13-HODE (P = 0.007), and 9-oxo-octadecadienoic acid (9-oxoODE) (P = 0.024) decreased after 12 wk. HFF% declined in both the not-at-risk (CC/CG) and at-risk (GG) PNPLA3 rs738409 genotype groups, with significant (P = 0.016) HFF% reduction in the GG group. Changes in 9-HODE (P = 0.023), 9-oxoODE (P = 0.009), and 13-oxoODE (P = 0.003) differed between the 2 genotype groups over time.ConclusionsThese data suggest that, independently of weight loss, a low n–6:n–3 PUFA diet ameliorates the metabolic phenotype of adolescents with fatty liver disease and that response to this diet is modulated by the PNPLA3 rs738409 genotype.This trial was registered at clinicaltrials.gov as NCT01556113.

Lipolysis modulates the biosynthesis of inflammatory lipid mediators derived from linoleic acid in adipose tissue of periparturient dairy cows

Oxidized linoleic acid metabolites (OXLAM) are products of adipocyte lipolysis with the potential to modulate adipose tissue (AT) lipid metabolism and inflammation. In periparturient cows, linoleic acid is preferentially mobilized from AT during lipolysis by hormone-sensitive lipase (HSL) compared with other polyunsaturated fatty acids. Enzymatic and nonenzymatic reactions generate OXLAM from linoleic acid. Among OXLAM, 9-, 10-, and 12-hydroxy-octadecadienoic acids (HODE) are associated with pro-inflammatory responses, whereas 9- and 13-oxo-octadecadienoic acids (oxoODE) and 13-HODE can facilitate inflammation resolution and promote lipogenesis. This study evaluated the effect of HSL activity on OXLAM biosynthesis using subcutaneous AT explants collected from multiparous dairy cows at 10 d before and again at 10 and 24 d after calving. Explants were treated for 3 h without or with the β-adrenergic agonist isoproterenol (ISO; 1 µM; MilliporeSigma, Burlington, MA) to induce HSL activity. The contribution of HSL to OXLAM biosynthesis was determined by inhibiting its activity with CAY10499 (2 µM; Cayman Chemical, Ann Arbor, MI). After treatments, media and explants were collected for lipidomic analysis using HPLC-tandem mass spectroscopy. Results indicated that ISO increased the biosynthesis of 9-, 12-, and 13-HODE and 9-oxoODE, and this effect was reduced at 24 d after calving. Inhibiting HSL activity partially reversed ISO effects on HODE and 9-oxoODE. Our ex vivo model demonstrated for the first time a direct effect of HSL activity on the biosynthesis of OXLAM in AT, especially at 10 d before and 10 d after calving. The biosynthesis of anti-inflammatory OXLAM is limited during the first weeks after parturition and may promote AT inflammation and lipolytic responses to negative energy balance. These results indicate that HSL activity releases linoleic acid for OXLAM biosynthesis in concentrations of a magnitude that may bypass the need for the activation of phospholipases linked with the inflammatory cascade and thus supports, in part, lipolysis-driven inflammation within AT of periparturient cows.

Dietary Linoleic Acid and Its Oxidized Metabolites Exacerbate Liver Injury Caused by Ethanol via Induction of Hepatic Proinflammatory Response in Mice

Alcoholic liver disease is a major human health problem leading to significant morbidity and mortality in the United States and worldwide. Dietary fat plays an important role in alcoholic liver disease pathogenesis. Herein, we tested the hypothesis that a combination of ethanol and a diet rich in linoleic acid (LA) leads to the increased production of oxidized LA metabolites (OXLAMs), specifically 9- and 13-hydroxyoctadecadienoic acids (HODEs), which contribute to a hepatic proinflammatory response exacerbating liver injury. Mice were fed unsaturated (with a high LA content) or saturated fat diets (USF and SF, respectively) with or without ethanol for 10 days, followed by a single binge of ethanol. Compared to SF+ethanol, mice fed USF+ethanol had elevated plasma alanine transaminase levels, enhanced hepatic steatosis, oxidative stress, and inflammation. Plasma and liver levels of 9- and 13-HODEs were increased in response to USF+ethanol feeding. We demonstrated that primarily 9-HODE, but not 13-HODE, induced the expression of several proinflammatory cytokines invitro in RAW264.7 macrophages. Finally, deficiency of arachidonate 15-lipoxygenase, a major enzyme involved in LA oxidation and OXLAM production, attenuated liver injury and inflammation caused by USF+ethanol feeding but had no effect on hepatic steatosis. This study demonstrates that OXLAM-mediated induction of a proinflammatory response in macrophages is one of the potential mechanisms underlying the progression from alcohol-induced steatosis to alcoholic steatohepatitis.

Oxidized Derivatives of Linoleic Acid in Pediatric Metabolic Syndrome: Is Their Pathogenic Role Modulated by the Genetic Background and the Gut Microbiota?

We tested whether oxidized linoleic acid metabolites (OXLAM) are associated with pediatric metabolic syndrome (MetS) and a proatherogenic lipoprotein profile in 122 obese adolescents. Furthermore, we examined whether genetic and metagenomic factors can modulate plasma OXLAM concentrations by genotyping the fatty acid desaturase 1/2 (FADS) gene and by characterizing the gut microbiota. Subjects with MetS (n = 50) showed higher concentrations of 9- and 13-oxo-octadecadienoic acid (9- and 13-oxo-ODE) than subjects without MetS (n = 72). Both metabolites were associated with an adverse lipoprotein profile that was characterized by elevated very small-dense low-density lipoprotein (p < 0.005) and large very low-density lipoprotein particles (p = 0.01). Plasma 9- and 13-oxo-ODE were higher in subjects carrying the haplotype AA of the FADS gene cluster (p = 0.030 and p = 0.048, respectively). Furthermore, the reduced gut bacterial load was associated with higher 9-oxo-ODE concentrations (p = 0.035). This is the first study showing that high plasma OXLAM concentrations are associated with MetS and suggesting that the leading factors for high plasma concentrations of OXLAM might be the genetic background and the composition of the gut microbiota. In conclusion, high concentrations of 9- and 13-oxo-ODE, which may be the result of a genetic predisposition and a reduced gut bacterial load, are associated with MetS and with a proatherogenic lipoprotein profile in obese adolescents.

Influence of Carbohydrate Ingestion from Bananas or Sugar Beverage on 9‐ and 13‐Hydroxy‐octadecadienoic acids (9 + 13 HODE) During Recovery from 75‐km Cycling

Linoleic acid, an essential polyunsaturated fatty acid (PUFA), is the direct precursor to oxidized linoleic acid metabolites (OXLAMs) including 9‐ and 13‐hydroxy‐octadecadienoic acid (9 + 13 HODE). 9 + 13 HODE are stable and abundant oxidation products in plasma, play a role in the regulation of inflammatory processes, and are elevated during recovery from intensive and long‐duration exercise. Carbohydrate intake has multiple benefits for the exercising athlete, including enhanced glucose availability to the working muscle, resulting in reduced post‐exercise cortisol and epinephrine levels, decreased leukocytosis and phagocytosis, and lower inflammatory cytokines. The effect of carbohydrate intake on post‐exercise HODEs is unknown, and the purpose of this study was to compare ingestion of Cavendish bananas with an equicaloric, sugar‐only beverage or water alone on plasma 9 + 13 HODE levels during recovery from an intensive 75‐km cycling bout (183.9±5.8 min across all trials). Participants included 16 male and female cyclists (age 39.9±2.4 years) who regularly competed in road races and were capable of cycling 75‐km at race pace. This study utilized a randomized crossover approach, and participants engaged in three 75‐km cycling time trials while ingesting water only, bananas and water, and a 6% sugar beverage in an overnight fasted state, with data analyzed using a repeated measures analysis of variance (ANOVA), within subjects approach. Before the exercise bout, participants ingested 5 ml/kg water alone, or the 6% sugar beverage or Cavendish banana (with water) adjusted to quantities providing 0.4 g carbohydrate per kilogram of body weight (g carb/kg). During the cycling bout, water was ingested at a rate of 3 ml/kg every 15 minutes, with carbohydrate intake from the 6% sugar beverage and banana adjusted to 0.2 g carb/kg every 15 minutes. Blood samples were collected pre‐ and post‐exercise, and then at 1.5 h and 21 h post‐exercise. Detection of plasma 9 + 13 HODE was conducted on an UPLC system coupled with Quattro Premier XE MS (Waters, Milford, MA) operated in electrospray ionization (ESI) negative mode. The overall pattern of change in 9 + 13 HODE was significantly different between treatment trials (interaction p‐value &lt;0.05), with 94%, 63%, and 72% increases immediately post‐exercise, and 22%, 4.2%, and 6.8% increases 1.5 h post‐exercise in the water alone, banana, and sugar beverage trials, respectively. Plasma 9 + 13 HODE was back to pre‐exercise levels in all trials by the next morning (21 h post‐exercise). In conclusion, these data indicate that carbohydrate ingestion from bananas with water or a 6% sugar beverage compared to water alone attenuates the post‐exercise increase in plasma 9 + 13 HODE following 75‐km of intensive cycling in overnight fasted athletes.Support or Funding InformationSupport provided by Dole Foods, Inc., Westlake Village, CA

The role of 12/15‐Lipoxygenase in a Mouse Model of Alcoholic Liver Disease

Introduction/Aim12/15‐Lipoxygenase (12/15‐LOX) catalyzes the oxidation of free and esterified polyunsaturated fatty acids, predominantly, linoleic and arachidonic, generating the whole spectrum of bioactive lipid mediators. This enzyme is involved in the regulation of various homeostatic processes as well as in the pathogenesis of multiple diseases. Recently published studies from our group and others have shown that experimental and clinical alcoholic liver disease was associated with elevated oxidized linoleic acid metabolites (OXLAMs), specifically 9‐ and 13‐hydroxy‐octadecadienoic acids (9‐ and 13‐HODEs). The aim of the present study was to examine the role of 12/15‐LOX in an animal model of ALD.Materials and MethodsEight‐week‐old male 12/15‐LOX knockout mice (12/15‐LOX−/−), and their genetically unaltered wild type (WT) counterparts were fed Lieber–DeCarli control (isocaloric maltose‐dextrin) or ethanol (5% w/v) liquid diets ad libitum for 10 days plus a single binge ethanol administration (5 g/kg) by gavage, whereas mice in control groups were gavaged with isocaloric maltose dextrin. Liver histopathological changes were estimated by hematoxylin and eosin staining. Liver triglyceride accumulation was assessed by biochemical assay and Oil Red O staining. Liver injury was evaluated by plasma ALT and AST activity. Liver inflammation was determined by hepatic proinflammatory gene expression. Chloroacetate esterase staining was used to evaluate necroinflammatory changes. TUNEL staining was used to determine hepatic apoptotic cell death. Plasma OXLAMs were analyzed on LC‐MS and GCxGC/TOF‐MS platforms.ResultsChronic‐binge alcohol administration resulted in a marked increase in plasma OXLAM levels, specifically 9‐HODE and 13‐HODE, in parallel with up‐regulation of hepatic 12/15‐LOX in WT animals. These effects were associated with hepatic steatosis, inflammation and injury. 12/15‐LOX genetic depletion did not reduced hepatic steatosis caused by EtOH, but did ameliorate hepatic injury as assessed by ALT (231.2+32.8 U/L in WT vs 148.4+48.6 U/L in 12/15‐LOX−/−) and AST (279.1+36.7 U/L in WT vs 177.1+28.3 U/L in 12/15‐LO−/−) levels. 12/15‐LOX deficiency did not affect hepatic apoptosis; while neutrophil infiltration was significantly reduced in 12/15‐LOX deficient mice compared to WT animals. 12/15‐LOX deficiency prevented EtOH‐induced pro‐inflammatory cytokine and chemokine expression, including Tnf‐α and Mcp1 mRNA levels.Conclusion12/15 LOX plays an important role in the pathogenesis of experimental ALD, in part, via increased production of OXLAMs. 12/15‐LOX and OXLAMs may represent novel therapeutic targets in ALD managementSupport or Funding InformationStudy supported by the NIH and Veterans Administration.

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.

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.

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.

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.

Identification and profiling of targeted oxidized linoleic acid metabolites in rat plasma by quadrupole time‐of‐flight mass spectrometry

Linoleic acid (LA) and LA-esters are the precursors of LA hydroperoxides, which are readily converted to 9- and 13-hydroxy-​octadecadienoic acid (HODE) and 9- and 13-oxo-​octadecadienoic acid (oxo ODE) metabolites in vivo. These four oxidized LA metabolites (OXLAMs) have been implicated in a variety of pathological conditions. Therefore, their accurate measurement may provide mechanistic insights into disease pathogenesis. Here we present a novel quadrupole time-of-flight mass spectrometry (Q-TOFMS) method for quantitation and identification of target OXLAMs in rat plasma. In this method, the esterified OXLAMs were base-hydrolyzed and followed by liquid-liquid extraction. Quantitative analyses were based on one-point standard addition with isotope dilution. The Q-TOFMS data of target metabolites were acquired and multiple reaction monitoring extracted-ion chromatograms were generated post-acquisition with a 10 ppm extraction window. The limit of quantitation was 9.7-35.9 nmol/L depending on the metabolite. The method was reproducible with a coefficient of variation of <18.5%. Mean concentrations of target metabolites in rat plasma were 57.8, 123.2, 218.1 and 57.8 nmol/L for 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE, respectively. Plasma levels of total OXLAMs were 456.9 nmol/L, which correlated well with published concentrations obtained by gas chromatography/mass spectrometry (GC/MS). The concentrations were also obtained utilizing a standard addition curve approach. The calibration curves were linear with correlation coefficients of >0.991. Concentrations of 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE were 84.0, 138.6, 263.0 and 69.5 nmol/L, respectively, which were consistent with the results obtained from one-point standard addition. Target metabolites were simultaneously characterized based on the accurate Q-TOFMS data. This is the first study of secondary LA metabolites using Q-TOFMS. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid in human diets, a major component of human tissues, and the direct precursor to the bioactive oxidized LA metabolites (OXLAMs), 9- and 13 hydroxy-octadecadienoic acid (9- and 13-HODE) and 9- and 13-oxo-octadecadienoic acid (9- and 13-oxoODE). These four OXLAMs have been mechanistically linked to pathological conditions ranging from cardiovascular disease to chronic pain. Plasma OXLAMs, which are elevated in Alzheimer's dementia and non-alcoholic steatohepatitis, have been proposed as biomarkers useful for indicating the presence and severity of both conditions. Because mammals lack the enzymatic machinery needed for de novo LA synthesis, the abundance of LA and OXLAMs in mammalian tissues may be modifiable via diet. To examine this issue in humans, we measured circulating LA and OXLAMs before and after a 12-week LA lowering dietary intervention in chronic headache patients. Lowering dietary LA significantly reduced the abundance of plasma OXLAMs, and reduced the LA content of multiple circulating lipid fractions that may serve as precursor pools for endogenous OXLAM synthesis. These results show that lowering dietary LA can reduce the synthesis and/or accumulation of oxidized LA derivatives that have been implicated in a variety of pathological conditions. Future studies evaluating the clinical implications of diet-induced OXLAM reductions are warranted.

Plasticity of cytochrome P450 isozyme expression in rat trigeminal ganglia neurons during inflammation

Recently, specific oxidized linoleic acid metabolites (OLAMs) have been identified as transient receptor potential vanilloid 1 (TRPV1) channel agonists that contribute to inflammatory and heat hyperalgesia mechanisms, yet the specific mechanism responsible for OLAM synthesis in sensory neurons is unknown. Here, we use molecular, anatomical, calcium imaging, and perforated patch electrophysiology methods to demonstrate the specific involvement of cytochrome P450 enzymes (CYPs) in the oxidation of linoleic acid leading to neuronal activation and show that this is enhanced under inflammatory conditions. Additional studies evaluated CYP expressions in the native rat trigeminal ganglia (TG) tissue and cultures as well as changes in their expression pattern following the induction of peripheral inflammation. Fourteen of 20 candidate transcripts were detected in native TG, and 7 of these displayed altered expression under cultured conditions. Moreover, complete Freund’s adjuvant-induced inflammation of vibrissal pad selectively increased expression of CYP3A23/3A1 and CYP2J4 transcripts in TG. In situ hybridization studies demonstrated broad expression pattern of CYP3A23/3A1 and CYP2J4 within TG neurons. Anatomical studies characterized the expression of CYP3A1 and the CYP2J families within TG sensory neurons, including those with TRPV1, with about half of all TRPV1-positive neurons showing more prominent CYP3A1 and CYP2J expression. Together, these findings show that CYP enzymes play a primary role in mediating linoleic acid-evoked activation of sensory neurons and furthermore, implicate the involvement of specific CYPs as contributing to the formation of OLAMs that act as TRPV1 agonists within this subpopulation of nociceptors.

Role of cytochrome P450 enzymes in oxidized linoleic acid metabolite-mediated inflammatory pain

Our group has recently demonstrated that oxidized linoleic acid metabolites (OLAMs) are released from injured tissues and activate TRPV1; a voltage gated ion channel that plays a pivotal role in inflammatory heat hyperalgesia and thermoregulation. However, the mechanism by which linoleic acid is converted to the OLAMs is unknown. Our recent results show the involvement of cytochrome P450 (CYPs) enzymes in the formation of these OLAMs. Importantly, treatment of animals with antibodies against the OLAMs or drugs that inhibit CYPs at peripheral sites results in reversal of thermal allodynia in a rat model of inflammatory pain. To further evaluate possible role of CYPs and/or LOX in the activation of sensory neurons, linoleic acid (LA, 1mM) was applied on to cultured sensory neurons to measure calcium influx as a measure of neuronal activation. LA application produced an I-RTX (100 nM) reversible increase in [Ca]i, indicating the formation of endogenous TRPV1 agonists under these conditions. Pretreatment with the CYP inhibitors: nordihydroguaiaretic acid, ketoconazole, diphenyliodonium and carbon monoxide all blocked LA-evoked increases in [Ca]i, suggesting that OLAM synthesis (from LA) is regulated via the CYP pathway. In addition, injection of linoleic acid caused significant spontaneous nocifensive behavior in inflamed rat hind paws but not in the uninflamed paws. Moreover, analysis of CYP expression in normal vs. inflamed rat tissue shows increased expression of certain CYP isoforms in the inflamed tissue. Taken, together, these results demonstrate that OLAMs may comprise a new family of physiologically relevant endogenous TRPV1 agonists during inflammatory pain and identifying the OLAM system (OLAMs and the enzymes that produce OLAMs) as potential new targets for analgesic drug development.

The Cytochrome P450 Inhibitor, Ketoconazole, Inhibits Oxidized Linoleic Acid Metabolite-Mediated Peripheral Inflammatory Pain

BackgroundOxidized linoleic acid metabolites (OLAMs) are a class of endogenous agonists to the transient receptor potential V1 (TRPV1) receptor. Although TRPV1 mediates inflammatory heat hyperalgesia, it is not known if the OLAMs contribute to the peripheral activation of this receptor during tissue inflammation. In the present study, we evaluated whether the OLAM system is activated during inflammation and whether cytochrome P450 enzymes mediate OLAM contributions to heat hyperalgesia using the complete Freund’s adjuvant (CFA) model of inflammation.ResultsOur results demonstrate that the intraplantar (ipl) injection of anti-OLAM antibodies significantly reversed CFA-induced heat hyperalgesia. Moreover, application of lipid extracts from inflamed rat skin to cultured sensory neurons triggered a significant release of iCGRP that is blocked by co-treatment with I-RTX, a TRPV1 antagonist. To determine the role of CYP enzymes in mediating OLAM effects, we used a broad spectrum CYP inhibitor, ketoconazole. Pretreatment with ketoconazole inhibited the release of TRPV1 agonists in lipid extracts from inflamed skin and significantly reversed CFA-induced heat hyperalgesia by a peripheral mechanism of action. Moreover, the ipl injection of linoleic acid to rats 24 hr after CFA evoked spontaneous nocifensive behaviors that were significantly reduced by capsazepine, by knockout of the TRPV1 gene, or by pretreatment with either anti-OLAM antibodies or ketoconazole.ConclusionsTaken together, our data suggests that OLAMs contribute to inflammatory nociception in the periphery and that cytochrome P450 enzymes play a crucial role in mediating OLAM contributions to inflammatory heat hyperalgesia.