Do meals contain a relevant amount of oxylipins? LC-MS-based analysis of oxidized fatty acids in food.

Several oxylipins are potent lipid mediators and are discussed to be absorbed after oral intake. However, information about their concentrations in oils and processed foods are scarce. Here, we analyzed the concentrations of mono-, di- and multihydroxy- as well as epoxy-PUFA in virgin and refined oils as well as in different foods/meals. Oil refining causes hydrolysis of epoxy-PUFA and thus high dihydroxy-PUFA concentrations (e.g. 15,16-DiHODE 290 µg/g in refined vs. 15 µg/g in virgin rapeseed oil), making the epoxy-to-diol ratio a potential marker for refined oils. Low oxylipin levels were found in foods with high amounts of saturated fatty acids such as Hamburger patties (around 30 µg/g). High concentrations (up to 1200 µg/g, 80 mg per serving) and high oxylipin/precursor-PUFA ratios were found in fried falafel and processed foods such as vegetarian sausage/fish fingers. Our study provides first insights in the oxylipin concentrations of our daily food, indicating a relevant intake.

Influence of Dietary n-3 Long Chain Polyunsaturated Fatty Acid Intake on Oxylipins in Erythrocytes of Women with Rheumatoid Arthritis.

Oxylipins derived from n-3 fatty acids are suggested as the link between these fatty acids and reduced inflammation. The aim of the present study was to explore the effect of a randomized controlled cross-over intervention on oxylipin patterns in erythrocytes. Twenty-three women with rheumatoid arthritis completed 2 × 11-weeks exchanging one cooked meal per day, 5 days a week, for a meal including 75 g blue mussels (source for n-3 fatty acids) or 75 g meat. Erythrocyte oxylipins were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results were analyzed with multivariate data analysis. Orthogonal projections to latent structures (OPLS) with effect projections and with discriminant analysis were performed to compare the two diets' effects on oxylipins. Wilcoxon signed rank test was used to test pre and post values for each dietary period as well as post blue-mussel vs. post meat. The blue-mussel diet led to significant changes in a few oxylipins from the precursor fatty acids arachidonic acid and dihomo-É£-linolenic acid. Despite significant changes in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and free EPA in erythrocytes in the mussel group, no concurrent changes in their oxylipins were seen. Further research is needed to study the link between n-3 fatty-acid intake, blood oxylipins, and inflammation.

Early detection of lipid oxidation in infant milk formula by measuring free oxylipins-Comparison with hydroperoxide value and thiobarbituric acid reactive substance methods.

Infant milk formula was used as a model food to compare the sensitivity of thiobarbituric acid reactive substances (TBARS) and hydroperoxide methods to UPLC-MS/MS oxylipin analysis for detecting early lipid oxidation. Two different infant milk formulas were tested during 21 days of storage at 4°C. Formulas 1 and 2 contained canola oil and canola oil + 1% docosahexaenoic acid ethyl ester, respectively. Formulas were sampled up to 21 days of storage. Formula 2 had higher peroxide values than Formula 1 across all time points. However, no significant differences over time in TBARS and peroxide values in either formula were observed. Several oxylipins increased in both formulas starting on day 7 (linoleic acid and alpha-linolenic acid-derived oxylipins in Formula 1 and DHA-derived oxylipins in Formula 2). These results indicate that free oxylipins are effective in detecting early lipid oxidation and distinguishing between formulations containing different fatty acids. PRACTICAL APPLICATION: We have recently shown that primary oxidation products known as oxylipins can be measured in their free form by ultra-high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to detect early lipid oxidation. However, a head-to-head comparison of the sensitivity of this approach to conventional spectrophotometric methods has not been evaluated. Our results indicate that free oxylipin measurements are better than conventional methods in detecting early lipid oxidation in milk infant formula  distinguishing between different formulations.

Omega-6 and Omega-3 Fatty Acid-Derived Oxylipins from the Lipoxygenase Pathway in Maternal and Umbilical Cord Plasma at Delivery and Their Relationship with Infant Growth.

Omega-3 and omega-6 fatty acids are important for neonatal development and health. One mechanism by which omega-3 and omega-6 fatty acids exert their effects is through their metabolism into oxylipins and specialized pro-resolving mediators. However, the influence of oxylipins on fetal growth is not well understood. Therefore, the objective of this study was to identify oxylipins present in maternal and umbilical cord plasma and investigate their relationship with infant growth. Liquid chromatography-tandem mass spectrometry was used to quantify oxylipin levels in plasma collected at the time of delivery. Spearman's correlations highlighted significant correlations between metabolite levels and infant growth. They were then adjusted for maternal obesity (normal body mass index (BMI: ≤30 kg/m2) vs. obese BMI (>30 kg/m2) and smoking status (never vs. current/former smoker) using linear regression modeling. A p-value < 0.05 was considered statistically significant. Our study demonstrated a diverse panel of oxylipins from the lipoxygenase pathway present at the time of delivery. In addition, both omega-3 and omega-6 oxylipins demonstrated potential influences on the birth length and weight percentiles. The oxylipins present during pregnancy may influence fetal growth and development, suggesting potential metabolites to be used as biomarkers for infant outcomes.

Quantitative analysis of n-3 polyunsaturated fatty acids and their metabolites by chemical isotope labeling coupled with liquid chromatography - mass spectrometry.

n-3 polyunsaturated fatty acids (PUFAs) and their metabolites play the crucial role in a wide range of physiologic and pathologic processes, including cardiovascular, neurodegenerative diseases, and inflammation-associated disorders. However, the quantitative analysis of n-3 PUFAs and their metabolites, oxylipins, is obstructed by high structural similarity, poor ionization efficiency and low abundance. In this study, a sensitive method was developed to quantify 28 n-3 PUFAs/oxylipins using chemical isotope labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Standards labeled with cholamine-d9 were used as one-to-one internal standards to achieve accurate quantification. The cholamine-d0-derivatized biological samples were mixed with cholamine d9-labeled standards for LC-MS/MS with multiple reaction monitoring. After cholamine derivatization, both MS sensitivity and chromatographic performance of n-3 PUFAs/oxylipins were substantially improved. Furthermore, the relationship between retention time and substituent position of regioisomers, and their fragmentation patterns were investigated, which may facilitate the identification of unknown oxylipins. Additionally, the developed method was applied to quantify the target n-3 PUFAs/oxylipins in serum and brain tissue from fish oil-supplemented mice, which exhibited its great potential and practicability. Collectively, this sensitive and reliable method may facilitate the elucidation of the roles of n-3 PUFAs/oxylipins in the physiological and pathological processes.

Quantitation of Oxylipins in Fish and Algae Oil Supplements Using Optimized Hydrolysis Procedures and Ultra-High Performance Liquid Chromatography Coupled to Tandem Mass-Spectrometry.

Fish and algae oil supplements are enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are precursors to oxidized fatty acids, known as oxylipins. Here, we optimized a base hydrolysis method for measuring oxylipins in oil with ultrahigh-performance liquid chromatography coupled to tandem mass-spectrometry (UPLC-MS/MS) and quantified them in fish and algae oil supplements. Hydrolysis of 2 µL of oil with sodium carbonate resulted in greater oxylipin concentrations and minimal matrix effects, compared to higher oil volumes (10, 20, and 30 µL). Oxylipin yield was higher when oil was hydrolyzed in methanol containing 0.1% acetic acid and 0.1% butylated hydroxytoluene, compared to no methanol, and using sodium hydroxide versus sodium carbonate. Oxylipins extracted from 2 µL of oil using sodium hydroxide in solvent showed that EPA-derived oxylipins were most abundant in fish oil (84-87%), whereas DHA-oxylipins were abundant in algae oil (83%). This study shows that fish and algae oils are direct sources of EPA- and DHA-derived oxylipins.

Spleen Oxylipin and Polyunsaturated Fatty Acid Profiles are Altered by Dietary Source of Polyunsaturated Fatty Acid and by Sex.

As the largest secondary lymphoid organ, the spleen plays an important role in immune responses. The role of arachidonic acid (ARA) and its 20-carbon eicosanoids in modulating immune function has long been of interest. However, recent advances have enabled the identification of numerous other n-6 and n-3 polyunsaturated fatty acid (PUFA)-derived oxylipins. Here, we investigate the effects of diet and sex on the spleen nonesterified oxylipin profiles and phospholipid and neutral lipid PUFA composition in Sprague-Dawley rats supplemented with oils rich in α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or linoleic acid. Dietary ALA, EPA, and DHA resulted in lower levels of ARA and ARA oxylipins. Oxylipins derived from other n-6 PUFA were also reduced despite no or opposite effect on their PUFA levels. Each diet also resulted in higher levels of oxylipins almost exclusively derived from the supplemented PUFA, despite PUFA in the same biosynthetic pathway also often being increased. Further, while oxylipin differences often reflected changes to phospholipid PUFA, there were instances where they corresponded more closely to changes in neutral lipid PUFA. With respect to sex effects, >50% of lipoxygenase ARA-derived oxylipins were higher in males in at least one diet group, while multiple DHA oxylipins were lower in males only in rats provided the DHA diet. This fundamental description of oxylipin composition in the spleen, including the influence of diet and sex and the relationship to PUFA composition, will help inform future studies examining the functions of these oxylipins under physiological and pathological conditions.

Actions of annatto-extracted tocotrienol supplementation on obese postmenopausal women: study protocol for a double-blinded, placebo-controlled, randomised trial.

INTRODUCTION: Obesity is a major health concern in postmenopausal women, and chronic low-grade inflammation contributes to the development of obesity. Cellular studies and high-fat-diet-induced obese mouse model mimicking obesity show the antiobesity effect of annatto-extracted tocotrienols (TT) with antioxidant capability. We aim to assess the safety and efficacy of TT consumption for lipid-related parameters in obese postmenopausal women. METHODS AND ANALYSIS: Eligible obese postmenopausal women will be randomly assigned to placebo group (430 mg olive oil) and TT group (DeltaGold Tocotrienol 70%) for 24 weeks. In the present study, the primary outcome is total/regional fat mass and visceral adipose tissue. The secondary outcomes include lipid profile in serum, mRNA expression of fatty acid synthase and carnitine palmitoyltransferase 1A in fat tissue, oxylipins and endocannabinoids in plasma and adipose tissue, abundance and composition of intestinal microbiome in faeces, high-sensitivity C-reactive protein (hs-CRP) in serum and leptin in serum. Every participant will be evaluated at 0 (prior to starting intervention) and 24 weeks of intervention, except for serum lipid profile and hs-CRP at 0, 12 and 24 weeks. 'Intent-to-treat' principle is employed for data analysis. Hierarchical linear modelling is used to estimate the effects of dietary TT supplementation while properly accounting for dependency of data and identified covariates. To our knowledge, this is the first randomised, placebo-controlled, double-blinded study to determine dietary TT supplementation on an obese population. If successful, this study will guide the future efficacy TT interventions and TT can be implemented as an alternative for obese population in antiobesity management. ETHICS AND DISSEMINATION: This study has been approved by the Bioethics Committee of the Texas Tech University Health Sciences Center, Lubbock. An informed consent form will be signed by a participant before enrolling in the study. The results from this trial will be actively disseminated through academic conference presentation and peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03705845.

Mechanisms, biomarkers and targets for therapy in alcohol-associated liver injury: From Genetics to nutrition: Summary of the ISBRA 2018 symposium.

The symposium "Mechanisms, Biomarkers and Targets for Therapy in Alcohol-associated Liver Injury: From Genetics to Nutrition" was held at the 19th Congress of International Society for Biomedical Research on Alcoholism on September 13th, 2018 in Kyoto, Japan. The goal of the symposium was to discuss the importance of genetics and nutrition in alcoholic liver disease (ALD) development from mechanistic and therapeutic perspectives. The following is a summary of this session addressing the gene polymorphisms in ALD, the role of zinc in gut-liver axis perturbations associated with ALD, highlighting the importance of dietary fat in ALD pathogenesis, the hepatic n6 and n3 PUFA oxylipin pattern associated with ethanol-induced liver injury, and finally deliberating on new biomarkers for alcoholic hepatitis and their implications for diagnosis and therapy. This summary of the symposium will benefit junior and senior faculty currently investigating alcohol-induced organ pathology as well as undergraduate, graduate, and post-graduate students and fellows.

Effect of Major Tea Insect Attack on Formation of Quality-Related Nonvolatile Specialized Metabolites in Tea ( Camellia sinensis) Leaves.

Insect attack is known to induce a high accumulation of volatile metabolites in tea ( Camellia sinensis). However, little information is available concerning the effect of insect attack on tea quality-related nonvolatile specialized metabolites. This study aimed to investigate the formation of characteristic nonvolatile specialized metabolites in tea leaves in response to attack by major tea insects, namely, tea green leafhoppers and tea geometrids, and determine the possible involvement of phytohormones in metabolite formation resulting from insect attack. Both tea green leafhopper and tea geometrid attacks increased the jasmonic acid and salicylic acid contents. The abscisic acid content was only increased under tea green leafhopper attack, perhaps due to special continuous piercing-sucking wounding. Tea green leafhopper attack induced the formation of theaflavins from catechins under the action of polyphenol oxidase, while tea geometrid attack increased the l-theanine content. Exogenous phytohormone treatments can affect the caffeine and catechin contents. These results will help to determine the influence of major tea pest insects on important tea quality-related metabolites and enhance understanding of the relationship of phytohormones and quality-related nonvolatile metabolite formation in tea exposed to tea pest insect attacks.

Oxylipins and Free Fatty Acids in Parenteral Lipid Emulsions Currently Used in Preterm Infant Care: An In Vitro Study.

Lipid emulsions used to support nutrition in preterm infants contain long-chain polyunsaturated fatty acids (LCPUFAs) as a source of essential fatty acids; these LCPUFAs and their parent polyunsaturated fatty acid (PUFA) can be oxidized by a variety of mechanisms to bioactive molecules called oxylipins, which are signaling molecules that initiate and/or resolve inflammation. The aim of this study was to explore levels of free LCPUFA and their related oxylipins in 3 commercially available lipid emulsions (Intralipid, SMOFlipid, and ClinOleic) using ultra high-performance liquid chromatography mass spectroscopy. Free LCPUFA were detected in all lipid emulsions tested. Seven, 8, and 9 different oxylipin compounds were detected in the 3 emulsions, respectively. The oxylipins detected were mainly derived from omega-6 PUFAs; these included 13-hydroxyoctadecadienoic acid from linoleic acid and 5-hydroxyeicosatetraenoic acid derived from arachidonic acid. It may be clinically important to know that oxylipins exist in lipid emulsions and to evaluate their potential effects on preterm infants.

Transcriptome and metabolome analysis reveal candidate genes and biochemicals involved in tea geometrid defense in Camellia sinensis.

Tea plant (Camellia sinensis (L) O. Kuntze) respond to herbivore attack through large changes in defense related metabolism and gene expression. Ectropis oblique (Prout) is one of the most devastating insects that feed on tea leaves and tender buds, which can cause severe production loss and deteriorate the quality of tea. To elucidate the biochemicals and molecular mechanism of defense against tea geometrid (TG), transcriptome and metabolome of TG interaction with susceptible (SG) and resistance (RG) tea genotypes were analyzed by using UPLC-Q-TOF-MS, GC-MS, and RNA-seq technologies. This revealed that jasmonic acid was highly induced in RG, following a plethora of secondary metabolites involved in defense against TG could be induced by jasmonic acid signaling pathway. However, the constitutively present of salicylic acid in SG might be a suppressor of jasmonate signaling and thus misdirect tea plants against TG. Furthermore, flavonoids and terpenoids biosynthesis pathways were highly activated in RG to constitute the chemical barrier on TG feeding behavior. In contrast, fructose and theanine, which can act as feeding stimulants were observed to highly accumulate in SG. Being present in the major hub, 39 transcription factors or protein kinases among putative candidates were identified as master regulators from protein-protein interaction network analysis. Together, the current study provides a comprehensive gene expression and metabolite profiles, which can shed new insights into the molecular mechanism of tea defense against TG. The candidate genes and specific metabolites identified in the present study can serve as a valuable resource for unraveling the possible defense mechanism of plants against various biotic stresses.

Sorting out the phytoprostane and phytofuran profile in vegetable oils.

Phytoprostanes (PhytoPs) and phytofurans (PhytoFs) are prostaglandin-like compounds, contributing to defense signaling and prevention of cellular damage. These plant oxylipins result from autoxidation of α-linolenic acid (ALA) and have been proposed as new bioactive compounds due to their structural analogies with isoprostanes (IsoPs) and prostanoids derived from arachidonic acid in mammals, which have demonstrated diverse biological activities. The present work assesses a wide range of vegetable oils - including extra virgin olive oils (n = 7) and flax, sesame, argan, safflower seed, grapeseed, and palm oils - for their content of PhytoPs and PhytoFs. Flax oil displayed the highest concentrations, being notable the presence of 9-epi-9-D1t-PhytoP, 9-D1t-PhytoP, 16-B1-PhytoP, and 9-L1-PhytoP (7.54, 28.09, 28.67, and 19.22 µg mL-1, respectively), which contributed to a total PhytoPs concentration of 119.15 µg mL-1, and of ent-16-(RS)-9-epi-ST-Δ14-10-PhytoF (21.46 µg mL-1). Palm and grapeseed oils appeared as the most appropriate negative controls, given the near absence of PhytoPs and PhytoFs (lower than 0.15 µg mL-1). These data inform on the chance to develop nutritional trials using flax and grapeseed oils as food matrices that would provide practical information to design further assays intended to determine the actual bioavailability/bioactivity in vivo.

Development of an efficient high-performance thin layer chromatography method for determination of jasmonic acid in leaf tissue of Stevia rebaudiana (Bertoni) Bertoni.

Determination of endogenous levels of jasmonic acid (JA) is essential, as it plays a pivotal role in the physiological processes during a plant's life cycle. A high performance thin layer chromatography (HPTLC) method was developed for the detection and quantification of JA in leaf extracts of medicinal plant, Stevia rebaudiana (Bertoni) Bertoni. The separation was achieved using the solvents ethyl acetate-benzene (1:1, v/v) as the mobile phase, followed by scanning and quantification at 295 nm. Densitometric analysis of leaf extract resulted in compact spots for JA at Rf = 0.45 ± 0.02. The linear regression analysis showed good relationship with r value. The recovery rate of JA indicated good reproducibility and repeatability of the assay. The statistical analysis proved the reproducibility of the method; therefore, it can be employed for routine quantification of JA in different tissue samples of S. rebaudiana and may also be extrapolated to other biological samples.

Quantitative profiling of oxylipins through comprehensive LC-MS/MS analysis of Fusarium verticillioides and maize kernels.

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize, even if frequently asymptomatic, producing a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins result as signals able to modulate the secondary metabolism in fungi. In keeping with this, a novel, quantitative LC-MS/MS method was designed to quantify up to 17 different oxylipins produced by F. verticillioides and maize kernels. By applying this method, we were able to quantify oxylipin production in vitro - F. verticillioides grown into Czapek-Dox/yeast extract medium amended with 0.2% w/v of cracked maize - and in vivo, i.e. during its growth on detached mature maize ears. This study pinpoints the role of oxylipins in a plant pathogen such as F. verticillioides and sets up a novel tool aimed at understanding the role oxylipins play in mycotoxigenic pathogens during their interactions with respective hosts.

Development of an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of salicylic acid, jasmonic acid, and abscisic acid in rose leaves.

This paper describes a method to detect and quantitate the endogenous plant hormones (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in hybrid rose leaf matrices. Deuterium-labeled [(2)H6] (+)-2-cis-4-trans-abscisic acid, [(2)H6] (±)-jasmonic acid, and [(2)H4]-salicylic acid were used as internal standards. Rose samples (10 mg) were extracted with methanol/water/acetic acid (10:89:1) and subsequently purified on an Oasis MCX 1 cm(3) Vac SPE cartridge. Performance characteristics were validated according to Commission Decision 2002/657/EC. Recovery, repeatability, and within-laboratory reproducibility were acceptable for all phytohormones tested at three different concentrations. The decision limit and detection capability for (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid were 0.0075 and 0.015 µg/g, 0.00015 and 0.00030 µg/g, and 0.0089 and 0.018 µg/g, respectively. Matrix effects (signal suppression or enhancement) appeared to be high for all substances considered, implying the need for quantitation based on matrix-matched calibration curves.

Quantification of selected endogenous hydroxy-oxylipins from tropical marine macroalgae.

The present study investigated the contents of hydroxy-oxylipins hydroxyoctadecadienoic acids (HODEs), hydroxyoctadecatrienoic acids (HOTrEs), and hydroxyeicosatetraenoic acids (HETEs) in 40 macroalgae belonging to the Chlorophyceae, Rhodophyceae and, Phaeophyceae. The hydroxy-oxylipin content was low and ranged from 0.14 ± 0.012 ng/g (Codium dwarkense) to 8,161.9 ± 253 ng/g (Chaetomorpha linum) among the Chlorophyceae, 345.4 ± 56.8 ng/g (Scytosiphon lomentaria) to 2,574.5 ± 155.5 ng/g (Stoechospermum marginatum) among the Phaeophyceae, and 19.4 ± 2.2 ng/g (Laurencia cruciata) to 1,753.1 ± 268.2 ng/g in Gracilaria corticata v. folifera) among the Rhodophyceae on fresh weight basis (p ≤ 0.01). The concentrations of C18-oxylipins were greater than C20-oxylipins in all the investigated macroalgae, except forUlva linza, Codium sursum, Dictyopteris deliculata, S. marginatum, Sargassum tenerrimum, Gracilaria spp. (except G. textorii), Rhodymenia sonderi, and Odonthalia veravalensis.The macroalgal species rich in HODEs, HOTrEs, and HETEs were segregated using principal component analysis. The red macroalgae showed the highest contents of HETEs, followed by brown and green macroalgae in consistent with their PUFA profiles. The relative contents of isomeric forms of oxylipins displayed the species-specific positional selectivity of lipoxygenase (LOX) enzyme in macroalgae. All the species exhibited 13-LOX specificity for linoleic acid analogous of higher plants, while 21 out of 40 species showed 9-LOX selectivity for the oxygenation of α-linolenic acid. No trend was observed for the oxygenation of arachidonic acid in macroalgae, except for in the Halymeniales, Ceramiales (except L. cruciata), and Corallinales. This study infers that LOX products, octadecanoids and eicosanoids, described in macroalgal taxa were similar to those of higher plants and mammals, respectively, and thus can be utilized as an alternative source of chemically synthesized oxylipin analogues in therapeutics, cosmetics, and nutritional oil supplements.

Dietary linoleic acid and α-linolenic acid differentially affect renal oxylipins and phospholipid fatty acids in diet-induced obese rats.

Analysis of oxylipins derived from fatty acids may provide insight into the biological effects of dietary lipids beyond their effects on tissue fatty acid profiles. We have previously observed that diets with higher amounts of α-linolenic acid (ALA; 18:3n3) are associated with reduced obesity-related glomerulopathy (ORG). Therefore, to examine the renal oxylipin profile, the effects of dietary linoleic acid (LA; 18:2n6) and ALA on oxylipins and renal phospholipid fatty acid composition, and the relationship between oxylipins and ORG, diet-induced obese rats displaying ORG were fed 8 different diets for 8 wk as follows (oil/oil = combination of two oils) [shown as ALA/LA (in g) per 100 g oil]: canola/flax (20/18), canola (8/18), soy (9/53), high-oleic canola/canola (5/16), high-oleic canola (2/15), lard/soy (1/8), and safflower (0.2/73). Targeted lipidomic analysis by HPLC-tandem mass spectrometry revealed that LA and ALA oxylipins comprised 60% of the total renal oxylipin profile examined. Of the >60 oxylipins screened, only those derived either directly or indirectly from ALA were associated with less glomerulomegaly, indicative of reduced ORG progression. Both the amount and ratio of dietary LA and ALA influenced renal polyunsaturated fatty acids (PUFAs); in contrast, only fatty acid amount altered oxylipins derived from these fatty acids, but there was no apparent competition by LA or ALA on their formation. Dietary LA incorporation into renal phospholipids was higher than for ALA, but ALA oxylipin:ALA ratios were higher than the analogous LA ratios for select lipoxygenase reactions. This indicates that the effect of dietary ALA on renal oxylipins exceeded what was reflected in renal PUFA composition. In conclusion, dietary LA and ALA have differential effects on renal oxylipins and PUFAs, and ALA-derived oxylipins are associated with renoprotection in this model of ORG.

Determination of chiral jasmonates in flowers by GC/MS after monolithic material sorptive extraction.

A GC/MS method with monolithic material sorptive extraction (MMSE) pretreatment was developed to determine contents of the enantiomers of jasmonic acid and methyl jasmonate in flowers. To optimize MMSE extraction, several MMSE parameters were investigated, including extraction temperature, extraction time, and extraction solvent. Under the optimal conditions, extraction efficiency was good. Using the selected-ion monitoring mode, the limit of detection (LOD, S/N = 3) for methyl jasmonates was 0.257 ng/mL. The limit of quantitation (LOQ, S/N = 10) was 0.856 ng/mL. The linearity range was 1-100 ng/mL. The average recovery of methyl jasmonate at lower concentration was 116.8% (2 ng/mL). The relative standard deviation of methyl jasmonate contents determined within the linear range of detection was less than or equal to 15% of the mean determined level. The proposed method is rapid, sensitive, and competently applied to the determination of jasmonic acid and methyl jasmonate enantiomers in flowers.

Metabolomic analysis of methyl jasmonate-induced triterpenoid production in the medicinal herb Centella asiatica (L.) urban.

Centella asiatica is an important source of biologically active pentacyclic triterpenoids. The enhancement of the biosynthesis of the centellosides by manipulation of associated metabolic pathways is receiving much attention. Jasmonates play critical roles in plant metabolism by up-regulating the expression of genes related to secondary metabolites. Here, we investigated the effect of methyl jasmonate (MeJa) in C. asiatica through targeted metabolomic profiling of asiaticoside and madecassoside as well as their aglycones, asiatic acid and madecassic acid. Cell suspensions were treated with 0.2 mM MeJa for 2, 4 and 6 days. Liquid chromatography coupled to mass spectrometry (LC-MS) was used to explore induced changes in metabolite profiles, both qualitatively and quantitatively. Principal component analysis (PCA)-derived scores plots revealed clusters of sample replicates for control and treated samples at 2, 4 and 6 days while loading plots aided in identifying signatory biomarkers (asiatic acid and madecassic acid, as well as asiaticoside and madecassoside) that clearly demonstrate the variability between samples. In addition to increased biosynthesis of the targeted centelloids, other differential changes in the intracellular metabolite profiles reflected the response of the C. asiatica cells to the MeJa-treatment as a reprogramming of the metabolome.