Fatty Acid Epoxides Research Articles

Dual Inhibitors of Cyclooxygenase‐2 and Soluble Epoxide Hydrolase: Studies of Binding Modes at the Active Sites and Time‐dependency of Inhibition, and Development of Water‐soluble Prodrugs

In recent years, there has been increasing interest in designing multi‐target agents that improve efficacy and safety compared to their single target counterparts.Cyclooxygenase‐2 (COX‐2) selective inhibitors (coxibs) are effective drugs for treating inflammation, pain, and cancer. However, their mechanism‐based side effects limit their use. Meanwhile, soluble epoxide hydrolase (sEH) is involved in the metabolism of fatty acid epoxides that are obtained by cytochrome P450 (CYP) from arachidonic acid. These CYP metabolites, epoxyeicosatrienoic acids (EETs), are anti‐hypertensive, anti‐inflammatory, analgesic and organ‐protective. Therefore, co‐inhibition of both enzymes with a combination treatment is synergistic and is maximized by combining both actions into a single molecule, such as PTUPB. These dual inhibitors showed superior efficacy in several rodent models of inflammatory pain, metabolic syndrome, and cancer.We found that they inhibit COX‐2 tightly and in a time‐dependent manner (25‐fold more potent) and EETs moderately inhibit COX‐2. Computational studies of their binding modes at the active site of enzymes using molecular docking confirm these results. In addition to these studies, water‐soluble prodrugs of these dual inhibitors were developed for their use in animal studies.Support or Funding InformationThis work was supported in part by NIEHS grant R01ES02710, NIEHS Superfund Basic Research Program grant P42 ES04699, National Institute of Neurological Disorders and Stroke (NINDS) U54 NS079202. NIH 5T32DC008072‐05.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oligomeric Composition of Polyols From Fatty Acid Methyl Ester: The Effect of Ring‐Opening Reactants of Epoxide Groups

AbstractCommercial availability of fatty acid methyl ester (FAME) from palm oil targeted for biodiesel offers a good feedstock for the production of structurally well‐defined polyols for polyurethane applications. The effect of molecular weight (MW), odd and even carbon numbers, and the linear and branched structure reactants used in the ring‐opening reaction of epoxidized fatty acid methyl ester (E‐FAME) on the properties of polyols was investigated. Conversions of E‐FAME to PolyFAME polyols were confirmed by Fourier transform infrared analysis, oxirane oxygen content, and hydroxyl number. Gel permeation chromatography (GPC) calibrated against polyether polyols as a standard and vapor pressure osmometry were used for MW determination. GPC chromatograms of PolyFAME polyols clearly demonstrated the formation of oligomers during ring‐opening reactions. MW, and odd and even carbon numbers in a structure of linear diols and branched diol used in the syntheses of PolyFAME polyols did not have an effect on crystallinity, glass transition, or melt temperatures measured using Differential scanning calorimetry (DSC). PolyFAME polyols ring‐opened with water, methanol, and 1,2‐propanediol contained secondary hydroxyl groups, whereas PolyFAME polyols ring‐opened with linear diols contained a mixture of primary and secondary hydroxyl groups. It was found that the concentration of primary hydroxyl groups increased significantly by increasing the number of carbons from C2 to C3 in the linear diols. The viscosity of PolyFAME polyols also increased with the MW of linear diols used in the E‐FAME ring‐opening reaction. These findings would be beneficial for formulators in choosing the most cost effective polyols for polyurethane formulations.

Epoxidation of Fatty Acids and Vegetable Oils Assisted by Microwaves Catalyzed by a Cation Exchange Resin

Epoxidation of oleic acid and cottonseed oil was conducted in a semibatch reactor with in-situ-formed percarboxylic acid (peracetic acid or perpropionic acid), using hydrogen peroxide as an oxidizi ...

Identification of Structural-Activity Features of Long Chain Polyunsaturated Fatty Acid Epoxides in Angiogenesis

Identification of Structural-Activity Features of Long Chain Polyunsaturated Fatty Acid Epoxides in Angiogenesis

Omega-3 fatty acid epoxides are autocrine mediators that control the magnitude of IgE-mediated mast cell activation.

Critical to the function of mast cells in immune responses including allergy is their production of lipid mediators, among which only omega-6 (ω-6) arachidonate-derived eicosanoids have been well characterized. Here, by employing comprehensive lipidomics, we identify omega-3 (ω-3) fatty acid epoxides as new mast cell-derived lipid mediators and show that they are produced by PAF-AH2, an oxidized-phospholipid-selective phospholipase A2. Genetic or pharmacological deletion of PAF-AH2 reduced the steady-state production of ω-3 epoxides, leading to attenuated mast cell activation and anaphylaxis following FcɛRI cross-linking. Mechanistically, the ω-3 epoxides promote IgE-mediated activation of mast cells by downregulating Srcin1, a Src-inhibitory protein that counteracts FcɛRI signaling, through a pathway involving PPARg. Thus, the PAF-AH2-ω-3 epoxide-Srcin1 axis presents new potential drug targets for allergic diseases.

Beyond detoxification: a role for mouse mEH in the hepatic metabolism of endogenous lipids

Microsomal and soluble epoxide hydrolase (mEH and sEH) fulfill apparently distinct roles: Whereas mEH detoxifies xenobiotics, sEH hydrolyzes fatty acid (FA) signaling molecules and is thus implicated in a variety of physiological functions. These epoxy FAs comprise epoxyeicosatrienoic acids (EETs) and epoxy-octadecenoic acids (EpOMEs), which are formed by CYP epoxygenases from arachidonic acid (AA) and linoleic acid, respectively, and then are hydrolyzed to their respective diols, the so-called DHETs and DiHOMEs. Although EETs and EpOMEs are also substrates for mEH, its role in lipid signaling is considered minor due to lower abundance and activity relative to sEH. Surprisingly, we found that in plasma from mEH KO mice, hydrolysis rates for 8,9-EET and 9,10-EpOME were reduced by 50% compared to WT plasma. This strongly suggests that mEH contributes substantially to the turnover of these FA epoxides—despite kinetic parameters being in favor of sEH. Given the crucial role of liver in controlling plasma diol levels, we next studied the capacity of sEH and mEH KO liver microsomes to synthesize DHETs with varying concentrations of AA (1–30 μM) and NADPH. mEH-generated DHET levels were similar to the ones generated by sEH, when AA concentrations were low (1 μM) or epoxygenase activity was curbed by modulating NADPH. With increasing AA concentrations sEH became more dominant and with 30 μM AA produced twice the level of DHETs compared to mEH. Immunohistochemistry of C57BL/6 liver slices further revealed that mEH expression was more widespread than sEH expression. mEH immunoreactivity was detected in hepatocytes, Kupffer cells, endothelial cells, and bile duct epithelial cells, while sEH immunoreactivity was confined to hepatocytes and bile duct epithelial cells. Finally, transcriptome analysis of WT, mEH KO, and sEH KO liver was carried out to discern transcriptional changes associated with the loss of EH genes along the CYP-epoxygenase–EH axis. We found several prominent dysregulations occurring in a parallel manner in both KO livers: (a) gene expression of Ephx1 (encoding for mEH protein) was increased 1.35-fold in sEH KO, while expression of Ephx2 (encoding for sEH protein) was increased 1.4-fold in mEH KO liver; (b) Cyp2c genes, encoding for the predominant epoxygenases in mouse liver, were mostly dysregulated in the same manner in both sEH and mEH KO mice, showing that loss of either EH has a similar impact. Taken together, mEH appears to play a leading role in the hydrolysis of 8,9-EET and 9,10-EpOME and also contributes to the hydrolysis of other FA epoxides. It probably profits from its high affinity for FA epoxides under non-saturating conditions and its close physical proximity to CYP epoxygenases, and compensates its lower abundance by a more widespread expression, being the only EH present in several sEH-lacking cell types.

O25 A novel synthetic ω-3 fatty acid epoxide analogue activates breast cancer cell killing in vitro and in vivo

Triple-negative breast cancer (TNBC) has a poor prognosis and new drugs are urgently needed. The CYP-derived ω-3 epoxyeicosapentaenoic acid and its saturated analogues were found to activate apoptosis in MDA-MB-231 TNBC cells [1] , [2] , but in vivo instability limits their drug potential. We prepared stable bioisosteres of ω-3 epoxy-fatty acids for testing in TNBC cells in vitro and in vivo. Cell viability was assessed by ATP formation, cell killing by caspase-3/7 activity and annexin V/7AAD staining, mitochondrial membrane potential with JC-1, gene profiling by RNA-seq and real-time RT-PCR, and protein expression by immunoblotting. In vivo tumour growth was evaluated in nude mice carrying intramammary xenografts. Of 11 bioisosteres, one agent – CTU – impaired ATP formation, activated apoptosis and disrupted the mitochondrion in TNBC cells. RNA-seq profiling identified 382 differentially-expressed genes (⩾2-fold) in CTU-treated cells with selective enrichment of genes for endoplasmic reticulum (ER) stress (XBP-1 and CHOP). The ER-stress inhibitors, AEBSF and toyocamycin, attenuated caspase-3/7 activation by CTU. MDA-MB-231 xenografts in nude mice were decreased to 42 ± 13% of control after CTU (40 mg/kg i.p., 7 weeks). Increased TUNEL and Ki-67 staining in tumours is consistent with CTU-mediated apoptosis and decreased proliferation in vivo. CTU acts by a unique mechanism to target the mitochondrion and activate ER-stress, and is the prototype of a new class of agents based on naturally occurring lipids with activity in TNBC.

Postprandial effect to decrease soluble epoxide hydrolase activity: roles of insulin and gut microbiota

Epoxides of free fatty acids (FFAs), especially epoxyeicosatrienoic acids (EETs), are lipid mediators with beneficial effects in metabolic and cardiovascular (CV) health. FFA epoxides are quickly metabolized to biologically less active diols by soluble epoxide hydrolase (sEH). Inhibition of sEH, which increases EET levels, improves glucose homeostasis and CV health and is proposed as an effective strategy for the treatment of diabetes and CV diseases. Here, we show evidence that sEH activity is profoundly reduced in postprandial states in rats; plasma levels of 17 sEH products (i.e., FFA diols), detected by targeted oxylipin analysis, all decreased after a meal. In addition, the ratios of sEH product to substrate (sEH P/S ratios), which may reflect sEH activity, decreased ~70% on average 2.5 h after a meal in rats (P<.01). To examine whether this effect was mediated by insulin action, a hyperinsulinemic–euglycemic clamp was performed for 2.5 h, and sEH P/S ratios were assessed before and after the clamp. The clamp resulted in small increases rather than decreases in sEH P/S ratios (P<.05), indicating that insulin cannot account for the postprandial decrease in sEH P/S ratios. Interestingly, in rats treated with antibiotics to deplete gut bacteria, the postprandial effect to decrease sEH P/S ratios was completely abolished, suggesting that a gut bacteria-derived factor(s) may be responsible for the effect. Further studies are warranted to identify such a factor(s) and elucidate the mechanism by which sEH activity (or sEH P/S ratio) is reduced in postprandial states.

Lipid-modifying enzymes in oat and faba bean

The aim was to study lipase, lipoxygenase (LOX) and peroxygenase (POX) activities in oat and faba bean samples to be able to evaluate their potential in formation of lipid-derived off-flavours. Lipase and LOX activities were measured by spectroscopy, and POX activities via the formation of epoxides. An ultra-high performance liquid chromatography method was developed to study the formation of fatty acid epoxides. The epoxides of esters were measured by gas chromatography. Mass spectroscopy was used to verify the identity of the epoxides. Both oat and faba bean possessed high lipase activities. In faba bean, LOX catalysed the formation of hydroperoxides, whose break-down products are the likely cause of off-flavours. Since oat had low LOX activity, autoxidation is needed to initiate lipid oxidation. Oat had high POX activity, which is able to convert hydroperoxides to epoxy and hydroxy fatty acids that could contribute significantly to off-flavours. POX activity in the faba bean was low. Thus, in faba bean volatile lipid oxidation products could rapidly be formed by LOX, whereas in oat reactions are slower due to the need of autoxidation prior to further reactions.

Identification and functional analysis of new peroxygenases in oat.

Two new peroxygenases for the biosynthesis of epoxy fatty acids in oat were identified and functionally analyzed by heterologous expression along with rationally designed site-directed mutagenesis. Oat (Avena sativa L.) contains a large family of peroxygenases, a group of heme-containing monooxygenases catalyzing hydroperoxide-dependent epoxidation of unsaturated fatty acids. Here, we report identification and functional analysis of two new peroxygenases AsPXG2 and AsPXG3 from oat. The open reading frame (ORF) of AsPXG2 contains 702 bps encoding a polypeptide of 233 amino acids, while the ORF of AsPXG3 is 627 bps coding for 208 amino acids. Both AsPXG2 and AsPXG3 comprise a single transmembrane domain, conserved histidines for heme binding and a conserved EF-hand motif for calcium binding, but they only share about 50% amino acid sequence identity with each other. When expressed in Escherichia coli and Pichia pastoris, AsPXG3 showed high epoxidation activity, while AsPXG2 exhibited no activity in E. coli and low activity in P. pastoris. AsPXG3 could effectively epoxidize both mono- and polyunsaturated fatty acids with linolenic acid being the most preferred substrate. Site-directed mutagenesis was employed to investigate the structure-function relationship of oat peroxygenase on 12 conserved residues of AsPXG3. Replacement of two conserved histidines, the ligands to the prosthetic heme group of the peroxygenase, by alanine resulted in complete loss of activity. Substitution of three conserved residues surrounding the two histidines resulted in reduction of the enzymatic activity by more than 80%. These results imply that these conserved residues might be located in or near the catalytic pocket, where the two histidine residues coordinate the heme group and the surrounding residues define the shape and size of the pocket for interaction with the heme as well as two substrates.

Arabidopsis thaliana EPOXIDE HYDROLASE1 (AtEH1) is a cytosolic epoxide hydrolase involved in the synthesis of poly-hydroxylated cutin monomers.

Epoxide hydrolases (EHs) are present in all living organisms. They have been extensively characterized in mammals; however, their biological functions in plants have not been demonstrated. Based on in silico analysis, we identified AtEH1 (At3g05600), a putative Arabidopsis thaliana epoxide hydrolase possibly involved in cutin monomer synthesis. We expressed AtEH1 in yeast and studied its localization invivo. We also analyzed the composition of cutin from A. thaliana lines in which this gene was knocked out. Incubation of recombinant AtEH1 with epoxy fatty acids confirmed its capacity to hydrolyze epoxides of C18 fatty acids into vicinal diols. Transfection of Nicotiana benthamiana leaveswith constructs expressing AtEH1 fused to enhanced green fluorescent protein (EGFP) indicated that AtEH1 is localized in the cytosol. Analysis of cutin monomers in loss-of-function Ateh1-1 and Ateh1-2 mutants showed an accumulation of 18-hydroxy-9,10-epoxyoctadecenoic acid and a concomitant decrease in corresponding vicinal diols in leaf and seed cutin. Compared with wild-type seeds, Ateh1 seeds showed delayed germination under osmotic stress conditions and increased seed coat permeability to tetrazolium red. This work reports a physiological role for a plant EH and identifies AtEH1 as a new member of the complex machinery involved in cutin synthesis.

Abstract 572: The Effect of Inflammation and Soluble Epoxide Hydrolase Inhibition on Fatty Acid Epoxide Incorporation Into VLDL

Objective: We have previously observed fatty acid epoxides, a class of potent anti-inflammatory oxylipins, in circulating VLDL. The source of these epoxides is unknown. Cytochrome P450 (CYP450) produces them via oxygenation of polyunsaturated fatty acids (PUFAs), and soluble epoxide hydrolase (sEH) converts them to diols. Our objectives were 1) to investigate if incorporation of epoxides into VLDL occurs via hepatic VLDL synthesis and 2) to determine if incorporation is modulated by inflammation or by inhibition of hepatic sEH. Approach and Results: A 2х2 factorial design was used for treatment assignment. Livers were isolated from rats treated with pro-inflammatory lipopolysaccharide (LPS, 10 mg/kg ip) or saline. AUDA, an inhibitor of sEH (10 μM), was included or excluded in the perfusate (Control, N=3; LPS, N=4; AUDA, N=4; LPS+AUDA, N=4). Livers were perfused for 180 minutes. VLDL was isolated by ultra-centrifugation, then analyzed by LC-MS/MS for oxylipin content. Analyzed epoxides and diols were derived from alpha-linolenic acid (ALA), linoleic acid (LA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Two-way ANOVA’s were used with triglyceride concentration as a covariate. Concentrations (nM) are reported as mean [95% CI]. DHA-derived epoxides increased with AUDA treatment (3.91 [3.01, 5.07]) compared to livers without AUDA (2.06 [1.58, 2.67]) (p=0.004), but other epoxides were unchanged by AUDA. EPA and ALA-derived epoxides decreased with LPS treatment (0.32 [0.22, 0.47]; 2.44 [2.07, 2.87]) compared to animals without LPS (0.73 [0.46, 1.16]; 3.28 [2.71, 3.96]) (p=0.01; 0.02). AA and DHA-derived diols decreased with LPS treatment (1.01 [0.82, 1.25]; 0.21 [0.17, 0.26]) compared to animals without LPS (1.46 [1.15, 1.86]; 0.31 [0.24, 0.39]) (p=0.03; 0.03). Conclusions: Treatment with LPS and AUDA have significant effects on incorporation of epoxides and diols into VLDL, supporting hepatic incorporation controlled by inflammation. Inflammation decreased select EPA- and ALA-derived epoxides. In contrast, sEH inhibition increased only DHA-derived epoxides. Surprisingly, in VLDL only epoxides derived from omega-3 fatty acids were affected by either inflammation or inhibition of sEH.

Generation and characterization of epoxide hydrolase 3 (EPHX3)-deficient mice.

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs) which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH) and soluble epoxide hydrolase (EPHX2, sEH) were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME) and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT). Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS) exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic disruption of Ephx3 does not result in an overt phenotype and has no significant effects on the metabolism of EETs or EpOMEs in vivo.

Lipidomic Profiling Identifies Cytochrome P450 as A Therapeutic Target for Colitis‐Associated Colorectal Cancer

Eicosanoids and associated lipid mediators play central roles in regulating inflammation and carcinogenesis. While most previous studies of lipid mediators in colorectal cancer have only analyzed single or limited number of lipid mediators, few systematic studies have been carried out. Here we used a LC‐MS/MS‐based lipidomics, which can analyze &gt;100 bioactive lipid mediators produced by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes, to profile azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced colorectal cancer in mice. Lipidomics showed that CYP‐derived fatty acid epoxides were the only lipid mediators which were increased in both plasma and colon tissues of AOM/DSS‐treated mice. RT‐PCR and western blotting validated that the expressions of CYP were increased, while the expression of soluble epoxide hydrolase (the dominant enzyme in degrading fatty acid epoxides) was reduced, in colon tissues of AOM/DSS‐treated mice. To study the functional roles of CYP enzymes, we treated mice with SKF‐525A and clotrimazole, which are two different selective inhibitors of CYP enzymes involved in biosynthesis of fatty acid epoxides, and found that CYP inhibitors significantly suppressed AOM/DSS‐induced colorectal cancer in mice. To elucidate the specific lipid mediator involved in the pro‐colorectal cancer effects of CYP, we treated mice with epoxyoctadecenoic acids (EpOMEs) which are metabolites of linoleic acid produced by CYP, and found that EpOMEs worsened DSS‐induced colitis. Together, these results suggest that the previously unappreciated CYP pathway plays critical roles in promoting progression of colorectal cancer through formation of pro‐inflammatory EpOMEs, and CYP enzymes could be novel therapeutic targets of colitis‐associated colorectal cancer.Support or Funding InformationUSDA NIFA 2016‐67017‐24423, NIEHS R01 ES002710 and Superfund Research Program P42 ES04699

Thermal Safety Assessment through the Concept of Structure–Reactivity: Application to Vegetable Oil Valorization

The concept of linear free-energy relationships, and more particularly the Taft equation, was extended to a multiphase system with several reactions and reaction centers: the epoxidation of vegetable oils and free fatty acids. The epoxidation of vegetable oils and free fatty acids by different percarboxylic acids produced in situ is an exothermic reaction system which can lead to a thermal runaway. The safety criterion time-to-maximum-rate under adiabatic conditions (TMRad), linked to the kinetics of the system, was determined with the advanced reactive system screening tool (ARSST). We have demonstrated that TMRad values for the epoxidation of vegetable oils and free fatty acids by using different carboxylic acids follow the Taft equation. The epoxidation reaction series by different carboxylic acids was found to be more sensitive to the polar effect than steric effect. It was demonstrated that, by knowing the carboxylic acid used for the epoxidation, it is possible to predict the values of TMRad.

Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.)

Uniqueness and diversity of mango flavour across various cultivars are well known. Among various flavour metabolites lactones form an important class of aroma volatiles in certain mango varieties due to their ripening specific appearance and lower odour detection threshold. In spite of their biological and biochemical importance, lactone biosynthetic pathway in plants remains elusive. Present study encompasses quantitative real-time analysis of 9-lipoxygenase (Mi9LOX), epoxide hydrolase 2 (MiEH2), peroxygenase, hydroperoxide lyase and acyl-CoA-oxidase genes during various developmental and ripening stages in fruit of Alphonso, Pairi and Kent cultivars with high, low and no lactone content and explains their variable lactone content. Study also covers isolation, recombinant protein characterization and transient over-expression of Mi9LOX and MiEH2 genes in mango fruits. Recombinant Mi9LOX utilized linoleic and linolenic acids, while MiEH2 utilized aromatic and fatty acid epoxides as their respective substrates depicting their role in fatty acid metabolism. Significant increase in concentration of δ-valerolactone and δ-decalactone upon Mi9LOX over-expression and that of δ-valerolactone, γ-hexalactone and δ-hexalactone upon MiEH2 over-expression further suggested probable involvement of these genes in lactone biosynthesis in mango.

Soluble epoxide hydrolase inhibition alleviates neuropathy in Akita (Ins2 Akita) mice

The soluble epoxide hydrolase (sEH) is a regulatory enzyme responsible for the metabolism of bioactive lipid epoxides of both omega-6 and omega-3 long chain polyunsaturated fatty acids. These natural epoxides mediate cell signaling in several physiological functions including blocking inflammation, high blood pressure and both inflammatory and neuropathic pain. Inhibition of the sEH maintains the level of endogenous bioactive epoxy-fatty acids (EpFA) and allows them to exert their generally beneficial effects. The Akita (Ins2Akita or Ins2C96Y) mice represent a maturity-onset of diabetes of the young (MODY) model in lean, functionally unimpaired animals, with a sexually dimorphic disease phenotype. This allowed for a test of male and female mice in a battery of functional and nociceptive assays to probe the role of sEH in this system. The results demonstrate that inhibiting the sEH is analgesic in diabetic neuropathy and this occurs in a sexually dimorphic manner. Interestingly, sEH activity is also sexually dimorphic in the Akita model, and moreover correlates with disease status particularly in the hearts of male mice. In addition, in vivo levels of oxidized lipid metabolites also correlate with increased sEH expression and the pathogenesis of disease in this model. Thus, sEH is a target to effectively block diabetic neuropathic pain but also demonstrates a potential role in mitigating the progression of this disease.

Chemo-enzymatic epoxidation of Sapindus mukurossi fatty acids catalyzed with Candida sp. 99–125 lipase in a solvent-free system

The non-edible Sapindus mukurossi oil, containing more than 80% unsaturated fatty acids, has potential applications in plastics manufacturing. In this research, a green and economically attractive chemo-enzymatic production of epoxidized fatty acids was performed in the presence of lipase Candida sp. 99–125 and H2O2 in different systems. The obtained experimental results demonstrated that the solvent-free and n-hexane systems yielded superior results compared to other organic solvent systems. A molecular dynamic simulation further proved the influence of different solvents on the structure of lipase. Maximization of the key parameters including enzyme loading, reaction time, molar ratio of H2O2 to the number of double bonds in the substrate, agitation speed, H2O2 addition frequency, and temperature were performed in a solvent-free system. The oxirane value reached 4.61% in 8h under a gradient temperature profile (first incubated at 30°C for 6h, then increased to 35°C and maintained for another 2h) with continuous addition of H2O2. The total molar amount of H2O2 added was equal to the number of CC bonds in the substrate (expressed in moles).

Specific Caleosin/Peroxygenase and Lipoxygenase Activities Are Tissue-Differentially Expressed in Date Palm (Phoenix dactylifera L.) Seedlings and Are Further Induced Following Exposure to the Toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Two caleosin/peroxygenase isoforms from date palm, Phoenix dactylifera L., PdCLO2 and PdCLO4, were characterized with respect to their tissue expression, subcellular localization, and oxylipin pathway substrate specificities in developing seedlings. Both PdCLO2 and PdCLO4 had peroxygenase activities that peaked at the mid-stage (radicle length of 2.5 cm) of seedling growth and were associated with the lipid droplet (LD) and microsomal fractions. Recombinant PdCLO2 and PdCLO4 proteins heterologously expressed in yeast cells were localized in both LD and microsomal fractions. Each of the purified recombinant proteins exhibited peroxygenase activity but they were catalytically distinct with respect to their specificity and product formation from fatty acid epoxide and hydroxide substrates. We recently showed that date palm CLO genes were upregulated following exposure to the potent toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Hanano et al., 2016), and we show here that transcripts of 9- and 13-lipoxygenase (LOX) genes were also induced by TCDD exposure. At the enzyme level, 9-LOX and 13-LOX activities were present in a range of seedling tissues and responded differently to TCDD exposure, as did the 9- and 13-fatty acid hydroperoxide reductase activities. This demonstrates that at least two branches of the oxylipin pathway are involved in responses to the environmental organic toxin, TCDD in date palm.

Amphiphilic dipyridinium-phosphotungstate as an efficient and recyclable catalyst for triphasic fatty ester epoxidation and oxidative cleavage with hydrogen peroxide

A novel amphiphilic dipyridinium peroxophosphotungstate ion pair achieved remarkable selectivity and recyclability towards epoxidation and oxidative cleavage of fatty acids and esters.