- Kinetic isotope effects in the oxidation of arachidonic acid by soybean lipoxygenase-1
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The reaction of soybean lipoxygenase-1 with linoleic acid has been extensively studied and displays very large kinetic isotope effects. In this work, substrate and solvent kinetic isotope effects as well as the viscosity dependence of the oxidation of arachidonic acid were investigated. The hydrogen atom abstraction step was rate-determining at all temperatures, but was partially masked by a viscosity-dependent step at ambient temperatures. The observed KIEs on kcat were large (~100 at 25 °C).
- Jacquot, Cyril,Peng, Sheng,van der Donk, Wilfred A.
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- PURIFICATION AND PROPERTIES OF LIPOXYGENASE IN MARCHANTIA POLYMORPHA CULTURED CELLS
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Lipoxygenase activity has been detected in cultured cells of a bryophyte, Marchantia polymorpha (liverwort).The activity was enhanced in the stationary phase.Most of the activity occurred in the cytosolic fraction.The lipoxygenase was purified to homogeneity by ammonium sulphate fractionation and hydrophobic, ion-exchange, and gel filtration chromatography.From the SDS-polyacrylamide gel electrophoresis, the Mr of the lipoxygenase was estimated as 109600.The pH optimum was ca 9.0 and the pI was 4.8.This enzyme formed mainly 13-(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid from linoleic acid.It showed twice the activity for γ-linolenic acid than for linoleic acid. Key Word Index - Marchantia polymorpha; Marchantiaceae; liverwort; purification; lipoxygenase.
- Matsui, Kenji,Narahara, Hiroko,Kajiwara, Tadahiko,Hatanaka, Akikazu
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- The autooxidation process in linoleic acid screened by Raman spectroscopy
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The chemical changes associated to the autooxidation process of linoleic acid (LA) were detected by Raman spectroscopy and interpreted in the light of density functional theory (DFT) calculations performed for both the fatty acid and its main oxidation products. The present methodology, applied for a six-day period upon induction of oxidation (through heating), allowed to understand the chemical modifications occurring during the oxidation process. Raman spectroscopy was shown to be a suitable and reliable technique for assessing the oxidation degree of fatty acid samples, particularly pure fatty acids, mainly when computational methods are used alongside to predict the spectral features of the distinct chemical entities involved. Screening of the oxidation process was mostly based on the loss of intensity of the bands assigned to LA cis-double bonds. Copyright
- MacHado,De Carvalho, L. A. E. Batista,Otero,Marques
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- Chemoenzymatic production of (+)-coriolic acid from trilinolein: Coupled synthesis and extraction
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Chemoenzymatic conversion of trilinolein to (+)-coriolic acid was investigated in this work. Lipase-catalyzed hydrolysis of trilinolein and lipoxygenation of liberated linoleic acid were coupled in a two-phase medium that consisted of a pH 9 borate buffer and a water-immiscible organic solvent (octane). High concentrations of trilinolein could be dissolved in the organic phase (up to 340 mM). Linoleic acid, liberated after hydrolysis, transferred to the aqueous phase and was enzymatically converted to the preferred 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid with soybean lipoxygenase-1. This product, which remained in the aqueous phase, could be recovered by centrifugation and then chemically reduced to (+)-coriolic acid (purity >95%). Recovery of this compound by liquid-liquid extraction was easy. The structure of (+)-coriolic acid has been confirmed by 1H nuclear magnetic resonance spectroscopy, mass spectrometry, and infrared spectroscopy. High yields were obtained with pure trilinolein or sunflower oil as initial substrates.
- Gargouri, Mohamed,Legoy, Marie Dominique
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- Enantioselective formation of an α, β-epoxy alcohol by reaction of methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate with titanium isopropoxide
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Methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate (threo isomer) was generated from linoleic acid by the sequential action of an enzyme and two chemical reagents. Linoleic acid was treated with lipoxygenase to yield its corresponding hydroperoxide [13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid]. After methylation with CH2N2, the hydroperoxide was treated with titanium (IV) isopropoxide [Ti(O-i-Pr)4] at 5 °C for 1 h. The products were separated by normal-phase high-performance liquid chromatography and characterized with gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Approximately 30% of the product was methyl 13(S)-hydroxy-9(Z),11(E)-octadecadienoate. Over 60% of the isolated product was methyl 11(R),12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate. After quenching Ti(O-i-Pr)4 with water, the spent catalyst could be removed from the fatty products by partitioning between CH2Cl2 and water. These results demonstrate that Ti(O-i-Pr)4 selectively promotes the formation of an α-epoxide with the threo configuration. It was critically important to start with dry methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate because the presence of small amounts of water in the reaction medium resulted in the complete hydrolysis of epoxy alcohol to trihydroxy products.
- Piazza,Foglia,Nunez
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- Reaction targets of antioxidants in azo-initiator or lipid hydroperoxide induced lipid peroxidation
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Lipid peroxidation (LPO) is reported to be involved in the pathogenesis of several oxidative diseases, and several therapeutic approaches using antioxidants have been proposed. LPO is thought to progress via a complicated series of multistep reactions suggesting that the activity of each antioxidant may be different, and depends on the reacting molecules. Hence, in this study, we evaluated the inhibitory mechanisms of several antioxidants toward arachidonic acid (AA) peroxidation induced by the azo initiator 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) or a lipid hydroperoxide, hydroperoxyoctadecadienoic acid (HpODE)/hemin. Edaravone, ferrostatin-1, TEMPO and trolox effectively inhibited the production of malondialdehyde (MDA) and several oxidised AAs generated in the AAPH-induced LPO because of their scavenging ability toward lipid peroxyl radicals. In contrast, ebselen and ferrostatin-1 showed strong antioxidative activity in the HpODE/hemin-induced peroxidation. Under this condition, ebselen and ferrostatin-1 were thought to reduce HpODE and its derived alkoxyl radicals to the corresponding lipid alcohols. In conclusion, we found that each antioxidant had different antioxidative activities that prevented the progression of LPO. We expect that these findings will contribute to the design of novel therapeutic strategies using an appropriate antioxidant targeted to each step of the development of oxidative stress diseases.
- Saito, Kota,Matsuoka, Yuta,Yamada, Ken-ichi
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- The hydroperoxide moiety of aliphatic lipid hydroperoxides is not affected by hypochlorous acid
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The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and spectrometric analysis revealed that the hydroperoxide moiety of 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HpETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) is not affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride system.
- Zschaler, Josefin,Arnhold, Juergen
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- Synthesis of 13R,20-dihydroxy-docosahexaenoic acid by site-directed mutagenesis of lipoxygenase derived from Oscillatoria nigro-viridis PCC 7112
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Lipoxygenases (LOXs) are implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators involved in immune cell signaling, most of which catalyze peroxidation of polyunsaturated fatty acids by distinct regio- and stereoselectivity. Current reports suggested that conserved amino acid, Gly in R-LOXs and Ala in S-LOXs, in the catalytic domain play an important role in determining the position as well as the stereochemistry of the functional group. Recently, we have confirmed that the catalytic specificity of cyanobacterial lipoxygenase, named Osc-LOX, with alanine at 296 was 13S-type toward linoleic acid, and producing a 17S- hydroxy-docosahexaenoic acid from docosahexaenoic acid (DHA). Here, we aimed to change the catalytic property of LOX from13S-LOX to 9R-LOX by replacing Ala with Gly and to produce a lipid mediators different from the wild-type using DHA. Finally, we succeeded in generating human endogenous a 13R-hydroxy-docosahexaenoic acid and a 13R,20-dihydroxy-docosahexaenoic acid from DHA through an enzymatic reaction using the Osc-LOX-A296G. Our study could enable physiological studies and pharmaceutical research for the 13R,20-dihydroxy-docosahexaenoic acid.
- Yi, Jong-Jae,Heo, Sun-Yeon,Ju, Jung-Hyun,Oh, Baek-Rock,Son, Woo Sung,Seo, Jeong-Woo
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- Lipoxygenase inhibitory activity of anacardic acids
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6[8′(2)-Pentadecenyl]salicylic acid, otherwise known as anacardic acid (C15:1), inhibited the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, type 1) with an IC50 of 6.8 μM. The inhibition of the enzyme by anacardic acid (C15:1) is a slow and reversible reaction without residual activity. The inhibition kinetics analyzed by Dixon plots indicates that anacardic acid (C15:1) is a competitive inhibitor and the inhibition constant, K1, was obtained as 2.8 μM. Although anacardic acid (C15:1) inhibited the linoleic acid peroxidation without being oxidized, 6[8′(Z),11′(Z)-pentadecadienyl]salicylic acid, otherwise known as anacardic acid (C15:2), was dioxygenated at low concentrations as a substrate. In addition, anacardic acid (C15:2) was also found to exhibit time-dependent inhibition of lipoxygenase-1. The alk(en)yl side chain of anacardic acids is essential to elicit the inhibitory activity. However, the hydrophobic interaction alone is not enough because cardanol (C15:1), which possesses the same side chain as anacardic acid (C15:1), acted neither as a substrate nor as an inhibitor.
- Ha, Tae Joung,Kubo, Isao
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- LOXPsa1, the first recombinant lipoxygenase from a basidiomycete fungus
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A dioxygenase from the edible basidiomycete Pleurotus sapidus, originally researched because of its distinct ability to convert the sequiterpene (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was identified as a potent lipoxygenase (LOXsu
- Plagemann, Ina,Zelena, Katerina,Arendt, Philipp,Ringel, Peter D.,Krings, Ulrich,Berger, Ralf G.
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- Preparation of fatty epoxy alcohols using oat seed peroxygenase in nonaqueous media
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Peroxygenase is an enzyme of higher plants that is capable of using hydroperoxide and hydrogen peroxide for oxidation of a double bond to an epoxide. A microsomal fraction was prepared from dry oat (Arena sativa) seeds. The peroxygenase activity of this fraction was tested using fatty acid hydroperoxide 2a [13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid] and its methyl ester 2b as sources of peroxygen. These were prepared by the action of soybean lipoxygenase on linoleic acid. A high-performance liquid chromatographic assay was used to differentiate between peroxygen cleavage and peroxygen cleavage with accompanying double-bond oxidation. Higher activity was obtained with 2b compared to 2a, and peroxygen cleavage activity was observed in both aqueous and organic solvent media. Double-bond oxidation activity was high only in aqueous media and nonpolar organic solvents. Structural elucidation of the epoxidized product showed it to be the oxylipid, methyl cis-9,10-epoxy-13(S)-hydroxy-11(E)-octade-cenoate 4b, demonstrating specificity for epoxidation of the cis double bond. Trihydroxy product was not detected, demonstrating that the epoxide was not hydrolyzed.
- Piazza, George J.,Foglia, Thomas A.,Nunez, Alberto
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- Role of structure and pH in cyclization of allene oxide fatty acids: Implications for the reaction mechanism
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Incubations of allene oxide synthases of flax or maize with the E,E-isomers of the 13- and 9-hydroperoxides of linoleic acid (E,E-13- and E,E-9-HPOD, respectively) at pH 7.5 afforded substantial yields of trans-disubstituted cyclopentenones. Under the conditions used, (Z,E)-HPODs were converted mainly into α-ketols and afforded only trace amount of cyclopentenones. These findings indicated that changing the double bond geometry from Z to E dramatically increased the rate of formation of the pericyclic pentadienyl cation intermediate necessary for electrocyclization of 18:2-allene oxides and thus the yield of cyclopentenones. The well-known cyclization of the homoallylic allene oxide (12,13-EOT) derived from α-linolenic acid 13-hydroperoxide (E,Z-13-HPOT) into cis-12-oxo-10,15-phytodienoic acid was suppressed at pH below neutral and was not observable at pH 4.5. In contrast, cyclization of the allene oxide ((9E)-12,13-EOD) derived from (E,E)-13-HPOD was slightly favoured at low pH. The finding that the cyclizations of 12,13-EOT and (9E)-12,13-EOD were differently affected by changes in pH suggested that the mechanisms of cyclization of these allene oxides are distinct.
- Grechkin, Alexander N.,Chechetkin, Ivan R.,Mukhtarova, Lucia S.,Hamberg, Mats
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- Reactivity of lysine moieties toward an epoxyhydroxylinoleic acid derivative: Aminolysis versus hydrolysis
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Epoxyols are generally accepted as crucial intermediates in lipid oxidation. The reactivity of tertbutyl (9R*,10S*,11E,13S)-9,10-epoxy-13- hydroxy-11-octadecenoate (11a,b) toward lysine moieties is investigated, employing N2-acetyllysine 4-methylcoumar-7-ylamide (12) as a model for protein-bound lysine. The prefixes R* and S* denote the relative configuration at the respective stereogenic centers. Independent synthesis and unequivocal structural characterization are reported for 11a,b, its precursors, and tert-butyl (9R*,10R*,11E,13S)-10-({5-(acetylamino)-6-[(4- methyl-2-oxo-2H-chromen-7-yl)amino]-6-oxohexyl}amino)-9,13-dihydroxy-11- octadecenoate (13a-d). Reactions of 11a,b and 12 in 1-methyl-2-pyrrolidone (MP) and MP/water mixtures at pH 7.4 and 37 °C for 56 days show formation of the aminols 13a-d to be favored by an increased water content. The same trend is observed for hydrolytic cleavage of 11a,b to tert-butyl (E)-9,10,13- trihydroxy-11-octadecenoate (14) and tert-butyl (E)-9,12,13-trihydroxy-10- octadecenoate (15). Under the given conditions, aminolysis proceeds via an S(N)2 substitution, in contrast with the S(N)1 process for hydrolysis. In the MP/water (8:2) incubation, 15.8% of 12 has been transformed to 13a-d and 10.5% of 11a,b hydrolyzed to the regioisomers 14 and 15 after 8 weeks, respectively. Aminolysis of α,β-unsaturated epoxides by lysine moleties therefore is expected to be an important mode of interaction between proteins and lipid oxidation products.
- Lederer, Markus O.,Schuler, Axel,Ohmenhaeuser, Marc
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- Spin adduct formation from lipophilic EMPO-derived spin traps with various oxygen- and carbon-centered radicals
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Free radicals are involved in the onset of many diseases, therefore the availability of adequate spin traps is crucial to the identification and localization of free radical formation in biological systems. In recent studies several hydrophilic compounds of 2-ethoxycarbonyl-2-methyl-pyrroline-N-oxide (EMPO) have been found to form rather stable Superoxide spin adducts with half-lives up to twenty minutes at physiological pH. This is a major improvement over DMPO (t1/2 = ca. 45 s), and even over DEPMPO (t1/2 = ca. 14 min), the best commercially available spin trap for the unambiguous detection of superoxide radicals. In order to allow the detection of superoxide and also other radicals in lipid environment a series of more lipophilic derivatives of EMPO was synthesized and their structure unambiguously characterized by 1H and 13C NMR spectroscopy. In this way, six different compounds with a n-butyl group in position 5 and either an ethoxy- (EBPO), propoxy- (PBPO), iso-propoxy- (iPBPO), butoxy- (BBPO), sec-butoxy- (sBBPO) or tert-butoxycarbonyl group (tBBPO) in position 5 of the pyrroline ring were obtained and fully analytically characterized (NMR, IR). The stability of the superoxide adducts of all investigated spin traps were comparable with EMPO (t1/2 = ca. 8 min), except for the two compounds bearing an additional methyl group in position 3 or 4 of the pyrroline ring, 5-butyl-5-ethoxycarbonyl-3-methyl-pyrroline-N-oxide (BEMPO-3) and 5-butyl-5-ethoxycarbonyl-4-methyl-pyrroline-N-oxide (BEMPO-4), of which the superoxide adducts were stable for more than 30 min. Spin adducts of other carbon- and oxygen-centered radicals were also investigated.
- Stolze, Klaus,Udilova, Natascha,Rosenau, Thomas,Hofinger, Andreas,Nohl, Hans
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- Development and Application of a Peroxyl Radical Clock Approach for Measuring Both Hydrogen-Atom Transfer and Peroxyl Radical Addition Rate Constants
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The rate-determining step in free radical lipid peroxidation is the propagation of the peroxyl radical, where generally two types of reactions occur: (a) hydrogen-atom transfer (HAT) from a donor to the peroxyl radical; (b) peroxyl radical addition (PRA) to a C=C double bond. Peroxyl radical clocks have been used to determine the rate constants of HAT reactions (kH), but no radical clock is available to measure the rate constants of PRA reactions (kadd). In this work, we modified the analytical approach on the linoleate-based peroxyl radical clock to enable the simultaneous measurement of both kH and kadd. Compared to the original approach, this new approach involves the use of a strong reducing agent, LiAlH4, to completely reduce both HAT and PRA-derived products and the relative quantitation of total linoleate oxidation products with or without reduction. The new approach was then applied to measuring the kH and kadd values for several series of organic substrates, including para- and meta-substituted styrenes, substituted conjugated dienes, and cyclic alkenes. Furthermore, the kH and kadd values for a variety of biologically important lipids were determined for the first time, including conjugated fatty acids, sterols, coenzyme Q10, and lipophilic vitamins, such as vitamins D3 and A.
- Do, Quynh,Lee, David D.,Dinh, Andrew N.,Seguin, Ryan P.,Zhang, Rutan,Xu, Libin
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supporting information
p. 153 - 168
(2020/12/23)
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- The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis
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The CYP74 family of cytochromes P450 includes four enzymes of fatty acid hydroperoxide metabolism: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), and epoxyalcohol synthase (EAS). The present work is concerned with catalytic specificities of three recombinant DESs, namely, the 9-DES (LeDES, CYP74D1) of tomato (Solanum lycopersicum), 9-DES (NtDES, CYP74D3) of tobacco (Nicotiana tabacum), and 13-DES (LuDES, CYP74B16) of flax (Linum usitatissimum), as well as their alterations upon the site-directed mutagenesis. Both LeDES and NtDES converted 9-hydroperoxides of linoleic and α?linolenic acids to divinyl ethers colneleic and colnelenic acids (respectively) with only minorities of HPL and EAS products. In contrast, LeDES and NtDES showed low efficiency towards the linoleate 13-hydroperoxide, affording only the low yield of epoxyalcohols. LuDES exhibited mainly the DES activity towards α?linolenate 13-hydroperoxide (preferred substrate), and HPL activity towards linoleate 13-hydroperoxide, respectively. In contrast, LuDES converted 9-hydroperoxides primarily to the epoxyalcohols. The F291V and A287G mutations within the I-helix groove region (SRS-4) of LuDES resulted in the loss of DES activity and the acquirement of the epoxyalcohol synthase activity. Thus, the studied enzymes exhibited the versatility of catalysis and its qualitative alterations upon the site-directed mutagenesis.
- Gorina, Svetlana S.,Grechkin, Alexander N.,Iljina, Tatiana M.,Mukhtarova, Lucia S.,Smirnova, Elena O.,Toporkova, Yana Y.
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- Catalytic production of oxo-fatty acids by lipoxygenases is mediated by the radical-radical dismutation between fatty acid alkoxyl radicals and fatty acid peroxyl radicals in fatty acid assembly
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Oxo-octadecadienoic acids (OxoODEs) act as peroxisome proliferator-activated receptor (PPAR) agonists biologically, and are known to be produced in the lipoxygenase/linoleate system. OxoODEs seem to originate from the linoleate alkoxyl radicals that are generated from (E/Z)-hydroperoxy octadecadienoic acids ((E/Z)HpODEs) by a pseudoperoxidase reaction that is catalyzed by ferrous lipoxygenase. However, the mechanism underlying the conversion of alkoxyl radical into OxoODE remains obscure. In the present study, we confirmed that OxoODEs are produced in the lipoxygenase/linoleate system in an oxygen-dependent manner. Interestingly, we revealed a correlation between the (E/Z)-OxoODEs content and the (E/E)-HpODEs content in the system. (E/E)-HpODEs could have been derived from (E/E)-linoleate peroxyl radicals, which are generated by the reaction between a free linoleate allyl radical and an oxygen molecule. Notably, the ferrous lipoxygenase-linoleate allyl radical (LOx(Fe2+)-L·) complex, which is an intermediate in the lipoxygenase/linoleate system, tends to dissociate into LOx(Fe2+) and a linoleate allyl radical. Subsequently, LOx(Fe2+) converts (E/Z)-HpODEs to an (E/Z)-linoleate alkoxyl radical through one-electron reduction. Taken together, we propose that (E/Z)-OxoODEs and (E/E)-HpODEs are produced through radical-radical dismutation between (E/Z)-linoleate alkoxyl radical and (E/E)-linoleate peroxyl radical. Furthermore, the production of (E/Z)OxoODEs and (E/E)-HpODEs was remarkably inhibited by a hydrophobic radical scavenger, 2,2,6,6-tetra-methylpiperidine 1-oxyl (TEMPO). On the contrary, water-miscible radical scavengers, 4-hydroxyl-2,2,6,6-tetramethylpiperidine 1-oxyl (OH-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmΔP) only modestly or sparingly inhibited the production of (E/Z)-OxoODEs and (E/E)-HpODEs. These facts indicate that the radical-radical dismutation between linoleate alkoxyl radical and linoleate peroxyl radical proceeds in the interior of micelles.
- Takigawa, Yuta,Koshiishi, Ichiro
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p. 258 - 264
(2020/11/26)
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- Characterization of Bitter-Tasting Oxylipins in Poppy Seeds (Papaver somniferum L.)
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Activity-guided fractionation of poppy seed (Papaver somniferum L.) extracts and analysis of fatty acid oxidation model experiments, followed by liquid chromatography time-of-flight mass spectrometry, tandem mass spectrometry, and one-/two-dimensional nuclear magnetic resonance experiments, revealed the chemical structures of five bitter-tasting fatty acids (1-5), three monoglycerides (6-8), six C18-lipidoxidation products (9-14), and four lipid oxidation degradation products (15 and 17-19) as well as two previously unreported monoglyceride oxidation degradation products, namely, 9-(2′,3′-dihydroxypropyloxy)-9-oxononaic acid (1-azeloyl-rac-glycerol, 16) and 1-(2′,3′-dihydroxypropyl)-8-(5″-oxo-2″,5″-dihydrofruan-2″-yl)-octonoate (1-ODFO-rac-glycerol, 20). Sensory studies exhibited low bitter taste threshold concentrations between 0.08 and 0.29 mmol/L, particularly for the higher oxidated C18-fatty acids trihydroxyoctadecenoic acid (THOE, 12), 12,13-dihydroxy-9-oxo-10-octadecenoic acid (12,13-diOH-9-oxo, 13), and 9,10-dihydroxy-13-oxo-11-octadecenoic acid (9,10-diOH-13-oxo, 14) as well as for the lipidoxidation degradation products 4-hydroxy-2-noneic acid (4-HNA, 17), 4-hydroxy-2-docecendienoic acid (HDdiA, 18), and 8-(5′-oxo-2′,5′-dihydrofuran-2′-yl)-octanoic acid (ODFO, 20).
- Lainer, Johanna,Dawid, Corinna,Dunkel, Andreas,Glaser, Peter,Wittl, Stephanie,Hofmann, Thomas
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p. 10361 - 10373
(2020/01/31)
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- Epoxyalcohol Synthase RjEAS (CYP74A88) from the Japanese Buttercup (Ranunculus japonicus): Cloning and Characterization of Catalytic Properties
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Cytochromes P450 of the CYP74 family play a key role in the lipoxygenase cascade generating oxylipins (products of polyunsaturated fatty acid oxidation). The CYP74 family includes allene oxide synthases, hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases. In this work, we cloned the CYP74A88 gene from the Japanese buttercup (Ranunculus japonicus) and studied the properties of the encoded recombinant protein. The CYP74A88 enzyme specifically converts linoleic acid 9-and 13-hydroperoxides to oxiranyl carbinols 9,10-epoxy-11-hydroxy-12-octadecenoic acid and 11-hydroxy-12,13-epoxy-9-octadecenoic acid, respectively, which was confirmed by GC-MS analysis and kinetic studies. Therefore, the CYP74A88 enzyme is a specific epoxyalcohol synthase.
- Toporkova,Fatykhova,Gorina,Mukhtarova,Grechkin
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p. 171 - 180
(2019/04/01)
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- Biosynthesis of Jasmonates from Linoleic Acid by the Fungus Fusarium oxysporum. Evidence for a Novel Allene Oxide Cyclase
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Fusarium oxysporum f. sp. tulipae (FOT) secretes (+)-7-iso-jasmonoyl-(S)-isoleucine ((+)-JA-Ile) to the growth medium together with about 10 times less 9,10-dihydro-(+)-7-iso-JA-Ile. Plants and fungi form (+)-JA-Ile from 18:3n-3 via 12-oxophytodienoic acid (12-OPDA), which is formed sequentially by 13S-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase (AOC). Plant AOC does not accept linoleic acid (18:2n-6)-derived allene oxides and dihydrojasmonates are not commonly found in plants. This raises the question whether 18:2n-6 serves as the precursor of 9,10-dihydro-JA-Ile in Fusarium, or whether the latter arises by a putative reductase activity operating on the n-3 double bond of (+)-JA-Ile or one of its precursors. Incubation of pentadeuterated (d5) 18:3n-3 with mycelia led to the formation of d5-(+)-JA-Ile whereas d5-9,10-dihydro-JA-Ile was not detectable. In contrast, d5-9,10-dihydro-(+)-JA-Ile was produced following incubation of [17,17,18,18,18-2H5]linoleic acid (d5-18:2n-6). Furthermore, 9(S),13(S)-12-oxophytoenoic acid, the 15,16-dihydro analog of 12-OPDA, was formed upon incubation of unlabeled or d5-18:2n-6. Appearance of the α-ketol, 12-oxo-13-hydroxy-9-octadecenoic acid following incubation of unlabeled or [13C18]-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid confirmed the involvement of AOS and the biosynthesis of the allene oxide 12,13(S)-epoxy-9,11-octadecadienoic acid. The lack of conversion of this allene oxide by AOC in higher plants necessitates the conclusion that the fungal AOC is distinct from the corresponding plant enzyme.
- Oliw, Ernst H.,Hamberg, Mats
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p. 543 - 556
(2019/08/12)
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- Oxygenation reactions catalyzed by the F557V mutant of soybean lipoxygenase-1: Evidence for two orientations of substrate binding
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Plant lipoxygenases oxygenate linoleic acid to produce 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPOD) or 9-hydroperoxy-10E,12Z-octadecadienoic acid (9(S)-HPOD). The manner in which these enzymes bind substrates and the mechanisms by which they control regiospecificity are uncertain. Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197) have identified an important residue, corresponding to phe-557 in soybean lipoxygenase-1 (SBLO-1). These authors proposed that large residues in this position favored binding of linoleate with the carboxylate group near the surface of the enzyme (tail-first binding), resulting in formation of 13(S)-HPOD. They also proposed that smaller residues in this position facilitate binding of linoleate in a head-first manner with its carboxylate group interacting with a conserved arginine residue (arg-707 in SBLO-1), which leads to 9(S)-HPOD. In the present work, we have tested these proposals on SBLO-1. The F557V mutant produced 33% 9-HPOD (S:R = 87:13) from linoleic acid at pH 7.5, compared with 8% for the wild-type enzyme and 12% with the F557V,R707L double mutant. Experiments with 11(S)-deuteriolinoleic acid indicated that the 9(S)-HPOD produced by the F557V mutant involves removal of hydrogen from the pro-R position on C-11 of linoleic acid, as expected if 9(S)-HPOD results from binding in an orientation that is inverted relative to that leading to 13(S)-HPOD. The product distributions obtained by oxygenation of 10Z,13Z-nonadecadienoic acid and arachidonic acid by the F557V mutant support the hypothesis that ω6 oxygenation results from tail-first binding and ω10 oxygenation from head-first binding. The results demonstrate that the regiospecificity of SBLO-1 can be altered by a mutation that facilitates an alternative mode of substrate binding and adds to the body of evidence that 13(S)-HPOD arises from tail-first binding.
- Hershelman, Dillon,Kahler, Kirsten M.,Price, Morgan J.,Lu, Iris,Fu,Plumeri, Patricia A.,Karaisz, Fred,Bassett, Natasha F.,Findeis, Peter M.,Clapp, Charles H.
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-
- Allene Oxide Synthase Pathway in Cereal Roots: Detection of Novel Oxylipin Graminoxins
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Young roots of wheat, barley, and sorghum, as well as methyl jasmonate pretreated rice seedlings, undergo an unprecedented allene oxide synthase pathway targeted to previously unknown oxylipins 1–3. These Favorskii-type products, (4Z)-2-pentyl-4-tridecene-1,13-dioic acid (1), (2′Z)-2-(2′-octenyl)-decane-1,10-dioic acid (2), and (2′Z,5′Z)-2-(2′,5′-octadienyl)-decane-1,10-dioic acid (3), have a carboxy function at the side chain, as revealed by their MS and NMR spectral data. Compounds 1–3 were the major oxylipins detected, along with the related α-ketols. Products 1–3 were biosynthesized from (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid, (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPOD), and (9S,10E,12Z,15Z)-9-hydroperoxy-10,12,15-octadecatrienoic acid, respectively, via the corresponding allene oxides and cyclopropanones. The data indicate that conversion of the allene oxide into the cyclopropanone is controlled by soluble cyclase. The short-lived cyclopropanones are hydrolyzed to products 1–3. The collective name “graminoxins” has been ascribed to oxylipins 1–3.
- Grechkin, Alexander N.,Ogorodnikova, Anna V.,Egorova, Alevtina M.,Mukhitova, Fakhima K.,Ilyina, Tatiana M.,Khairutdinov, Bulat I.
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p. 336 - 343
(2018/06/04)
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- N-linoleoylamino acids as chiral probes of substrate binding by soybean lipoxygenase-1
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Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-L-valine (LLV) and N-linoleoyl-D-valine (LDV) are both good substrates with kcat/Km values that are equal within error and about 40% higher than kcat/Km for linoleic acid. Experiments with N-linoleoyl-L-tryptophan (LLT) and N-linoleoyl-D-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6–8 μM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the D enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly.
- Clapp, Charles H.,Pachuski, Justin,Bassett, Natasha F.,Bishop, Kathleen A.,Carter, Gillian,Young, Megan,Young, Thomas,Fu, Yuhan
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p. 170 - 177
(2018/03/24)
-
- Calcium modulates membrane association, positional specificity, and product distribution in dual positional specific maize lipoxygenase-1
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This study investigates how calcium modulates the properties of dual positional specific maize lipoxygenase-1, including its interaction with substrate, association with subcellular membrane and alteration of product distribution. Bioinformatic analyses identified Asp38, Glu127 and Glu201 as putative calcium binding residues and Leu37 as a flanking hydrophobic residue also potentially involved in calcium-mediated binding of the enzyme to subcellular membranes. Asp38 and Leu37 were shown to be important but not essential for calcium-mediated association of maize lipoxygenase-1 to subcellular membranes in vitro. Kinetic studies demonstrate that catalytic efficiency (Vmax/Km) shows a bell-shaped dependence on log of the molar ratio of substrate to unbound calcium. Calcium also modulates product distribution of the maize lipoxygenase-1 reaction, favoring 13-positional specificity and increasing the relative amount of (E,Z)-isomeric products. The results suggest that calcium regulates the maize lipoxygenase-1 reaction by binding to substrate, and by promoting binding of substrate to enzyme and association of maize lipoxygenase-1 to subcellular membranes.
- Cho, Kyoungwon,Han, Jihoon,Rakwal, Randeep,Han, Oksoo
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-
- Ascorbic acid 6-palmitate: A potent inhibitor of human and soybean lipoxygenase-dependent lipid peroxidation
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Objectives Lipoxygenases (LOX) are the key enzymes involved in the biosynthesis of leukotrienes and reactive oxygen species, which are implicated in pathophysiology of inflammatory disorders. This study was conducted to evaluate the inhibitory effect of water-soluble antioxidant ascorbic acid and its lipophilic derivative, ascorbic acid 6-palmitate (Vcpal) on polymorphonuclear lymphocyte 5-LOX and soybean 15-LOX (sLOX) in vitro. Methods LOX activity was determined by measuring the end products, 5-hydroperoxy eicosatetraenoic acid (5-HETE) and lipid hydroperoxides, by spectrophotometric and high performance liquid chromatography methods. The substrate-dependent enzyme kinetics and docking studies were carried out to understand the nature of inhibition. Key findings Vcpal potently inhibited 5-LOX when compared with its inhibitory effect on sLOX (IC50; 2.5 and 10.3μm respectively, P= 0.003). Further, Vcpal inhibited 5-LOX more strongly than the known synthetic drugs: phenidone and nordihydroguaiaretic acid (P= 0.0007). Enzyme kinetic studies demonstrated Vcpal as a non-competitive reversible inhibitor of 5-LOX. In-silico molecular docking revealed high MolDock and Rerank score for Vcpal than ascorbic acid, complementing in-vitro results. Conclusion Both in-vitro and docking studies demonstrated Vcpal but not ascorbic acid as a non-competitive inhibitor of 5-LOX- and sLOX-induced lipid peroxidation, suggesting a key role for lipophilic nature in bringing about inhibition.
- Mohamed, Riyaz,Tarannum, Shaista,Yariswamy, Manjunath,Vivek, Hamse K.,Siddesha, Jalahalli M.,Angaswamy, Nataraju,Vishwanath, Bannikuppe S.
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p. 769 - 778
(2014/06/09)
-
- Inhibitory and mechanistic investigations of oxo-lipids with human lipoxygenase isozymes
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Oxo-lipids, a large family of oxidized human lipoxygenase (hLOX) products, are of increasing interest to researchers due to their involvement in different inflammatory responses in the cell. Oxo-lipids are unique because they contain electrophilic sites that can potentially form covalent bonds through a Michael addition mechanism with nucleophilic residues in protein active sites and thus increase inhibitor potency. Due to the resemblance of oxo-lipids to LOX substrates, the inhibitor potency of 4 different oxo-lipids; 5-oxo-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-oxo-ETE), 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE), 12-oxo-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid (12-oxo-ETE), and 13-oxo-9,11-(Z,E)-octadecadienoic acid (13-oxo-ODE) were determined against a library of LOX isozymes; leukocyte 5-lipoxygenase (h5-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), human platelet 12-lipoxygenase (h12-LOX), human epithelial 15-lipoxygenase-2 (h15-LOX-2), soybean 15-lipoxygenase-1 (s15-LOX-1), and rabbit reticulocyte 15-LOX (r15-LOX). 15-Oxo-ETE exhibited the highest potency against h12-LOX, with an IC50 = 1 ± 0.1 μM and was highly selective. Steady state inhibition kinetic experiments determined 15-oxo-ETE to be a mixed inhibitor against h12-LOX, with a Kic value of 0.087 ± 0.008 μM and a Kiu value of 2.10 ± 0.8 μM. Time-dependent studies demonstrated irreversible inhibition with 12-oxo-ETE and h15-LOX-1, however, the concentration of 12-oxo-ETE required (Ki = 36.8 ± 13.2 μM) and the time frame (k2 = 0.0019 ± 0.00032 s-1) were not biologically relevant. These data are the first observations that oxo-lipids can inhibit LOX isozymes and may be another mechanism in which LOX products regulate LOX activity.
- Armstrong, Michelle M.,Diaz, Giovanni,Kenyon, Victor,Holman, Theodore R.
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p. 4293 - 4297
(2014/08/18)
-
- Investigating inner-sphere reorganization via secondary kinetic isotope effects in the C-H cleavage reaction catalyzed by soybean lipoxygenase: Tunneling in the substrate backbone as well as the transferred hydrogen
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This work describes the application of NMR to the measurement of secondary deuterium (2° 2H) and carbon-13 (13C) kinetic isotope effects (KIEs) at positions 9-13 within the substrate linoleic acid (LA) of soybean lipoxygenase-1. The KIEs have been measured using LA labeled with either protium (11,11- h2-LA) or deuterium (11,11-d2-LA) at the reactive C11 position, which has been previously shown to yield a primary deuterium isotope effect of ca. 80. The conditions of measurement yield the intrinsic 2° 2H and 13C KIEs on kcat/Km directly for 11,11-d2-LA, whereas the values for the 2° 2H KIEs for 11,11-h2-LA are obtained after correction for a kinetic commitment. The pattern of the resulting 2° 2H and 13C isotope effects reveals values that lie far above those predicted from changes in local force constants. Additionally, many of the experimental values cannot be modeled by electronic effects, torsional strain, or the simple inclusion of a tunneling correction to the rate. Although previous studies have shown the importance of extensive tunneling for cleavage of the primary hydrogen at C11 of LA, the present findings can only be interpreted by extending the conclusion of nonclassical behavior to the secondary hydrogens and carbons that flank the position undergoing C-H bond cleavage. A quantum mechanical method introduced by Buhks et al. [J. Phys. Chem. 1981, 85, 3763] to model the inner-sphere reorganization that accompanies electron transfer has been shown to be able to reproduce the scale of the 2° 2H KIEs.
- Meyer, Matthew P.,Klinman, Judith P.
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supporting information; experimental part
p. 430 - 439
(2011/04/16)
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- Formation of a cyclopropyl epoxide via a leukotriene A synthase-related pathway in an anaerobic reaction of soybean lipoxygenase-1 with 15S-hydroperoxyeicosatetraenoic acid: Evidence that oxygen access is a determinant of secondary reactions with fatty acid hydroperoxides
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The further conversion of an arachidonic acid hydroperoxide to a leukotriene A (LTA) type epoxide by specific lipoxygenase (LOX) enzymes constitutes a key step in inflammatory mediator biosynthesis. Whereas mammalian 5-LOX transforms its primary product (5S-hydroperoxyeicosatetraenoic acid; 5S-HPETE) almost exclusively to LTA4, the model enzyme, soybean LOX-1, normally produces no detectable leukotrienes and instead further oxygenates its primary product 15S-HPETE to 5,15- and 8,15-dihydroperoxides. Mammalian 15-LOX-1 displays both types of activity. We reasoned that availability of molecular oxygen within the LOX active site favors oxygenation, whereas lack of O2 promotes LTA epoxide synthesis. To test this, we reacted 15S-HPETE with soybean LOX-1 under anaerobic conditions and identified the products by high pressure liquid chromatography, UV, mass spectrometry, and NMR. Among the products, we identified a pair of 8,15-dihydroxy diastereomers with all-trans-conjugated trienes that incorporated 18O from H 218O at C-8, indicative of the formation of 14,15-LTA4. A pair of 5,15-dihydroxy diastereomers containing two trans,trans-conjugated dienes (6E,8E,11E,13E) and that incorporated 18O from H 218O at C-5 was deduced to arise from hydrolysis of a novel epoxide containing a cyclopropyl ring, 14,15-epoxy-[9,10,11-cyclopropyl]- eicosa-5Z,7E,13E-trienoic acid. Also identified was the δ-lactone of the 5,15-diol, a derivative that exhibited no 18O incorporation due to its formation by intramolecular reaction of the carboxyl anion with the proposed epoxide intermediate. Our results support a model in which access to molecular oxygen within the active site directs the outcome from competing pathways in the secondary reactions of lipoxygenases.
- Zheng, Yuxiang,Brash, Alan R.
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experimental part
p. 13427 - 13436
(2011/03/22)
-
- In vitro inhibition of linoleic acid peroxidation by primary S-Nitrosothiols
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Nitric oxide (*NO) is an effective chain-breaking antioxidant in the inhibition of lipid peroxidation and circulates in vivo mainly as primary S-nitrosothiols (RSNOs). In this work, the in vitro peroxidation of linoleic acid-SDS comicelles (LA-SDS) catalyzed by soybean lipoxygenase (SLO) and Fe II ions was monitored in the presence and absence of three primary RSNOs: S-nitrosocysteine, S-nitroso-N-acetylcysteyne and S-nitrosoglutathione. Kinetic measurements based on the formation of conjugated double bonds and fluorescent oxidized LA-lysine adducts, showed that RSNOs are more potent antioxidants than their corresponding free thiols (RSHs) in equimolar conditions. These results are consistent with the blocking of LA-SDS peroxidation by RSNOs through the inactivation of peroxyl/alkoxyl (LOO*/LO*) radicals, leading to nitrogen-containing products of oxidized LA, which release free *NO. These results indicate that endogenous RSNOs may play a major role in the blocking of lipid peroxidation in vivo, through the primary inactivation of alkoxyl/peroxyl radicals and also of preformed lipid hydroperoxides.
- Simplicio, Fernanda I.,Seabra, Amedea B.,De Souza, Gabriela F. P.,De Oliveira, Marcelo G.
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experimental part
p. 1885 - 1895
(2011/01/12)
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- Proteins modified by the lipid peroxidation aldehyde 9,12-dioxo-10(E)- dodecenoic acid in MCF7 breast cancer cells
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The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic acid (DODE), which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by DODE. To reduce false positives, three methods were used to identify DODE-modified proteins. First, cells were treated with a synthetically biotinylated 13-HPODE (13-HPODE-biotin). Modified proteins were enriched by neutravidin affinity and identified by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Second, cells were treated with native 13-HPODE. Protein carbonyls were biotinylated with an aldehyde reactive probe, and modified proteins were enriched by neutravidin affinity and identified by 2D LC-MS/MS. Third, using a newly developed DODE antibody, DODEmodified proteins were located by 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized by all three methods revealed a significant overlap and identified 32 primary proteins modified by DODE in MCF7 cells. These results demonstrated the feasibility for the cellular formation of DODE protein-carbonyl adducts that may be future indicators of oxidative stress.
- Slade, Peter G.,Williams, Michelle V.,Brahmbhatt, Viral,Dash, Ajit,Wishnok, John S.,Tannenbaum, Steven R.
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experimental part
p. 557 - 567
(2011/02/24)
-
- Quantitation of hydroperoxy-, keto- and hydroxy-dienes during oxidation of FAMEs from high-linoleic and high-oleic sunflower oils
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The objective of this work was to study the quantitative formation of hydroperoxydienes, ketodienes and hydroxydienes during autoxidation at 40 °C of fatty acid methyl esters derived from two sunflower oils with different degree of unsaturation, high-linoleic sunflower oil and high-oleic sunflower oil. The analysis of the oxidation compounds was carried out by NP-HPLC-UV and results were compared to the specific extinction at 232 nm (K 232) and the peroxide value (PV). Analysis of FAME polymers by HPSEC was also performed to discard samples of advanced oxidation. Results showed that the contents of hydroperoxydienes with respect to the PV were higher for the high linoleic (HL) sample. At the end of the period of slow polymerization (ΔPol ≤ 1 wt%), the content of hydroperoxydienes was found to be 86.0 and 30.7 μg/mg for the HL and high oleic (HO) samples, respectively. Throughout this period, hydroperoxydienes constituted around 90 and 50 wt% of the total hydroperoxides in the HL and HO samples, respectively, suggesting that a significant oxidation of oleic acid also occurred in both samples. The contents of ketodienes and hydroxydienes as a whole constituted 2-3 wt% of the diene compounds analyzed at the end of the period of slow polymerization. Higher contents of ketodienes than of hydroxydienes were found throughout the oxidation time, and the ratio between the contents of ketodienes and hydroxydienes increased with a factor that changed from 1 to 2 throughout the period of slow polymerization.
- Morales, Arturo,Dobarganes, Carmen,Marquez-Ruiz, Gloria,Velasco, Joaquin
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experimental part
p. 1271 - 1279
(2011/08/21)
-
- Rate constants for peroxidation of polyunsaturated fatty acids and sterols in solution and in liposomes
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Rate constants for autoxidation propagation of several unsaturated lipids in benzene solution at 37°C and in phosphatidylcholine liposomes were determined by a linoleate radical clock. This radical clock is based on competition between hydrogen atom abstraction by an intermediate peroxyl radical derived from linoleic acid that leads to a trans,cis-conjugated hydroxyoctadecadienoic product and β-fragmentation of the same peroxyl that gives the trans,trans-product hydroxyoctadecadienoic acid. Rate constants determined by this approach in solution relative to linoleic acid (k p) 62 M-1 s-1) were: arachidonic acid (k p = 197 ± 13 M-1 s-1), eicosapentaenoic acid (kp = 249 ± 16 M-1 s-1), docosahexaenoic acid (kp = 334 ± 37 M-1 s -1), cholesterol (kp= 11 ± 2 M-1 s -1), and 7-dehydrocholesterol (kp = 2260 ± 40 M-1 s-1). Free radical oxidations of multilamellar and unilamellar liposomes of various mixtures of glycerophosphatidylcholine molecular species were also carried out. In some experiments, cholesterol or 7-dehydrocholesterol was incorporated into the lipid mixture undergoing oxidation. A phosphatidylcholine bearing a linoleate ester at sn-2 was a component of each liposome peroxidation reaction and the ratio of trans,cis/trans,trans (t,c/t,t)-conjugated diene oxidation products formed from this phospholipid was determined for each oxidation reaction. This t,c/t,t-product ratio from linoleate was used to "clock" liposome constituents as hydrogen atom donors in the lipid bilayer. Application of this lipid bilayer radical clock gives relative autoxidation propagation rate constants of arachidonate (20:4), eicosapentaenoate (20:5), docosahexaenoate (22:6), and 7-dehydrocholesterol to be 115 ± 7, 145 ± 8, 172 ± 13, and 832 ± 86, respectively, a reactivity trend that parallels the one in solution. We also conclude from the liposome oxidations that linoleate peroxyl radicals at different positions on the eighteen-carbon chain (at C-9 and C-13) have different kinetic properties. This is in contrast to the results of solution oxidations of linoleate in which the C-9 and C-13 peroxyl radicals have similar reactivities. We suggest that peroxyl radical β-scission depends on solvent polarity and the polarity of the local environment of peroxyl radicals in liposomal oxidations depends on the position of the peroxyl radical on the 18-carbon chain.
- Xu, Libin,Davis, Todd A.,Porter, Ned A.
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experimental part
p. 13037 - 13044
(2009/12/08)
-
- Development of a screening system for the evaluation of soybean volatiles
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Flavor properties are important factors of soybean seeds in their utilization as food materials. In order to isolate novel varieties and mutants of soybean having preferable flavor properties, a simple and efficient screening system was established using
- Matsui, Kenji,Kakumyan, Pattana,Kato, Marie,Hajika, Makita
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experimental part
p. 1844 - 1848
(2010/03/30)
-
- Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways
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Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering p
- Anterola, Aldwin,G?bel, Cornelia,Hornung, Ellen,Sellhorn, George,Feussner, Ivo,Grimes, Howard
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experimental part
p. 40 - 52
(2009/07/11)
-
- Detection of divinyl ether synthase in Lily-of-the-Valley (Convallaria majalis) roots
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Incubations of linoleic acid with cell-free preparations from Lily-of-the-Valley (Convallaria majalis L., Ruscaceae) roots revealed the presence of 13-lipoxygenase and divinyl ether synthase (DES) activities. Exogenous linoleic acid was metabolized predominantly into (9Z,11E,1′E)-12-(1′-hexenyloxy)-9,11-dodecadienoic (etheroleic) acid. Its identification was confirmed by the data of ultraviolet spectroscopy, mass spectra, 1H NMR, COSY, catalytic hydrogenation. The isomeric divinyl ether (8E,1′E,3′Z)-12-(1′,3′-nonadienyloxy)-8-nonenoic (colneleic) acid was detected as a minor product. Incubations with linoleic acid hydroperoxides revealed that 13-hydroperoxide was a preferential substrate, while the 9-hydroperoxide was utilized with lesser efficiency.
- Ogorodnikova, Anna V.,Latypova, Larisa R.,Mukhitova, Fahima K.,Mukhtarova, Lucia S.,Grechkin, Alexander N.
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experimental part
p. 2793 - 2798
(2009/04/11)
-
- Contribution of lipid oxidation products to acrylamide formation in model systems
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The reactions of asparagine with methyl linoleate (1), methyl 13-hydroperoxyoctadeca-9,11-dienoate (2), methyl 13-hydroxyoctadeca-9,11- dienoate (3), methyl 13-oxooctadeca-9,11-dienoate (4), methyl 9,10-epoxy-13-hydroxy-11-octadecenoate (5), methyl 9,10-epoxy-13-oxo-11- octadecenoate (6), 2,4-decadienal (7), 2-octenal (8), 4,5-epoxy-2-decenal (9), and benzaldehyde (10) were studied to determine the potential contribution of lipid derivatives to acrylamide formation in heated foodstuffs. Reaction mixtures were heated in sealed tubes for 10 min at 180°C under nitrogen. The reactivity of the assayed compounds was 7 ? 9 > 4 > 2 ? 8 ~ 6 ? 10 ~ 5. The presence of compounds 1 and 3 did not result in the formation of acrylamide. These results suggested that α,β,γ, δ-diunsaturated carbonyl compounds were the most reactive compounds for this reaction followed by lipid hydroperoxides, more likely as a consequence of the thermal decomposition of these last compounds to produce α,β,γ,δ-diunsaturated carbonyl compounds. However, in the presence of glucose this reactivity changed, and compound 1/glucose mixtures showed a positive synergism (synergism factor = 1.6), which was observed neither in methyl stearate/glucose mixtures nor in the presence of antioxidants. This synergism is proposed to be a consequence of the formation of free radicals during the asparagine/glucose Maillard reaction, which oxidized the lipid and facilitated its reaction with the amino acid. These results suggest that both unoxidized and oxidized lipids are able to contribute to the conversion of asparagine into acrylamide, but unoxidized lipids need to be oxidized as a preliminary step.
- Zamora, Rosario,Hidalgo, Francisco J.
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experimental part
p. 6075 - 6080
(2010/04/02)
-
- Characterisation of lipoxygenase isoforms from olive callus cultures
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Two lipoxygenase isoforms from olive callus cultures were separated from each other. Acetone powders were made to stabilise activity and remove lipids. Separation was then achieved by salt precipitation and ion-exchange chromatography. Both isoforms had comparable activity with linoleic and α-linolenic acid substrates, a basic pH optimum and had molecular masses of around 95 kDa. The callus extracts preferentially formed the 13-hydroperoxy products, in keeping with the pattern of volatile derivatives found in olive tissues and oils derived therefrom.
- Williams, Mark,Harwood, John L.
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experimental part
p. 2532 - 2538
(2009/04/04)
-
- A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves
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Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1′Z)-12-(1′-hexenyloxy)-9,11-dodecadienoic [(ω5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of α-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1′Z,3′Z)-12-(1′,3′-hexadienyloxy)-9,11-dodecadienoic [(ω5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, 1H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.
- Chechetkin, Ivan R.,Blufard, Alexander,Hamberg, Mats,Grechkin, Alexander N.
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p. 2008 - 2015
(2008/12/21)
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- Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia
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Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.
- Hornung, Ellen,Kunze, Susan,Liavonchanka, Alena,Zimmermann, Grit,Kuehn, Diana,Fritsche, Kathrin,Renz, Andreas,Kuehn, Hartmut,Feussner, Ivo
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scheme or table
p. 2774 - 2780
(2009/04/10)
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- Properties of a mini 9R-lipoxygenase from Nostoc sp. PCC 7120 and its mutant forms
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Lipoxygenases (LOXs) consist of a class of enzymes that catalyze the regio- and stereospecific dioxygenation of polyunsaturated fatty acids. Current reports propose that a conserved glycine residue in the active site of R-lipoxygenases and an alanine residue at the corresponding position in S-lipoxygenases play a crucial role in determining the stereochemistry of the product. Recently, a bifunctional lipoxygenase with a linoleate diol synthase activity from Nostoc sp. PCC7120 with R stereospecificity and the so far unique feature of carrying an alanine instead of the conserved glycine in the position of the sequence determinant for chiral specificity was identified. The recombinant carboxy-terminal domain was purified after expression in Escherichia coli. The ability of the enzyme to use linoleic acid esterified to a bulky phosphatidylcholine molecule as a substrate suggested a tail-fist binding orientation of the substrate. Site directed mutagenesis of the alanine to glycine did not cause alterations in the stereospecificity of the products, while mutation of the alanine to valine or isoleucine modified both regio- and enantioselectivity of the enzyme. Kinetic measurements revealed that substitution of Ala by Gly or Val did not significantly influence the reaction characteristics, while the A162I mutant showed a reduced vmax. Based on the mutagenesis data obtained, we suggest that the existing model for stereocontrol of the lipoxygenase reaction may be expanded to include enzymes that seem to have in general a smaller amino acid in R and a bulkier one in S lipoxygenases at the position that controls stereospecificity.
- Andreou, Alexandra-Zoi,Vanko, Marian,Bezakova, Lydia,Feussner, Ivo
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p. 1832 - 1837
(2008/09/20)
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- Antioxidative activities of galloyl glucopyranosides from the stem-bark of Juglans mandshurica
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Two phenolics, 1,2,6-trigalloylglucose (1) and 1,2,3,6-tetragalloylglucose (2), isolated from the stem-bark of Juglans mandshurica were evaluated for their antioxidative activities. The results showed that compounds 1 and 2 exhibited strong scavenging activities against 1,1′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzenthiazoline-6-sulphonic) acid (ABTS?+), and superoxide radicals (O2 ?-), and also had a significant inhibitory effect on lipid peroxidation and low-density lipoprotein (LDL) oxidation. The strong superoxide radical scavenging of 1 and 2 resulted from the potential competitive inhibition with xanthine at the active site of xanthine oxidase (OX). In addition, compounds 1 and 2 displayed significant lipoxygenase inhibitory activity, the mode of inhibition also being identified as competitive. In comparison, the antioxidative activities of compounds 1 and 2, together with gallic acid, indicated that the number of galloyl moieties could play an important role in the antioxidative activity.
- Ngoc, Tran Minh,Hung, Tran Manh,Thuong, Phuong Thien,Kim, Jin-Cheon,Choi, Jae Sue,Bae, Kihwan,Hattori, Masao,Choi, Chung-Sig,Lee, Joon Seok,Min, Byung-Sun
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p. 2158 - 2163
(2008/12/23)
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- Cyclization of natural allene oxide in aprotic solvent: formation of the novel oxylipin methyl cis-12-oxo-10-phytoenoate
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Allene oxide, (9Z,11E)-12,13-epoxy-9,11-octadecadienoic acid (12,13-EOD), was prepared by incubation of linoleic acid (13S)-hydroperoxide with flaxseed allene oxide synthase (AOS) and purified (as methyl ester) by low temperature HPLC. Identification of pure 12,13-EOD was substantiated by its UV and 1H NMR spectra and by GC-MS data for its methanol trapping product. The methyl ester of 12,13-EOD (but not the free carboxylic acid) is slowly cyclized in hexane solution, affording a novel cyclopentenone cis-12-oxo-10-phytoenoic acid. Free carboxylic form of 12,13-EOD does not cyclize due to the exceeding formation of macrolactone (9Z)-12-oxo-9-octadecen-11-olide. The spontaneous cyclization of pure natural allene oxide (12,13-EOD) into cis-cyclopentenone have been observed first time.
- Medvedeva, Natalia V.,Mukhtarova, Lucia S.,Mukhitova, Faina K.,Balandina, Alsu A.,Latypov, Shamil K.,Grechkin, Alexander N.
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- Regiochemical and stereochemical evidence for enzyme-initiated catalysis in dual positional specific maize lipoxygenase-1
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Dual positional specific maize lipoxygenase-1 catalyzed the formation of racemic mixtures of four possible regioisomers and was strongly inhibited by the radical scavenger, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinoxy radical. Molecular modeling studies indicated that the oxygen-binding cavity is segregated from the substrate-binding cavity. The data suggest that a bis-allylic radical reaction intermediate is generated enzymatically, released from the enzyme active site, and subsequently oxygenated outside of the enzyme active site by a nonenzymatic mechanism.
- Jang, Sungkuk,Huon, Thavrak,Kim, Keumhwa,Um, Eunji,Han, Oksoo
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p. 3113 - 3116
(2008/02/10)
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- Linoleic acid 10-hydroperoxide as an intermediate during formation of 1-octen-3-ol from linoleic acid in Lentinus decadetes.
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In order to confirm the biosynthetic pathway to 1-octen-3-ol from linoleic acid, a crude enzyme solution was prepared from the edible mushroom, Lentinus decadetes. When the reaction was performed in the presence of glutathione peroxidase, which can reduce
- Matsui, Kenji,Sasahara, Satomi,Akakabe, Yoshihiko,Kajiwara, Tadahiko
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p. 2280 - 2282
(2007/10/03)
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- Mechanism of lipid peroxidation photosensitized by tiaprofenic acid: Product studies using linoleic acid and 1,4-Cyclohexadienes as model substrates
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A careful study of the linoleic acid hydroperoxide (LOOH) profile obtained upon peroxidation of linoleic acid (LA) photosensitized by tiaprofenic acid (TPA) and analogous ketones has been undertaken to distinguish between type-I and type-II photoperoxidation mechanisms. 1,4-Cyclohexadiene and 1,2-dimethylcyclohexa-2,5-diene-carboxylic acid (CHDCA) have also been used as models for LA since they also have double allylic systems. Coir-radiation of LA with TPA and decarboxytiaprofenic acid (DTPA) in acetonitrile and micellar media produced significant amounts of conjugated dienic LOOH. The cis,trans to trans,trans ratio depended on the irradiation time; thus, this parameter is an ambiguous tool for mechanistic assignment. An interesting finding was the decrease of the LOOH level after long irradiation times in mixtures photooxidized by DTPA, which is attributed to quenching of the DTPA triplet by the generated dienic LOOH. High-performance liquid chromatography analyses confirmed that the main pathway operating in photodynamic lipid peroxidation sensitized by (D)TPA is a type-I mechanism. However, product studies using CHDCA have clearly shown that a type-II mechanism is also operating and might contribute to the overall photooxidation process in a significant way.
- Samadi, Abdelouahid,Martinez, Luis A.,Miranda, Miguel A.,Morera, Isabel M.
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p. 359 - 365
(2007/10/03)
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- Antioxidant activities of major components of γ-oryzanol from rice bran using a linoleic acid model
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The change in hydroperoxides of linoleic acid incubated with constant micro air flow at 37°C was used to evaluate the antioxidant activities of three major components of γ-oryzanol from rice bran (cycloartenyl ferulate, 24-methylene cycloartanyl ferulate, and campesteryl ferulate) compared with α-tocopherol and ferulic acid. The four hydroperoxide isomers of linoleic acid, 9-hydroperoxy-10-trans, 12-cis-octadecadienoic acid [9HPODE(t,c)], 9-hydroperoxy-10-trans,12-trans-octadecadienoic acid, 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid [13HPODE(c,t)], and 13-hydroperoxy-9-trans, 11-trans-octadecadienoic acid, were measured using normal-phase high-performance liquid chromatography with an ultraviolet detector. The three components of γ-oryzanol evidenced significant antioxidant activity when they were mixed with linoleic acid in a molar ratio of 1:100 and 1:250 but not in a molar ratio of 1:500 (P 0.05). α-Tocopherol and ferulic acid also demonstrated significant antioxidant activity at all three molar ratios (P 0.05). The highest molar ratio (1:100) of α-tocopherol, however, caused greater levels of 9HPODE(t,c) and 13HPODE(c,t) than the other two less concentrated treatments.
- Xu,Samuel Godber
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p. 645 - 649
(2007/10/03)
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- Characterization of 4-oxo-2-nonenal as a novel product of lipid peroxidation
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Fe (II)-mediated decomposition of 13-[S-(Z,E)]-9,11-hydroperoxyoctadecadienoic (hydroperoxy-linoleic) acid resulted in the formation of three α,β-unsaturated aldehydes. At low Fe (II) concentrations or at early time points after the addition of Fe(II), two major products were observed. The least polar product had chromatographic properties that were identical with those of 4-oxo-2-nonenal. Conversion of this product to its bis-oxime derivative with hydroxylamine hydrochloride resulted in two syn- and two anti-oxime isomers that had chromatographic and mass spectral properties identical with the properties of products derived from an authentic standard of 4-oxo-2-nonenal. This confirmed for the first time that 4-oxo-2-nonenal is a major product of the Fe(II)-mediated breakdown of lipid hydroperoxides. The more polar product had chromatographic properties that were similar to those of 4-hydroperoxy-2-nonenal. LC/MS analysis of its syn- and anti-oxime isomers confirmed this structural assignment. Thus, 4-hydroperoxy-2-nonenal is a previously unrecognized major product of lipid hydroperoxide decomposition. At high Fen concentrations and at longer incubation times, a third more polar product was observed with chromatographic properties that were identical to those of 4-hydroxy-2-nonenal. The syn- and anti-oxime isomers had chromatographic and mass spectral properties identical with the properties of products derived from an authentic standard of 4-hydroxy-2-nonenal. It appears that 4-hydroperoxy-2-nonenal is formed initially and that it is then converted to 4-hydroxy-2-nonenal in the presence of high Fe (II) concentrations or by extended incubations in the presence of low Fen concentrations. It is conceivable that some of the 4-hydroperoxy-2-nonenal is also converted to 4-oxo-2-nonenal. However, we cannot rule out the possibility that it is also formed by a concerted mechanism from a rearrangement product of 13-[S-(Z,E)]-9,11-hydroperoxyoctadecadienoic acid.
- Hwa Lee,Blair
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p. 698 - 702
(2007/10/03)
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- Quantitative HPLC determination of the antioxidant activity of capsaicin on the formation of lipid hydroperoxides of linoleic acid: A comparative study against BHT and melatonin
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The antioxidant activity of capsaicin, as compared to BHT and melatonin, was determined by the direct measurement of lipid hydroperoxides formed upon linoleic acid autoxidation initiated by AIBN. The formation of four isomeric lipid hydroperoxides was detected after reverse-phase HPLC separation. Data from three detectors, UV absorption, glassy carbon electrode electrochemical detection, and postcolumn chemiluminescence using luminol, were compared. Capsaicin was more effective than melatonin in suppressing the formation of lipid hydroperoxides but not as effective as BHT. The formation of capsaicin and BHT dimers was observed during oxidation, and the dimers were characterized using APCI MS(n).
- Henderson, David E.,Slickman, Adam M.,Henderson, Susan K.
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p. 2563 - 2570
(2007/10/03)
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- Biological activity and biosynthesis of pentacyclic oxylipins: The linoleic acid pathway
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The relevance of the postulated pathway from linoleic acid to dihydrojasmonic acid is analysed. Pentacyclic oxylipins having pentenyl or pentyl side chains were tested for their secondary metabolite inducing activity in seven different plant cell culture species which all responded well to 12-oxo-phytodienoic acid and jasmonic acid. The response towards the dihydro-derivatives 15,16-dihydro-12-oxo-phytodienoic acid and 9,10- dihydrojasmonic acid ranged from strong activity in Eschscholzia californica to no activity in Lycopersicon esculentum. 15,16-Dihydro-12-oxo-phytodienoic acid can be formed from linoleic acid (18:2) by a linseed acetone powder enzyme preparation. Application experiments with linoleic (18:2) and linolenic acid (18:3) showed that the bottleneck of the 18:2 pathway is most likely the cyclization of the intermediate allene oxide when compared to the ease by which 15,16-dihydro-12-oxo-phytodienoic acid is converted to dihydrojasmonic acid in plant systems. The metabolism of potential precursors of jasmonic and dihydrojasmonic acid was extensively studied in various cell cultures.
- Gundlach, Heidrun,Zenk, Meinhart H.
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p. 527 - 537
(2007/10/03)
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- Enantioselective conversion of linoleate hydroperoxide to an α, β-epoxy alcohol by niobium ethoxide
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Niobium (V) ethoxide [Nb(OC2H5)5] catalyzed the rearrangement of methyl 13(S)-hydroperoxy-9(Z),11 (E)-octadecadienoate (Me-HPODE) to epoxy hydroxy isomers. At low temperature (5°C) in aprotic solvent, Me-HPODE was converted to the diastereomeric α, β-epoxy alcohols, methyl 11(R, S), 12(R, S)-epoxy-13(S)-hydroxy-9(Z)octadecenoate. These products are referred to as oxylipids and structurally resemble those obtained from the vanadium- and epoxygenase-catalyzed rearrangement of Me-HPODE but are distinct from products obtained from ferrous iron-, hematin-, and hemoglobin-catalyzed rearrangements. Because the product of the niobium-catalyzed rearrangement of Me-HPODE was predominantly the erythro diastereomer, the rearrangement is distinguished from that produced by a titanium catalyst, in which the threo diastereomer [methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate] predominates, and from that produced by a vanadium catalyst, in which both diastereomers are produced in equal proportion. The synthesis of alcohol epoxide by Nb(OC2H5)5 was inhibited by traces of water, but inclusion of molecular sieves in the reaction medium did not improve yield, as the alcohol epoxide rearranged to ketonic materials.
- Piazza,Foglia,Nunez
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p. 939 - 943
(2007/10/03)
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- Retinoic acid-dependent stimulation of 2,2'-azobis(2-amidinopropane)- initiated autoxidation of linoleic acid in sodium dodecyl sulfate micelles: A novel prooxidant effect of retinoic acid
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(E)-Retinoic acid (RA) was shown to stimulate the rate of 2,2'-azobis(2- amidinopropane) (AAPH)-initiated autoxidation of linoleic acid (18:2) in sodium dodecyl sulfate (SDS) micelles. RA-dependent stimulation of 18:2 autoxidation was characterized by enhanced rates of dioxygen uptake which were linear with retinoid concentration. In contrast, 5,6-epoxy-RA, a major oxidation product of RA, failed to affect the rate of dioxygen consumption at all concentrations tested. RA was also shown to stimulate peroxyl radical- dependent oxidation of styrene to the corresponding oxirane when styrene was included in the micellar system as a molecular probe. Furthermore, unequivocal evidence of RA-dependent stimulation of 18:2 autoxidation was obtained by relative quantitation of 13-hydroxy-(9Z, 11E)-octadecadienoic acid (13-HODE) plus 9-hydroxy-(10E,12Z)-octadecadienoic acid (9-HODE) production. In addition, enhanced carboncentered radical formation was demonstrated in the presence of RA by EPR spectroscopy using α-(4-pyridyl 1- oxide)-N-tert-butylnitrone (4-POBN) as a spin trap. Analysis and quantitation of RA oxidation products indicated that RA was oxidized to one primary product, 5,6-epoxy-RA, which was identified on the basis of cochromatography with synthetic standard (in a reverse-phase HPLC system), electronic absorption spectroscopy, and positive chemical ionization mass spectrometry of the corresponding methyl ester. Other minor oxidation products were also detected but not characterized. In contrast, reaction mixtures devoid of 18:2 failed to demonstrate significant retinoid oxidation. Mechanisms are proposed to account for the prooxidant effects of RA in this system.
- Freyaldenhoven, Mary Ann,Lehman, Paul A.,Franz, Thomas J.,Lloyd, Roger V.,Samokyszyn, Victor M.
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p. 102 - 110
(2007/10/03)
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