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Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction.
Teder T, Boeglin WE, Brash AR
(2014) J Lipid Res 55: 2587-96
MeSH Terms: 8,11,14-Eicosatrienoic Acid, Animals, Arachidonate 12-Lipoxygenase, Arachidonate 15-Lipoxygenase, Biocatalysis, Blood Platelets, Chromatography, High Pressure Liquid, Eicosanoids, Epoxy Compounds, Gas Chromatography-Mass Spectrometry, Humans, Hydroxylation, Linolenic Acids, Lipid Peroxides, Lipoxygenase, Mice, Molecular Structure, Nuclear Magnetic Resonance, Biomolecular, Oxidation-Reduction, Recombinant Proteins, Soybean Proteins, Spectrometry, Mass, Electrospray Ionization, Stereoisomerism
Show Abstract · Added January 21, 2015
Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purified by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic substitution (SNi) between the hydroperoxy (nucleophile) and epoxy group (electrophile). Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the autoxidation reactions of polyunsaturated fatty acids.
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.
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23 MeSH Terms
Demonstration of HNE-related aldehyde formation via lipoxygenase-catalyzed synthesis of a bis-allylic dihydroperoxide intermediate.
Jin J, Zheng Y, Brash AR
(2013) Chem Res Toxicol 26: 896-903
MeSH Terms: Aldehydes, Animals, Anthozoa, Biocatalysis, Hydrogen Peroxide, Lipid Peroxides, Lipoxygenase, Molecular Structure
Show Abstract · Added March 7, 2014
One of the proposed pathways to the synthesis of 4-hydroxy-nonenal (HNE) and related aldehydes entails formation of an intermediate bis-allylic fatty acid dihydroperoxide. As a first direct demonstration of such a pathway and proof of principle, herein we show that 8R-lipoxygenase (8R-LOX) catalyzes the enzymatic production of the HNE-like product (11-oxo-8-hydroperoxy-undeca-5,9-dienoic acid) via synthesis of 8,11-dihydroperoxy-eicosa-5,9,12,14-tetraenoic acid intermediate. Incubation of arachidonic acid with 8R-LOX formed initially 8R-hydroperoxy-eicosatetraenoic acid (8R-HPETE), which was further converted to a mixture of products including a prominent HPNE-like enone. A new bis-allylic dihydroperoxide was trapped when the incubation was repeated on ice. Reincubation of this intermediate with 8R-LOX successfully demonstrated its conversion to the enone products, and this reaction was greatly accelerated by coincubation with NDGA, a reductant of the LOX iron. These findings identify a plausible mechanism that could contribute to the production of 4-hydroxy-alkenals in vivo.
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8 MeSH Terms
Tyrosine-lipid peroxide adducts from radical termination: para coupling and intramolecular Diels-Alder cyclization.
Shchepin R, Möller MN, Kim HY, Hatch DM, Bartesaghi S, Kalyanaraman B, Radi R, Porter NA
(2010) J Am Chem Soc 132: 17490-500
MeSH Terms: Chromatography, High Pressure Liquid, Cyclization, Cycloaddition Reaction, Free Radicals, Linoleic Acids, Lipid Peroxides, Mass Spectrometry, Oxidation-Reduction, Peroxides, Phosphatidylcholines, Silver, Tyrosine
Show Abstract · Added May 29, 2014
Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR spectroscopy as well as by mass spectrometry (MS). The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic (13)C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl (13)C chemical shifts at ~198 ppm. All of the NMR HMBC and HSQC correlations support the structure assignments of the primary and Diels-Alder adducts, as does MS collision-induced dissociation data. Kinetic rate constants and activation parameters for the IMDA reaction were determined, and the primary adducts were reduced with cuprous ion to give a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found in either the primary or cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts, which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein cross-links via interprotein Michael adducts.
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12 MeSH Terms
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.
Zheng Y, Brash AR
(2010) J Biol Chem 285: 13427-36
MeSH Terms: Animals, Arachidonate 5-Lipoxygenase, Epoxy Compounds, Lipid Peroxides, Lipoxygenase, Mammals, Models, Chemical, Oxidation-Reduction, Oxygen, Plant Proteins, Soybeans
Show Abstract · Added December 10, 2013
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 LTA(4), 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 O(2) 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 (18)O from H(2)(18)O at C-8, indicative of the formation of 14,15-LTA(4). A pair of 5,15-dihydroxy diastereomers containing two trans,trans-conjugated dienes (6E,8E,11E,13E) and that incorporated (18)O from H(2)(18)O 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 delta-lactone of the 5,15-diol, a derivative that exhibited no (18)O 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.
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11 MeSH Terms
Studies on biomarkers for oxidative stress in patients with chronic myeloid leukemia.
Singh RK, Tripathi AK, Tripathi P, Singh S, Singh R, Ahmad R
(2009) Hematol Oncol Stem Cell Ther 2: 285-8
MeSH Terms: Adult, Biomarkers, Tumor, Female, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive, Lipid Peroxides, Male, Middle Aged, Oxidative Stress, Protein Carbonylation, Thiobarbituric Acid Reactive Substances, Young Adult
Show Abstract · Added July 12, 2013
BACKGROUND - Chronic myeloid leukemia (CML) is a myeloproliferative disorder with a unique genetic rearrangement, the Philadelphia chromosome. High reactive oxygen species (ROS) levels favor oxidative stress, which could play a vital role in normal processes and various pathophysiologies including neoplasm. Biomarkers of oxidative stress are measured as products of oxidized proteins and lipids. Plasma levels of protein carbonyl (PC), thiobarbituric acid reactive substances (TBARS) and total lipid hydroperoxide (LOOH) were used as biomarkers of oxidative stress in the past. The aim of this study was to evaluate the products of protein oxidation and lipid peroxidation in plasma as biomarkers of oxidative stress in CML patients.
PATIENTS AND METHODS - The study included 40 CML patients and 20 age- and sex-matched healthy volunteers. Of 40 CML patients, 28 were in chronic phase (CML-CP) and 12 in accelerated phase (CML-AP). Plasma levels of PC, TBARS and LOOH as biomarkers of oxidative stress were evaluated by spectrophotometric methods.
RESULTS - There were significant differences (P < .05) in plasma levels of PC, TBARS and LOOH in CML, CML-CP and CML-AP patients as compared to controls.
CONCLUSION - PC, TBARS and LOOH might reflect oxidative stress in CML patients and might be used as biomarkers in such patients.
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12 MeSH Terms
EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase.
Rapp J, Xu S, Sharp AM, Griffith WP, Kim YW, Funk MO
(2009) Arch Biochem Biophys 490: 50-6
MeSH Terms: Amino Acid Sequence, Animals, Arachidonate 12-Lipoxygenase, Electron Spin Resonance Spectroscopy, Escherichia coli, Hydrogen-Ion Concentration, Iron, Leukocytes, Lipid Peroxides, Molecular Weight, Oxidation-Reduction, Recombinant Proteins, Spectrometry, Mass, Electrospray Ionization, Sus scrofa, Temperature, Transformation, Genetic
Show Abstract · Added April 14, 2017
The procedure for the expression and purification of recombinant porcine leukocyte 12-lipoxygenase using Escherichia coli [K.M. Richards, L.J. Marnett, Biochemistry 36 (1997) 6692-6699] was updated to make it possible to produce enough protein for physical measurements. Electrospray ionization tandem mass spectrometry confirmed the amino acid sequence. The redox properties of the cofactor iron site were examined by EPR spectroscopy at 25K following treatment with a variety of fatty acid hydroperoxides. Combination of the enzyme in a stoichiometric ratio with the hydroperoxides led to a g4.3 signal in EPR spectra instead of the g6 signal characteristic of similarly treated soybean lipoxygenase-1. Native 12-lipoxygenase was also subjected to electrospray ionization mass spectrometry. There was evidence for loss of the mass of an iron atom from the protein as the pH was lowered from 5 to 4. Native ions in these samples indicated that iron was lost without the protein completely unfolding.
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16 MeSH Terms
Evidence for an ionic intermediate in the transformation of fatty acid hydroperoxide by a catalase-related allene oxide synthase from the Cyanobacterium Acaryochloris marina.
Gao B, Boeglin WE, Zheng Y, Schneider C, Brash AR
(2009) J Biol Chem 284: 22087-98
MeSH Terms: Catalase, Catalysis, Chromatography, Gas, Cloning, Molecular, Cyanobacteria, Fatty Acids, Gene Expression Regulation, Bacterial, Intramolecular Oxidoreductases, Lipid Peroxides, Magnetic Resonance Spectroscopy, Mass Spectrometry, Models, Biological, Models, Chemical, Oxygen, Solvents
Show Abstract · Added December 10, 2013
Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4omega3 and 18.3omega3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted approximately 70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only approximately 30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H2(18)O indicated that approximately 72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.
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15 MeSH Terms
An update on products and mechanisms of lipid peroxidation.
Schneider C
(2009) Mol Nutr Food Res 53: 315-21
MeSH Terms: Antioxidants, Fatty Acids, Unsaturated, Kinetics, Lipid Peroxidation, Lipid Peroxides, Peroxides, Thermodynamics, alpha-Tocopherol
Show Abstract · Added January 20, 2015
The free radical reaction of polyunsaturated fatty acids with molecular oxygen leads to hydroperoxides as the first stable products. From linoleic acid the two conjugated diene hydroperoxides at carbons 9 and 13 were considered the only primary products until the recent discovery of the bis-allylic 11-hydroperoxide. The 11-carbon is the site of the initial hydrogen abstraction, and the 11-hydroperoxide is formed without isomerization of the 9,10 and 12,13 cis double bonds. In the autoxidation reaction, bis-allylic hydroperoxides are obtained only in the presence of an efficient antioxidant, for example, alpha-tocopherol. The antioxidant functions as a hydrogen atom donor, necessary to trap the fleeting bis-allylic peroxyl radical intermediate as the hydroperoxide. Understanding of the mechanism of formation of bis-allylic hydroperoxides has led to increased appreciation of the central role of the intermediate peroxyl radical in determining the outcome of lipid peroxidation.
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8 MeSH Terms
Identification of intact lipid peroxides by Ag+ coordination ion-spray mass spectrometry (CIS-MS).
Yin H, Porter NA
(2007) Methods Enzymol 433: 193-211
MeSH Terms: Cholesterol Esters, Humans, Lipid Peroxides, Mass Spectrometry, Phospholipids, Silver
Show Abstract · Added May 29, 2014
Free radical-induced autoxidation of lipids containing polyunsaturated fatty acids (PUFA) has been implicated in numerous human diseases including atherosclerosis, neurodegenerative diseases, and cancer. Autoxidation of PUFAs generates hydroperoxides as primary oxidation products and further oxidation leads to cyclic peroxides as secondary oxidation products. It is challenging to identify these peroxides by conventional electro-spray ionization (ESI) mass spectrometry (MS) method because of their thermal and chemical instability under most analytical conditions. Ag(+) coordination ion-spray CIS-MS has proven to be a powerful tool to analyze these intact lipid peroxides. Ag(+) preferentially complexes with the double bonds and induces characteristic fragmentation in the gas phase. Monocyclic peroxides, bicyclic endoperoxides, serial cyclic peroxides, and dioxolane-isoprostane peroxides have been identified from the oxidation of cholesteryl arachidonate and phospholipids containing arachidonate. This technique has been widely used for structural identification but it is difficult to make it a quantitative tool because of the formation of multiple silver adducts.
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6 MeSH Terms
Synthesis of dihydroperoxides of linoleic and linolenic acids and studies on their transformation to 4-hydroperoxynonenal.
Schneider C, Boeglin WE, Yin H, Ste DF, Hachey DL, Porter NA, Brash AR
(2005) Lipids 40: 1155-62
MeSH Terms: Aldehydes, Linoleic Acids, Linolenic Acids, Lipid Peroxides, Oxidation-Reduction
Show Abstract · Added December 10, 2013
The cytotoxic aldehydes 4-hydroxynonenal, 4-hydroperoxynonenal (4-HPNE), and 4-oxononenal are formed during lipid peroxidation via oxidative transformation of the hydroxy or hydroperoxy precursor fatty acids, respectively. The mechanism of the carbon chain cleavage reaction leading to the aldehyde fragments is not known, but Hock-cleavage of a suitable dihydroperoxide derivative was implicated to account for the fragmentation [Schneider, C., Tallman, K.A., Porter, N.A., and Brash, A.R. (2001) Two Distinct Pathways of Formation of 4-Hydroxynonenal. Mechanisms of Nonenzymatic Transformation of the 9- and 13-Hydroperoxides of Linoleic Acid to 4-Hydroxyalkenals, J. Biol. Chem. 275, 20831-20838]. Both 8,13- and 10,13-dihydroperoxyoctadecadienoic acids (diHPODE) could serve as precursors in a Hock-cleavage leading to 4-HPNE via two different pathways. Here, we synthesized diastereomeric 9,12-, 10,12-, and 10,13-diHPODE using singlet oxidation of linoleic acid. 8,13-Dihydroperoxyoctadecatrienoic acid was synthesized by vitamin E-controlled autoxidation of gamma-linolenic acid followed by reaction with soybean lipoxygenase. The transformation of these potential precursors to 4-HPNE was studied under conditions of autoxidation, hematin-, and acid-catalysis. In contrast to 9- or 13-HPODE, neither of the dihydroperoxides formed 4-HPNE on autoxidation (lipid film, 37 degrees C), regardless of whether the free acid or the methyl ester derivative was used. Acid treatment of 10,13-diHPODE led to the expected formation of 4-HPNE as a significant product, in accord with a Hock-type cleavage reaction. We conclude that, although the suppression of 4-H(P)NE formation from monohydroperoxides by alpha-tocopherol indicates peroxyl radical reactions in the major route of carbon chain cleavage, the dihydroperoxides previously implicated are not intermediates in the autoxidative transformation of monohydroperoxy fatty acids to 4-HPNE and related aldehydes.
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5 MeSH Terms