linoleic-acid and 13-hydroperoxy-9-11-octadecadienoic-acid

To elucidate the role of enzymatic lipid peroxidation in disease pathogenesis and in food deterioration, we recently achieved stereoselective analysis of phosphatidylcholine hydroperoxide (PCOOH) possessing 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-9Z,11E-HPODE) using HPLC-MS/MS with a CHIRALPAK OP (+) column. Because enzymatic oxidation progresses concurrently with auto-oxidation, we need to distinguish them further. Here, we attempted such an analysis. First, we used lipoxygenase, linoleic acid, and lysophosphatidylcholine (LPC) to synthesize the enzymatic oxidation product 13(S)-9Z,11E-HPODE PC, and the auto-oxidation products 13(RS)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC, which were used as standards to test the ability of various columns to separate the enzymatic oxidation product from auto-oxidation products. Separation was achieved by connecting in series two columns with different properties: CHIRALPAK OP (+) and CHIRALPAK IB-3. The CHIRALPAK OP (+) column separated 13(R)-9Z,11E-HPODE PC and 13(S)-9Z,11E-HPODE PC, whereas CHIRALPAK IB-3 enabled separation of 13(S)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC. The results for the analysis of both enzymatically oxidized and auto-oxidized lecithin (an important phospholipid mixture in vivo and in food) indicate that our method would be useful for distinguishing enzymatic oxidation and auto-oxidation reactions. Such information will be invaluable for elucidating the involvement of PCOOH in disease pathogenesis and in food deterioration.

Topics: Chemical Fractionation; Chromatography, High Pressure Liquid; Glycine max; Lecithins; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipoxygenase; Lysophosphatidylcholines; Phosphatidylcholines; Stereoisomerism; Tandem Mass Spectrometry

15-Lipoxygenases (15-LOs) catalyze the peroxidation reaction of linoleic acid (LA) in mammals producing almost exclusively 13-(S)-hydroperoxyoctadecadienoic acid (13-(S)-HPODE). Although several hypotheses have been formulated, the molecular basis of such enzymatic regiospecificity is unclear. We have here combined quantum mechanics/molecular mechanics (QM/MM) calculations with molecular dynamics simulations to analyze the peroxidation mechanism using a complete rabbit 15-LO-1/LA solvated model. C9 and C13 being equivalent for planarity and spin density, the QM/MM potential energy profiles of the O2 addition to those two atoms were calculated. The difference in the potential energy barrier heights is clear enough to justify that O2 selectively attacks C13 giving 13-(S)-HPODE. Oxygenation at C9 is hindered by two steric-shielding residues (Leu597 and Gln548). The calculated free energy profile at 300 K for the O2 addition to C13 confirms that the peroxidation on C13 is a reversible viable process in agreement with experiments. Thus, the subsequent reduction of the peroxyl radical to give the final hydroperoxidated product is expected to give the irreversibility character to the overall process.

Topics: Animals; Arachidonate 15-Lipoxygenase; Biocatalysis; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Molecular Dynamics Simulation; Oxygen; Quantum Theory; Rabbits; Stereoisomerism; Thermodynamics

Human reticulocyte 15-lipoxygenase-1 (15-hLO-1) and human platelet 12-lipoxygenase (12-hLO) have been implicated in a number of diseases, with differences in their relative activity potentially playing a central role. In this work, we characterize the catalytic mechanism of these two enzymes with arachidonic acid (AA) as the substrate. Using variable-temperature kinetic isotope effects (KIE) and solvent isotope effects (SIE), we demonstrate that both k(cat)/K(M) and k(cat) for 15-hLO-1 and 12-hLO involve multiple rate-limiting steps that include a solvent-dependent step and hydrogen atom abstraction. A relatively low k(cat)/K(M) KIE of 8 was determined for 15-hLO-1, which increases to 18 upon the addition of the allosteric effector molecule, 12-hydroxyeicosatetraenoic acid (12-HETE), indicating a tunneling mechanism. Furthermore, the addition of 12-HETE lowers the observed k(cat)/K(M) SIE from 2.2 to 1.4, indicating that the rate-limiting contribution from a solvent sensitive step in the reaction mechanism of 15-hLO-1 has decreased, with a concomitant increase in the C-H bond abstraction contribution. Finally, the allosteric binding of 12-HETE to 15-hLO-1 decreases the K(M)[O(2)] for AA to 15 microM but increases the K(M)[O(2)] for linoleic acid (LA) to 22 microM, such that the k(cat)/K(M)[O(2)] values become similar for both substrates (approximately 0.3 s(-1) microM(-1)). Considering that the oxygen concentration in cancerous tissue can be less than 5 microM, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Allosteric Regulation; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Biocatalysis; Blood Platelets; Carbon Isotopes; Humans; Kinetics; Leukotrienes; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Models, Chemical; Oxygen; Recombinant Proteins; Reticulocytes; Solvents; Temperature

In rats, oxidized fats activate the peroxisome proliferator-activated receptor alpha (PPARalpha), leading to reduced triglyceride concentrations in liver, plasma and very low density lipoproteins. Oxidation products of linoleic acid constitute an important portion of oxidized dietary fats. This study was conducted to check whether the primary lipid peroxidation product of linoleic acid, 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), might be involved in the PPARalpha-activating effect of oxidized fats. Therefore, we examined the effect of 13-HPODE on the expression of PPARalpha target genes in the rat Fao and the human HepG2 hepatoma cell lines. In Fao cells, 13-HPODE increased the mRNA concentration of the PPARalpha target genes acyl-CoA oxidase (ACO), cytochrome P450 4A1 and carnitine-palmitoyltransferase 1A (CPT1A). Furthermore, the concentration of cellular and secreted triglycerides was reduced in Fao cells treated with 13-HPODE. Because PPARalpha mRNA was not influenced, we conclude that these effects are due to an activation of PPARalpha by 13-HPODE. In contrast, HepG2 cells seemed to be resistant to PPARalpha activation by 13-HPODE because no remarkable induction of the PPARalpha target genes ACO, CPT1A, mitochondrial HMG-CoA synthase and delta9-desaturase was observed. Consequently, cellular and secreted triglyceride levels were not changed after incubation of HepG2 cells with 13-HPODE. In conclusion, this study shows that 13-HPODE activates PPARalpha in rat Fao but not in human HepG2 hepatoma cells.

Topics: Acyl-CoA Oxidase; Animals; Carcinoma, Hepatocellular; Carnitine O-Palmitoyltransferase; Cell Line, Tumor; Cytochrome P-450 CYP4A; Gene Expression; Humans; Linoleic Acid; Linoleic Acids; Lipid Metabolism; Lipid Peroxides; Lipids; Liver; Liver Neoplasms; Liver Neoplasms, Experimental; PPAR alpha; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Triglycerides

The lipoxygenase isoenzymes LOX1 and LOX2 from green malt were separated by isoelectric focusing, and their catalytic properties regarding complex lipids as substrates were characterized. The regio- and stereoisomers of hydroperoxy octadecadienoates (HPODE) resulting from LOX1 and LOX2 enzymatic transformations of linoleic acid, methyl linoleate, linoleic acid glycerol esters monolinolein, dilinolein, and trilinolein, and 1-palmitoyl-2-linoleoyl-glycero-3-phosphocholine (PamLinGroPCho) were determined. In addition, biotransformations of polar and nonpolar lipids extracted from malt were performed with LOX1 and LOX2. The results show that LOX2 catalyzes the oxidation of esterified fatty acids at a higher rate and is more regioselective than LOX1. The dual position specificity of LOX2 (9-HPODE:13-HPODE) with trilinolein as the substrate (6:94) was higher than the resultant ratio (13:87) when free linoleic acid was transformed. A high (S)-enantiomeric excess of 13-HPODE was analyzed with all esterified substrates confirming the formation of 13-HPODE through the LOX2 enzyme; however, 9-HPODE detected after LOX2 biotransformations showed (R)-enantiomeric excesses. PamLinGroPCho was oxygenated by LOX1 with the highest regio- and stereoselectivities among the applied substrates.

Topics: Esterification; Germination; Hordeum; Isoelectric Focusing; Isoenzymes; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Seeds; Stereoisomerism; Substrate Specificity

The oxidation of linoleic acid produces several products with biological activity including the hydroperoxy fatty acid 13-hydroperoxyoctadecadienoic acid (13-HPODE), the hydroxy fatty acid 13-hydroxyoctadecadienoic acid (13-HODE), and the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). In the present work, the peroxidase activity of glutathione transferases (GST) A1-1, M1-1, M2-2, and P1-1(Val 105) toward 13-HPODE has been examined. The alpha class enzyme is the most efficient peroxidase while the two enzymes from the mu class exhibit weak peroxidase activity toward 13-HPODE. It was also determined that the conjugated diene 13-HODE is not a substrate for GST from the alpha and mu classes but that 13-HODE does inhibit the GST-catalyzed conjugation of CDNB by enzymes from the alpha, mu, and pi classes. Finally, both 13-HODE and 13-OXO were shown to be inducers of GST activity in HT-29 and HCT-116 colon tumor cells. These data help to clarify the role of GST in the metabolic disposition of linoleic acid oxidation products.

Topics: Acetonitriles; Cell Line, Tumor; Dinitrochlorobenzene; Dose-Response Relationship, Drug; Glutathione; Humans; Kinetics; Linoleic Acid; Linoleic Acids; Linolenic Acids; Lipid Peroxides; Models, Chemical; Oxygen; Peroxidase

Intracellular Fe(II), which is up-regulated during oxidative stress and during iron overload, induces the formation of a hydroxyl radical by Fenton chemistry. The hydroxyl radical can convert the prototypic omega-6 polyunsaturated fatty acid, linoleic acid, to 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (13-HPODE). Cyclooxygenases can also convert linoleic acid to 13(S)-HPODE during oxidative stress. Subsequent Fe(II)-mediated decomposition to protein- and DNA-reactive bifunctional electrophiles was examined by normal-phase liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)/mass spectrometry. The potential individual bifunctional electrophiles trans-4,5-epoxy-2(E)-decenal (EDE), cis-EDE, 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) exhibited protonated molecular ions at m/z 169, 169, 155 and 157, respectively. The MH(+) ion at m/z 173 for 4-hydroperoxy-2(E)-nonenal (HPNE) was very weak with an ion corresponding to the loss of OH at m/z 156 as the major ion in the APCI mass spectrum. The bifunctional electrophiles were all separated under normal-phase LC conditions. Interestingly, ions corresponding to ONE and HNE were detected at the same retention time as HPNE, suggesting that it decomposed in the source of the mass spectrometer to ONE and HNE. All five bifunctional electrophiles were formed when 13-HPODE was treated with 50 microM Fe(II). At this concentration of Fe(II), the addition of vitamin C resulted in increased bifunctional electrophile formation. At higher concentrations of Fe(II) (500 microM to 2 mM), no HPNE was detected and there was no additive effect of vitamin C. Additional experiments with synthetic HPNE revealed that it was quantitatively converted to a mixture of ONE and HNE by Fe(II). The HNE is thought to arise from a one-electron reduction of an alkoxy radical derived from HPNE. In contrast, ONE can arise through an alpha-cleavage of the HPNE-derived alkoxy radical or by direct dehydration of HPNE.

Topics: Gas Chromatography-Mass Spectrometry; Hydrogen Peroxide; Iron; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Molecular Structure

There is very little information available on the kinetic characteristics of fungal lipoxygenases (LOXs) because most data on the mechanism of this enzyme concern soybean LOX. In this paper, the kinetic properties of LOX from Terfezia claveryi Chatin ascocarps were studied for the first time. The enzyme did not show the "substrate aggregation-dependent activity" described for other LOXs and presented a K(m) for linoleic acid of 41 microM at pH 7.0. The effect of different inhibitors was also studied. The enzyme presented the characteristic lag phase of other LOXs, and the influence of different factors on its duration was analyzed. The lag period was reduced not only by the product of the reaction (13-HPOD) but also by 9-HPOD. Calculation of the activation constant is proposed for the first time as a useful tool for the characterization of LOX because this method makes it possible to quantify the effectiveness of different hydroperoxides as LOX activators. The activation constants obtained were 0.3 and 6.4 microM for 13- and 9-HPOD, respectively; thus, the product of the reaction was approximately 21-fold more effective than 9-HPOD as a T. claveryi LOX activator.

Topics: Ascomycota; Enzyme Activation; Hydrogen-Ion Concentration; Kinetics; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Lipoxygenase Inhibitors

Oxygenation of linoleic acid by the enzyme lipoxygenase (LOX) that is present in the microalga Chlorella pyrenoidosa is known to produce the corresponding 9- and 13-hydroperoxide derivatives of linoleic acid (9- and 13-HPOD, respectively). Previous work with this microalga indicated that partially purified LOX, present in the 30-45 and 45-80% saturated (NH4)2SO4 precipitate fractions, produced both HPOD isomers but in different ratios. It was not clear, however, if the observed activity in the two isolates represented the presence of one or more isozymes. In the present work, LOX isolated from the intracellular fraction of Chlorella by (NH4)2SO4 precipitation (35-80% saturated) was purified by ion exchange and hydrophobic interaction chromatography to apparent homogeneity. Analysis of the purified protein by SDS-PAGE and subsequent native size exclusion chromatography demonstrated that LOX in Chlorella is a single monomeric protein with a molecular mass of approximately 47 kDa. The purified LOX produced both the 9-HPOD and 13-HPOD isomers from linoleic acid in equal amounts, and the isomer ratio was not altered over the pH range of 6 to 9. Optimal activity of LOX was at pH 7.5.

Topics: Chlorella; Hydrogen Peroxide; Hydrogen-Ion Concentration; Isomerism; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Molecular Weight; Spectrophotometry, Atomic

Lipid peroxidation (LPO) processes observed in diseases connected with inflammation involve mainly linoleic acid. Its primary LPO products, 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) and 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), decompose in multistep degradation reactions. These reactions were investigated in model studies: decomposition of either 9-HPODE or 13-HPODE by Fe(2+) catalyzed air oxidation generates (with the exception of corresponding hydroxy and oxo derivatives) identical products in often nearly equal amounts, pointing to a common intermediate. Pairs of carbonyl compounds were recognized by reacting the oxidation mixtures with pentafluorobenzylhydroxylamine. Even if a pure lipid hydroperoxide is subjected to decomposition a great variety of products is generated, since primary products suffer further transformations. Therefore pure primarily decomposition products of HPODEs were exposed to stirring in air with or without addition of iron ions. Thus we observed that primary products containing the structural element R-CH=CH-CH=CH-CH=O add water and then they are cleaved by retroaldol reactions. 2,4-Decadienal is degraded in the absence of iron ions to 2-butenal, hexanal and 5-oxodecanal. Small amounts of buten-1,4-dial were also detected. Addition of m-chloroperbenzoic acid transforms 2,4-decadienal to 4-hydroxy-2-nonenal. 4,5-Epoxy-2-decenal, synthetically available by treatment of 2,4-decadienal with dimethyldioxirane, is hydrolyzed to 4,5-dihydroxy-2-decenal.

Topics: Air; Aldehydes; Arteriosclerosis; Cations, Divalent; Chromatography, High Pressure Liquid; Epoxy Compounds; Gas Chromatography-Mass Spectrometry; Humans; Hydroxylamines; Iron; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipoxygenase; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Structure; Oxidation-Reduction

The oxidation of linoleic acid by soybean lipoxygenase-1 (LOX-1) was inhibited in a time-dependent manner by 4-hydroxy-2(E)-nonenal (HNE). Kinetic analysis indicated the effect was due to slow-binding inhibition conforming to an affinity labeling mechanism-based inhibition. After 25 min of preincubation of LOX-1 with and without HNE, Lineweaver-Burk reciprocal plots indicated mixed noncompetitive/competitive inhibition. Low concentrations of HNE influenced the electron paramagnetic resonance (EPR) signal of 13(S)-hydroperoxy-9(Z), 11 (E)-octadecadienoic acid (13-HPODE)-generated Fe3+-LOX-1 slightly, but higher concentrations completely eliminated the EPR signal indicating an active site hindered from access by 13-HPODE. HNE may compete for the active site of LOX-1 because its precursor, 4-hydroperoxy-(2E)-nonenal, is a product of LOX-1 oxidation of (3Z)-nonenal. Also, it was an attractive hypothesis to suggest that HNE may disrupt the active site by forming a Michael adduct with one or more of the three histidines that ligate the iron active site of LOX-1.

Topics: Aldehydes; Buffers; Catalytic Domain; Cysteine Proteinase Inhibitors; Electron Spin Resonance Spectroscopy; Enzyme Activation; Histidine; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Lipoxygenase Inhibitors

Manganese porphyrin complexes serve to catalytically scavenge superoxide, hydrogen peroxide, and peroxynitrite. Herein, reactions of manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) with lipids and lipid hydroperoxides (LOOH) are examined. In linoleic acid and human low-density lipoprotein (LDL), MnTE-2-PyP(5+) promotes oxidative reactions when biological reductants are not present. By redox cycling between Mn(+3) and Mn(+4) forms, MnTE-2-PyP(5+) initiates lipid peroxidation via decomposition of 13(S)hydroperoxyoctadecadienoic acid [13(S)HPODE], with a second-order rate constant of 8.9 x 10(3) M(-1)s(-1)and k(cat) = 0.32 s(-1). Studies of LDL oxidation demonstrate that: (i) MnTE-2-PyP(5+) can directly oxidize LDL, (ii) MnTE-2-PyP(5+) does not inhibit Cu-induced LDL oxidation, and (iii) MnTE-2-PyP(5+) plus a reductant partially inhibit lipid peroxidation. MnTE-2-PyP(5+) (1-5 microM) also significantly inhibits FeCl(3) plus ascorbate-induced lipid peroxidation of rat brain homogenate. In summary, MnTE-2-PyP(5+) initiates membrane lipid and lipoprotein oxidation in the absence of biological reductants, while MnTE-2-PyP(5+) inhibits lipid oxidation reactions initiated by other oxidants when reductants are present. It is proposed that, as the Mn(+3) resting redox state of MnTE-2-PyP(5+) becomes oxidized to the Mn(+4) redox state, LOOH is decomposed to byproducts that propagate lipid oxidation reactions. When the manganese of MnTE-2-PyP(5+) is reduced to the +2 state by biological reductants, antioxidant reactions of the metalloporphyrin are favored.

Topics: Animals; Brain Chemistry; Catalysis; Chromatography, Thin Layer; Free Radical Scavengers; Humans; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipids; Lipoproteins; Lipoproteins, LDL; Male; Manganese; Mass Spectrometry; Metalloporphyrins; Oxidation-Reduction; Rats; Rats, Sprague-Dawley

Lipid peroxidation by managanese peroxidase (MnP) is reported to decompose recalcitrant polycyclic aromatic hydrocabon (PAH) and nonphenolic lignin models. To elucidate the oxidative process, linoleic acid and 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid [13(S)-HPODE] were reacted with MnPs from Ceriporiopsis subvermispora and Bjerkandera adusta and the free radicals produced were analyzed by ESR. When the MnPs were reacted with 13(S)-HPODE in the presence of Mn(II), H2O2 and tert-nitrosobutane (t-NB), the ESR spectrum contained a sharp triplet of acyl radical (aN = 0.81 mT). Formation of acyl radical was also observed in the reactions of Mn(III)-tartrate with 13(S)-HPODE and with linoleic acid, but the latter reaction occurred explosively after an induction period of around 30 min. Reactions of MnP with linoleic acid in the presence of Mn(II), H2O2 and t-NB gave no spin adducts while addition of t-NB after preincubation of linoleic acid with MnP/Mn(II)/H2O2 for 2 h gave spin adducts of carbon-centered (aN = 1.53 mT, aH = 0.21 mT) and acyl (aN = 0.81 mT) radicals. In contrast to linoleic acid, methyl linoleate and oleic acid were not peroxidized by MnP and chelated Mn(III) within a few hours, indicating that structures containing both the 1,4-pentadienyl moiety and a free carboxyl group are necessary for inducing the peroxidation in a short reaction time. These results indicate that MnP-dependent lipid peroxidation is not initiated by direct abstraction of hydrogen from the bis-allylic position during turnover but proceeds by a Mn(III)-dependent hydrogen abstraction from enols and subsequent propagation reactions involving the formation of acyl radical from lipid hydroperoxide. This finding expands the role of chelated Mn(III) from a phenol oxidant to a strong generator of free radicals from lipids and lipid hydroperoxides in lignin biodegradation.

Topics: Electron Spin Resonance Spectroscopy; Free Radicals; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Magnetic Resonance Spectroscopy; Peroxidases; Polyporaceae

Gastrointestinal glutathione peroxidase (GI-GPx), 1 of the 4 types of selenium-dependent glutathione peroxidases, is expressed exclusively in the gastrointestinal system and has therefore been suggested to function as a barrier against the absorption of dietary hydroperoxides.. The selenium-dependent expression of GI-GPx and cytosolic GPx (cGPx) was analyzed by Western blotting. Transport of 13-hydroperoxy octadecadienoic acid (13-HPODE) was investigated in a CaCo-2 cell monolayer modulated in GI-GPx and cGPx by selenium restriction or repletion. Localization of GI-GPx in rat intestine was visualized by immunohistochemistry.. Low but significant GI-GPx levels were detected in selenium-deficient CaCo-2 cells and in the gastrointestinal tract of selenium-deficient rats, whereas cGPx was completely absent. Selenium supplementation of CaCo-2 cells resulted in a 5-fold increase of GI-GPx protein, whereas total GPx activity increased by a factor of 13, with most of the GPx activity under selenium-adequate conditions being cGPx. Irrespective of the selenium status, 13-HPODE did not reach the basolateral side of an intact CaCo-2 cell monolayer. Depending on the selenium status, hydroperoxides damaged the monolayer as evidenced by loss of transepithelial resistance and paracellular diffusion of lucifer yellow. Only under these conditions was unmetabolized 13-HPODE detectable at the basolateral side.. Low GI-GPx levels, as present in selenium deficiency, suffice to prevent transport of 13-HPODE. GI-GPx may thus function as a barrier against hydroperoxide absorption. cGPx contributes to balance major oxidative challenge.

Topics: Animals; Caco-2 Cells; Carbon Radioisotopes; Cell Polarity; Diet; Enzyme Activation; Glutathione; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Humans; Hydrogen Peroxide; Intestinal Mucosa; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Liver; Rats; Rats, Wistar; Selenium

Various forms of oxidized low-density lipoproteins (Ox-LDL) are thought to play a major role in the development of atherosclerosis. The lipid components of Ox-LDL present a plethora of proatherogenic effects in in vitro cell culture systems, suggesting that oxidative stress could be an important risk factor for coronary artery disease. However, buried among these effects are those that could be interpreted as antiatherogenic. The present study demonstrates that various oxidants, including oxidized fatty acids and mildly oxidized forms of LDL (MO-LDL), are able to induce catalase (an antioxidant enzyme) expression in rabbit femoral arterial smooth muscle cells (RFASMC), RAW cells (macrophages), and human umbilical vein endothelial cells (HUVEC). In RFASMC, catalase protein, mRNA, and the enzyme activity are increased in response to oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid [13-HPODE] and 13-hydroxy-9,11-octadecadienoic acid [13-HODE]), MO-LDL, or hydrogen peroxide (H(2)O(2)). Such an increase in catalase gene expression cannot totally be attributed to the cellular response to an intracellular generation of H(2)O(2) after the addition of 13-HPODE or 13-HODE because these agents induce a further increase of catalase as seen in catalase-transfected RFASMC. Taken together with the induction of heme oxygenase, NO synthase, manganese superoxide dismutase (Mn-SOD), and glutathione synthesis by oxidative stress, our results provide yet more evidence suggesting that a moderate oxidative stress can induce cellular antioxidant response in vascular cells, and thereby could be beneficial for preventing further oxidative stress.

Topics: Animals; Catalase; Cells, Cultured; Endothelium, Vascular; Fatty Acids; Femoral Artery; Gene Expression; Humans; Hydrogen Peroxide; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoproteins, LDL; Muscle, Smooth, Vascular; Oxidants; Oxidation-Reduction; Rabbits; RNA, Messenger; Transfection; Umbilical Veins

Reactive nitrogen species derived from nitric oxide are potent oxidants formed during inflammation that can oxidize membrane and lipoprotein lipids in vivo. Herein, it is demonstrated that several of these species react with unsaturated fatty acid to yield nitrated oxidation products. Using HPLC coupled with both UV detection and electrospray ionization mass spectrometry, products of reaction of ONOO- with linoleic acid displayed mass/charge (m/z) characteristics of LNO2 (at least three products at m/z 324, negative ion mode). Further analysis by MS/MS gave a major fragment at m/z 46. Addition of a NO2 group was confirmed using [15N]ONOO- which gave a product at m/z 325, fragmenting to form a daughter ion at m/z 47. Formation of nitrated lipids was inhibited by bicarbonate, superoxide dismutase (SOD), and Fe3+-EDTA, while the yield of oxidation products was decreased by bicarbonate and SOD, but not by Fe3+-EDTA. Reaction of linoleic acid with both nitrogen dioxide (*NO2) or nitronium tetrafluoroborate (NO2BF4) also yielded nitrated lipid products (m/z 324), with HPLC retention times and MS/MS fragmentation patterns identical to the m/z 324 species formed by reaction of ONOO- with linoleic acid. Finally, reaction of HPODE, but not linoleate, with nitrous acid (HONO) or isobutyl nitrite (BuiONO) yielded a product at m/z 340, or 341 upon reacting with [15N]HONO. MS/MS analysis gave an NO2- fragment, and 15N NMR indicated that the product contained a nitro (RNO2) functional group, suggesting that the product was nitroepoxylinoleic acid [L(O)NO2]. This species could form via homolytic dissociation of LOONO to LO* and *NO2 and rearrangement of LO* to an epoxyallylic radical L(O)* followed by recombination of L(O)* with *NO2. Since unsaturated lipids of membranes and lipoproteins are critical targets of reactive oxygen and nitrogen species, these pathways lend insight into mechanisms for the formation of novel nitrogen-containing lipid products in vivo and provide synthetic strategies for further structural and functional studies.

Topics: Chromatography, High Pressure Liquid; Chromatography, Liquid; Fatty Acids, Unsaturated; Hydrogen-Ion Concentration; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Magnetic Resonance Spectroscopy; Mass Spectrometry; Nitrates; Nitric Oxide; Nitrogen Dioxide; Nitrous Acid; Oxidants; Oxidation-Reduction

Aspergillus spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus is affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotium production in some A. flavus strains in which sclerotium production decreases in the light but increases in the dark. In A. nidulans, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on A. nidulans required an intact veA gene. The sporogenic effects of light and linoleic acid on Aspergillus spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.

Topics: Aspergillus; Aspergillus flavus; Aspergillus nidulans; Fatty Acids, Unsaturated; Light; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Spores, Fungal

[1-14C]Linoleic acid was incubated with a whole homogenate preparation of potato leaves (Solanum tuberosum L., var. Bintje). The methyl-esterified product was subjected to straight-phase high-performance liquid chromatography and was found to contain four major radioactive oxidation products, i.e., the epoxy alcohols methyl 10(S),11(S)-epoxy-9(S)-hydroxy-12(Z)-octadecenoate (14% of the recovered radioactivity) and methyl 12(R), 13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoate (14%), and the trihydroxy derivatives methyl 9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoate (18%)and methyl 9(S), 12(S),13(S)-trihydroxy-10(E)-octadecenoate (30%). The structures and stereochemical configurations of these oxylipins were determined by chemical and spectral methods using the authentic compounds as references. Incubations performed in the presence of glutathione peroxidase revealed that lipoxygenase activity of potato leaves generated the 9- and 13-hydroperoxides of linoleic acid in a ratio of 95:5. Separate incubations of these hydroperoxides showed that linoleic acid 9(S)-hydroperoxide was metabolized into epoxy alcohols by particle-bound epoxy alcohol synthase activity, whereas the 13-hydroperoxide was metabolized into alpha- and gamma-ketols by a particle-bound allene oxide synthase. It was concluded that the main pathway of linoleic acid metabolism in potato leaves involved 9-lipoxygenase-catalyzed oxygenation into linoleic acid 9(S)-hydroperoxide followed by rapid conversion of this hydroperoxide into epoxy alcohols and a slower, epoxide hydrolase-catalyzed conversion of the epoxy alcohols into trihydroxy-octadecenoates. Trihydroxy derivatives of linoleic and linolenic acids have previously been reported to be growth-inhibitory to plant-pathogenic fungi, and a role of the new pathway of linoleic acid oxidation in defense reactions against pathogens is conceivable.

Topics: alpha-Linolenic Acid; Antifungal Agents; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Epoxide Hydrolases; Glutathione Peroxidase; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Molecular Structure; Oleic Acids; Oxidation-Reduction; Plant Leaves; Solanum tuberosum

The effect of oxygen concentration on the regiospecificity of the soybean lipoxygenase-1 dioxygenation reaction was studied. At low oxygen concentrations (<5 microM), a dramatic change in the regiospecificity of the enzyme was observed with the hydroperoxy-octadecadienoic acid (HPOD) 13:9 ratio closer to 50:50 instead of the generally reported 95:5. This alteration of regiospecificity is not an isolated phenomenon, since it occurs during a reaction carried out under "classical" conditions, i.e. in a buffer saturated with air before the reaction. beta-carotene bleaching and electronic paramagnetic resonance findings provided evidence that substrate-derived free radical species are released from the enzyme. The kinetic scheme proposed by Schilstra et al. (Schilstra, M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1994) Biochemistry 33, 3974-3979) was thus expanded to account for the observed variations in specificity. The equations describing the branched scheme show two different kinetic pathways: a fully enzymatic one leading to a regio-isomeric composition of 13-HPOD:9-HPOD = 95:5, and a semienzymatic one leading to a regio-isomeric composition of 13-HPOD:9-HPOD = 50:50. The ratio between the two different pathways depends on oxygen concentration, which thus determines the overall specificity of the reaction.

Topics: beta Carotene; Computer Simulation; Electron Spin Resonance Spectroscopy; Glycine max; Isomerism; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Models, Chemical; Oxygen; Potentiometry; Substrate Specificity

Linoleic acid, a polyunsaturated C18 fatty acid, is one of the major fatty acids in the coronary arterial wall. Although diets rich in linoleic acid reduce blood pressure and prevent coronary artery disease in both humans and animals, very little is known about its mechanism of action. We believed that its beneficial effects might be mediated by changes in vascular tone. We investigated whether linoleic acid induces relaxation of porcine coronary artery rings and the mechanism involved in this process. Linoleic acid and two of its metabolites, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), induced dose-dependent relaxation of prostaglandin (PG) F2alpha-precontracted rings that was not affected by indomethacin (10[-5] mol/L), a cyclooxygenase inhibitor, or cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC; 10[-5] mol/L), a lipoxygenase inhibitor. Removal of endothelial cells had no effect on vasorelaxation, suggesting a direct effect on the vascular smooth muscle cells (VSMC). When rings were contracted with KCl, linoleic acid failed to induce relaxation. Although tetrabutylammonium (5 x 10[-3] mol/L), a nonselective K+ channel blocker, slightly inhibited the relaxation caused by linoleic acid, glibenclamide (10[-6] mol/L), an ATP-sensitive K+ channel blocker, and charybdotoxin (7.5x10[-8] mol/L) or tetraethylammonium (5x10[-3] mol/L), two different Ca2+-activated K+ channel blockers, had no effect. However, relaxation was completely blocked by ouabain (5x10[-7] mol/L), a Na+/K+-ATPase inhibitor, or by a K+-free solution. In addition, linoleic acid (10[-6] mol/L) caused sustained hyperpolarization of porcine coronary VSMC (from -49.5+/-2.0 to -60.7+/-4.2 mV), which was also abolished by ouabain. We concluded that linoleic acid induces relaxation and hyperpolarization of porcine coronary VSMC via a mechanism that involves activation of the Na+/K+-ATPase pump.

Topics: Animals; Coronary Vessels; Electrophysiology; Fatty Acids; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Membrane Potentials; Swine; Vasodilation

The Cu(II)-promoted oxidation of lipids is a lipid hydroperoxide (LOOH)-dependent process that has been used routinely to assess the oxidizability of low-density lipoprotein (LDL) in human subjects. Metal-dependent redox reactions, including those mediated by copper, have been implicated in the pathogenesis ofatherosclerosis. Despite its widespread use and possible biological significance, key elements of the mechanism are not clear. For example, although it is evident that copper acts as a catalyst, which implies a redox cycle between the Cu(II) and Cu(I) redox states, the reductants remain uncertain. In LDL these could include alpha-tocopherol, amino acid residues on the protein and LOOH. However, both alpha-tocopherol and amino acid residues are probably consumed before the most rapid phase of lipid peroxidation occurs, suggesting that another reductant must be donating electrons to Cu(II), the most likely candidate being LOOH. This role has been disputed, since LDLs nominally devoid of LOOH are still capable of reducing Cu(II) to Cu(I) and thermodynamic calculations for this reaction are not favourable. Direct investigation of the role of LOOH as reductant has not been reported and in the present study, using simple lipid systems and LDL, we have re-examined this issue using the Cu(I) chelator bathocuproine. We have shown that Cu(II) may promote lipid peroxidation in liposomes, which do not contain either protein or alpha-tocopherol, and that this is associated with reduction to Cu(I). The data also indicate that an equilibrium between free Cu(II) and LOOH exists, which only in the presence of an oxidizable substrate, i.e. unsaturated fatty acids, is shifted towards formation of Cu(I) and lipid-derived peroxyl radicals. We propose that reduction of Cu(II) by LOOH is a necessary component in sustaining the propagation of lipid peroxidation and that the formation of peroxyl radicals and their products in a lipid environment is sufficient to overcome thermodynamic barriers to the reaction.

Topics: Copper; Copper Sulfate; Electron Spin Resonance Spectroscopy; Humans; Indicators and Reagents; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoproteins, LDL; Liposomes; Models, Chemical; Oxidation-Reduction; Peroxides; Phenanthrolines; Phosphatidylcholines; Spectrophotometry

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cricetinae; Embryo, Mammalian; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Hybrid Cells; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Mesocricetus; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction; Stimulation, Chemical

We have used stopped-flow rapid reaction methods, employing both fluorescence and absorbance monitoring, together with HPLC analysis of the products to study the activation of soybean 15-lipoxygenase by 13(S)-hydroperoxy-9, 11(E,Z)-octadecadienoic acid (13-HPOD). When lipoxygenase is mixed with an equimolar concentration of 13-HPOD, the enzyme undergoes a rapid change in fluorescence. The rate of the change of fluorescence is dependent on the concentration of the 13-HPOD (k = 6.7 x 10(6) M-1 s-1) and is accompanied by activation of the enzyme. The fluorescence change is not accompanied by any change in the UV absorbance of the 13-HPOD, suggesting no loss of the conjugated diene during enzyme activation, and HPLC analysis of the products of the reaction confirms that the 13-HPOD can be recovered unchanged following this reaction. In the presence of an inhibitor (BWA4C, a hydroxamate inhibitor) that reduces the active-site iron, the 13-HPOD and the inhibitor are destroyed in a peroxidase-like reaction. On the basis of these observations we propose that 13-HPOD binds to the enzyme and facilitates activation of the enzyme, possibly through the formation of a protein radical, and that the 13-HPOD is not changed chemically in this process.

Topics: Arachidonate 15-Lipoxygenase; Benzeneacetamides; Borates; Chromatography, High Pressure Liquid; Enzyme Activation; Glycine max; Hydroxamic Acids; Kinetics; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipoxygenase Inhibitors; Models, Chemical; Spectrometry, Fluorescence

Previous studies from other laboratories suggest that linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, play an important role in modulating the growth of some cells. A correlation has been demonstrated between hydroperoxyoctadecadienoic acids and conditions characterized by abnormal cell growth such as atherosclerosis and psoriasis. To determine if linoleic acid and its metabolites modulate cell growth in atherosclerosis, we measured DNA synthesis, protooncogene mRNA expression, and mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (VSMC). Linoleic acid induces DNA synthesis, c-fos, c-jun, and c-myc mRNA expression and MAPK activation in VSMC. Furthermore, nordihydroguaiaretic acid, a potent inhibitor of the lipoxygenase system, significantly reduced the growth-response effects of linoleic acid in VSMC, suggesting that conversion of linoleic acid to hydroperoxyoctadecadienoic acids (HPODEs) is required for these effects. HPODEs also caused significant induction of DNA synthesis, protooncogene mRNA expression, and MAPK activation in growth-arrested VSMC, suggesting that linoleic acid and its metabolic products, HPODEs, are potential mitogens in VSMC, and that conditions such as oxidative stress and lipid peroxidation which provoke the production of these substances may alter VSMC growth.

Topics: Animals; Aorta, Thoracic; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Enzyme Activation; Gene Expression Regulation; Genes, fos; Genes, jun; Genes, myc; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipoxygenase; Male; Masoprocol; Muscle Development; Muscle, Smooth, Vascular; Oxidative Stress; Rats; Rats, Sprague-Dawley

Incubations of [1-14C]linoleic acid or [1-14C]-(9Z,11E, 13S)-13-hydropero xy-9,11-octadecadienoic acid (13-HPOD) with juice of garlic bulbs lead to the formation of one predominant labelled product, viz., the novel divinyl ether (9Z,11E, 1'E)-12-(1'-hexenyloxy)-9,11-dodecadienoic acid ('etheroleic acid'). With lesser efficiency [1-14C]alpha-linolenic acid or [1-14C](9Z,11E, 13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid (13-HPOT) are converted in this way into (9Z,11E,1'E,1'E,3'Z)-12-(1',3'-hexadienyloxy)-9,11- dodecadienoic acid ('etherolenic acid'). Thus, garlic bulbs possess the activity of a new 13-hydroperoxide-specific divinyl ether synthase.

Topics: alpha-Linolenic Acid; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Garlic; Linoleic Acid; Linoleic Acids; Linolenic Acids; Lipid Peroxides; Lipoxygenase; Magnetic Resonance Spectroscopy; Plants, Medicinal

A new method designed to monitor lipid peroxidation in plants has been set up with soybean hypocotyl/radicles. The hydroperoxy fatty acids present in situ are converted by rapid thermal treatment (80 s and 210 J g-1) of the biological sample into ethane and n-pentane, which are analyzed by gas chromatography. The method has been directly calibrated by quantification of the hydroperoxy fatty acids by silica-phase HPLC analysis of their reduced hydroxy derivatives. Hypocotyl/radicles from the two soybean cultivars Argenta and Soriano were submitted to various chemical oxidative treatments and were analyzed for both thermally produced volatile alkanes and hydroperoxy fatty acid levels. Our results showed that ethane and n-pentane production are in both cases closely correlated with linolenic as well as linoleic acid hydroperoxide levels (P < 0.001). Within a given plant material, thermal conversion of both hydroperoxides into alkanes occurred with yields which were not dependent on the oxidative treatment. These yields are however functions of the biological material since in Soriano and Argenta cultivars they were around 6 and 25%, respectively. Taking into account the last point, the alkane test cannot be used to directly quantify the absolute lipid hydroperoxide levels of plant tissues but it is convenient to monitor the peroxidative phenomenon as it occurs. The assay is easy and rapid to perform (analysis of 50 samples per day) since no sample preparation is needed, and the low detection limit (20 pmol of alkane g-1) permits the analysis of small samples.

Topics: Alkanes; alpha-Linolenic Acid; Chromatography, High Pressure Liquid; Glycine max; Heating; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Pentanes; Plants; Seeds

Treatment of soybean lipoxygenase isozyme 1 with its substrates, linoleic acid and oxygen, or product, 13(S)-hydroperoxy-9,11(Z,E)-octadecadienoic acid (13-HPOD), leads to the appearance of a purple color. Although the structure of the chromophore has not been determined, we present strong evidence that it is an Fe(3+)-OOR complex between the enzyme and 13-HPOD. Irradiation of frozen purple solutions of lipoxygenase causes the reversible production of a radical, shown by the effects of 2H and 17O enrichment on its EPR spectrum to be derived from 13-HPOD. The action spectrum of the photolysis reaction corresponds to the visible spectrum of the purple species, strongly implying that the purple chromophore contains 13-HPOD (or a product thereof) as part of its structure. Concomitant with the production of this radical there is a decrease in the intensity of an EPR signal corresponding to enzyme-bound Fe3+ and characteristic of the purple species. Taken together, these observations support the suggestion that the purple species is a complex between ferric lipoxygenase and 13-HPOD, likely the ferric peroxide.

Topics: Chromogenic Compounds; Glycine max; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Oxygen; Photolysis; Spectrum Analysis

The effect of a hydroperoxy adduct of linoleic acid, 13-hydroperoxyoctadecadienoic acid (13-HPODE), on 15-hydroxy prostaglandin dehydrogenase activity in rabbit kidney cortex was examined. 13-HPODE inhibited the 15-hydroxy prostaglandin dehydrogenase activity at concentrations ranging from 1 to 10 microM. The effect was concentration-dependent and the concentration required for 50% inhibition was approximately 3 microM. Linoleic acid and 13-hydroxyoctadecadienoic acid (13-HODE) exhibited weaker inhibition of the enzyme activity than did 13-HPODE (linoleic acid, 30% inhibition at 10 microM; 13-HODE, 45% inhibition at 10 microM). Studies utilizing Fe2+ (a catalyst of peroxide decomposition), and mannitol or dimethylsulfoxide (a hydroxy radical scavenger) revealed that the inhibitory effect of 13-HPODE on the 15-hydroxy prostaglandin dehydrogenase activity is not due to the hydroxy radicals which are expected to be formed from 13-HPODE and that the hydroperoxy functional group is a prerequisite. The inhibition by 13-HPODE was uncompetitive and non-competitive with regard to NAD+ and prostaglandin E2, respectively. These results suggest that 13-HPODE has the potential to modulate the prostaglandin catabolism by affecting the 15-hydroxy prostaglandin dehydrogenase activity.

Topics: Animals; Dimethyl Sulfoxide; Ferrous Compounds; Hydroxyprostaglandin Dehydrogenases; Kidney Cortex; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Male; Mannitol; Rabbits

An assay for the detection of hydroperoxy derivatives of linoleic acid formed by the action of 15-lipoxygenase is described. The assay developed is based on a method first reported by Ohishi et al. (1985) Biochem. Int. 10, 205-211) with some important modifications. The assay described herein takes advantage of the ability of (9Z,11E)-13-hydroperoxyoctadecadienoic acid (13-HPODE), the product of the action of 15-lipoxygenase on linoleic acid, to oxidize N-benzoyl leucomethylene blue to methylene blue in the presence of hemoglobin. The resultant blue color is stable to light and air and can be quantified spectrophometrically at 660 nm. The linear range of the assay is 1.6-32 nmol (0.5-10 micrograms) of 13-HPODE. The utility of the assay can be extended to detect other peroxides as well as inhibitors of 15-lipoxygenase. The assay is a rapid, reliable method for the detection of lipid hydroperoxide production.

Topics: Animals; Arachidonate 15-Lipoxygenase; Chromatography, High Pressure Liquid; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipoxygenase Inhibitors; Methylene Blue; Rabbits; Spectrophotometry

A pure lipoxygenase from dried green pea seeds (isoenzyme 1) oxygenates linoleic acid to 9(S/R)-hydroperoxy-10E,12Z-octadecadienoic acid (9-HPODE) and 13(S/R)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPODE). Furthermore (10E,12Z)-9-keto-10,12-octadecadienoic acid (9-KODE) and (9Z,11E)-13-keto-9,11-octadecadienoic acid (13-KODE) in a ratio of 1:1 were formed. Uv-spectroscopic measurements and HPLC data indicated a hydroperoxy fatty acid: keto fatty acid ratio of about 2:1. The product mixture formed from arachidonic acid was even more complex. 15-, 11-, 9- and 5-H(P)ETE1 and their corresponding keto derivatives have been detected. The chemical structures of the compounds have been identified by HPLC analysis, by uv- and ir-spectroscopy and gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The data presented indicate that a pure lipoxygenase catalyzes the formation of both hydroperoxypolyenoic fatty acids and ketopolyenoic fatty acids from linoleic acid and arachidonic acid. The possible mechanism of the formation of the keto compounds is discussed.

Topics: Arachidonic Acids; Chromatography, High Pressure Liquid; Fabaceae; Gas Chromatography-Mass Spectrometry; Kinetics; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Molecular Structure; Oxygen; Plants, Medicinal; Spectrophotometry, Ultraviolet

When linoleic acid was incubated with the purified potato lipoxygenase under O2 atmosphere, a mixture of 9 and 13-hydroperoxyoctadecadienoic acids was formed. Stereochemical analysis of the respective methyl-hydroxyoctadecadienoic acids revealed that the 9-isomer was in S-configuration whereas 13-hydroxyoctadecadienoic acid was a mixture of S (39%) and R (61%). Exactly the opposite was the case with the soybean lipoxygenase products, where the 13-isomer was found to be in S-configuration and 9-hydroxyoctadecadienoic acid - a mixture of S (73%) and R (27%). A general scheme is proposed for the stereochemical nature of oxidation products of enzymes which are predominantly either [+2] or [-2] lipoxygenases.

Topics: Chromatography, High Pressure Liquid; Glycine max; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Solanum tuberosum; Stereoisomerism

Soybean lipoxygenase-1 produces a preponderance of two chiral products from linoleic acid, (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid and (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid. The former of these hydroperoxides was generated at all pH values, but in the presence of Tween 20, the latter product did not form at pH values above 8.5. As the pH decreased below 8.5, the proportion of (9S)-hydroperoxide increased linearly until at pH 6 it constituted about 25% of the chiral products attributed to enzymic action. Below pH 6, lipoxygenase activity was barely measurable, and the hydroperoxide product arose mainly from autoxidation and possibly non-enzymic oxygenation of the pentadienyl radical formed by the enzyme. The change in percent enzymically formed 9-hydroperoxide between pH 6.0 and 8.5 paralleled the pH plot of a sodium linoleate/linoleic acid titration. It was concluded that the (9S)-hydroperoxide is formed only from the nonionized carboxylic acid form of linoleic acid. Methyl esterification of linoleic acid blocked the formation of the (9S)-hydroperoxide by lipoxygenase-1, but not the (13S)-hydroperoxide. Since the hydroperoxydiene moieties of the (9S)- and (13S)-hydroperoxides are spatially identical when the molecules are arranged head to tail in opposite orientations, it is suggested that the carboxylic acid form of the substrate can arrange itself at the active site in either orientation, but the carboxylate anion can be positioned only in one orientation. These observations, as well as others in the literature, suggest and active-site model for soybean lipoxygenase-1.

Topics: Binding Sites; Hydrogen-Ion Concentration; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Models, Chemical

In a previous paper we reported that arachidonic acid (20:4(n-6] strongly enhances the endothelial cell synthesis of prostaglandin I3 (PGI3) from eicosapentaenoic acid (20:5(n-3], in stimulating the cyclooxygenase rather than the prostacyclin synthase (Bordet et al. (1986) Biochem. Biophys. Res. Commun. 135, 403-410). In the present study, endothelial cell monolayers were co-incubated with exogenous 20:5(n-3) or docosatetraenoic acid (22:4(n-6], and n-6 lipoxygenase products of 20:4(n-6) or linoleic acid (18:2(n-6], namely 15-HPETE and 13-HPOD, respectively. Prostaglandins or dihomoprostaglandins were then measured by gas chromatography-mass spectrometry. Both hydroperoxides, up to 20 microM, stimulated the cyclooxygenation of 20:5(n-3) and 22:4(n-6), in particular the formation of PGI3 and dihomo-PGI2, respectively. Higher concentrations inhibited prostacyclin synthetase. In contrast, the reduced products of hydroperoxides, 15-HETE and 13-HOD, failed to stimulate these cyclooxygenations, 13-HPOD appeared more potent than 15-HPETE and the cyclooxygenation of 22:4(n-6) seemed to require higher amounts of hydroperoxides to be efficiently metabolized than 20:5(n-3). These data suggest that prostacyclin potential of endothelium might be enhanced by raising the peroxide tone.

Topics: Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Endothelium, Vascular; Epoprostenol; Erucic Acids; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Humans; Kinetics; Leukotrienes; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Mass Spectrometry; Prostaglandins F; Umbilical Veins

Soybean lipoxygenase is rapidly inactivated when incubated with arachidonic acid and any of several lipoxygenase inhibitors, including NDGA, the aminopyrazolines BW 755C and BW 540C, and the acetohydroxamic acid derivatives BW A4C and BW A137C. Little or no inactivation was found when the enzyme was incubated with substrate or with inhibitors alone. 15-HPETE was as effective as arachidonic acid in promoting inactivation, but linoleic acid and 13-HPOD were much less effective. The UV absorption at 235 nm, due to the conjugated diene in 15-HPETE or 13-HPOD, was rapidly destroyed in the presence of soybean lipoxygenase and inhibitor in a presumed pseudoperoxidase reaction. The products of the reaction between linoleic acid, BW A137C and soybean lipoxygenase have been partially characterized. A derivative of arachidonic acid is postulated to be the inactivating agent.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Arachidonic Acid; Arachidonic Acids; Benzeneacetamides; Ethanol; Ethylene Glycol; Ethylene Glycols; Glycine max; Hydroxamic Acids; Leukotrienes; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase Inhibitors; Masoprocol; Pyrazoles

From a comparison of 9Ds-HPODE and 13Ls-HPODE and their methyl esters as substrates and inactivating agents of reticulocyte lipoxygenase it is concluded that the compounds inactivate the enzyme independently of any hydroperoxidase reaction. The protective effect of 4-nitrocatechol indicates the formation of Fe(III) complexes of the enzyme with the hydroperoxyfatty acid compounds prior to inactivation.

Topics: Anaerobiosis; Animals; Catechols; Ferric Compounds; Isomerism; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase Inhibitors; Methylation; Rabbits; Reticulocytes

The breakage of double-strand (ds) DNA by 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid (LAHPO) was investigated by agarose gel electrophoresis of supercoiled pBR322 DNA and the site of cleavage on the DNA molecule was determined by the method of DNA sequence analysis using 3'-end and 5'-end-labeled DNA fragments as substrates. LAHPO caused cleavage at the position of guanine nucleotide in dsDNA. LAHPO caused dsDNA breaks at specific sites, but linoleic acid (LA) and 13-L-hydroxy-cis-9,trans-11-octadecadienoic acid (LAHO) have no such effects on dsDNA. The active oxygen atom of the hydroperoxy group of LAHPO was perhaps responsible for the site-specific cleavage of dsDNA.

Topics: Animals; DNA, Superhelical; Drosophila melanogaster; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Piperidines; Plasmids