Unsaturated Fatty Acid Hydroperoxides Research Articles

Heme-catalyzed degradation of linoleate 9-hydroperoxide (9-HPODE) forms two allylic epoxy-ketones via a proposed pseudo-symmetrical diepoxy radical intermediate

Heme-initiated decomposition of unsaturated fatty acid hydroperoxides creates alkoxyl radicals that propagate a complex series of reactions to hydroxy, keto, epoxy and aldehydic products. Herein, among the products from the hematin-catalyzed degradation of 9-hydroperoxy-linoleic acid (9-HPODE), we observed a double peak on normal-phase HPLC that resolved on RP-HPLC into equal proportions of two epoxy-allylic ketones with identical UV spectra. Their proton NMR spectra were also indistinguishable and consistent with 9,10-trans-epoxy-11E-13-keto- and 9-keto-10E-12,13-trans-epoxy-octadecenoic acids. Acid hydrolysis to the corresponding dihydroxy-ketones and GC-MS analysis identified the earlier eluting product on RP-HPLC as the 9,10-epoxy regio-isomer. Starting from the C9-hydroperoxide, recovery of the two epoxy-ketones in equal proportions suggests their formation from a common intermediate. Earlier work has proposed formation of a pseudo-symmetrical diepoxy radical (9,10-epoxy-11(•)-12,13-epoxy, derived from an epoxy allylic hydroperoxide precursor) in the carbon chain fragmentation leading to aldehydic products. This intermediate in pathways of alkoxyl radical reactions forms equal pairs of aldehydes, and now also a pair of epoxy-ketones, and based on mechanism the same products arise from either 9-HPODE or 13-HPODE. Our results point to the intermediacy of this diepoxy-carbinyl radical in the origin of at least two classes of linoleate peroxidation products, and it should be considered as a viable intermediate for homo-conjugated diene peroxidation in general. The reactions could contribute to the aldehydes and epoxy-ketones in tissues undergoing oxidative transformations of polyunsaturated fatty acids.

Comparison of the Catabolic Rates of Linoleic and Oleic Acid Hydroperoxides Using 13CO2 Expired from Mice.

Unsaturated fatty acids, such as oleic and linoleic acids, are easily oxidized by exposure to temperature and light in the presence of air to form unsaturated fatty acid hydroperoxides as primary oxidation products. However, the catabolic rates of unsaturated fatty acid hydroperoxides in the human body remain unknown. In this study, ethyl esters of 13C-labeled linoleic acid (*C18:2-EE) and oleic acid (*C18:1-EE) and their hydroperoxides (*C18:2-EE-OOH and *C18:1-EE-OOH, respectively) prepared by the photo-oxidation of *C18:2-EE and *C18:1-EE, respectively, were administered to mice and their catabolic rates were determined by measuring the expired 13CO2 levels. *C18:2-EE-OOH and *C18:1-EE-OOH were β-oxidized faster than *C18:2-EE and *C18:1-EE, respectively. Notably, rapid β-oxidation of *C18:2-EE-OOH and *C18:1-EE-OOH was similar to that of medium-chain fatty acids, such as octanoic acid. Then, degradation products of C18:2-EE-OOH and C18:1-EE-OOH were analyzed under gastric conditions by gas chromatography/mass spectrometry. Major decomposition products of C18:2-EE-OOH and C18:1-EE-OOH were medium-chain compounds, such as octanoic acid ethyl ester, 9-oxo-nonanoic acid ethyl ester, and 10-oxo-8-decenoic acid ethyl esters, indicating that C18:2-EE-OOH and C18:1-EE-OOH isomers formed during photo-oxidation were decomposed under acidic conditions. These findings support previous reports that dietary lipid hydroperoxides are not absorbed into the intestine as lipid hydroperoxides but as degradation products. This is the first study to suggest that dietary lipid hydroperoxides decompose during gastric digestion to form medium-chain compounds that are directly absorbed into the liver via the portal vein and rapidly catabolized via β-oxidation.

Intramolecular H-Atom Transfers in Alkoxyl Radical Intermediates Underlie the Apparent Oxidation of Lipid Hydroperoxides by Fe(II).

The one-electron reduction of lipid hydroperoxides by low-valent iron species is believed to be a driver of cellular lipid peroxidation and associated ferroptotic cell death. We investigated reactions of cholesterol 7α-OOH, the primary cholesterol autoxidation product, with Fe2+ to find that 7-ketocholesterol (7-KC, an oxidation product) is the major product under these (reducing) conditions. Mechanistic studies reveal the intervention of a 1,2-H-atom shift upon formation of the 7-alkoxyl radical to yield a ketyl radical that can be oxidized by either Fe3+ or O2 to give 7-KC, the most abundant oxysterol in vivo. We also investigated the corresponding reduction of the isomeric cholesterol 5α-OOH and again found that an oxidation product (5-hydroxycholesten-3-one) predominates under reducing conditions. An intramolecular H-atom shift (this time 1,4-) in the initially formed 5-alkoxyl radical is suggested to yield a ketyl radical that is oxidized to give the observed product. It would appear that a 1,2-H shift also accounts for the predominance of ketones over alcohols when unsaturated fatty acid hydroperoxides are exposed to iron-based reductants, which had previously been reported with hematin and demonstrated here with Fe2+. The predominance of 7-KC over the corresponding alcohol is maintained when cholesterol 7α-OOH embedded in phospholipid liposomes is treated with Fe2+ or when ferroptosis is induced in mouse embryonic fibroblasts. Our observation that 7-KC accumulates in ferroptotic cells suggests that it may be a good biomarker for ferroptosis.

Kinetics of oxidation of iron(II) ions with lipid hydroperoxides

AbstractOne of the pathways for excessive production of free radicals is the decomposition of lipid hydroperoxides catalyzed by iron. A number of hydroperoxides of unsaturated fatty acids (LOOH), some prepared in our laboratory and others extracted from biological materials, were used to determine the rate constants of Fe2+ oxidation by measuring the formation rate of Fe3+ ions in the presence of simple unidentate ligands, chloride, and thiocyanate as the [FeCl]2+ and [FeNCS]2+ complexes, in a deoxygenated dichloromethane:methanol (2:1, v/v) mixture. The rates of Fe2+ oxidation with prepared LOOHs via the [FeNCS]2+ complex were approximately the same‐the average second‐order reaction rate constant was 1390 ± 340 dm3 mol−1 s−1; the rate constants of LOOHs from different biological materials were in the same range. The rates measured as the [FeNCS]2+ complex were somewhat higher than the rates measured as the [FeCl]2+ complex, indicating that ligands could interact in the transition state, thus affecting the disruption of the intermediate complex. Since there were no significant differences in the activation thermodynamic parameters for reactions within the reaction series of studied hydroperoxides, it was assumed that the oxidation proceeded by an inner sphere mechanism, considering that the breakdown of the successor inner sphere complex with the homolytic cleavage of peroxide bonds of hydroperoxides forming reactive alkoxyl radicals was the rate‐limiting step. Based on this research, an indirect spectrophotometric method for quantitative determination of LOOH was reestimated. The microprocedure proposed for the lipid hydroperoxide assay could be applied to follow the early stages of lipid peroxidation processes in real biological samples.

Exposure assessment of epoxy fatty acids through consumption of specific foods available in Belgium

ABSTRACTEpoxy fatty acids (EFAs) are secondary oxidation products formed from unsaturated fatty acid hydroperoxides. Seventeen food categories were analysed for C18 monoEFAs of food products available on the Belgian market. A quantitative exposure assessment was performed based on deterministic and probabilistic approaches combining these concentration data with consumption data obtained from the Belgian National Food Consumption Survey of 2004. A preliminary evaluation of any potential risk related to the intake of the studied EFAs through the studied foods was performed by applying the threshold of toxicological concern (TTC) concept. Three food categories out of 17 foods, mayonnaise, butter–margarine and ready-to-eat meals were found to contribute most to the intake of EFAs. According to probabilistic determination, these foods had P50 intakes of 0.4085, 0.3328 and 0.2997 mg kg–1 bw day–1 respectively. They had P99.5 intakes of 3.7183, 2.7921 and 38.6068 mg kg–1 bw day–1 respectively. The intake below the TTC was from the consumption of cooked meat, smoked salmon and raw cured ham, with P50 intakes of 0.0006, 0.0007 and 0.0011 mg kg–1 bw day–1 respectively, and the other foods were above the TTC. Based on the TTC concept, a risk to human health could be identified related to the consumption of cheese, snacks foods, plant oils, French fries, dry nuts, chips, cured minced raw meat, cookies, fresh and frozen salmon and bacon.

Kinetic and spectroscopic studies of N694C lipoxygenase: a probe of the substrate activation mechanism of a nonheme ferric enzyme.

Lipoxygenases (LOs) comprise a class of substrate activating mononuclear nonheme iron enzymes which catalyze the hydroperoxidation of unsaturated fatty acids. A commonly proposed mechanism for LO catalysis involves H-atom abstraction by an FeIII-OH- site, best described as a proton coupled electron transfer (PCET) process, followed by direct reaction of O2 with the resulting substrate radical to yield product. An alternative mechanism that has also been discussed involves the abstraction of a proton from the substrate by the FeIII-OH leading to a sigma-organoiron intermediate, where the subsequent sigma bond insertion of dioxygen into the C-Fe bond completes the reaction. H-atom abstraction is favored by a high E(o) of the FeII/FeIII couple and high pK(a) of water bound to the ferrous state, while an organoiron mechanism would be favored by a low E(o) (to keep the site oxidized) and a high pK(a) of water bound to the ferric state (to deprotonate the substrate). A first coordination sphere mutant of soybean LO (N694C) has been prepared and characterized by near-infrared circular dichroism (CD) and variable-temperature, variable-field (VTVH) magnetic circular dichroism (MCD) spectroscopies (FeII site), as well as UV/vis absorption, UV/vis CD, and electron paramagnetic resonance (EPR) spectroscopies (FeIII site). These studies suggest that N694C has a lowered E degrees of the FeII/FeIII couple and a raised pKa of water bound to the ferric site relative to wild type soybean lipoxygenase-1 (WT sLO-1) which would favor the organoiron mechanism. However, the observation in N694C of a significant deuterium isotope effect, anaerobic reduction of iron by substrate, and a substantial decrease in k(cat) (approximately 3000-fold) support H-atom abstraction as the relevant substrate-activation mechanism in sLO-1.

Glutathione peroxidase‐like protein of Synechocystis PCC 6803 confers tolerance to oxidative and environmental stresses in transgenic Arabidopsis

Glutathione peroxidase (GPX)‐like proteins (GPX‐1 and GPX‐2) of Synechocystis PCC 6803 (S. PCC 6803) reduce unsaturated fatty acid hydroperoxides using NADPH, but not reduced glutathione (GSH), as an electron donor. Here, we generated transgenic Arabidopsis plants overexpressing S. PCC 6803 GPX‐2 in the cytosol (AcGPX2) or chloroplasts (ApGPX2). The activities toward α‐linolenic acid hydroperoxide in ApGPX2 and AcGPX2 plants were 6.5–11.5 and 8.2–16.3 nmol min−1 mg protein−1, respectively, while no activity (<0.1 nmol min−1 mg protein−1) was detected in the wild‐type plants. Both transgenic lines (AcGPX2 and ApGPX2) showed enhanced tolerance to oxidative damage caused by treatment with H2O2 (10 mM), Fe ions (200 μM) or methylviologen (50 μM) and environmental stress conditions, such as chilling with high light intensity (4°C, 1000 μmol photons m−2 s−1), high salinity (100 mM NaCl) or drought. The degree of tolerance of the transgenic plants to all types of stress was correlated with the levels of lipid peroxide suppressed by the overexpression of S. PCC 6803 GPX‐2. Under conditions of oxidative stress due to the H2O2 treatment, the NADPH/(NADP++ NADPH) ratio in the transgenic plants was lower than that in the wild‐type plants. The data reported here indicate that the expression of S. PCC 6803 GPX‐2 contributes to the reduction in unsaturated fatty acid hydroperoxides using NADPH in situ under stress conditions in the transgenic plants.

Induction and functional analysis of two reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione peroxidase-like proteins in Synechocystis PCC 6803 during the progression of oxidative stress.

Synechocystis PCC 6803 contains two types of glutathione peroxidase-like proteins (GPX-1 and GPX-2) that utilize NADPH but not reduced glutathione and unsaturated fatty acid hydroperoxides or alkyl hydroperoxides. The steady-state transcript level of gpx-1 gradually increased under oxidative stress conditions imposed by high light intensity, high salinity, or application of methylviologen or t-butyl hydroperoxide in the wild-type and GPX-2 knock-out mutant (gpx-2Delta) cells. To examine the ability of GPX-1, GPX-2, and thioredoxin peroxidase to scavenge lipid hydroperoxide in vivo, we measured the photosynthetic evolution of O(2) and the level of lipid peroxidation in the wild-type and each type of mutant cell after the application of t-butyl hydroperoxide or H(2)O(2). The data reported here indicate that GPX-1 and GPX-2 are essential for the removal of lipid hydroperoxides under normal and stress conditions, leading to the protection of membrane integrity.

Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol.

To evaluate the physiological potential of the defense system against hydroperoxidation of membrane-lipid components caused by environmental stresses in higher plants, we generated transgenic tobacco plants expressing a glutathione peroxidase (GPX)-like protein in the cytosol (TcGPX) or chloroplasts (TpGPX). The activities toward alpha-linolenic acid hydroperoxide in TcGPX and TpGPX plants were 47.5-75.3 and 32.7-42.1 nM min(-1) mg(-1) protein, respectively, while no activity was detected in wild-type plants. The transgenic plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV: 50 microM) under moderate light intensity (200 micro E m(-2) sec(-1)), chilling stress under high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)), or salt stress (250 mM NaCl). Under these stresses, the lipid hydroperoxidation (the production of malondialdehyde (MDA)) of the leaves of TcGPX and TpGPX plants was clearly suppressed compared with that of wild-type plants. Furthermore, the capacity of the photosynthetic and antioxidative systems in the transgenic plants remained higher than those of wild-type plants under chilling or salt stress. These results clearly indicate that a high level of GPX-like protein in tobacco plants functions to remove unsaturated fatty acid hydroperoxides generated in cellular membranes under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress caused by various stress conditions.

Spectroscopic characterization of soybean lipoxygenase-1 mutants: the role of second coordination sphere residues in the regulation of enzyme activity.

Lipoxygenases are non-heme iron enzymes, which catalyze the stereo- and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five- and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe(II) was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe(II) systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.

Molecular characterization of glutathione peroxidase-like protein in halotolerant Chlamydomonas sp. W80.

A cDNA clone encoding a glutathione peroxidase (GPX)-like protein was isolated from the cDNA library from halotolerant Chlamydomonas W80 (C. W80) by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 163 amino acids with a calculated molecular mass of 18 267 Da. No potential signal peptide was found. The deduced amino acid sequence of the cDNA showed 40-63% and 37-46% homology to those of GPX-like proteins from higher plants and mammalian GPXs, respectively. The C. W80 GPX-like protein contained a normal cysteine residue instead of a selenocysteine at the catalytic site. However, it contained amino acid residues (glutamine and tryptophan) that are involved in three protein loops and are important for the catalytic activity in the mammalian GPX. Interestingly, the native and recombinant GPX-like proteins showed activities towards unsaturated fatty acid hydroperoxides, but not towards either H2O2 or phospholipid hydroperoxide. Transformed E. coli cells expressing the C. W80 GPX-like protein showed enhanced tolerance to 5% NaCl or 0.2 mM paraquat treatments. Accession number: The nucleotide sequence data reported have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession number AB009083.

Occurrence and biochemistry of hydroperoxidases in oxygenic phototrophic prokaryotes (cyanobacteria)

Blue-green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant-type photosynthetic organisms. They were the first which produced molecular oxygen as a byproduct of photosynthetic activity. Also today they live in habitats with potentially damaging photooxidative conditions due to high irradiation and oxygen concentrations. Therefore, the cells must have evolved protective mechanisms to cope with reactive oxygen species produced by incomplete reduction of molecular oxygen via electron transport processes to prevent damage of biologically important macromolecules. Hydrogen peroxide and organic peroxides can be removed by enzymes called hydroperoxidases which on the one hand disproportionate it (catalases and catalase-peroxidases) and on the other hand use electron donors to reduce it to water or the corresponding alcohols. Until now the sequenced or partially sequenced genomes of six cyanobacteria are available in databases. Based on similarity searches and multiple sequence alignments, several cyanobacterial hydroperoxidases can be detected. All the cyanobacteria possess peroxiredoxins which use thioredoxin or other reduced thiols to get rid of hydrogen peroxide and lipid peroxides. Nearly all cyanobacteria contain an NADPH-dependent glutathione peroxidase-like protein which uses NADPH to reduce unsaturated fatty acid hydroperoxides. The best analyzed cyanobacterial antioxidative enzyme is the hemoprotein catalase-peroxidase which has a high catalase activity but concerning the sequence it is a typical peroxidase. Two species seem to encode a manganese-containing catalase and Nostoc punctiforme could use a monofunctional catalase. There are as well additional peroxidases encoded in cyanobacteria whose physiological relevance is unknown.

Detection of docosahexaenoic acid hydroperoxides in retina by gas chromatography/mass spectrometry.

AbstractIn the retina, as well as in other organs, inflammation-mediated membrane lipid peroxidation is regarded as the major source of unsaturated fatty acid hydroperoxides. It is generally accepted that inflammatory infiltrates, such as polymorphonuclear leukocytes and macropahges, simultaneously generate superoxide and nitric oxide (NO) (1). Although both radicals display low chemical reactivity, their combination product, peroxynitrite, is a potent oxidant capable of oxidizing cellular macromolecules, including membrane lipids (2). Photoreceptor membranes in the retina contain an unusually high concentration of docosahexaenoic acid (22:6, omega 3), amounting to nearly 50% of the total fatty acid pool (3). By virtue of its structure, 22:6 is particularly susceptible to lipid peroxidation induced by peroxynitrite (2,2).Peroxidation of photoreceptor membranes in the retina has been recognized as a key component in various types of photoreceptor degeneration and pathogenesis (5). Interest in the pathological consequences of photoreceptor membrane lipid peroxidation has led to the development of several analytical approaches designed to detect peroxidized lipids in the retina.KeywordsDocosahexaenoic AcidButylate HydroxytoluenePositional IsomerTrimethylsilyl EtherFatty Acid MoietyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

NADPH-dependent glutathione peroxidase-like proteins (Gpx-1, Gpx-2) reduce unsaturated fatty acid hydroperoxides in Synechocystis PCC 6803

Here we isolated and characterized two genes ( slr1171, slr1992) designated gpx-1 and gpx-2, respectively, encoding glutathione peroxidase (GPX)-like proteins (Gpx-1, Gpx-2) from Synechocystis PCC 6803. The deduced amino acid sequences for gpx-1 and gpx-2 showed high similarity to those of GPX-like proteins from higher plants and mammalian GPXs, respectively. Surprisingly, both recombinant proteins in Escherichia coli were able to utilize NADPH, but not reduced glutathione, as an electron donor and unsaturated fatty acid hydroperoxides or alkyl hydroperoxides as an acceptor. It seems accurate to refer to Gpx-1 and Gpx-2 as NADPH-dependent GPX-like proteins that serve as a new defense system for the reduction of unsaturated fatty acid hydroperoxides.

Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.

Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln(495) and Gln(697), which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn(694)). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln(495). The nature of the C-H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH(-) is the active site base. A new transition state model for hydrogen atom abstraction is proposed.

Effect of cytochrome c on the linoleic acid–degrading activity of porcine leukocyte 12-lipoxygenase

Hemoproteins are known to have quasilipoxygenase activity that converts linoleic acid (LA) to its hydroperoxides. However, it is not still clear whether, like lipoxygenases, hemoproteins can produce LA hydroperoxides when the LA is part of a mixture containing many different saturated and unsaturated fatty acids. In this study, we found that such hemoprotein as cytochrome c (Cyt c) did not produce LA hydroperoxides from the phospholipase A 2 (PL-A 2) hydrolysis products of egg yolk phosphatidylcholine (PC). We also found that traces of hydroperoxides and a high concentration of the target unsaturated fatty acid (LA) needs to be present in a fatty acid mixture before the quasi-lipoxygenase activity of Cyt c becomes apparent. We also attempted to elucidate how Cyt c interact with porcine leukocyte 12-lipoxygenase (12-LOX). Hemoproteins are known to possess pseudo-lipohydroperoxidase activity, and can remove the hydroperoxides of unsaturated fatty acids from a reaction mixture. However, we found that Cyt c catalyzed the reaction by which hydroperoxides degrade LA, and thus enhanced the LA-degrading activity of 12-LOX. This hemoprotein-induced promotion of the ability of 12-LOX to degrade LA was observed even when the reaction mixture contained many different saturated and unsaturated fatty acids.

Spectroscopic and Functional Characterization of a Ligand Coordination Mutant of Soybean Lipoxygenase-1: First Coordination Sphere Analogue of Human 15-Lipoxygenase

Lipoxygenases are an important class of non-heme iron enzymes which catalyze the hydroperoxidation of unsaturated fatty acids. This study utilizes a combination of electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD to probe the ground state and excited states of the active site of an iron coordination mutant of soybean lipoxygenase-1 (SLO-1), N694H, and relate the structural modifications with kinetic changes. The sixth coordination ligand for SLO-1, Asn694, is proposed to be a primary factor in the coordination flexibility of the site and is reflected in both its ferrous MCD and ferric EPR spectroscopic properties. In the present study, we compare the spectroscopic properties of the N694H SLO-1 mutant with those of wild-type (WT) SLO-1 and WT human 15-lipoxygenase (15-HLO) and determine that its properties are intermediate between the WT enzymes. WT 15-HLO has a His substitution in the Asn694 equivalent position. The low-temperatu...

Biocatalytic hydroxylation of linoleic acid in a double-fed batch system with lipoxygenase and cysteine

The enzymatic large-scale preparation of unsaturated fatty acid hydroperoxides is the first step in the preparation of the corresponding fatty acid hydroxides. Since hydroxides are more suitable than hydroperoxides as precursors of fine chemicals like certain flavour compounds, a convenient and effective reduction step of hydroperoxides is required. In this paper a double-fed batch procedure for the enzymatic preparation of fatty acid hydroperoxides including the application of cysteine as a mild reducing agent is described. A crude extract from defatted soybean flour was used as the source of lipoxygenase, while the primary substrate was linoleic acid from hydrolyzed safflower oil. Enzyme and substrate were fed synchronously into the bioreactor that already contained buffer solution and cysteine. Typically, reactions with 40 g/l substrate in a 7 l reactor were done on a 2 l scale. Without any intermediate work-up, a final product yield of 90% could be achieved consisting for nearly 96% of 13(S)-hydroxy-(9Z,11E)-octadeca-9,11-dienoic acid.

Separation of isomeric hydroperoxides of unsaturated fatty acids by capillary electrophoresis

Nearly all the isomeric hydroperoxides from the reaction of oleic, linoleic and linolenic acids with singlet oxygen are separated in a single analysis by micellar electrokinetic chromatography with polyoxyethylene lauryl ether (Brij 35) as surfactant. Fatty acids tend to adsorb strongly onto the silica, so that coating of the capillary is essential. Brij 35 is used for dynamic coating of either octadecylsilane-derivatised or fused-silica capillaries. A diode array is used for the detection.

Increase of Caryophyllene Oxide in Ageing Lemon Balm Leaves (Melissa officinalis L.) -A Consequence of Lipid Peroxidation?

Oxidative processes, especially lipid peroxidation (LPO), are assumed to increase during ageing. In an attempt to provide experimental evidence for this assumption lemon balm plants (Melissa officinalis L.) of different age and location were analyzed for oxidatively caused changes in the etheric oil composition. The investigation revealed that the caryophyllene oxide (CarO) content of lemon balm plants - a main constituent - depends on two factors: age and habitat. The content of CarO increased with age continuously up to a factor of 3. Poor nutritive conditions, as growth on unfertilized soil, also caused an increase in CarO content. Addition of Fe2+/ascorbate - enhancing oxidative processes - promoted the formation of CarO. Hydroperoxides of unsaturated fatty acids (LOOHs) in the lipid extract of lemon balm leaves were converted to corresponding hydroxy acids (LOHs) by sodium borohydride reduction. These were hydrogenated and subjected to GC/MS analysis after derivatisation. A surplus of 9-hydroxy-octadecanoic acid over the 13 isomer indicated at least in part enzymatic lipid peroxidation. Polarographie determination of the oxygen consumption revealed a generally low but in ageing plants increased lipoxygenase activity. This indicates a contribution of lipid peroxidation in the epoxidation process of caryophyllene