Regio- and stereochemical analysis of trihydroxyoctadecenoic acids derived from linoleic acid 9- and 13-hydroperoxides

The methyl esters of 9S,10S,13R-trihydroxy-11E-octadecenoic acid, 9S,10R,13S-trihydroxy-11E-octadecenoic acid, and 9S,10R,13R-trihydroxy-11E-octadecenoic acid were prepared from 9S-hydroperoxy-10E,12Z-octadecadienoic acidvia the epoxy alcohols methyl 10R,11R-epoxy-9S-hydroxy-12Z-octadecenoate and methyl 10S,11S-epoxy-9S-hydroxy-12Z-octadecenoate. The trihydroxyesters, as well as four stereoisomeric methyl 9,12,13-trihydroxy-10E-octadecenoates earlier prepared [Hamberg, M.,Chem. Phys. Lipids 43, 55–67 (1987)], were characterized by thin-layer chromatography, gas-liquid chromatography, mass spectrometry, and by chemical degradation. Availability of these chemically defined trihydroxyoctadecenoates made it possible to design a method for regio- and stereochemical analysis of 9,10,13- and 9,12,13-trihydroxyoctadecenoic acids obtained from various sources. Application of the method revealed that the mixture of 9,10,13- and 9,12,13-trihydroxyoctadecenoic acids formed during autoxidation of linoleic acid in aqueous medium contained comparable amounts of the sixteen possible regio- and stereoisomers. Furthermore, hydrolysis of the allylic epoxy alcohol, methyl 9S,10R-epoxy-13S-hydroxy-11E-octadecenoate, yielded a major trihydroxyoctadecenoate,i.e., methyl 9S,10S,13S-trihydroxyl-11E-octadecenoate, together with smaller amounts of methyl 9S,10R,13S-trihydroxy-11E-octadecenoate, methyl 9S,12R,13S-trihydroxy-10E-octadecenoate, and methyl 9S,12S,13S-trihydroxy-10E-octadecenoate.     

Efficient and Specific Conversion of 9-Lipoxygenase Hydroperoxides in the Beetroot. Formation of Pinellic Acid

The linoleate 9-lipoxygenase product 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid was stirred with a crude enzyme preparation from the beetroot (Beta vulgaris ssp. vulgaris var. vulgaris) to afford a product consisting of 95% of 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid (pinellic acid). The linolenic acid-derived hydroperoxide 9(S)-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid was converted in an analogous way into 9(S),12(S),13(S)-trihydroxy-10(E),15(Z)-octadecadienoic acid (fulgidic acid). On the other hand, the 13-lipoxygenase-generated hydroperoxides of linoleic or linolenic acids failed to produce significant amounts of trihydroxy acids. Short-time incubation of 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid afforded the epoxy alcohol 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid as the main product indicating the sequence 9-hydroperoxide → epoxy alcohol → trihydroxy acid catalyzed by epoxy alcohol synthase and epoxide hydrolase activities, respectively. The high capacity of the enzyme system detected in beetroot combined with a simple isolation protocol made possible by the low amounts of endogenous lipids in the enzyme preparation offered an easy access to pinellic and fulgidic acids for use in biological and medical studies.     

9-Hydroxy-traumatin, a new metabolite of the lipoxygenase pathway

9-Hydroxy-traumatin, 9-hydroxy-12-oxo-10E-dodecenoic acid, was isolated as a product of 13S-hydroperoxy-9Z, 11E-octadecadienoic acid as catalyzed by enzyme preparations of both soybean and alfalfa seedlings. This suggested that 9Z-traumatin, 12-oxo-9Z-dodecenoic acid, was being converted into 9-hydroxy-traumatin in an analogous manner to the previously identified enzymic conversion of 3Z-nonenal and 3Z-hexenal into 4-hydroxy-2E-nonenal and 4-hydroxy-2E-hexenal, respectively. Other metabolites of 13S-hydroperoxy-9Z,11E-octadecadienoic acid were similar for both soybean and alfalfa seedling preparations, and they are briefly described.     

Metabolic profile of linoleic acid in stored apples: Formation of 13(R)-hydroxy-9(Z),11(E)-octadecadienoic acid

During our ongoing project on the biosynthesis of R-(+)-octane-1,3-diol the metabolism of linoleic acid was investigated in stored apples after injection of [1-¹⁴C]-, [9,10,12,13-³H]-, ¹³C₁₈- and unlabeled substrates. After different incubation periods the products were analyzed by gas chromatography-mass spectroscopy (MS), high-performance liquid chromatography-MS/MS, and HPLC-radiodetection. Water-soluble compounds and CO₂ were the major products whereas 13(R)-hydroxy- and 13-keto-9(Z),11(E)-octadecadienoic acid, 9(S)-hydroxy-and 9-keto-10(E),12(Z)-octadecadienoic acid, and the stereoisomers of the 9,10,13- and 9,12,13-trihydroxyoctadecenoic acids were identified as the major metabolites found in the diethyl ether extracts. Hydroperoxides were not detected. The ratio of 9/13-hydroxy- and 9/13-keto-octadecadienoic acid was 1∶4 and 1∶10, respectively. Chiral phase HPLC of the methyl ester derivatives showed enantiomeric excesses of 75% (R) and 65% (S) for 13-hydroxy-9(Z),11(E)-octadecadienoic acid and 9-hydroxy-10(E),12(Z)-octadecadienoic acid, respectively. Enzymatically active homogenates from apples were able to convert unlabeled linoleic acid into the metabolites. Radiotracer experiments showed that the transformation products of linoleic acid were converted into (R)-octane-1,3-diol. 13(R)-Hydroxy-9(Z), 11(E)-octadecadienoic acid is probably formed in stored apples from 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid. It is possible that the S-enantiomer of the hydroperoxide is primarily degraded by enzymatic side reactions, resulting in an enrichment of the R-enantiomer and thus leading to the formation of 13(R)-hydroxy-9(Z),11(E)-octadecadienoic acid.     

Enantioselective formation of an α, β-epoxy alcohol by reaction of methyl 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate with titanium isopropoxide

Methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate (threo isomer) was generated from linoleic acid by the sequential action of an enzyme and two chemical reagents. Linoleic acid was treated with lipoxygenase to yield its corresponding hydroperoxide [13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid]. After methylation with CH₂N₂, the hydroperoxide was treated with titanium (IV) isopropoxide [Ti(O-i-Pr)₄] at 5°C for 1 h. The products were separated by normal-phase high-performance liquid chromatography and characterized with gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Approximately 30% of the product was methyl 13(S)-hydroxy-9(Z), 11(E)-octadecadienoate. Over 60% of the isolated product was methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate. After quenching Ti(O-i-Pr)₄ with water, the spent catalyst could be removed from the fatty products by partitioning between CH₂Cl₂ and water. These results demonstrate that Ti(O-i-Pr)₄ selectively promotes the formation of an α-epoxide with the threo configuration. It was critically important to start with dry methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate because the presence of small amounts of water in the reaction medium resulted in the complete hydrolysis of epoxy alcohol to trihydroxy products.     

A pathway for biosynthesis of divinyl ether fatty acids in green leaves

[1-¹⁴C]α-Linolenic acid was incubated with a particulate fraction of homogenate of leaves of the meadow buttercup (Ranunculus acris L.). The main product was a divinyl ether fatty acid, which was identified as 12-[1′(Z),3′(Z)-hexadienyloxy]-9(Z), 11(E)-dodecadienoic acid. Addition of glutathione peroxidase and reduced glutathione to incubations of α-linolenic acid almost completely suppressed formation of the divinyl ether acid and resulted in the appearance of 13(S)-hydroxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid as the main product. This result, together with the finding that 13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid served as an efficient precursor of the divinyl ether fatty acid, indicated that divinyl ether biosynthesis in leaves of R. acris occurred by a two-step pathway involving an ω6-lipoxygenase and a divinyl ether synthase. Incubations of isomeric hydroperoxides derived from α-linolenic and linoleic acids with the enzyme preparation from R. acris showed that 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid was transformed into the divinyl ether 12-[1′(Z)-hexenyloxy]-9(Z), 11(E)-dodecadienoic acid. In contrast, neither the 9(S)-hydroperoxides of linoleic or α-linolenic acids nor the 13(R)-hydroperoxide of α-linolenic acid served as precursors of divinyl ethers.     

Method to produce 9(S)-hydroperoxides of linoleic and linolenic acids by maize lipoxygenase

Seed from maize (corn) Zea mays provides a ready source of 9-lipoxygenase that oxidizes linoleic acid and linolenic acid into 9(S)-hydroperoxy-10(F), 12(Z)-octadecadienoic acid and 9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid, respectively. Corn seed has a very active hydro-peroxide-decomposing enzyme, allene oxide synthase (AOS), which must be removed prior to oxidizing the fatty acid. A simple pH 4.5 treatment followed by centrifugation removes most of the AOS activity. Subsequent purification by ammonium sulfate fractional precipitation results in negligible improvement in 9-hydroperoxide formation. This facile alternative method of preparing 9-hydroperoxides has advantages over other commonly used plant lipoxygenases.     

Fatty acid hydroperoxide isomerase inSaprolegnia parasitica: Structural studies of epoxy alcohols formed from isomeric hydroperoxyoctadecadienoates

The major part (80%) of the fatty acid hydroperoxide isomerase activity present in homogenates of the fungus,Saprolegnia parasitica, was localized in the particle fraction sedimenting at 105,000×g. 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid and 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid were both good substrates for the particle-bound hydroperoxide isomerase. The products formed from the 13(S)-hydroperoxide were identified as an α,β- and a γ,δ-epoxy alcohol, i.e., 11(R),12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoic acid and 9(S),10(R)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid, respectively. The 9(S)-hydroperoxide was converted in an analogous way into an α,β-epoxy alcohol, 10(R),11(R)-epoxy-9(S)-hydroxy-12(Z)-octadecenoic acid and a γ,δ-epoxy alcohol, 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid. 9(R,S)-Hydroperoxy-10(E),12(E)-octadecadienoic acid and 13(R,S)-hydroperoxy-9(E),11(E)-octadecadienoic acid were poor substrates for theS. parasitica hydroperoxide isomerase. Experiments with 13(R,S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid showed that the 13(R)-hydroperoxy enantiomer was slowly isomerized by the enzyme. The major product was identified as α,β-epoxy alcohol 11(R),12(R)-epoxy-13(R)-hydroxy-9(Z)-octadecenoic acid.     

Conversion of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid to the corresponding hydroxy fatty acid by KOH: A kinetic study

Transformation of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-HPOD) to 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid (13S-HOD) under alkaline conditions (0.05 to 5 M KOH) occurred first-order with respect to 13S-HPOD concentration. Overall yield was about 80%. The energy of activation at higher concentrations (3.75 to 5 M KOH) was determined to be in the range of 15.3 to 15.6 kcal. Compared to the 13S-HPOD conversion, 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13S-HPOT) was converted at a faster rate to the corresponding hydroxy fatty acid (13S-HOT), with the reaction also being first-order. Chiral phase high-performance liquid chromatography demonstrated that in the transformation the stereochemistry of both the 13S-HPOD and 13S-HPOT reactants was preserved. Manometric analyses of the KOH/13S-HPOD reaction showed an uptake of gas, which amounted to 11% of the mols of reactant 13S-HPOD on the assumption that the gas was O₂. As there is a theoretical loss of 1 oxygen atom in the reaction, the fate of this oxygen (possiblyvia active oxygen species) may involve reaction with 13S-HPOD/13SHOD to form the 20% by-products.     

Effect of 4-hydroxy-2(E)-nonenal on soybean lipoxygenase-1

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.     

Biosynthesis of new divinyl ether oxylipins in Ranunculus plants

[1-¹⁴C]Linolenic acid was incubated with homogenates of leaves from the aquatic plants Ranunculus lingua (greater spearwort) or R. peltatus (pond water-crowfoot). Analysis by reversed-phase high-performance liquid radiochromatography demonstrated the formation of a new divinyl ether FA, i.e., 12-[1′(E), 3′(Z)-hexadienyloxy]-9(Z), 11(Z)-dodecadienoic acid [11(Z)-etherolenic acid] as well as a smaller proportion of ω5(Z)-etherolenic acid previously identified in terrestrial Ranunculus plants. The same divinyl ethers were formed upon incubation of 13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid, a lipoxygenase metabolite of linolenic acid, whereas the isomeric hydroperoxide, 9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid, was not converted into divinyl ethers in R. lingua or R. peltatus. Incubation of [1-¹⁴C]linoleic acid or 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid produced the divinyl ether 12-[1′(E)-hexenyloxy]-9(Z), 11(Z)-dodecadienoic acid [11(Z)-etheroleic acid] and a smaller amount of ω5(Z)-etheroleic acid. The experiments demonstrated the existence in R. lingua and R. peltatus of a divinyl ether synthase distinct from those previously encountered in higher plants and algae.     

Fatty acid allene oxides. III. Albumin-induced cyclization of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid

Recently, corn (Zea mays L.) hydroperoxide dehydrase was found to catalyze the conversion of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid into an unstable fatty acid allene oxide, 12,13(S)-epoxy-9(Z),11-octadecadienoic acid. This study is concerned with the chemistry of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid in the presence of vertebrate serum albumins. Albumins were found to greatly enhance the aqueous half-life of the allene oxide, i.e. 14.1±1.8 min, 11.6±1.2 min and 4.8±0.5 min at 0 C in the presence of 15 mg/ml of bovine, human and equine serum albumins, respectively, as compared with ca. 33 sec in the absence of albumin. Degradation of allene oxide in the presence of bovine serum albumin led to the formation of a novel cyclization product, i.e. 3-oxo-2-pentyl-cyclopent-4-en-1-octanoic acid (12-oxo-10-phytoenoic acid, in which the relative configuration of the side chains attached to the five-membered ring istrans). Steric analysis of the cyclic derivative showed that the compound was largely racemic (ratio between enantiomers, 58∶42). 12-Oxo-10,15(Z)-phytodienoic acid, needed for reference purposes, was prepared by incubation of 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid with corn hydroperoxide dehydrase. Steric analysis showed that the 12-oxo-10,15(Z)-phytodienoic acid thus obtained was not optically pure but a mixture of enantiomers in a ratio of 82∶18. The first paper in this series is Reference 1.     

Preparation of fatty epoxy alcohols using oat seed peroxygenase in nonaqueous media

Peroxygenase is an enzyme of higher plants that is capable of using hydroperoxide and hydrogen peroxide for oxidation of a double bond to an epoxide. A microsomal fraction was prepared from dry oat (Avena sativa) seeds. The peroxygenase activity of this fraction was tested using fatty acid hydroperoxide 2a [13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid] and its methyl ester 2b as sources of peroxygen. These were prepared by the action of soybean lipoxygenase on linoleic acid. A high-performance liquid chromatographic assay was used to differentiate between peroxygen cleavage and peroxygen cleavage with accompanying double-bond oxidation Higher activity was obtained with 2b compared to 2a, and peroxygen cleavage activity was observed in both aqueous and organic solvent media. Double-bond oxidation activity was high only in aqueous media and nonpolar organic solvents. Structural elucidation of the epoxidized product showed it to be the oxylipid, methyl cis-9,10-epoxy-13(S)-hydroxy-11(E)-octade-cenoate 4b, demonstrating specificity for epoxidation of the cis double bond. Trihydroxy product was not detected, demonstrating that the epoxide was not hydrolyzed.     

Chemoenzymatic production of (+)-coriolic acid from trilinolein: Coupled synthesis and extraction

Chemoenzymatic conversion of trilinolein to (+)-coriolic acid was investigated in this work. Lipase-catalyzed hydrolysis of trilinolein and lipoxygenation of liberated linoleic acid were coupled in a two-phase medium that consisted of a pH 9 borate buffer and a water-immiscible organic solvent (octane). High concentrations of trilinolein could be dissolved in the organic phase (up to 340 mM). Linoleic acid, liberated after hydrolysis, transferred to the aqueous phase and was enzymatically converted to the preferred 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid with soybean lipoxygenase-1. This product, which remained in the aqueous phase, could be recovered by centrifugation and then chemically reduced to (+)-coriolic acid (purity >95%). Recovery of this compound by liquid-liquid extraction was easy. The structure of (+)-coriolic acid has been confirmed by ¹H nuclear magnetic resonance spectroscopy, mass spectrometry, and infrared spectroscopy. High yields were obtained with pure trilinolein or sunflower oil as initial substrates.     

Soybean lipoxygenase is active on nonaqueous media at low moisture: a constraint to xerophilic fungi and aflatoxins?

Previous workers have reported that certain products of the lipoxygenase pathway are detrimental either to the development and growth of Aspergillus species or to aflatoxin production by these organisms. Since Aspergillus often thrives on “dry” stored grains, depending on the level of the relative humidity, we sought to determine if lipoxygenase could catalyze the oxidation of linoleic acid on these “dry” substrates equilibrated at various relative humidities. A desiccated model system, previously adjusted to pH 7.5, was composed of soybean extract, linoleic acid, and cellulose carrier. The model system was incubated for up to 24 h at four relative humidities ranging between 52 and 95% to determine the extent of oxidation catalyzed by lipoxygenase, compared with heat-inactivated controls. Oxidation in the active samples was much greater than in the controls at all relative humidities, and oxidation was principally enzymatic as demonstrated by chiral analysis of the linoleate hydroperoxides formed. The main product was 13S-hydroperoxy-9Z,11E-octadecadienoic acid, accompanied by a significant percentage of 9S-hydroperoxy-10E,12Z-octadecadienoic acid. Since the products became more racemic with time of incubation, autoxidation appeared to be initiated by the lipoxygenase reaction in dry media. Additionally, the biological relevance of lipoxygenase activity was tested under these xerophilic conditions. Thus, enzyme-active and heat-inactivated defatted soy flour amended either with or without 3.5% by weight linoleic acid was inoculated with fungal spores and incubated at 95% relative humidity. Although fungal growth occurred on all treatments, samples inoculated with Aspergillus parasiticus showed significantly less aflatoxin in the enzyme-active samples, compared to inactivated flour. Addition of linoleic acid had little effect, possibly because the defatted soy flour was found to contain 1.7% residual linoleic acid as glyceride lipid.     

Catalytic properties of allene oxide synthase from flaxseed (Linum usitatissimum L.)

We investigated the catalytic and kinetic properties of allene oxide synthase (AOS; E.C. 3.2.1.92) from flaxseed (Linum usitatissimum L.). Both Michaelis constant and maximal initial velocity for the conversion of 9(S)-and 13(S)-hydroper-oxides of linoleic and linolenic acid were determined by a photometric assay, 13(S)-Hydroperoxy-9(Z), 11(E)-octadecadienoic acid [13(S)-HPOD] as the most effective substrate was converted at 116.9±5.8 nkat/mg protein by the flax enzyme extract. The enzyme was also incubated with a series of variable conjugated hydroperoxy dienyladipates. Substrates with a shape similar to the natural hydroperoxides showed the best reactivity. Monoenoic substrates as oleic acid hydroperoxides were not converted by the enzyme. In contrast, 12-hydroperoxy-9(Z), 13(E)-octadecadienoic acid was a strong competitive inhibitor for AOS catalyzed degradation of 13(S)-HPOD. The inhibitor constant was determined to be 0.09 μM. Based on these results, we concluded that allene oxide synthase requires conjugated diene hydroperoxides for successful catalysis. Studying the enantiomeric preference of the enzyme, we found that AOS was also able to metabolize (R)-configurated fatty acid hydroperoxides. Conversion of these substrates into labile allene oxides was confirmed by steric analysis of the stable α-ketol hydrolysis products.     

Analysis of oxylipins by high-performance liquid chromatography with evaporative light-scattering detection and particle beam-mass spectrometry

The metabolism of 13 S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid was investigated in a crude enzyme extract from mung bean seedlings (Phaseolus radiatus L.). Hydroperoxide-metabolizing activity was mainly due to a hydroperoxide lyase and, to a lesser extent, to an allene oxide synthase and a peroxygenase. Oxylipins originating from hydrolysis and cyclization of the allene oxide synthase product 12,13-epoxy-9Z,11,15Z-octadecatrienoic acid and from peroxygenase catalysis were identified by high-performance liquid chromatography (HPLC) particle beam-mass spectrometry (PB-MS) and quantified by normal-phase HPLC with an evaporative light-scattering detector (ELSD). An advantage of this methodology was the possibility to avoid extensive derivatization procedures commonly used for the gas chromatographic analysis of oxylipins. Owing to a comparable sample inlet system, the ELSD served an important analytical pilot function for the PB-MS: Qualitatively identical chromatographic patterns were obtained with both detection systems. The HPLC system enabled the separation of methyl 12-oxo-phytodienoate, methyl 11-hydroxy-12-oxo-9Z,15Z-octadecadienoate, methyl 12-oxo-13-hydroxy-9Z,15Z-octadecadienoate, methyl 9-hydroxy-12-oxo-10E,15Z-octadecadienoate, methyl 13-hydroxy-9Z,11E,15Z-octadecatrienoate, methyl 15,16-epoxy-13-hydroxy-9Z,11E,15Z-octadecatrienoate, and methyl 13-hydroperoxy-9Z,11E,15Z-octadecatrienoate on a Lichrospher DIOL column within 33 min. Compared with a diode array detector, the ELSD proved to be more sensitive, in the case of methyl 12-oxo-13-hydroxy-9Z, 15Z-octadecadienoate by a factor of about 15. In addition, volatile metabolites were analyzed by capillary gas chromatography. The yield of the hydroperoxide lyase product 2E-hexenal was 49%, whereas the sum of oxylipins reached about 15%.     

Volatiles from thermal decomposition of isomeric methyl (12S, 13S)-(E)-12,13-epoxy-9-hydroperoxy-10-octadecenoates

Two epimers of methyl (12S,13S)-(E)-12,13-epoxy-9-hydroperoxy-10-octadecenoate were isolated after esterification of a mixture of fatty acids obtained from decomposition of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid by an Fe⁺⁺-cysteine catalyst. These epimeric epoxyhydro-peroxyoctadecenoates were decomposed by heat (210 C) in the injection port of a gas chromatograph, and the cleavage fragments were subsequently separated by gas chromatography (GC) and identified by mass spectrometry (MS). Among the scission products obtained, the most prominent in the GC peak profile were methyl octanoate and methyl 9-oxononanoate. Other peaks were identified as pentane, 1-pentanol, hexanal, 2-heptanone, 2-pentylfuran, methyl heptanoate, 2-octenal, 4,5-epoxy-2-decenal, methyl 8-(2-furyl)-octanoate, 11-oxo-9-undecenoate and methyl 13-oxo-9,11-tridecadienoate. In addition, 3,4-epoxynonanal, methyl 8-oxooctanoate, 3-hydroxy-2-pentyl-2,3-dihydrofuran and methyl 10-oxodecanoate were tentatively identified. Except for the furan compounds, the formation of the fragmentation products could be explained by conventional free-radical scission mechanisms. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Linoleic acid metabolism in the red algaLithothamnion corallioides: Biosynthesis of 11(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid

Incubation of [1-¹⁴C]linoleic acid with an enzyme preparation obtained from the red algaLithothamnion corallioides Crouan resulted in the formation of 11-hydroxy-9(Z),12(Z)-octadecadienoic acid as well as smaller amounts of 9-hydroxy-10(E),12(Z)-octadecadienoic acid, 13-hydroxy-9(Z),11(E)-octadecadienoic acid and 11-keto-9(Z),12(Z)-octadecadienoic acid. Steric analysis showed that the 11-hydroxyoctadecadienoic acid had the (R) configuration. The 9- and 13-hydroxyoctadecadienoic acids were not optically pure, but were due to mixtures of 75% (R) and 25% (S) enantiomers (9-hydroxyoctadecadienoate), and 24% (R) and 76% (S) enantiomers (13-hydroxy-octadecadienoate). 11-Hydroxyoctadecadienoic acid was unstable at acidic pH. In acidified water, equal parts of 9(R,S)-hydroxy-10(E),12(Z)-octadecadienoate and 13(R,S)-hydroxy-9(Z),11(E)-octadecadienoate, plus smaller amounts of the corresponding (E),(E) isomers were produced. In aprotic solvents, acid treatment resulted in dehydration and in the formation of equal amounts of 8,10,12- and 9,11,13-octadecatrienoates. The enzymatic conversion of linoleic acid into the hydroxyoctadecadienoic acids and the ketooctadecadienoic acid was oxygen-dependent; however, inhibitor experiments indicated that neither lipoxygenase nor cytochrome P-450 were involved in the conversion. This conclusion was supported by experiments with¹⁸O₂ and H₂ ¹⁸O, which demonstrated that the hydroxyl oxygen of the hydroxy-octadecadienoic acids and the keto oxygen of the 11-ketooctadecadienoic acid were derived from water and not from molecular oxygen. The term “oxylipin” was introduced recently (ref. 1) as an encompassing term for oxygenated compounds which are formed from fatty acids by reaction(s) involving at least one step of mono- or dixoygenase-catalyzed oxygenation.     

Formation of the intense flavor compoundtrans-4,5-epoxy-(E)-2-decenal in thermally treated fats

Thermal degradation of several possible precursors of the intense flavor compoundtrans-4,5-epoxy-(E)-2-decenal in model experiments revealed that the odorant is formed in significant yields from 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD) and 9-hydroperoxy-10,12-octadecadienoic acid (9-HPOD). Of these hydroperoxides, arising in equal amounts during autoxidation of linoleic acid, the 9-HPOD was established as the more effective precursor. The key intermediates in the generation of the epoxyaldehyde were found to be 2,4-decadienal, arising from 9-HPOD, and 12,13-epoxy-9-hydroperoxy-10-octadecenoic acid, a degradation product of 13-HPOD. Isolation and characterization of the precursors from a baking margarine confirmed glycerine-bound 9- and 13-HPOD as the intermediates in the formation of the epoxyaldehyde during heating of fats that contain linoleic acid.