Lack of regioselectivity in formation of oxohydroxyoctadecenoic acids from the 9- or 13-hydroperoxide of linoleic acid

Either 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid or 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid was treated with the catalyst, cysteine-FeCl₃, in the presence of oxygen. Oxohydroxyoctadecenoic acids were among the many products formed as a result of hydroperoxide decomposition. A mixture of 9(13)-oxo-13(9)-hydroxy-trans-11(10)-octadecenoic acids (δ-ketols) was produced from either isomeric hydroperoxide. The formation of isomeric δ-ketols from 9-hydroxy-trans-12,13-epoxy-trans-10-octadecenoic acid (epoxyol), a known product of 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid decomposition, implies that the epoxyol is an intermediate. The mechanism was elucidated by the facile conversion of the epoxyol (methyl ester_ to methyl 9(13)-oxo-13(9)-hydroxy-trans-11(10)-octadecenoates with a Lewis acid, BF₃-etherate. Presented at the 14th World Congress, International Society for Fat Research, Brighton, U.K., September 17–22, 1978. The mention of firm names or trade products does not imply that they are endorsed or recomended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

9-cis β-carotene in human plasma and blood cells after ingestion of β-carotene

For 44 wk, thirty male volunteers were given daily either 60 mg of synthesized all-trans β-carotene, a naturally-occurring β-carotene derived fromDunaliella bardawil, or a placebo. Basal levels of 9-cis β-carotene in plasma, platelets, and mononuclear cells were 10, 20 and 25% of those of the all-trans form, respectively. The plasma levels reached a maximum after two weeks of administration and plateaued thereafter in the subjects who took the β-carotene preparations. The all-trans β-carotene level in the subjects given the synthesized all-trans form was almost twice that for theDunaliella preparation. The plasma 9-cis level was found to be higher in the all-trans β-carotene group than in theDunaliella group, despite no intake of the 9-cis form in the all-trans group and the higher intake of the 9-cis form in theDunaliella group. This finding suggests that isomerization of the all-trans form to the 9-cis form may occur in the body either during or after absorption.     

Isolation of isomeric hydroperoxides from the peanut lipoxygenase- linoleic acid reaction

Hydroperoxides were isolated from the peanut lipoxygenase-linoleic acid reaction mixture and were separated as their methyl esters by high performance liquid chromatography. Mass spectrometry and infra-red analysis indicated the isolated hydroperoxides to be 13-hydroperoxy-cis-9,trans- 11-octadecadienoic acid; 13-hydroperoxy-trans- 9,trans- 11-octadeca-dienoic acid; and 9-hydroperoxy-trans-l0,trans- 12- octadecadienoic acid. The percentages of the hydro-peroxides in the reaction mixture were 72.8%, 3.6%, and 23.6% under the conditions used. 1 Paper No. 4973 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607. Paper No. 4973 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607.     

Evidence for [1,5] sigmatropic rearrangements of CLA in heated oils

Linoleic acid was heated at 200°C under helium. Analysis of degradation products by GC on a long polar open tubular capillary column showed the presence of CLA isomers. The identified mono trans CLA isomers were cis-9, trans-11, trans-9, cis-11, trans-10, cis-12, cis-10, trans-12, trans-8, cis-10, and cis-11, trans-13 18:2 acids. Oils containing different levels of linoleic acid (peanut, sesame seed, and safflower seed oils) were also heat treated, resulting in similar CLA distributions. Elution order was confirmed using cis-9, trans-11 and trans-10, cis-12 acid methyl esters standards and their respective configuration isomers (trans-9, cis-11, cis-10, trans-12), obtained after mild selenium-catalyzed isomerization. These results indicated that two conjugated mono trans isomers of 18:2 acid, cis-8, trans-10 and trans-11, cis-13 18:2 were absent from the series, thus strongly suggesting that some constraints were preventing their formation. By heating pure methyl rumenate (cis-9, trans-11 18:2) under similar conditions, isomerization resulted principally in a nearly equimolar mixture of methyl rumenate and trans-8, cis-10 18:2. Similarly, the methyl ester of trans-10, cis-12 18:2 acid was partially transformed into cis-11, trans-13 18:2 acid. Respective geometrical isomers were also formed in trace amounts. A concerted pericyclic isomerization mechanism, a [1,5] sigmatropic rearrangement, is proposed that limits the conjugated system to isomerization from a cis-trans acid to a trans-cis acid, and vice versa. This mechanism is consistent with undetected cis-8, trans-10 and trans-11, cis-13 18:2 isomers in heated oils containing linoleic acid.     

Photo-oxidation of lipids impregnated on the surface of dried seaweed (<Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Ueda). Hydroperoxide distribution

The distribution of hydroperoxide isomers generated by photo-oxidation of natural lipids impregnated on the surface of dried seaweed previously exposed to visible light and without added photosensitizer were studied. The surface of dried seaweed was impregnated with linoleic acid methyl ester, and the sample was divided into two parts. One part was exposed to light from a 100-W tungsten bulb (4500 lux) in a low-temperature room (5°C). The other part was kept in the dark as a control. Positional isomers of the hydroperoxides generated from the impregnated linoleic acid methyl ester were separated individually by HPLC and further identified by MS. The dried seaweed kept in the dark contained four hydroperoxide isomers, namely, 13-hydroperoxy-cis-9, trans-11-octadecadienoate, 13-hydroperoxy-trans-9, trans-11-octadecadienoate, 9-hydroperoxy-trans-10,cis-12-octadecadienoate, and 9-hydroperoxy-trans-10, trans-12-octadecadienoate. For the dried seaweed exposed to light, the oxidized lipids contained not only the same four isomers, but also 12-hydroperoxy-cis-9, trans-13-octadecadienoate and 10-hydroperoxy-trans-8,cis-12-octadecadienoate. When fresh seaweed was dried in the sunlight, the formation of 12-cis,trans- and 10-cis,trans-hydroperoxides of naturally occurring methyl linoleate was verified. Dried seaweed was then impregnated with eicosapentaenoic acid ethyl ester and exposed to light. Light exposure also generated certain hydroperoxide isomers attributable to singlet oxygen oxidation, namely, 6-hydroperoxy-trans-4,cis-8, cis-11,cis-14,cis-17-ethyl and 17-hydroperoxy-cis-5,cis-8,cis-11, cis-14,trans-18-ethyl eicosapentaenoate. When dried sea-weed without any impregnated lipids was exposed to the light for 24 h in a cold room (5°C), characteristic isomers, including both the 20-carbon FA isomers 6-OOH and 17-OOH as well as the 18-carbon FA isomers 10-OOH and 12-OOH, were detected in the light-exposed sample but were not found in the control. These results clearly show that singlet oxygen oxidation of lipids occurred in the seaweed exposed to light. We concluded that this lipid oxidation was catalyzed by chlorophyll as a photosensitizer in seaweed.     

Radical addition of linoleic hydroperoxides to α-tocopherol or the analogous hydroxychroman

Either linoleic acid hydroperoxide (LOOH) or methyl linoleate hydroperoxide react anaerobically with either α-tocopherol (TOH) or its model compound-2,2,5,7,8-pentamethyl-6-hydroxychroman (COH)-to form principally an addition compound of the two reactants. The reaction can be catalyzed either by 1.28 X 10⁻⁵ M Fe(III) or by proflavin (0.01%) sensitized by visible light. The presence of air in the reaction terminates the addition, and quinones become the major products from TOH or its model compound. The addition compound synthesized from COH and LOOH (a 4.9∶1 ratio of 13-hydroperoxy-cis-9,trans-12-octadecadienoic acid and 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid) was used to solve structural details of the bridging function. Three isomers of the addition compound (methyl esterified) were isolated and identified as methyl 11-(2,2,5,7,8-pentamethyl-6-oxychroman)-cis-12,13-epoxy-trans-9-octadecenoate; methyl 11-(2,2,5,7,8-pentamethyl-6-oxychroman)-trans-12,13-epoxy-trans-9-octadecenoate; and methyl 11-(2,2,5,7,8-pentamethyl-6-oxychroman)-cis-9,10-epoxy-trans-12-octadecenoate in order of decreasing abundance. The mechanism appears to be free radical addition brought about by the catalytic formation of alkoxy radicals from the hydroperoxide and chromanoxy radicals from TOH or its model. Presented at the AOCS Meeting, Atlantic City, N.J. October 1971. N. Market. Nutr. Res. Div., ARS, USDA.     

Homolytic decomposition of linoleic acid hydroperoxide: Identification of fatty acid products

An isomeric mixture of linoleic acid hydroperoxides, 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid (79%) and 9-hydroperoxy-cis-12,trans-10-octadecadienoic acid (21%), was decomposed homolytically by Fe(II) in an ethanol-water solution. In one series of experiments, the hydroperoxides were decomposed by catalytic concentrations of Fe(II). The 10⁻⁵ M Fe(III) used to initiate the decomposition was kept reduced as Fe(II) by a high concentration of cysteine added to the reaction in molar excess of the hydroperoxides. Nine different monomeric (no detectable dimeric) fatty acids were identified from the reaction. Analyses of these fatty acids revealed that they were mixtures of positional isomers identified as follows: (I) 13-oxo-trans,trans-(andcis,trans-) 9,11-octadecadienoic and 9-oxo-trans,trans- (andcis,trans-) 10,12-octadecadienoic acids; (II) 13-oxo-trans-9,10-epoxy-trans-11-octadecenoic and 9-oxo-trans-12, 13-epoxy-trans-10-octadecenoic acids; (III) 13-oxo-cis-9,10-epoxy-trans-11-octadecenoic and 9-oxo-cis-12, 13-epoxy-trans-10-octadecenoic acids; (IV) 13-hydroxy-9,11-octadecadienoic and 9-hydroxy-10,12-octadecadienoic acids; (V) 11-hydroxy-trans-12, 13-epoxy-cis-9-octadecenoic and 11-hydroxy-trans-9, 10-epoxy-cis-12-octadecenoic acids; (VI) 11-hydroxy-trans-12, 13-epoxy-trans-9-octadecenoic and 11-hydroxy-trans-9,10-epoxy-trans-12-octadecenoic acids; (VII) 13-oxo-9-hydroxy-trans-10-octadecenoic acids; (VIII) isomeric mixtures of 9, 12, 13-dihydroxyethoxy-trans-10-octadecenoic and 9, 10, 13-dihydroxyethoxy-trans-11-octadecenoic acids; and (IX) 9, 12, 13-trihydroxy-trans-10-octadecenoic and 9, 10, 13-trihydroxy-trans-11-octadecenoic acids. In another experiment, equimolar amounts of Fe(II) and hydroperoxide were reacted in the absence of cysteine. A large proportion of dimeric fatty acids and a smaller amount of monomeric fatty acids resulted. The monomeric fatty acids were examined by gas liquid chromatography-mass spectroscopy. Spectra indicated that the monomers were largely similar to those produced by the Fe(III)-cysteine reaction. Presented in part at the American Chemical Society Meeting, Los Angeles, March 1974. ARS, USDA.     

Oxidation of lipids: III. Oxidation of methyl linoleate in solution

The effects of oxygen pressure, substrate concentration and solvent on the rate and products of oxidation of methyl linoleate were studied at 50 C with azobisisobutyronitrile as a radical initiator. The absolute and quantitative numbers for oxygen uptake, substrate disappearance, and formation of conjugated diene and hydroperoxides were measured. Under the present conditions, 4 conjugated diene hydroperoxides, 13-hydroperoxy-9-cis, 11-trans-(2a), 13-hydroperoxy-9-trans, 11-trans-(3a), 9-hydroperoxy-10-trans, 12-cis-(4a), and 9-hydroperoxy-10-trans, 12-trans-(5a) octadecadienoic acid methyl esters, were formed almost quantitatively. The rate of oxidation decreased with decreasing oxygen pressure. However, the ratio ofcis,trans totrans,trans hydroperoxides, (2a+4a)/(3a+5a), was independent of oxygen pressure, and this ratio increased with increasing methyl linoleate concentration, as found recently by Porter. Further, the rate of oxidation and the ratio ofcis,trans/trans,trans hydroperoxides were dependent on solvent and increased with an increase in dielectric constant of solvent. A mechanism of methyl linoleate oxidation consistent with these results is discussed. Presented at the 15th Symposium on Oxidation Reactions, Nagoya, October 1981.     

Effects of dietary 9-trans, 12-trans linoleate on arachidonic acid metabolism in rat platelets

In order to determine the minimal amount of dietary 9-trans,12-trans-linoleate which can decrease endoperoxide metabolites synthesized and their precursor in rat platelets, graded amounts (0, 0.1, 0.5, 1.0, 2.5%) of thetrans-linoleate were fed to rats with a constant amount of all-cis-linoleate (2.5%) for 12 weeks. Arachidonic acid levels in platelet phospholipids of groups receiving thetrans-linoleate at 2.5 and 1.0% were significantly (p<0.01) lower than that of the control receiving notrans-linoleate. Concentrations of TXB₂ and PGF_2α in sera of the group receiving 2.5%trans-linoleate were significantly (p<0.05) lower than those of the control; however, there was no difference between the group receiving 1.0%trans-linoleate and the control. To determine whether the difference in serum concentrations of endoperoxide metabolites could be manifested if rats were fed for longer period of time, 2 groups of rats were again fed diets containing 0 and 1.0%trans-linoleate, respectively, for 16 weeks. Arachidonic acid in platelet phospholipids of the group receiving thetrans-linoleate was again significantly (p<0.01) lower than that of the control group. Concentrations of TXB₂ and PGF_2α, and 12-hydroxyeicosatetraenoic acid formed in platelets, were smaller in the group receivingtrans-linoleate than the control group; however, the difference was not statistically significant. These results indicated that all-trans-linoleate can reduce arachidonic acid metabolites formed in rat platelets when its dietary level is equal to or exceeds the level of all-cis-linoeate.     

High-performance liquid chromatography and spectroscopic studies on fish oil oxidation products extracted from frozen atlantic mackerel

The formation of stable hydroxy derivatives from hydroperoxides produced during the oxidation of linoleic acid methyl ester and fish oil were studied by reverse-phase high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The oxidation products identified were mixtures of four isomeric hydroxy derivatives: 13-hydroxy-9-cis,11-trans-octadecadienoic, 13-hydroxy-9-trans,11-trans-octadecadienoic, 9-hydroxy-10-trans,12-cis-octadecadienoic, and 9-hydroxy-10-trans,12-trans-octadecadienoic acids. The presence of hydroxy compounds was confirmed by ¹³C NMR, which gave rise to a hydroxy carbon peak at 87 ppm, and by GC-MS, which showed three peaks corresponding to isomeric mixtures of trimethylsilyl ethers of the oxidized linoleic acid methyl ester. The mass spectra scans of the three peaks showed that they represent isomers of molecular weight 382 and are consistent with the molecular formula C₂₂H₄₂O₃Si. In oil extracted from stored frozen mackerel, 13-hydroxy-9-cis,11-trans-octadecadienoic acid was more prominent compared to the model lipid systems. HPLC offered a sensitive means of detection of hydroxy compounds produced both in the initiation and latter stages of oxidation. The effect of antioxidants added to the fish mince prior to storage can also be monitored by HPLC. Thus, the monitoring of lipid oxidation hydroxy derivatives by HPLC is of practical value in the efficient processing and quality control of fish, fish oils, and other fatty foodstuffs in order to enhance the acceptability, nutritional, and safety aspects.     

Lipase-catalyzed fractionation of conjugated linoleic acid isomers

The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11–18∶2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11–18∶2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9,trans-11–18∶2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11–18∶2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11–18∶2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12–18∶2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11–18∶2, and a corresponding CLA fraction depleted of this isomer.     

Isolation of a natural isomer of linoleic acid from a seed oil1,2

trans-10-trans-12-Octadecadienoic acid was found to be a component of the glyceride oil of the seeds ofChilopsis linearis (Cav.) Sweet. It was isolated by fractional crystallization of the acids at low temperatures. Identification was made by absorption spectra, by the adduct with maleci anhydride, and by identification of degralative products. The conjugated triene of the oil was also isolated and identified astrans-9-trans-11-cis-13octadecatrienoic acid. in two samples of seed, the diene acid constituted about 9% of the oil and the triene acid 18% and 25%. Presented at the AOCS meeting in Toronto, Canada, 1962. Issued as NRC No. 7592.     

Oxidative degradation kinetics of lycopene, lutein, and 9-cis and all-trans β-carotene

The thermal and oxidative degradation of carotenoids was studied in an oil model system to determine their relative stabilities and the major β-carotene isomers formed during the reaction. All-trans β-carotene, 9-cis β-carotene, lycopene, and lutein were heated in safflower seed oil at 75, 85, and 95°C for 24, 12, and 5 h, respectively. The major isomers formed during heating of β-carotene were 13-cis, 9-cis, and an unidentified cis isomer. The degradation kinetics for the carotenoids followed a first-order kinetic model. The rates of degradation were as follows: lycopene>all-trans β-carotene≈9-cis β-carotene>lutein. The values for the thermodynamic parameters indicate that a kinetic compensation effect exists between all of the carotenoids. These data suggest that lycopene was most susceptible to degradation and lutein had the greatest stability in the model system of the carotenoids tested. Furthermore, there was no significant difference in the rates of degradation for 9-cis and all-trans β-carotene under the experimental conditions.     

Stereochemistry of the hydroperoxides formed during autoxidation of CLA methyl ester in the presence of α-tocopherol

The initial steps in the autoxidation of CLA methyl ester are poorly understood. The aim of this study was to determine the stereochemistry of the hydroperoxides formed during autoxidation of CLA methyl ester in the presence of a good hydrogen atom donor. For this purpose, 9-cis, 11-trans CLA methyl ester was autoxidized in the presence of α-tocopherol under atmospheric oxygen at 40°C in the dark. The CLA methyl ester hydroperoxides were isolated, reduced to the corresponding hydroxy derivatives, and separated by HPLC. The stereochemistry of seven hydroxy-CLA methyl esters was investigated. The position of the hydroxy group was determined by GC-MS. The geometry as well as the position of the double bonds in the alkyl chain was determined by NMR. In addition, the ¹³C NMR spectra of six hydroxy-CLA methyl esters were assigned using COSY, gradient heteronuclear multiple bond correlation, gradient heteronuclear single quantum correlation, and total correlation spectroscopy experiments. The autoxidation of 9-cis, 11-trans CLA methyl ester in the presence of a good hydrogen atom donor is stereoselective in favor of one geometric isomer, namely the 13-(R,S)-hydroperoxy-9-cis, 11-trans-octadecadienoic acid methyl ester. Three types of conjugated diene hydroperoxides are formed as primary hydroperoxides: trans,trans hydroperoxides (12-OOH-8t,10t and 9-OOH-10t,12t), a cis,trans hydroperoxide with the trans double bond adjacent to the hydroperoxide-bearing carbon atom (13-OOH-9c,11t), and a new type of cis,trans lipid hydroperoxide with the cis double bond adjacent to the hydroperoxide-bearing carbon atom (8-OOH-9c,11t). In addition, three nonkinetic hydroperoxides (13-OOH-9t,11t, 8-OOH-9t,11t, and 9-OOH-10t,12c) are formed. This study supports the theory that CLA methyl ester autoxidizes at least partly through an autocatalytic free radical reaction. The complexity of the hydroperoxide mixture is due to formation of two different pentadienyl radicals. Moreover, the stereoslectivity in favor of one geometric isomer can be explained by the selectivity of the two previous steps: the preferential formation of a W-conformer of the pentadienyl radical over the Z-conformer, and regioselectivity of the oxygen addition to the pentadienyl radical.     

13C nuclear magnetic resonance spectral confirmation of Δ6-and Δ7-trans-18:1 fatty acid methyl ester positional isomers

Trans octadecenoic acid methyl ester isomers were obtained from a partially hydrognated soybean oil and isolated by silver-ion high-performance liquid chromatography. Recently, the double-bond positions for nine individual trans octadecenoic acid positional isomers (Δ8 through Δ16) were confirmed by gas chromatography-electron ionization mass spectrometry after derivatization to 2-alkenyl-4,4-dimethyloxazoline. In this communication, the presence of two additional trans-18:1 fatty acid methyl ester positional isomers (Δ6 and Δ7) in the same mixture is confirmed by ¹³C nuclear magnetic resonance spectroscopy. The identity of the Δ5-trans-18:1 fatty acid methyl ester positional isomer is inferred. Summer student researcher.     

A simple method for the preparation of pure 9-d-hydroperoxide of linoleic acid and methyl linoleate based on the positional specificity of lipoxygenase in tomato fruit

Incubation of linoleic acid with crude homogenate of tomato fruit gave a high yield (69%) of linoleic acid hydroperoxides with a ratio of 9- to 13-hydroperoxide isomers of 96∶4. After chromatography of the products, as free acids or methyl esters, hydroperoxides with 9- to 13-isomeric ratios of >99∶1 were obtained. The major product was characterized as 9-d-hydroperoxy-octadeca-trans-10,cis-12-dienoic acid. The results demonstrate the positional specificity of lipoxygenase from tomato fruit.     

Positional and geometrical isomers of linoleic acid in partially hydrogenated oils

The geometrical and positional isomers of linoleic acid of a partially hydrogenated canola oil-based spread were isolated and identified. Through partial hydrazine reduction and mass spectral studies,cis-9,trans-13 octadecadienoic acid was identified as the major isomer. Other quantitatively important isomers characterized werecis-9,trans-12;trans-9,cis-12 andcis-9,cis-15. These four were also the major isomers in margarine based on common vegetable oils. A number of minor isomers were detected and some structures identified weretrans-9,trans-12;trans-8,cis-12;trans-8,cis-13;cis-8,cis-13;trans-9,cis-15;trans-10,cis-15 andcis-9,cis-13. The proportions of the various isomers are given for some margarines in the Canadian retail market. The amounts oftrans-9,trans-12 isomer in Canadian margarines were generally below 0.5% of the total fatty acids.     

Infrared studies on the isomers of kamlolenic acid

Summary Thecis-trans and positional configuration of α-and β-kamlolenic acids have been investigated. Infrared data of the two isomers, their acetyl derivatives and maleic anhydride adducts, and the permanganate oxidation products of the addition compounds, together with the selective epoxidation of the exocyclic double bond of the maleinated derivatives, have been used to confirm the structure of the three conjugated double bonds in α-kamlolenic acid ascis-9-trans 11-trans 13. and β-kamlolenic acid astrans 9-trans 11-trans 13.     

Hydroperoxide formation during autoxidation of conjugated linoleic acid methyl ester

The aim of this study was to investigate whether hydroperoxides are formed in the autoxidation of conjugated linoleic acid (CLA) methyl ester both in the presence and absence of α‐tocopherol. The existence of hydroperoxide protons was confirmed by D₂O exchange and by chemoselective reduction of the hydroperoxide groups into hydroxyl groups using NaBH₄. These experiments were followed by nuclear magnetic resonance (NMR) spectroscopy. The ¹³C and ¹HNMR spectra of a mixture of 9‐hydroper‐oxy‐10‐trans,12‐cis‐octadecadienoic acid methyl ester (9‐OOH) and 13‐hydroperoxy‐9‐cis, 11‐trans‐octadecadienoic acid methyl ester (13‐OOH), which are formed during the autoxidation of methyl linoleate, were studied in detail to allow the comparison between the two linoleate hydroperoxides and the CLA methyl ester hydroperoxides. The ¹³CNMR spectra of samples enriched with one of the two linoleate hydroperoxide isomers were assigned using 2D NMR techniques, namely Correlated Spectroscopy (COSY), gradient Heteronuclear Multiple Bond Correlation (gHMBC), and gradient Heteronuclear Single Quantum Correlation (gHSQC). The ¹³C and ¹H NMR experiments performed in this study show that hydroperoxides are formed during the autoxidation of CLA methyl ester both in the presence and absence of α‐tocopherol and that the major isomers of CLA methyl ester hydroperoxides have a conjugated monohydroperoxydiene structure similar to that in linoleate hydroperoxides.     

Gas chromatographic analysis of diels-alder adducts of geometrical and positional isomers of conjugated linoleic acid

This research demonstrates the gas chromatographic analysis of the 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) adducts derived from standards of cis,trans-9,11-octadecadienoic acid, trans,trans-9,11-octadecadienoic acid, and cis,cis-9,11-octadecadienoic acid. Methyl cis,trans-9,11-octadecadienoate and methyl trans,trans-9,11-octadecadienoate formed Diels-Alder addition products with MTAD to produce adducts with similar mass spectral fragmentation patterns but different retention times determined by gas chromatography/ mass spectrometry. Methyl cis,cis-9,11-octadecadienoate reacted slowly and produced two adducts with similar fragmentation patterns and different retention times. These results were comparable to those reported for an analogous series of conjugated hexadienes. Based on hexadiene reactions, methyl cis,trans-9,11-octadecadienoate produced a trans adduct as a major product while methyl trans,trans-9,11-octadecadienoate formed a cis adduct. Methyl cis,cis-9,11-octadecadienoate reacted slowly under the conditions used leaving mostly unreacted material. Of the adducts observed from this isomer, a major trans adduct and a minor cis adduct were formed.