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.     

A soy extract catalyzes formation of 9-oxo-trans-12,13-epoxy-trans-10-octadecenoic acid from 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid

In the presence of oxygen, a crude soy extract converted 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid into numerous products, from which 9-oxo-trans-12,13-epoxy-trans-10-octadecenoic acid was isolated. Additionally, the soy extract oxidized linoleic acid to the oxo-epoxyoctadecenoic acid, presumably via a sequential reaction involving lipoxygenase oxidation of linoleic acid followed by degradation of the resultant linoleic acid hydroperoxide. However, the linoleic acid substrate yielded two isomeric linoleic acid hydroperoxides and because of this, two isomeric oxoepoxyoctadecenoic acids. Presented in part at the 13th Congress, International Society for Fat Research, Marseilles, France, August 30–September 4, 1976.     

Positional specificity of γ-ketol formation from linoleic acid hydroperoxides by a corn germ enzyme

We have shown unequivocally that the positional specificity of γ-ketol formation by a corn germ enzyme was different from that observed previously by others with an alfalfa seedling enzyme. When the pure positional isomers of linoleic acid hydroperoxide served as substrates, the corn germ enzyme formed one of two γ-ketols: 12-oxo-9-hydroxy-trans-10-octadecenoic acid from 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid (99+% pure) and 10-oxo-13-hydroxy-trans-11-octadecenoic acid from 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid (96% pure). Also isolated from these reactions was one of two α-ketols commonly found as a result of catalysis by linoleic acid hydroperoxide isomerase: 12-oxo-13-hydroxy-cis-9-octadecenoic acid from the 13-hydroperoxide and 10-oxo-9-hydroxy-cis-12-octadecenoic acid from the 9-hydroperoxide. Evidence is offered that γ-ketol formation is catalyzed by linoleic acid hydroperoxide isomerase, the same enzyme responsible for α-ketol production. Presented at the AOCS Spring Meeting, Dallas, Texas, April, 1975.     

Formation oftrans-12,13-epoxy-9-hydroperoxy-trans-10-octadecenoic acid from 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid catalyzed by either a soybean extract or cysteine-FeC13

A soybean extract or an ethanolic solution of cysteine and ferric chloride catalyzed the conversion of 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid to numerous products among which wastrans-12,13-epoxy-9-hydroperoxy-trans-10-octadecenoic acid. When this fatty acid was treated further with the cysteine-ferric chloride solution, 9-hydroxy-12,13-epoxy-10-octadecenoic and 9-oxo-12,13-epoxy-10-octadecenoic acids were formed. Thus,trans-12,13-epoxy-9-hydroperoxy-trans-10-octadecenoic acid probably is an intermediate in the formation of the latter two compounds. Additionally, theerythro andthreo isomers oftrans-12,13-epoxy-11-hydroperoxy-cis-9-octadecenoic acid tenatatively were identified as products. Presented in part at the 13th World Congress, International Society for Fat Research, Marseilles, France, August 30-September 4, 1976, and the AOCS Meeting, Chicago, September 1976.     

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.     

Oxygenated fatty acids of oil from sunflower seeds after prolonged storage

Chemical analysis of a number of sunflower (Helianthus annuus) seed oil samples revealed a low and variable percentage of hydrogen bromide-reactive material. To characterize the compounds responsible for this reactivity, oil was extracted from selected introductions from Uruguay, Turkey, and Yugoslavia that had been subjected to prolonged storage. Two epoxy fatty acids and two conjugated dienolic acids were isolated from the methyl esters derived from these sunflower seed oils by using a combination of column chromatography and countercurrent distribution. The epoxy acids arecis-9,10-epoxystearic acid (0.5%) andcis-9,10-epoxy-cis-12-octadecenoic (coronaric) acid (2.2%). Characterization of the dienols revealed that they are 9-hydroxy-trans-10,cis-12-octadecadienoic acid (1.2%) and 13-hydroxy-cis-9,trans-11-octadecadienoic acid (1.3%). Fresher seed of some of these introductions contained less of the oxygenated components. Oil from recently produced seed of selected high-oil Russian sunflower varieties, including some currently grown in the United States, contained no more than trace amounts of oxygenated acids. Though the relative contributions of genetic and environmental factors toward genesis of oxygenated acids are not established, increase of those acids in some sunflower lines as a result of storage has been demonstrated. Presented at the AOCS-AACC Joint Meeting, Washington, D. C., April 1968. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Epoxy oils from plant seeds

Epoxy acids have been reported in seed oils from more than 60 species in 12 plant families. The discovery of 9,10-epoxyoctadec-12-ynoic and 9,10-epoxy-trans-3,cis-12-octadecadienoic acids brings to six the number of natural epoxy acids now known to occur in seed oils. These latest epoxy acids and 15,16-epoxy-cis-9,cis-12-octadecadienoic acid have been found in only one species each and at levels lower than 5% of the oil. Coronaric (9,10-epoxy-cis-12-octadecenoic) acid and 9,10-epoxystearic acid have been encountered in several seed oils, the first as much as 15% of the oil and the latter in only small amounts. Vernolic (12,13-epoxy-cis-9-octadecenoic) acid, which has been identified in numerous oils, is the only epoxy acid known to occur in seed oils at levels above 15%, and it may constitute as much as 75%. On the basis of data available to date,Vernonia anthelmintica appears to have the best potential for commercial production of an epoxy oil. Although one improved line has been selected, continued improvement is needed. Formation of epoxy acids in oilseeds during storage after harvest has been demonstrated, and may be partly responsible for the small amounts of epoxide detected in oils from a wide variety of seeds. Presented at the AOCS Meeting, New Orleans, April 1970. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Stereospecificity of linoleic acid hydroperoxide isomerase from corn germ

Linoleic acid hydroperoxide isomerase from corn germ inverted the stereoconfiguration of its substrate. 9-D(S)-Hydroperoxy-trans-10,cis-12-octadecadienoic acid was converted to 10-oxo-9-L(R)-hydroxy-cis-12-octadecenoic acid. Presumably, the H₂O solvent of OH⁻ acted as a nucleophile. In the presence of another nucleophile, linoleate, the 9-D(S)-hydroperoxide was transformed into 9-L(R)-linoleoyloxy-10-oxo-cis-12-octadecenoic acid. The substitution of nucleophiles from the incubation solution and the inversion of stereoconfiguration at carbon-9 are consistent with a bimolecular nucleophilic substitution (S_N2) mechanism. 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.     

Cysteine adds to lipid hydroperoxide

Cysteine reacts with linoleic acid hydroperoxide to yield several products, some of which were identified as fatty acid-cysteine adducts. The addition was catalyzed by ferric chloride (10⁻⁵ M) by initiating free radical reactions. When isomerically pure 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid and cysteine were reacted in 80% ethanol under N₂, the major adducts were 9-S-cysteine-13-hydroxy-10-ethoxy-trans-11-octadecenoic acid (I) and 9-S-cysteine-10,13-dihydroxy-trans-11-octadecenoic acid (II). When the reaction included both isomers of the hydroperoxide (13-and 9-hydroperoxide) and air, an adduct of 9-oxononanoic acid and cysteine also was isolated. Additional experiments gave information about possible mechanisms of I and II formation.     

OxygenatedTrans-3-olefinic acids in aStenachaenium seed oil

Interesting differences were found in oils from two samples ofStenachaenium macrocephalum (Compositae) seed with dissimilar storage histories. One contained significant amounts of epoxy acids (6.5%) and hydroxy conjugated dienoic acids (5.6%), but the other contained no more than 1% of these oxygenated acids. Characterization of components in the former oil established that the principal epoxy acid (4.0%) is the previously unknowncis-9, 10-epoxy-trans-3,cis-12-octadecadienoic acid. The conjugated dienols include two additional new acids with Δ3 unsaturation (2.5%): 9-hydroxy-trans-3,-trans-10,cis-12-octadecatrienoic and 13-hydroxy-trans-3,cis-9,trans-11-octadecatrienoic acoids. The nonoxygenated acids, except for the large amount (40%) oftrans-3,cis-9,cis-12-octadecatrienoic, are those that commonly occur in seed oils. Presented at the AOCS Meeting, San Francisco, April 1969. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Therans-3-enoic acids ofAster alpinus andArctium minus seed oils

Thetrans-3-enoic acids ofAster alpinus (dwarf aster, rock aster) andArctium minus (burdock) seed oils have been isolated and characterized.Arctium seed oil containstrans-3,cis-9,cis-12-octadecatrienoic acid (9.9%), andAster oil containstrans-3-hexadecenoic (7.1%),rans-3-octadecenoic (1.9%),trans-3,cis-9-octadecadienoic (3.0%),a ndtrans-3,cis-9,cis-12-octadecatrienoic (13.7%) acids.Aster oil also has an epoxy acid as a minor constituent (ca. 2.0%), which has been identified ascis-9,10-epoxy-cis-12-octadecenoic acid.     

Addition ofN-acetylcysteine to linoleic acid hydroperoxide

Catalyzed by 10⁻⁵ M ionic iron in 80% ethanol,N-acetylcysteine added to linoleic acid hydroperoxide, forming a thiobond. Reaction of a specific isomer of the hydroperoxide, 13-hydroperoxy-trans-11,cis-9-octadecadienoic acid, andN-acetylcysteine, forms a number of products, of which two were identified as addition compounds. One addition product was 9-S-(N-acetylcysteine)-13-hydroxy-10-ethoxy-trans-11-octadecenoic acid, and the other was 9-S-(N-acetylcysteine)-10,13-dihydroxy-trans-11-octadecenoic acid. Presented at the AOCS 49th Annual Fall Meeting, Cincinnati, September–October, 1975.     

cis-5,cis-9,cis-12-octadecatrienoic and some unusual oxygenated acids inXeranthemum annuum seed oil

Seed oil ofXeranthemum annuum (family Compositae) contains a number of unusual fatty acids in addition to palmitic, stearic, oleic, linoleic and linolenic. These acids includecis-5,cis-9,cis-12-octadecatrienoic, 5%;cis-9-l,10-l-epoxyoctadecanoic, 3%;cis-9-l,10-l-epoxy-cis-12-octadecenoic (coronaric), 8%; andcis-12-d,13-d-epoxy-cis-9-octadecenoic (vernolic), 2%; as well as a mixture of two hydroxy acids, 11%. The absolute configurations of the two 9,10-epoxy acids are established for the first time. Presented in part at the AOCS Meeting in Cincinnati, October 1965. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Enzymatic oxidation of linolenic acid in aqueous wheat flour suspensions

Linolenic acid oxidation by the enzyme lipoxygenase in an aqueous wheat flour suspension does not lead to accumulation of linolenic acid hydroperoxides but immediately to secondary oxidation products. The 3 most important products among these were identified as 9-hydroxy-trans-10,cis-12,cis-15-octadecatrienoic acid, 9-hydroxy-10-oxo,cis-12,cis-15-octadecadienoic acid, and 9,12,13-trihydroxy-trans-10,cis-15-octadecadienoic acid.     

Transformation of fatty acid hydroperoxides by alkali and characterization of products

It has previously been determined that (13S,9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid was mainly converted into (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid by 5 N KHO with preservation of the stereochemistry of the reactant [Simpson, T.D., and Gardner, H.W. (1993)Lipids 28, 325–330]. In addition, about 20–25% of the reactant was converted into several unknown by-products. In the present work it was confirmed that the stereochemistry was conserved during the hydroperoxy-diene to hydroxydiene transformation, but also, novel by-products were identified. It was found that after only 40 min reaction (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid accumulated to as much as 7% of the total. Later, (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid began to disappear, and several other compounds continued to increase in yield. Two of these compounds, 2-butyl-3,5-tetradecadienedioic acid and 2-butyl-4-hydroxy-5-tetradecenedioic acid, were shown to originate from (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid, and they accumulated up to 2–3% each after 4 to 6 h. Some other lesser products included 11-hydroxy-9,12-heptadecadienoic acid, 3-hydroxy-4-tridecenedioic acid, 13-oxo-9,11-octadecadienoic acid and 12,13-epoxy-11-hydroxy-9-octadecenoic acid. Except for the latter two, most or all of the compounds could have originated from Favorskii rearrangement of the early product, (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid, through a cyclopropanone intermediate.     

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.     

A new reaction of unsaturated fatty acid hydroperoxides: Formation of 11-hydroxy-12,13-epoxy-9-octadecenoic acid from 13-hydroperoxy-9,11-octadecadienoic acid

One of the main compounds formed from 13L-hydroperoxy-9cis,11trans-octadecadienoic acid anaerobically at 100 C in aqueous ethanol was found to bethreo-11-hydroxy-12,13-epoxy-9-octadecenoic acid. The major part (ca. 90%) of this compound was formed from the fatty acid hydroperoxide in a reaction involvingcis-addition to the Δ¹¹ double bond of the proximally linked hydroperoxide oxygen and hydroxyl ion or hydroxyl radical from the solvent. A small part (ca. 10%) was formed bycis-addition of the two hydroperoxide oxygens to the Δ¹¹ double bond. 11-Hydroxy-12,13-epoxy-9-octadecenoic acid and its isomer, tentatively identified as 11-hydroxy-9,10-epoxy-12-octadecenoic acid, also were isolated from a sample of autoxidized linoleic acid.     

Fluorescent pigments by covalent binding of lipid peroxidation by-products to protein and amino acids

The fluorescent products formed on reaction of 12-oxo-cis-9-octadecenoic acid (12-keto-oleic acid) with about 20 different amino acids, polylysine and bovine serum albumin (BSA) were studied. Besides glycine, only the basic amino acids histidine, lysine and arginine gave products with strong fluorescence. N-Acetylation of amino acids greatly reduced the fluorescence of their reaction products. The formation of fluorescent products was inhibited strongly by SH-amino acids such as N-acetyl-cysteine and glutathione. Polyacrylamide gel electrophoresis showed that BSA treated with 12-keto-oleic acid was more acidic than untreated or ricinoleic acid-treated BSA, indicating that basic amino acid residues in BSA were modified by reaction with the keto fatty acid. None of the structural analogs of 12-keto-oleic acid tested–12-oxo-trans-10-octadecenoic acid, 12-oxo-octadecanoic acid, 12-hydroxy-cis-9-octadecenoic acid (ricinoleic acid),cis-9-octadecenoic acid (oleic acid) and linoleic acid—reacted with glycine to give a fluorescent product. The fluorescent products formed on reaction of 12-keto-oleic acid methyl ester with benzyl amine and glycine methyl ester were shown to be 8-(N-substituted-4,5-dihydro-4-oxo-5-hexyl-5-hydroxy-2-pyrrolyl) octanoic acid methyl esters. The fluorescence properties of these compounds were attributed to the chromophobic system NC=CC=O which contains 6π electrons. This investigation contributes to insight of the mechanism of formation of fluorescent pigments, probably by a similar reaction of other compounds of the β,γ-unsaturated carbonyl type.     

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.     

Dimorphotheca sinuata lipoxygenase: Formation of 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid from linoleic acid

Lipoxygenase (EC 1.13.1.13) from the seed ofDimorphotheca sinuata oxidized linoleic acid to predominantly 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid. When the reaction proceeded at pH 6.9, the 13-hydroperoxide was the only isomer detected; but at pH 5.1, the 13-isomer was 92% of the total, the remaining 8% being the 9-hydroperoxide. At both pH's small amounts of hydroxyoctadecadienoic acid accumulated during the reaction. This acid from the pH 6.9 reaction was analyzed as 13-hydroxy-cis,trans-octadecadienoic. The postulate advanced by many workers that dimorphecolic acid, 9-D-hydroxy-trans-10,trans-12-octadecadienoic acid, is biosynthesized via a lipoxygenase product was not proved. Although the product specificity ofD. sinuata lipoxygenase is like that of lipoxygenase type 1 from soybeans, its inactivity at pH 9 demonstrated that it is a novel enzyme. ARS, USDA.