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.     

Calcium activation of peanut lipoxygenase

Peanut lipoxygenase isozyme 1 (pH optimum, 8.3) was strongly activated by 0.5–1.0 mM Ca⁺⁺, and the rate of activation was maximum when the ratio of substrate to Ca⁺⁺ was ca. 2∶1. Peanut lipoxygenase isozymes 2 and 3 (pH optima, 6.2) were activated by calcium but did not have an optimum level of activity. Calcium differentially activated peanut lipoxygenase causing the rate of pentane production to increase much more rapidly than the rate of oxygen consumed by the enzyme reaction. At pH 6.2, in the absence of calcium, the percentages of the hydroperoxide isomers produced by peanut lipoxygenase were 74.9% 13-hydroperoxycis-9,trans-11-octadecadienoic acid (13 LOOHcis-trans), 2.6% 13-hydroperoxytrans-9,trans-11-octadecadienoic acid (13 LOOHtrans-trans) and 22.5% 9-hydroperoxy 10, 12-octadecadienoic acid (9 LOOH). The presence of 1 mM Ca⁺⁺ at pH 6.2 did not significantly affect the percentage distribution of the hydroperoxides produced. However, at pH 8.3, the percentage distribution of hydroperoxides produced was 45.2% 13 LOOHcis-trans, 10.9% 13 LOOHtrans-trans and 43.9% 9 LOOH in the absence of Ca⁺⁺ and 57.0% 13 LOOHcis-trans, 8.0% 13 LOOHtrans-trans and 35.0% 9 LOOH in the presence of 1 mM Ca⁺⁺. Paper No. 5110 of the Journal of the North Carolina Agricultural Experiment Station, Raleight, NC 27607.     

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.     

Isolation of a pure isomer of linoleic acid hydroperoxide

A mixture of positional isomers of linoleic acid hydroperoxide was produced from the oxidation of linoleic acid by lipoxygenase from corn or soybean. Chromatography on a column of silicic acid separated 13-hydroperoxy-11,9-octadecadienoic acid in 99+% purity from the mixture obtained by soybean lipoxygenase oxidation of linoleic acid. Attempts at isolation of pure 9-hydroperoxy-10,12-octadecadienoic acid from hydroperoxides obtained by corn lipoxygenase oxygenation of linoleic acid were partially successful with isolation of the 9-hydroperoxide in 97% purity.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Formation of γ-ketols from 13- and 9-hydroperoxides of linolenic acid by flaxseed hydroperoxide isomerase

A reexamination of the flaxseed hydroperoxide isomerase reaction showed that a minor enzymic product (ca. 5%), identified as a γ-ketol, was present. The substrates were the 13- or 9-hydroperoxides of linolenic acid, which were converted to 9-hydroxy-12-oxo-cis-15-trans-11-octadecadienoic acid, respectively. These compounds were formed in addition to the major products reported earlier: a 12,13-α-ketol and 12-oxo-cis-10,15-phytodienoic acid from the 13-isomer, and a 9,10-α-ketol from the 9-isomer.     

Conjugated linoleic acid production from linoleic acid by lactic acid bacteria

After screening 14 genera of lactic acid bacteria, Lactobacillus plantarum AKU 1009a was selected as a potential strain for CLA production from linoleic acid. Washed cells of L. plantarum with high levels of CLA production were obtained by cultivation in a nutrient medium with 0.06% (wt/vol) linoleic acid (cis-9,cis-12-octadecadienoic acid). Under the optimal reaction conditions with the free form of linoleic acid as the substrate, washed cells of L. plantarum produced 40 mg CLA/mL reaction mixture (33% molar yield) from 12% (wt/vol) linoleic acid in 108 h. The resulting CLA was a mixture of two CLA isomers, cis-9,trans-11 (or trans-9,cis-11)-octadecadienoic acid (CLA1, 38% of total CLA) and trans-9,trans-11-octadecadienoic acid (CLA2, 62% of total CLA), and accounted for 50% of the total FA obtained. A higher yield (80% molar yield to linoleic acid) was attained with 2.6% (wt/vol) linoleic acid as the substrate in 96 h, resulting in CLA production of 20 mg/mL reaction mixture [consisting of CLA1 (2%) and CLA2 (98%)] and accounting for 80% of total FA obtained. Most of the CLA produced was associated with the cells (ca. 380 mg CLA/g dry cells), mainly as FFA.     

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.     

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.     

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.     

Partial reduction of α-Eleostearic acid with hydrazine

The following products are formed during partial reduction of α-eleostearic acid with hydrazine:cis,trans-9,11-octadecadienoic andtrans,trans-11,13-octadecadienoic acids;cis-9-,trans-11- andtrans-13-octadecenoic acids; and stearic acid. The double bonds are reduced individually in the conjugated triene and also in the conjugated dienes that are formed. However, the reduction is selective since thetrans-11 double bonds in the conjugated triene is reduced only slightly to yield the isolated 9,13-diene. Thetrans double bond of thecis,trans conjugated diene reduces at a faster rate than thecis bond. No differences were observed in the rate of reduction of thecis-9 andtrans-13 bonds in the triene or of the bonds in thetrans,trans conjugated diene. No. Utiliz. Res. & Dev. Div., ARS, USDA.     

CLA production from ricinoleic acid by lactic acid bacteria

The ability to produce CLA from ricinoleic acid is widely distributed in lactic acid bacteria. Washed cells of Lactobacillus plantarum JCM 1551 were selected as a potential catalyst for CLA production from ricinoleic acid. Cells cultivated in medium supplemented with a mixture of α-linolenic acid and linoleic acid showed enhanced CLA productivity. Under optimal reaction conditions, with the free acid form of ricinoleic acid as the substrate and washed cells of L. plantarum as the catalyst, 2.4 mg/mL CLA was produced from 3.4 mg/mL ricinoleic acid in 90 h, with a molar yield with respect to ricinoleic acid of 71%. The CLA produced, which was obtained in the FFA form, consisted of a mixture of two CLA isomers, cis-9,trans-11-octadecadienoic acid (21% of total CLA) and trans-9,trans-11-octadecadienoic acid (79% of total CLA), and accounted for 72% of the total FA obtained.     

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.     

Catalytic hydrogenation of linoleic acid on nickel,copper, and palladium

The catalytic activity and selectivity for hydrogenation of linoleic acid were studied on Ni, Cu, and Pd catalysts. A detailed analysis of the reaction product was performed by a gas-liquid chromatograph, equipped with a capillary column, and Fourier transform-infrared spectroscopy. Geometrical and positional isomerization of linoleic acid occurred during hydrogenation, and many kinds of linoleic acid isomers (trans-9,trans-12; trans-8,cis-12 orcis-9,trans-13; cis-9,trans-12; trans-9,cis-12 andcis-9,cis-12 18∶2) were contained in the reaction products. The monoenoic acids in the partial hydrogenation products contained eight kinds of isomers and showed different isomer distributions on Ni, Cu, and Pd catalysts, respectively. The positional isomers of monoenoic acid were produced by double-bond migration during hydrogenation. On Ni and Pd catalysts, the yield ofcis-12 andtrans-12 monoenoic acids was larger than that ofcis-9 andtrans-9 monoenoic acids. On the contrary, the yield ofcis-9 andtrans-9 monoenoic acids was larger than that ofcis-12 andtrans-12 monoenoic acids on Cu catalyst. From these results, it is concluded that the double bond closer to the methyl group (Δ12) and that to the carboxyl group (Δ9) show different reactivity for hydrogenation on Ni, Cu, and Pd catalysts. Monoenoic acid formation was more selective on Cu catalyst than on Ni and Pd catalysts.