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.     

Taxus baccata seed oil: A new source ofcis-5,cis-9-octadecadienoic acid

Methyl esters prepared from the seed oil of the coniferTaxus baccata L. were found by gas liquid chromatography to contain 12% of a component which, when isolated by preparative thin layer chromatography and characterized by mass spectrometry, ozonolysis and nuclear magnetic resonance, was identified ascis-5,cis-9-octadecadienoic acid.     

A new acid fromcalea urticaefolia seed oil:trans-3,cis-9,cis-12-octadecatrienoic acid

A major constituent fatty acid (31.2%) fromCalea urticaefolia (Mill.) DC. seed oil is the previously unknowntrans-3,cis-9,cis-12-octadecatrienoic acid. The oil also contains 2.2% of an unidentified acid and others with gas-liquid chromatographic characteristics that correspond to the conventional fatty acids: myristic, 0.1%; palmitic, 9.3%; stearic, 2.9%; oleic, 5.3%; and linoleic, 48.9%. Presented at the AOCS Meeting in New Orleans, 1964. No. Utiliz. Res. & Dev. Div., ARS, USDA.     

Evidence that the trans-10,cis-12 isomer of conjugated linoleic acid induces body composition changes in mice

We investigated the effects of conjugated linoleic acid (CLA) preparations, which were enriched for the cis-9,trans-11 CLA isomer or the trans-10,cis-12 CLA isomer, on body composition in mice. Body composition changes (reduced body fat, enhanced body water, enhanced body protein, and enhanced body ash) were associated with feeding the trans-10,cis-12 CLA isomer. In cultured 3T3-L1 adipocytes, the trans-10,cis-12 isomer reduced lipoprotein lipase activity, intracellular triacylglycerol and glycerol, and enhanced glycerol release into the medium. By contrast, the cis-9,trans-11 and trans-9,trans-11 CLA isomers did not affect these biochemical activities. We conclude that CLA-associated body composition change results from feeding the trans-10,cis-12 isomer.     

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.     

Hydrogenation ofcis-9,cis-12-,cis-9,trans-12- andtrans-9,trans-12- Octadecadienoates

The products formed by hydrogenation of methylcis-9,trans-12- andtrans-9,trans-12-octadecadienoates with nickel and platinum catalysts have been compared with those from methyl esters of the naturally occurring all-cis linoleate. Hydrogen uptake is slower for thetrans isomers. Much of the monoene consisted of esters with double bonds at the 9 and 12 positions with their original geometric configurations. Monoenoic esters with double bonds at the 10 and 11 positions were predominatelytrans and apparently formed by conjugation before hydrogenation. Nickel produced more isomerization than platinum but less than previously reported for copper. With both catalysts hydrogenation proceeded both directly and through conjugated intermediates, in contrast to copper in which all hydrogenation is believed to follow conjugation. Presented at the AOCS Meeting, Los Angeles, April 1972. ARS, USDA.     

Metabolism in humans ofcis-12,rans-15-octadecadienoic acid relative to palmitic,stearic, oleic and linoleic acids

Mixtures of triglycerides containing deuterium-labeled hexadecanoic acid (16∶0), octadecanoic acid (18∶0),cis-9-octadecenoic acid (9c–18∶1),cis-9,cis-12-octadecadienoic acid (9c, 12c–18∶2) andcis-12,trans-15-octadecadienoic acid (12c,15t–18∶2) were fed to two young-adult males. Plasma lipid classes were isolated from samples collected periodically over 48 hr. Incorporation and turnover of the deuterium-labeled fats in plasma lipids were followed by gas chromatography-mass spectrometry (GC-MS) analysis of the methyl ester derivatives. Absorption of the deuterated fats was followed by GC-MS analysis of chylomicron triglycerides isolated by ultracentrifugation. Results were the following: (i) endogenous fat contributed about 40% of the total fat incorporated into chylomicron triglycerides; (ii) elongation, desaturation and chain-shortened products from the deuterated fats were not detected; (iii) the polyunsaturated isomer 12c,15t–18∶2 was metabolically more similar to saturated and 9c–18∶1 fatty acids than to 9c,12c–18∶2 (iv) relative incorporation of 9c,12c–18∶2 into phospholipids did not increase proportionally with an increase of 9c,12c–18∶2 in the mixture of deuterated fats fed; (v) absorption of 16∶0, 18∶0, 9c–18∶1, 9c,12c–18∶2 and 12c,15t–18∶2 were similar; and (vi) data for the 1- and 2-acyl positions of phosphatidylcholine and for cholesteryl ester fractions reflected the known high specificity of phosphatidylcholine acyltransferase and lecithin:cholesteryl acyltransferase for 9c,12c–18∶2. These results illustrate that incorporation of dietary fatty acids into human plasma lipid classes is selectively controlled and that incorporation of dietary 9c,12c–18∶2 is limited. These results suggest that nutritional benefits of diets high in 9c,12c–18∶2 may be of little value to normal subjects and that the 12c,15t–18∶2 isomer in hydrogenated fat is not a nutritional liability at the present dietary level.     

Evidence that commercial calf and horse sera can contain substantial amounts of trans-10,cis-12 conjugated linoleic acid

We analyzed fetal calf, newborn calf, horse, and adult cow sera for conjugated linoleic acid (CLA). All sera samples contained CLA, but the amounts varied. The predominant isomer was cis-9,trans-11 CLA but some samples appeared to contain substantial amounts of an isomer with the retention time of trans-10,cis-12 CLA.     

Effects of the individual isomers cis-9,trans-11 vs. trans-10,cis-12 of conjugated linoleic acid (CLA) on inflammation parameters in moderately overweight subjects with LDL-phenotype B

Immune-modulating effects of CLA have been reported in animals, but results are inconsistent. In humans, CLA has shown no effects or only minor effects on immune function. The objective of this study was to evaluate the immune-modulating effects of 3 g cis-9,trans-11 (c9,t11) vs. trans-10,cis-12 (t10,c12) CLA isomers in a population with a high risk of coronary heart disease characterized by moderate overweight (body-mass index, 25–32.5 kg/m²) in combination with LDL-phenotype B (≥35% small LDL cholesterol, density≥1.040 g/mL). After a run-in period of 1 wk, 42 men and women were randomly allocated to the c9,t11 CLA group, the t10,c12 CLA group, or the placebo group. Effects of 13 wk of consumption of 3 g of CLA isomers on cytokine production by ex vivo lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) and whole blood, and on plasma C-remononuclear protein (CRP) concentrations were evaluated. To generate hypotheses for future studies, protein expression patterns of 42 cytokines, chemokines, and growth factors were evaluated with an antibody array in pooled, nonstimulated, fasting plasma samples. LPS induced interleukin (IL)-6, IL-8, and tumor necrosis factor-α production by PBMC, and whole blood as well as plasma CRP concentrations were not significantly changed by the c9,t11, and the t10,c12 CLA isomers. The cytokine expression profile in nonstimulated plasma suggested that both CLA isomers induced a specific inflammatory signature, in which the c9,t11 CLA group showed more activity in terms of numbers of proteins regulated. We conclude that daily consumption of 3 g of c9,t11 or t10,c12 CLA isomer did not affect LPS-stimulated cytokine production by PBMC or whole blood and plasma CRP levels. Inflammatory signatures in fasting, nonstimulated plasma as determined by an antibody array may indicate enhanced immune function by both CLA isomers.     

Preparation of methylcis-9,cis-12,cis-15-octadecatrienoate-15,16-d 2 and methylcis-9,cis-12,cis-15-octadecatrienoate-6,6,7,7-d 4

Methylcis-9,cis-12,cis-15-octadecatrienoate-15,16-d ₂ was obtained from Wittig coupling of methyl 12-oxo-cis-9-dodecenoate,18, and 3,4-dideutero-cis-3-hexenyltriphenylphosphonium bromide,16. Compound18 was obtained by periodic acid oxidation of methyl 12,13-dihydroxy-cis-9-octadecenoate,17, obtained fromVernonia oil. Compound18 also was synthesized from methyl oleate as the starting material. The deuterated fragment,16, was prepared from 3-hexynol and using Lindlar's catalyst and deuterium gas to introduce the deuterium atoms. Methylcis-9,cis-12,cis-15-octadecatrienoate-6,6,7,7-d ₄ was prepared by Wittig coupling of 3,6-nonadienyltriphenylphosphonium iodide,5, with methyl 9-oxononanoate-6,6,7,7-d ₄,11. Deuterium atoms were introduced during the synthesis of11 from 3-butynol and 5-bromopentanoic acid with deuterium gas in the presence of [Ph₃P]₃-RhCl. For the preparation of5, the 3,6-nonadiynol intermediate was reduced to 3,6-nonadienol with P-2 Nickel and hydrogen. The final products were separated from isomers formed during the synthetic sequences by silver resin chromatography.     

Desaturation indices in liver, muscle, and bone of growing male and female mice fed trans-10, cis-12 conjugated linoleic acid

trans-10, cis-12-CLA (t10,c12-CLA) inhibits lipid deposition in adipose tissue of many species, but it also enhances lipid deposition in liver. We evaluated effects of dietary t10,c12-CLA content and gender on carcass composition, FA profile of selected tissues, and expression of FA synthase (FAS) and stearoyl-CoA desaturase-1 (SCD) mRNA in adipose tissue. Male and female (63 of each) CD-1 mice were assigned a diet containing 0.0, 0.15, or 0.30% t10,c12-CLA at 4 wk of age. Seven mice per dietary group within gender were sacrificed after 2,4 or 6 wk. The CLA isomer caused dose-dependent reductions in dry carcass weight and fat content, without altering protein content, but carcass fat and epididymal fat pad weights of males were reduced to a greater extent than carcass fat and inguinal fat pad weights of females. FAS and SCD mRNA in adipose tissue was more abundant in females than males, but expression in both genders decreased as the t10, c12-CLA content of the diet increased. Although the weight of gastrocnemius muscle was not influenced by diet, total FA content of the muscle of both genders decreased in response to dietary t10,c12-CLA content. Femur weight of male mice increased as the t10,c12-CLA content of the diet increased, but the weight increase was associated with a reduction in total FA content. The Δ9 desaturation indices for muscle and femur suggested a linear reduction in SCD activity, whereas Δ9 indices for liver indicated linear enhancement of SCD activity. Overall, results suggested that growing male mice were more susceptible than females to t10,c12-CLA inhibition of lipid deposition.     

A simple method of preparation of methyl trans-10,cis-12- and cis-9,trans-11-octadecadienoates from methyl linoleate

Pure conjugated isomers of linoleic acid were prepared on a large scale by alkali-isomerization of purified methyl linoleate. The methyl esters of alkali-isomerized linoleic acid contained mainly the methyl cis-9,trans-11- and trans-10,cis-12-octadecadienoates (44 and 47%, respectively). These two isomers were then separated and purified by a series of low-temperature crystallizations from acetone. The isomeric purity obtained for the cis-9,trans-11-octadecadienoate isomer was >90% and that of the trans-10,cis-12-octadecadienoate isomer was 89 to 97%. The isolated yield of the two isomers corresponded to 18 and 25.7%, respectively, of the starting material. The structure of the two isomers was confirmed using partial hydrazine reduction, silver nitrate-thin-layer chromatography of the resulting monoenes and gas chromatography coupled with mass spectrometry of the 4,4-dimethyloxazoline derivatives. Fourier transform infrared spectroscopy of the monoenes gave the confirmation of the geometry of each double bond.     

Effects of cis-9, trans-11 and trans-10, cis-12 CLA isomers on liver and adipose tissue fatty acid profile in hamsters

The aim of the present study was to investigate the effect of cis-9, trans-11 and trans-10, cis-12 CLA on FA composition of TAG in epididymal adipose tissue and liver, and of hepatic phospholipids PL. Twenty-four Syrian Golden hamsters were randomly divided into three groups of eight animals each and fed semipurified atherogenic diets supplemented with either 0.5 g/100g diet of linoleic acid or cis-9, trans-11 or trans-12, cis-9 CLA for 6 wk. Total lipids were extracted, and TAG and PL were separated by TLC. FA profile in lipid species from liver and adipose tissue, as well as in feces, was determined by GC. Trans-10, cis-12 CLA feeding significantly reduced linoleic and linolenic acids in TAG from both tissues, leading to reduced total PUFA content. Moreover, in the epididymal adipose tissue docosenoic and arachidonic acids were significantly increased. In liver PL, although no changes in individual FA were observed, total saturated FA (SFA) were decreased. No changes in TAG and PL FA profiles were induced by the cis-9, trans-11 CLA. TAG and PL incorporated cis-9, trans-11 more readily than trans-11, cis-12 CLA. This difference was not due to differential intestinal absorption, as shown by the analysis of feces. We concluded that only trans-10, cis-12 CLA induces changes in FA composition. Whereas increased PUFA content was observed in either liver or adipose tissue TAG, decreased SFA were found in liver PL. Incorporation of cis-9, trans-11 CLA in TAG is greater than that of trans-10, cis-12 CLA, but this is not due to differences in intestinal absorption.     

Infrared absorption of methylcis-9,trans-11-, andtrans-10,cis-12-octadecadienoates

The ratio of absorptivity at 10.2 µm and 10.6 µm differs between methylcis-9,trans-11-, andtrans-10,cis-12-octadecadienoates. For thecis-9,trans-11-ester, a_10.2 µm/a_10.6 µm is in the range of 1.1–1.2; for thetrans-10,cis-12-ester, it is 1.3–1.4. These differences in absorptivities are great enough to affect significantly compositions calculated from IR absorption.     

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.     

Dimer acid structures: Cyclic structures of clay catalyzed dimers of normal linoleic acid, 9-cis, 12-cis-Octadecadienoic acid

The clay catalyzed dimer of linoleic acid has been examined by mass spectrometry of the unhydrogenated, the partially hydrogenated and completely hydrogenated dimer. The results show that monocyclic, bicyclic and tricyclic structures are present. Monocyclic structures predominate, bicyclic structures are also prominent, and tricyclic structures are relatively minor. The monocyclic structure is believed to arise from a Diels-Alder type addition reaction. The bicyclic structure may result from a free radical coupling followed by intramolecular ring closure. The monocyclic structure in the unhydrogenated dimer appears to be mostly a benzene ring with saturated and unsaturated side-chains. It probably is formed by hydrogen transfer from the Diels-Alder cyclohexene structure first formed. Little, if any, of the Diels-Alder dimer structure as such is present. The catalytic linoleate dimer has a higher ratio of monocyclic to bicyclic dimer than does the noncatalytic (thermal) dimer made from normal (nonconjugated) linoleate, while the thermal dimer of a conjugatedtrans-trans linoleate is exclusively monocyclic. It is suggested that the clay catalyzes conjugation and hence favors the Diels-Alder reaction, and then catalyzes hydrogen transfer to aromatize the cyclohexene ring.     

Synthesis of 9Z,11E-octadecadienoic and 10E,12Z-octadecadienoic acids, the major components of conjugated linoleic acid

Linoleic acid was efficiently converted into the two major components of conjugated linoleic acid, 9Z,11E-octadecadienoic (1a) and 10E,12Z-octadecadienoic acid (1b) using either the superbase (n-butyllithium/potassium tert-butoxide) or by simply refluxing with KOH in 1-butanol. In turn, 1a and 1b were separated from each other using the lipase from Aspergillus niger via stereoselective esterification in 1-butanol. This enzyme has a preference for the 9Z,11E isomer, 1a, and has excellent selectivity. This method has allowed the ready preparation of gram quantities of 1a and 1b in their highly purified forms, which are not readily accessible by current methods.     

A convenient laboratory method for preparingtrans,trans-9, 11-octadecadienoic acid

A convenient laboratory method to preparetrans,trans- 9,11-octadecadienoic acid (TTA) via a polyester intermediate is described. Ricinelaidic acid was heated at 235C under vacuum for 3-4 hr to form a polyester having a mol wt of 1,500-1,600. Pyrolysis of this polyester and simultane-ous distillation of the products gave crude dehy-drated acids. TTA was crystallized from a 95% ethanol solution of these acids, in a yield of 35%. Of the variables affecting pyrolysis, the mol wt of polyester had the greatest effect on yield of TTA. Paper II in the series on “Reactions of Conjugated Fatty Acids,” presented at AOCS meeting in Atlanta, 1963. A laboratory of the No. Utiliz. Res. & Dev. Div., ARS, USDA.     

Isomerization during hydrogenation. II. Methyl cis-10, cis-12-octadecadienoate

Summary Methylcis-10,cis-12-octadecadienoate was prepared and found to have an ultraviolet absorption peak at 235 mμ, α=95. The infrared spectrum revealed no diagnostic peaks for thecis,cis conjugated system. The hydrogenation of the ester showed that when one mole of hydrogen was added, 1–2, 1–4, and 3–4 addition took place with equal ease to produce an equimolar mixture ofcis-10-,trans-11-, andcis-12-octadecenoates. An explanation of the reaction is offered on the basis of the structure of the diene system.