New Geometric Isomers of Oxooctadecadienoate in Copper-catalyzed Decomposition Products of Linoleate Hydroperoxide

Methyl linoleate hydroperoxide produced by autoxidation was refluxed with 10^-4 M Cu-naphthenate in benzene. Two new geometrical isomers of oxooctadecadienoate (compounds I and II) were found in addition to the four known isomers. They were isolated by a Sephadex LH-20 column chromatography with chloroform-hexane (2:1) and purified by HPLC on Nucleosil ®100-5 and Zorbax ODS columns. UV, IR, MS, and ¹H-NMR spectra were measured. The geometry of conjugated dienes were assigned from the coupling constants of the olefinic protons. Compounds I and II were identified as 13-oxo-trans-9, cis-11- and 9-oxo-cis-10, trans-12-octadecadienoate, respectively. Each of them had a cis double bond adjacent to the oxo group. The hydroperoxides of the same geometry as compounds I and II were also detected in autoxidation products.     

Synthesis and properties of methyl 9,12,15-octadecatrienoate geometric isomers

The eight geometrically isomeric methyl 9,12,15-octadecatrienoates were prepared by using the Wittig reaction to couple cis- or trans-3-hexyenyltriphenylphosphonium bromide and methyl 12-oxo-cis- or trans-9-dodecenoate. Pairs of geometric triene isomers formed were separated by partial silver resin chromatography. Physical constants including melting points, percent trans by infrared, equivalent chain lengths (ECL), and ¹³C nuclear magnetic resonance (NMR) chemcial shifts are tabulated for the individual isomers.     

Geometrical Isomers of Monohydroperoxides Formed by Autoxidation of Methyl Linoleate

Isomeric monohydroperoxides produced from autoxidized methyl linoleate were separated into two geometrical isomers (cis-trans and trans-trans) by silver nitrate TLC. Purified monohydroperoxides were converted into hydroxy octadecadienoates. Trimethylsilyl (TMS) derivatives of these compounds (four components) were separated into three peaks in the gas chromatogram; the mixture of 9-hydroxy-cis,trans-isomer and 13-hydroxy-cis,trans-isomer, 9-hydroxy-trans,trans-isomer and 13-hydroxy-trans,trans-isomer. The trans-trans isomers became more dominant than the cis-trans isomers in the later stage of autoxidation and with the rise of temperature. At the degradation of monohydroperoxides, the decrease of trans- trans isomers was apparently slower than that of cis-trans isomers. It is proposed that cis,trans isomerization of monohydroperoxides takes place at the process of autoxidation of methyl linoleate.     

Identification of enriched conjugated linoleic acid isomers in cultures of ruminal microorganisms after dosing with 1-<Superscript>13</Superscript>C-linoleic acid

Most studies of linoleic acid biohydrogenation propose that it converts to stearic acid through the production of cis-9 trans-11 CLA and trans-11 C18:1. However, several other CLA have been identified in ruminai contents, suggesting additional pathways may exist. To explore this possibility, this research investigated the linoleic acid biohydrogenation pathway to identify CLA isomers in cultures of ruminai microorganisms after dosing with a ¹³C stable isotope. The ¹³C enrichment was calculated as [(M+1/M)×100] in labeled minus unlabeled cultures. After 48 h incubation, significant ¹³C enrichment was observed in seven CLA isomers, indicating their formation from linoleic acid. All enriched CLA isomers had double bonds in either the 9,11 or 10,12 position except for trans-9 cis-11 CLA. The cis-9 trans-11 CLA exhibited the highest enrichment (30.65%), followed by enrichments from 21.06 to 23.08% for trans-10 cis-12, cis-10 trans-12, trans-9 trans-11, and trans-10 trans-12 CLA. The remaining two CLA (cis-9 cis-11 and cis-10 cis-12 CLA) exhibited enrichments of 18.38 and 19.29%, respectively. The results of this study verified the formation of cis-9 trans-11 and trans-10 cis-12 CLA isomers from linoleic acid biohydrogenation. An additional five CLA isomers also contained carbons originating from linoleic acid, indicating that pathways of linoleic acid biohydrogenation are more complex than previously described.     

Metabolism of Fatty Acid Hydroperoxides by Chlorella pyrenoidosa

The green alga Chlorella pyrenoidosa was examined for its ability to metabolize 13-hydroperoxylinoleic and 13-hydroperoxylinolenic acids. The study showed that Chlorella extracts possessed hydroperoxide dehydrase and other enzymes of the jasmonic acid pathway. However, under normal laboratory conditions for culture growth, neither jasmonic acid nor metabolites of the jasmonic acid pathway were present in Chlorella. In vitro enzyme studies also revealed the presence of hydroperoxide lyase activity that cleaved 13-hydroperoxylinoleic or 13-hydroperoxylinolenic acid into two products, 13-oxo-cis-9,trans-11-tridecadienoic acid and pentane (from linoleic acid) or pentene (from linolenic acid). The lyase was heat-labile, insensitive to 50 millimolar KCN, and had an approximate molecular weight of 48,000 as estimated by gel filtration. Two other products, 13-hydroxy-cis-9,trans-11,cis-15-octadecatrienoic acid and 12, 13-trans-epoxy-9-oxo-trans-10,cis-15-octadecadienoic acid, were also observed. Because these compounds are also products of nonenzymic, Fe(II)-catalyzed hydroperoxide decomposition reactions, their presence suggested that the observed lyase activity may occur via a homolytic decomposition mechanism.     

Volatile C6-Aldehyde Formation via Hydroperoxides from C18-Unsaturated Fatty Acids in Etiolated Alfalfa and Cucumber Seedlings

1. Etiolated seedlings of alfalfa and cucumber evolved n-hexanal from linoleic acid and cis-3-hexenal and trans-2-hexenal from linolenic acid when they were homogenized.

2. The activities for n-hexanal formation from linoleic acid, lipoxygenase and hydro-peroxide lyase were maximum in dry seeds and 1~2 day-old etiolated seedlings of alfalfa, and in 6~7 day-old etiolated seedlings of cucumber.

3. n-Hexanal was produced from linoleic acid and 13-hydroperoxylinoleic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. cis-3-Hexenal and trans-2-hexenal were produced from linolenic acid and 13-hydroperoxylinolenic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. But these extracts, particulariy cucumber one, showed a high isomerizing activity from cis-3-hexenal to trans-2-hexenal.

4. When the C₈-aldehydes were produced from linoleic acid and linolenic acid by the crude extracts, formation of hydroperoxides of these C₁₈-fatty acids was observed.

5. When 9-hydroperoxylinoleic acid was used as a substrate, trans-2-nonenal was produced by the cucumber homogenate but not by the alfalfa homogenate.

6. As the enzymes concerned with C₆-aldehyde formation, lipoxygenase was partially purified from alfalfa and cucumber seedlings and hydroperoxide lyase, from cucumber seedlings. Lipoxygenase was found in a soluble fraction, but hydroperoxide lyase was in a membrane bound form. Alfalfa lipoxygenase catalyzed formation of 9- and 13-hydroperoxylinoleic acid (35: 65) from linoleic acid and cucumber one, mainly 13-hydroperoxylinoleic acid formation. Alfalfa hydroperoxide lyase catalyzed n-hexanal formation from 13-hydroperoxylinoleic acid, but cucumber one catalyzed formation of n-hexanal and trans-2-nonenal from 13- and 9-hydroperoxylinoleic acid, respectively.

7. From the above results, the biosynthetic pathway for C₆-aldehyde formation in etiolated alfalfa and cucumber seedlings is established that C₆-aldehydes (n-hexanal, cis-3-hexenal and trans-2-hexenal) are produced from linoleic acid and linolenic acid via their 13-hydroperoxides by lipoxygenase and hydroperoxide lyase.     

Decomposition products of Dimers Arising from Secondary Oxidation of Methyl Linoleate Hydroperoxides

Dimers formed in aerated methyl linoleate hydroperoxides were decomposed in liquid paraffin by bubbling with dry air at 30°C for 24 hr to identify the decomposition products. The aerated dimers were fractionated according to their molecular weights by gel permeation chromatography. Identification of the monomeric (25.6%) and low molecular fission products (10.8%) by gas chromatography-mass spectrometry showed the major monomers as methyl hydroxy-octadecadienoate, methyl hydroxy (or hydroperoxy)-epoxy-octadecenoate, methyl dihydroxy (or hydroperoxy)-octadecenoate, methyl trihydroxy (or hydroperoxy)-octadecenoate; and the major fission products as methyl 8-hydroxy-octanoate, 4-hydroxy (or hydroperoxy)-nonanal or -2-nonenal, methyl 12-oxo-9-hydroxy (or hydroperoxy)-dodecanoate or -10-dodecenoate, and methyl 11-oxo-9-undecenoate.

The monomeric products were presumed to be derived from alkoxy radicals generated by the cleavage of peroxy linkages in the dimers, whereas the low molecular products were suggested to be raised by the direct carbon-carbon scission of oxygenated ester moieties on both sides of the peroxy bonds.     

The partition of cis-parinaric acid and trans-parinaric acid among aqueous,fluid lipid,and solid lipid phases

Summary In this article, I review the current information concerning the partition of the fluorescent probes, cis-parinaric acid (9, 11, 13, 15-cis, trans, trans, cis-octadecatetraenoic acid) and trans-parinaric acid (9, 11, 13, 15-all trans-octadecatetraenoic acid) among aqueous, solid lipid, and fluid lipid phases. The association of these probes with lipid is described by a mole fraction partition coefficient whose value is typically in the range of 1–5 × 10⁶, a reasonable value in light of partition coefficients for other fatty acids between hydrophobic phases and water. The partition coefficient, in the absence of lipid phase changes, is relatively independent of temperature and only slightly dependent on the total aqueous probe concentration.In lipid samples which contain coexisting fluid and solid phases, trans-parinaric acid preferentially partitions into the solid phase, while cis-parinaric acid distributes nearly equally between fluid and solid phases. This partition behavior probably arises from the molecular shape of the cis and trans parinaric acid isomers. From measurements of the polarization of fluorescence of cis and trans parinaric acid in mixed lipid systems or membranes it is possible to evaluate the proportion of lipid components involved in phase changes or phase separation. From fluorescence energy transfer between protein typtophan residues and the parinaric acid isomers it is possible to gain information about the organization of lipids and proteins in membranes and model systems. I close the review by considering some of the membrane research areas where these probes and their various lipid derivatives may be particularly useful.     

Partial purification and characterization of the linoleate hydroperoxide isomerase from grains of Hordeum distichum

Linoleate hydroperoxide isomerase was mainly located in the embryos of barley grains and its activity decreased during germination. The enzyme partially purified from embryos converted 9-hydroperoxy, trans-10, cis-12-octadecadienoic acid to 9-hydroxy, 10-oxo, cis-12-octadecenoic acid and 13-hydroxy, 10-oxo, trans-11-octadecenoic acid in the ratio of ca 2:1.     

Elaidic and trans-vaccenic acids in plasma phospholipids as indicators of dietary intake of 18:1 trans-fatty acids

Octadecenoic (18:1) trans-fatty acid fractions from margarine, butter and plasma phospholipids (PL) were isolated by silver ion TLC, and nine positional isomers (n-11-n-3) were identified by GC-MS based on their ozonolysis products. The GC analysis of the isolated fractions gave similar peak profiles and separated seven trans-isomers (n-11-n-6 and n-3). Without a preceding isolation step, the reproducibility of the Gc method for plasma PL elaidic (18:1 n-9 trans) and trans-vaccenic acids (n-7) was 3.4 and 2.7% (R.S.D.), respectively. These trans-isomers were rapidly incorporated and cleared in plasma PL and they closely reflected both increased and decreased intake of 18:1 trans-fatty acids during moderate fat substitutions. Significant associations between high-density lipoprotein cholesterol (HDL-C) and PL elaidic and trans-vaccenic acids appeared in habitual margarine users only.     

The formation of cis-3-nonenal, trans-2-nonenal and hexanal from linoleic acid hydroperoxide isomers by a hydroperoxide cleavage enzyme system in cucumber (Cucumis sativus) fruits.

1. A particulate enzyme fraction and an acetone powder preparation from cucumber fruits cleaved 9- and 13-hydroperoxyoctadecadienoic acids to form volatile aldehydes and oxoacid fragments. 2. From the 9-hydroperoxide, the major volatile fragments were cis-3-nonenal and trans-2-nonenal using particulate enzyme and acetone powder preparations, respectively. 3. Hexanal was the only significant volatile fragment from the 13-hydroperoxide. 4. The particulate enzyme system was equally effective on both 9- and 13-hydroperoxide isomers and was fully active under anaerobic conditions and at pH 6.4. 5. An enzymic pathway for the biogenesis of hexanal, cis-3- and trans-2-nonenal (components of the characteristic flavour volatiles of cucumber) from linoleic acid is proposed. This involves the sequential activity of lipoxygenase, hydroperoxide cleavage and cis-3-: trans-2-enal isomerase enzymes.     

One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum

Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.     

Steric Enhancement of Imidazole Basicity incis-Urocanic Acid Derivatives: Models for the Action of Chymotrypsin

To test the hypothesis that substrate-induced steric compression between His 57 and Asp 102 at the active site of chymotrypsin can increase the basicity of His 57, we have synthesized thecis- andtrans-isomers of 2-bromo-3-(N-tritylimidazole)-2-propenoic acid and 2-chloro-3-(N-tritylimidazole)-2-propenoic acid and compared selected properties with those ofcis-andtrans-urocanic acids. Thecis-isomers display low field¹H NMR signals at 17 ppm in dimethylsulfoxide, similar tocis-urocanic acid; whereas thetrans-isomers do not show strong hydrogen bonds. Increasing the size of the C2 substituent (H < Cl < Br) in thecis-isomers increases the pK_aof the imidazolium group from 6.78 for H to 7.81 and 9.10 for Cl and Br, respectively; whereas the pK_as of thetransisomers are all 6.0 ± 0.1. The results indicate that thecis-urocanic acid derivatives with large substituents at C2 act as proton sponges in water, and they support the concept that steric compression in the catalytic triad of chymotrypsin can increase the basicity of His 57.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

Enzymatic Improvement of Food Flavor. V. Oxidation of Aldehydes in Soybean Extracts by an NAD+-Regenerating System Made up of Aldehyde Dehydrogenase and Diaphorase

Fourteen new and three known o-arylethenylbenzoic acids (10 ~ 18), required for testing as potential plant growth regulators, have been synthesized by the Wittig reaction. The cis- and trans-isomers were separated by fractional crystallization and identified by NMR spectroscopy. The preliminary assay results on cress seedlings are reported and show that both geometric isomers appear to have nearly identical anit-geotropic activity in this test system.     

Quantitative changes of free-base,riboside, ribotide and glucoside cytokinins in developing rice grains

Rice grains at various growth stages were analysed for endogenous free-base, riboside, ribotide and glucoside cytokinins on the basis of GC/MS and GC/SIM using deuterium-labeled internal standards. Cytokinins identified were trans- and cis-zeatins, trans- and cis-ribosylzeatins, isopentenyladenosine, isopentenyladenosine monophosphate, trans- and cis-ribosylzeatin monophosphates, trans- and cis-zeatin-O-glucosides, trans- and cis-ribosylzeatin-O-glucosides and zeatin-9-glucoside (trans/cis geometry was not determined). The highest amounts of cytokinins were recorded at the early growth stage, namely either heading, anthesis or milk stage, suggesting that cytokinins may play important roles in the development of the grain. Cis isomers of zeatin derivatives were always present and more abundant than trans isomers. It seemed unlikely that cis isomers were released from t-RNAs during the extraction procedure.     

Substrate selectivity of lipases in the esterification of cis/trans-isomers and positional isomers of conjugated linoleic acid (CLA)

The substrate selectivity of several microbial lipases has been examined in the esterification of the conjugated linoleic acid (CLA) isomers cis-9,trans-11-, cis-9,cis-11-, trans-9,trans-11- and trans-10,cis-12-octadecadienoic acid with n-butanol in n-hexane. Lipases from Candida cylindracea and Mucor miehei had a preference for the cis-9,trans-11-octadecadienoic acid, while Chirazyme L-5, a Candida antarctica lipase A, accepted the trans-9,trans-11-fatty acid with a high selectivity. Moreover, lipase from Candida cylindracea and Chirazyme L-5 catalysed the esterification of the cis-9,trans-11-octadecadienoic acid with n-butanol faster than the corresponding reaction of the trans-10,cis-12-fatty acid.     

Isolation and spectral characterization of thermally generated multi-Z-isomers of lycopene and the theoretically preferred pathway to di-Z-isomers

Lycopene has a large number of geometric isomers caused by E/Z isomerization at arbitrary sites within the 11 conjugated double bonds, offering varying characteristics related to features such as antioxidant capacity and bioavailability. However, the geometric structures of only a few lycopene Z-isomers have been thoroughly identified from natural sources. In this study, seven multi-Z-isomers of lycopene, (9Z,13′Z)-, (5Z,13Z,9′Z)-, (9Z,9′Z)-, (5Z,13′Z)-, (5Z,9′Z)-, (5Z,9Z,5′Z)-, and (5Z,9Z)-lycopene, were obtained from tomato samples by thermal isomerization, and then isolated by elaborate chromatography, and fully assigned using proton nuclear magnetic resonance. Moreover, the theoretically preferred pathway from (all-E)-lycopene to di-Z-isomers was examined with a computational approach using a Gaussian program. Fine-tuning of the HPLC separation conditions led to the discovery of novel multi-Z-isomers, and whose formation was supported by advanced theoretical calculations.     

Molecular dynamic simulation study of cholesterol and conjugated double bonds in lipid bilayers

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is believed to have anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be associated with anti-obesity effects. In this paper we extend earlier molecular dynamics (MD) simulations of pure CLA–phosphatidylcholine bilayers to investigate the comparative effects of cholesterol on bilayers composed of the two respective isomers. Simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed in which, for each isomer, the simulated bilayers contained 10% and 30% cholesterol (Chol). From MD trajectories we calculate and compare structural properties of the bilayers, including areas per molecule, thickness of bilayers, tilt angle of cholesterols, order parameter profiles, and one and two-dimensional radial distribution function (RDF), as functions of Chol concentration. While the structural effect of cholesterol is approximately the same for both isomers, we find differences at an atomistic level in order parameter profiles and in two-dimensional radial distribution functions.     

Conjugated double bonds in lipid bilayers: a molecular dynamics simulation study

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is attributed to have the anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be responsible for the anti-obesity effects. Since dietary CLA are incorporated into membrane phospholipids, we have used Molecular Dynamics (MD) simulations to investigate the comparative effects of the two isomers on lipid bilayer structure. Specifically, simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed. Force field parameters for the torsional potential of double bonds were obtained from ab initio calculations. From the MD trajectories we calculated and compared structural properties of the two lipid bilayers, including areas per molecule, density profiles, thickness of bilayers, tilt angle of tail chains, order parameters profiles, radial distribution function (RDF) and lateral pressure profiles. The main differences found between bilayers of the two CLA isomers, are (1) the order parameter profile for C9T11 has a dip in the middle of sn-2 chain while the profile for T10C12 has a deeper dip close to terminal of sn-2 chain, and (2) the lateral pressure profiles show differences between the two isomers. Our simulation results reveal localized physical structural differences between bilayers of the two CLA isomers that may contribute to different biological effects through differential interactions with membrane proteins or cholesterol.