Cutins of Malus pumila fruits and leaves

The cutins of fruits and leaves of four apple cultivars have been analysed using TLC, GLC and GC-MS. They are similarly composed of saturated, monounsaturated and diunsaturated fatty, hydroxy-fatty and epoxyhydroxy-fatty acids. The most abundant monomers are 18-hydroxyoctadeca-9,12-dienoic, 10,16-dihydroxyhexadecanoic, 9,10-epoxy-18-hydroxyoctadec-12-enoic, 9,10-epoxy-18-hydroxyoctadecanoic and 9,10,18-trihydroxyoctadecanoic acids. The fruit cutins have high contents of epoxides (35–40%) and unsaturated components ( > 40%) and C₁₈ compounds predominate over C₁₆. The leaf cutins contain smaller amounts of unsaturated components than the fruits and higher proportions of C₁₆ compounds. The adaxial leaf cutin differs in composition from the abaxial. 10,16-Dihydroxyhexadecanoic and 9,10-epoxy-18-hydroxoctadecanoic acids are the major constituents (each ca. 30%) of the adaxial leaf cutin and 10,16-dihydroxyhexadecanoic acid (55–65%) predominates in the abaxial.     

Resin acids of Pinus resinosa needles

The principal resin acids in the needles of Pinus resinosa are the labdane diterpenes, the new 8,13-epoxy-14-labden-19-oic acid (epimanoyl oxide acid), 8,13β-epoxy-14-labden-19-oic acid (manoyl oxide acid), 8(17),E-12,14-labdatrien-19-oic acid (communic acid) and 15-oxo-8(17)-labden-19-oic acid (imbricataloic acid). A survey of needles from representative populations of P. resinosa showed a limited variability in resin acid composition consistent with the uniformity of other traits. The composition of needle resin acids for putative P. nigra x resinosa hybrids strongly suggests the improbability of P. resinosa as the pollen parent.     

Direct evidence for the involvement of epoxide intermediates in the biosynthesis of the C18 family of cutin acids

Cutin, the structural component of plant cuticle, is a polymer of C₁₆ and C₁₈ hydroxy fatty acids. Previous results have suggested that oleic acid undergoes ω-hydroxylation, epoxidation of the double bond, and, finally, hydration of the epoxide to give rise to the three major components of the C₁₈ family of cutin acids. 18-Hydroxy [18-³H]oleic acid and 18-hydroxy-9,10-epoxy[18-³H]stfaric acid have been synthesized and, with these synthetic substrates, the conversion of 18-hydroxyoleic acid to 18-hydroxy-9,10-epoxystearic acid and the hydrolysis of 18-hydroxy-9,10-epoxystearic acid to 9,10,18-trihydroxystearic acid were directly demonstrated in apple fruit skin and in the leaves of apple and Senecio odoris. Trichloropropene oxide, an inhibitor of microsomal epoxide hydrases of animals, specifically inhibited the conversion of [1-¹⁴C]oleic acid into 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid, while it had no effect on the conversion of [1-¹⁴C]palmitic acid into hydroxylated palmitic acid, a process which does not involve epoxy acid intermediates. Therefore, it appears that this inhibitor affects epoxidation and or epoxide hydration steps involved in cutin biosynthesis.     

Conversion of epoxyeicosatrienoic acids (EETs) to chain-shortened epoxy fatty acids by human skin fibroblasts

Epoxyeicosatrienoic acids (EETs), the eicosanoid biomediators synthesized from arachidonic acid by cytochrome P450 epoxygenases, are inactivated in many tissues by conversion to dihydroxyeicosatrienoic acids (DHETs). However, we find that human skin fibroblasts convert EETs mostly to chain-shortened epoxy-fatty acids and produce only small amounts of DHETs. Comparative studies with [5,6,8,9,11,12,14,15-(3)H]11,12-EET ([(3)H]11,12-EET) and [1-(14)C]11,12-EET demonstrated that chain-shortened metabolites are formed by removal of carbons from the carboxyl end of the EET. These metabolites accumulated primarily in the medium, but small amounts also were incorporated into the cell lipids. The most abundant 11, 12-EET product was 7,8-epoxyhexadecadienoic acid (7,8-epoxy-16:2), and two of the others that were identified are 9, 10-epoxyoctadecadienoic acid (9,10-epoxy-18:2) and 5, 6-epoxytetradecaenoic acid (5,6-epoxy-14:1). The main epoxy-fatty acid produced from 14,15-EET was 10,11-epoxyhexadecadienoic acid (10, 11-epoxy-16:2). [(3)H]8,9-EET was converted to a single metabolite with the chromatographic properties of a 16-carbon epoxy-fatty acid, but we were not able to identify this compound. Large amounts of the chain-shortened 11,12-EET metabolites were produced by long-chain acyl CoA dehydrogenase-deficient fibroblasts but not by Zellweger syndrome and acyl CoA oxidase-deficient fibroblasts. We conclude that the chain-shortened epoxy-fatty acids are produced primarily by peroxisomal beta-oxidation. This may serve as an alternate mechanism for EET inactivation and removal from the tissues. However, it is possible that the epoxy-fatty acid products may have metabolic or functional effects and that the purpose of the beta-oxidation pathway is to generate these products.     

Naturally occurring conjugated octadecatrienoic acids are strong inhibitors of prostaglandin biosynthesis

Fatty acids from natural sources (mostly seed oils) were isolated and assayed for their effect on the bioconversion of arachidonic acid into prostaglandin E2, using sheep vesicular gland microsomes. Homologues and isomers of the naturally occurring fatty acids, obtained by chemical modification and/or organic synthetic methods, were also tested. Two very active cyclooxygenase inhibitors were discovered, namely jacarandic acid (8Z, 10E, 12Z-octadecatrienoic acid), isolated from Jacaranda mimosifolia, the concentration which gives 50% inhibition ([I]50) being 2.4 microM and the synthetic 8Z, 10E, 12E-octadecatrienoic acid, having an [I]50 of 1.0 microM. Under the conditions of the assay (75 microM substrate), earlier described potent inhibitors showed the following [I]50's: indomethacin: 1.3 microM; 9,12-octadecadiynoic acid: 1.3 microM, 8Z, 12E, 14Z-eicosatrienoic acid: 2.7 microM; 5,8,11,14-eicosatetraynoic acid: 4.4 microM. At a concentration of about half that of the substrate, the following naturally occurring fatty acids revealed inhibition ([I]50): columbinic acid (29 microM), calendulic acid (31 microM), liagoric acid (31 microM), ximenynic acid (39 microM), crepenynic acid (40 microM) and timnodonic acid (43 microM). Other fatty acids, and some of the above acids, were converted themselves more or less rapidly, mostly into conjugated monohydroxy fatty acids.     

Biosynthesis of cutin monomers: involvement of a lipoxygenase/peroxygenase pathway

Cutin is synthesized from oxygenated fatty acids derived preponderantly from oleic acid. The enzymatic pathways involved in the biosynthesis of such cutin monomers have been studied, i.e. 18-hydroxyoleic acid, 9,10-epoxy-18-hydroxystearic acid (the major constituent) and 9,10,18-trihydroxystearic acid. This was approached by studying (i) the substrate specificity and stereoselectivity of purified peroxygenase, which epoxidizes unsaturated fatty acids, and fatty acid epoxide hydrolase, i.e. two enzyme activities that have been found recently in higher plants, and (ii) the transformation of oleic acid into cutin monomers by a cell free system, i.e. soybean microsomes. These two enzymes, along with a ω-hydroxylating activity, can account for the biosynthesis of the oleic acid-derived cutin monomers and their precursors. A new biosynthetic scheme is proposed, whose pathways take into account the dynamic aspects of the expression of the different enzyme activities involved. Importantly, since peroxygenase, for its activity, is strictly dependent on fatty acid hydroperoxides, which act as co-substrates, the biosynthesis of cutin monomers is also dependent on the activity of lipoxygenases.     

Synthesis and spectral characteristics of some unusual fatty esters of podophyllotoxin

Five fatty ester derivatives of podophyllotoxin have been prepared by reacting the corresponding fatty acids with the hydroxy group of podophyllotoxin in the presence of dimethylaminopyridine and N,N-dicyclohexylcarbodiimide. The fatty acids incorporated are: 9,12-epoxy-9,11-octadecadienoic acid, octadec-11E-en-9-ynoic acid, 11,12-E-epoxy-octadec-9-ynoic acid, octadeca-9Z,11E-dienoic acid and 9,10-dibromooctadecanoic acid. The average yield of esterification was >95% and the structures of the products were confirmed by a combination of nuclear magnetic resonance spectroscopic and mass spectrometric analyses.     

Fatty acids,part 21: ring opening reactions of synthetic and natural furanoid fatty esters

Methyl 2,5-disubstituted C₁₈ furanoid fatty ester (viz. methyl 9,12-epoxyoctadeca-9,11-dienoate) was readily converted to methyl 9,12-dioxostearate using mineral or maleic acid. Conversion of the naturally occurring 2,3,5-trisubstituted furanoid fatty ester (viz. methyl 10,13-epoxy-11-methyloctadeca-10,12-dienoate) to the corresponding methyl 10,13-dioxo-11-methylstearate was much slower in rate under similar reaction conditions. The case of separating the dioxo derivatives from a mixture of other common fatty esters was demonstrated and the cyclodehydration of the isolated dioxo derivatives to the parent furanoid ester was rapidly achieved using dilute BF₃-methanol complex.     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.     

Distribution and chemotaxonomic significance of acetylenic fatty acids in mosses of the dicranales

Thirty-eight moss species from four families of the order Dicranales were analysed for the fatty acid composition of their acyl lipids. In the Ditrichaceae and the Dicranaceae numerous species were found to contain acetylenic fatty acids in their triglycerides, 9,12,15-Octadecatrien-6-ynoic acid was the major component, often accounting for more than 80 mol%, whereas 9,12-octadecadien-6-ynoic acid was found in small amounts of less than 5 mol%. In some genera, all the species examined contained acetylenic fatty acids, e.g.Dicranella andDicranum, whereas in the genusCampylopus all five species tested were free of acetylenic compounds. Two genera, Ditrichum andDicranoweisia, were found to have a non-homogeous distribution of acetylenic fatty acids. The chemotaxonomic significance of the fatty acid composition in relation to morphological characters is discussed.     

On the Specificity of a Fatty Acid Epoxygenase in Broad Bean (Vicia faba L.)

Seeds of broad bean (Vicia faba L.) contain a hydroperoxide-dependent fatty acid epoxygenase. Hydrogen peroxide served as an effective oxygen donor in the epoxygenase reaction. Fifteen unsaturated fatty acids were incubated with V. faba epoxygenase in the presence of hydrogen peroxide and the epoxy fatty acids produced were identified. Examination of the substrate specificity of the epoxygenase using a series of monounsaturated fatty acids demonstrated that (Z)-fatty acids were rapidly epoxidized into the corresponding cis-epoxy acids, whereas (E)-fatty acids were converted into their trans-epoxides at a very slow rate. In the series of (Z)-monoenoic acids, the double bond position as well as the chain length influenced the rate of epoxidation. The best substrates were found to be palmitoleic, oleic, and myristoleic acids. Steric analysis showed that most of the epoxy acids produced from monounsaturated fatty acids as well as from linoleic and α-linolenic acids had mainly the (R),(S) configuration. Exceptions were C₁₈ acids having the epoxide group located at C-12/13, in which cases the (S),(R) enantiomers dominated. 13(S)-Hydroxy-9(Z),11(E)-octadecadienoic acid incubated with epoxygenase afforded the epoxy alcohol 9(S),10(R)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as the major product. Smaller amounts of the diastereomeric epoxy alcohol 9(R),10(S)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as well as the α,β-epoxy alcohol 11(R),12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoic acid were also obtained. The soluble fraction of homogenate of V. faba seeds contained an epoxide hydrolase activity that catalyzed the conversion of cis-9,10-epoxyoctadecanoic acid into threo-9,10-dihydroxyoctadecanoic acid.     

Oxygenation of unsaturated fatty acids in seeds during storage

Seeds of Cichorium intybus L., Crepis thomsonii Babc, and Crepis vesicaria L, were stored from 4 to 8 years at 5°C and then for 18 months under a variety of conditions. Oxygenated acids in Cichorium intybus oil increased from approximately 1% initially to 3% in the first storage period and to 17% while stored at room temperature during the second period. The corresponding levels at these three stages for Crepis thomsonii were 2, 6 and 18%. By gas chromatography (GC) and GC-mass spectrometry, the major oxygenated acids formed during storage were identified as hydroxy acids with conjugated unsaturation and 9,10-epoxy acids. In Crepis vesicaria seed, oil of which contained 53% vernolic (12,13-epoxy-9-octadecenoic) acid originally, approximately 2% of 9,10-epoxides were formed during the storage at room temperature. Levels of hydroxy acids with conjugated unsaturation in this species were 0.3% initially, 2% after 5 years at 5°C, and 9% after 18 months at room temperature. Primary substrates from which oxygenated acids were formed in the three species were crepenynic and linoleic acids, and the almost exclusive formation of 9,10-epoxide from linoleic acid indicated enzymatic involvement.     

Payne rearrangement during analysis of epoxyalcohols of linoleic and alpha-linolenic acids by normal phase liquid chromatography with tandem mass spectrometry

Hydroperoxides of polyunsaturated fatty acids can be transformed to epoxyalcohols and keto fatty acids by metal enzymes, hematin, and various catalysts. In the current study, we used hematin to transform 9-hydroperoxy-10E,12Z-octadecadienoic acid and 13-hydroperoxy-9Z,11E-octadecadienoic acid to epoxyalcohols (with trans epoxide configuration) and to keto fatty acids. The products were separated by normal phase high-performance liquid chromatography (NP-HPLC) and analyzed using postcolumn addition of isopropanol/water and online negative ion electrospray ionization mass spectrometry (MS). The tandem MS (MS/MS) spectra were studied using analogs prepared from [9,10,12,13-²H₄]linoleic acid (18:2n−6) and from α-linolenic acid (18:3n−3). We also studied the MS/MS spectra of epoxyalcohols formed from 11-hydroperoxy- and 8-hydroperoxy-9Z,12Z-octadecadienoic acids. Results were confirmed by MS/MS analysis of a series of authentic standards. MS/MS ions of 9-keto-10E,12Z-octadecadienoic acid and 13-keto-9Z,11E-octadecadienoic acid could be explained by keto-enol tautomerism. MS/MS spectra of regioisomeric allylic epoxyalcohols differed in relative intensities of characteristic ions. The MS/MS spectra of the epoxyalcohols with 1-hydroxy-2,3-epoxy-4Z-pentene or 3-hydroxy-1,2-epoxy-4Z-pentene elements were virtually identical and showed two characteristic ions that differed by 30 in m/z values (CH(OH)). The results suggested that epoxide migration (Payne rearrangement) occurred during collision-induced dissociation. We conclude that regioisomeric allylic epoxyalcohols can be identified by their MS/MS spectra, whereas regioisomeric epoxyalcohols can be identified by MS/MS in combination with their retention times on NP-HPLC.     

Naturally occurring conjugated octadecatrienoic acids are strong inhibitors of prostaglandin biosynthesis

Fatty acids from natural sources (mostly seed oils) were isolated and assayed for their effect on the bioconversio of arachidonic acid into prostaglandin E₂, using sheep vesicular gland microsomes. Homologues and isomers of the naturally occurring fatty acids, obtained by chemical modification and/or organic synthetic methods, were also tested. Two very active cyclooxygenase inhibitors were discovered, namely jacarandic acid (8 , 10 , 12 -octadecatrienoic acid), isolated from , the concentration which gives 50% inhibition ([I]₅₀) being 2.4 μM and the synthetic 8 , 10 , 12 -octadecarienoic acid, having an [I]₅₀ of 1.0 μM. Under the conditions of the assay (75 μM substrate), earlier described potent inhibitors showed the following [I]₅₀′s: indomethacin: 1.3 μM; 9,12-octadecadiynoic acid: 1.3 μM, 8 , 12 , 14 -eicosatrienoic acid: 2.7 μM; 5,8,11,14-eicosatetraynoic acid: 4.4 μM. At a concentration of about half that of the substrate, the following naturally occurring fatty acids revealed inhibition ([I]₅₀): columbinic acid (29 μM), calendulic acid (31 μM), liagoric acid (31 μM), ximenynic acid (39 μM), crepenynic acid (40 μM) and timnodonic acid (43 μM). Other fatty acids, and some of the above acids, were converted themselves more or less rapidly, mostly into conjugated monohydroxy fatty acids.     

Micro-analysis of amino acids

An automated amino acid analyzer has been developed for the analysis of amino acids with the sensitivity at the 10–100 pmol level except for proline which requires >50 pmol. o-Phthalaldehyde, in the presence of 2-mercaptoethanol, is used for the fluorometric detection of amino groups (Roth, M. (1971) Anal. Chem. 43, 880–882). A post-column reaction of the amino acid with sodium hypochlorite (Bohlen, P. and Mellet, M. (1979) Anal. Biochem. 94, 313–321) gives oxidation products amenable to detection with o-phthalaldehyde. The instrument uses high-performance liquid chromatographic pumps capable of micro-flow rates with a minimum pulsation. The method is suitable for routine analyses of amino acids at picomole levels with reproducibility and accuracy comparable to the ninhydrin-based amino acid analysis.     

Fatty acid constituents of Peganum harmala plant using Gas Chromatography–Mass Spectroscopy

Fatty acid contents of the Peganum harmala plant as a result of hexane extraction were analyzed using GC–MS. The saturated fatty acid composition of the harmal plant was tetradecanoic, pentadecanoic, tridecanoic, hexadecanoic, heptadecanoic and octadecanoic acids, while the saturated fatty acid derivatives were 12-methyl tetradecanoic, 5,9,13-trimethyl tetradecanoic and 2-methyl octadecanoic acids. The most abundant fatty acid was hexadecanoic with concentration 48.13% followed by octadecanoic with concentration 13.80%. There are four unsaturated fatty acids called (E)-9-dodecenoic, (Z)-9-hexadecenoic, (Z,Z)-9,12-octadecadienoic and (Z,Z,Z)-9,12,15-octadecatrienoic. The most abundant unsaturated fatty acid was (Z,Z,Z)-9,12,15-octadecatrienoic with concentration 14.79% followed by (Z,Z)-9,12-octadecadienoic with concentration 10.61%. Also, there are eight non-fatty acid compounds 1-octadecene, 6,10,14-trimethyl-2-pentadecanone, (E)-15-heptadecenal, oxacyclohexadecan-2 one, 1,2,2,6,8-pentamethyl-7-oxabicyclo[4.3.1]dec-8-en-10-one, hexadecane-1,2-diol, n-heneicosane and eicosan-3-ol.     

A peroxygenase pathway involved in the biosynthesis of epoxy fatty acids in oat

While oat (Avena sativa) has long been known to produce epoxy fatty acids in seeds, synthesized by a peroxygenase pathway, the gene encoding the peroxygenase remains to be determined. Here we report identification of a peroxygenase cDNA AsPXG1 from developing seeds of oat. AsPXG1 is a small protein with 249 amino acids in length and contains conserved heme-binding residues and a calcium-binding motif. When expressed in Pichia pastoris and Escherichia coli, AsPXG1 catalyzes the strictly hydroperoxide-dependent epoxidation of unsaturated fatty acids. It prefers hydroperoxy-trienoic acids over hydroperoxy-dienoic acids as oxygen donors to oxidize a wide range of unsaturated fatty acids with cis double bonds. Oleic acid is the most preferred substrate. The acyl carrier substrate specificity assay showed phospholipid and acyl-CoA were not effective substrate forms for AsPXG1 and it could only use free fatty acid or fatty acid methyl esters as substrates. A second gene, AsLOX2, cloned from oat codes for a 9-lipoxygenase catalyzing the synthesis of 9-hydroperoxy-dienoic and 9-hydroperoxy-trienoic acids, respectively, when linoleic (18:2-9c,12c) and linolenic (18:3-9c,12c,15c) acids were used as substrates. The peroxygenase pathway was reconstituted in vitro using a mixture of AsPXG1 and AsLOX2 extracts from E. coli. Incubation of methyl oleate and linoleic acid or linolenic acid with the enzyme mixture produced methyl 9,10-epoxy stearate. Incubation of linoleic acid alone with a mixture of AsPXG1 and AsLOX2 produced two major epoxy fatty acids, 9,10-epoxy-12-cis-octadecenoic acid and 12,13-epoxy-9-cis-octadecenoic acid, and a minor epoxy fatty acid, probably 12,13-epoxy-9-hydroxy-10-transoctadecenoic acid. AsPXG1 predominately catalyzes intermolecular peroxygenation.     

Suberins of Malus pumila stem and root corks

The suberin contents of the isolated superficial cork layers of Malus pumila stems and root ranged from 15 to 35% of the dry weight. The qualitative composition of the aliphatic monomers obtained after alkaline depolymerization of the extractive-free corks was similar but some quantitative differences were found according to cultivar and age of the cork layer. 1-Alkanols (mainly 22:0, 24:0 and 26:0), alkanoic acids (mainly 22:0 and 24:0), α, ω-alkanedioic acids (mainly 16:0, 18:1 (9) and 18:0) and ω-hydroxyalkanoic acids (mainly 18:1 (9) and 22:0) were major constituents of all the samples examined and together they comprised 40–50% of the total monomeric mixture. The remainder was composed mainly of 9,10-epoxy-18-hydroxy-and 9,10,18-trihydroxyoctadecanoic acids. The corresponding dibasic acids, 9,10-epoxy- and 9,10-dihydroxyoctadecane-1,18-dioic, were minor components as were C₁₆ and C₁₈ dihydroxyalkanoic acids (mainly 10,16-dihydroxyhexadecanoic and 10,18-dihydroxyoctadecanoic acids, respectively). The root suberin dittered from that of the stem in containing larger amounts of 9,10-epoxy- 18-hydroxyoctadecanoic and 18-hydroxyoctadec-9-enoic acids.     

Fatty acids,part 54. Some reactions of long-chain oxygenated acids with special reference to those furnishing furanoid acids

C₁₈ furanoid acids are prepared from natural oxygenated acids by palladium (II)-catalysed cyclodehydrogenation, by rearrangement of epoxides with iodopropane-sodium iodide-dimethylsulphoxide, and by dehydration of endoperoxides. Some reactions give mixed products but routes to the individual 10,13-, 9,12- and 8,11-furans are reported. The endoperoxide route leads to speculation about the biosynthesis of natural furanoid acids.