The biosynthesis of cyclopropane and cyclopropene fatty acids in higher plants (Malvaceae)

The biosynthesis of cyclopropane and cyclopropene fatty acids has been investigated in immature seeds, leaves and callus tissue cultures of several species of Malvaceae. Chemical characterization of labeled cyclopropane and cyclopropene fatty acids obtained from incubations withl-[¹⁴CH₃] methionine confirmed that the ring methylene group was derived from the methyl group of methionine. The variation with time in the distribution of radioactivity in the products of incubations with [¹⁴CH₃] methionine and [2-¹⁴C] acetate suggested that the pathway involved initial formation of dihydrosterculic acid from oleic acid with subsequent desaturation to sterculic acid and α-oxidation to malvalic and dihydromalvalic acids. Direct evidence in favor of this pathway was provided by the conversion of [1-¹⁴C] oleic acid to dihydrosterculic and sterculic acids and by the desaturation of [1-¹⁴C] dihydrosterculic acid to sterculic acid, the first time that these processes have been demonstrated in higher plants. No conversion of [1-¹⁴C] stearolic acid to sterculic acid could be obtained under the same conditions. The presence of an active fatty acid α-oxidation system was demonstrated in the callus cultures.     

The biosynthesis of cyclopropane and cyclopropene fatty acids in plant tissues

The biosynthesis of cyclopropane and cyclopropene fatty acids was investigated in seeds of several species of the order Malvales, including species with a high content of sterculic acid, a high content of malvalic acid, and a low content of these cyclopropene fatty acids. The fatty acid composition of the lipids in young and developing seeds is compared with particular attention to variations in cyclopropane and cyclopropene fatty acid contents. Incubation studies employing several¹⁴C compounds indicate that the methyl group of methionine is the most likely precursor of the ring-methylene carbon. A pathway for the synthesis of the cyclopropane and cyclopropene fatty acids is postulated. The origin of malvalic acid is also considered.     

Gas-liquid chromatographic analysis of cyclopropene fatty acids

A gas-liquid chromatographic method is described for the quantitative estimation of cyclopropene fatty acids as their methyl mercaptan derivatives. This method estimates individual cyclopropene acids as well as normal and cyclopropane acids. Nine seed oils were analyzed for their cyclopropene fatty acid content. Evidence was obtained for the presence of a cyclopropene fatty acid of shorter chain length than malvalic inAlthaea rosea cav and one with a higher chain length than sterculic inBombacopsis glabra seed oil. This method is less accurate for cottonseed oil than for the other oils tested because of the appearance of some unsymmetrical peaks of unknown origin. The mercaptan derivatives of the cyclopropene acids may be isolated by silver ion thin-layer chromatography. Small amounts of cyclopropane fatty acids were found in a number of the oils analyzed for cyclopropene fatty acids. Presented at the AOCS meeting, Chicago, October 1964.     

Substituted fatty acids in the leaves of some higher plants

Substituted (hydroxy-, epoxy- and dicarboxy) fatty acids have been analyzed in the needles of two gymnosperms (Pinus sylvestris andJuniperus communis) and the leaves of four angiosperms (Betula verrucosa, Populus nigra, Quercus petraea andCorylus avellana) growing in the Lake Léman basin. The differences in the distributions of the acids that have been analyzed are greater between the various species than between the spring and autumn samples of a given species. Moreover, the isomeric composition of the 8,16-, 9,16- and 10,16-dihy-droxypalmitic acid mixture is specific for each species.     

Fat metabolism in higher plants: Metabolism of medium chain fatty acids

Cell free preparations of avocado mesocarp and spinach leaf tissue rapidly convert lauryl CoA to DL-3-hydroxyl lauric acid as well as 2-, and 3-dodecanoic acids. The conversion does not occur under anaerobic conditions unless a suitable redox carrier such as ferredoxin is present. H2 18O is incorporated into the 3-hydroxyl function, but O2(18) is not. The characteristics of this system are presented and a possible function of this system is proposed.     

Ring position in cyclopropene fatty acids and stearic acid desaturation in hen liver

Four cyclopropene fatty acids, having the double bond of the cyclopropene ring at the 8,9, 9,10, 10,11 and 11,12 positions, respectively, were tested as inhibitors of stearic acid desaturation by the desaturase enzyme system of hen liver. The first three were powerful inhibitors, but the last was not. The cyclopropene acids with the 9,10 and 10,11 double bonds were equally strong inhibitors, while the acid with the 8,9 double bond was less effective. To account for the specificity of those cyclopropene fatty acids in which the C9 or C10 carbon atom is included in the cyclopropene ring, it is suggested that the conformation and structure of the CoA derivatives of these acids is such that they can irreversibly occupy the site on the enzyme responsible for 9,10-desaturation.     

Fatty acid composition of seed oils from sixAdansonia species with particular reference to cyclopropane and cyclopropene acids

The oil content of sixAdansonia species (Bombacaceae family) of Madagascar (Adansonia grandidieri, A. za, A. digitata, A. fony, A. madagascariensis andA. suarenzensis) and Africa (A. digitata) ranges from 8 to 46%. All the oils give a positive response to the Halphen test. Malvalic, sterculic and dihydrosterculic acids were detected using gas liquid chromatography-mass spectrometry (GLC-MS). Epoxy or hydroxy fatty acids were not found in these oils. Fatty acid composition was determined by GLC using glass capillary columns coated with BDS and Carbowax 20 M. Results obtained for cyclopropenic fatty acids (CPEFA) were compared to those given by glass capillary GLC after derivatization with silver nitrate in methanol, by hydrogen bromide titration and by proton magnetic resonance (PMR). Good agreement was observed for the results given by the various methods. Malvalic acid content ranges from 3 to 28%, sterculic acid from 1 to 8% and dihydrosterculic acid from 1.5 to 5.1%. Odd-numbered fatty acids (Pentadecanoic and hepatadecanoic) were also observed in minute amounts (0.1–1.1%). Among the normal fatty acids, we observed mainly palmitic (21–46%), oleic (15–40%) and linoleic (12–32%). The relationship between fatty acid composition andAdansonia species is discussed.     

The biosynthesis of polyunsaturated fatty acids in plants

This paper is a review of some of the work being done at the author's laboratory. The phospholipids and glycolipids of the alga,Chlorella vulgaris, have been implicated in fatty acid transformations such as chain elongation and desaturation. Labeling studies with [¹⁴C] acetate have shown that newly synthesized galactosyl glycerides have mainly saturated fatty acids. Subsequent to de novo synthesis, a series of alterations of fatty acid structure takes place within the same glycolipid molecules. The specific incorporation of [¹⁴C] oleic acid intoChlorella phosphatidyl choline provides a convenient model system for studying the lipid dependent desaturation of oleic to linoleic acid. The inhibitor of fatty acid desaturation, sterculic acid, only inhibits the conversion of oleate into linoleate if added before the precursor fatty acid has been incorporated into a complex lipid. Studies with isomeric monoenoic fatty acids have suggested that there are two enzymes which catalyze the formation of linoleic from oleic acid. One measures the position of the second double bond from the carboxyl group, the other, from the methyl end of the chain. The latter enzyme probably requires the complex lipid substrate.     

Fatty acids of Thespesia populnea: Mass spectrometry of picolinyl esters of cyclopropene fatty acids

Thespesia populnea belongs to the plant family of Malvaceae which contain cyclopropane and cyclopropene fatty acids. However, previous literature reports vary regarding the content of these compounds in Thespesia populnea seed oil. In this work, the content of malvalic acid (8,9‐methylene‐9‐heptadecenoic acid) in the fatty acid profile of Thespesia populnea seed oil was approximately 7% by GC. Two cyclopropane fatty acids were identified, including dihydrosterculic acid. The methyl and picolinyl esters of Thespesia populnea seed oil were also prepared. The mass spectrum of picolinyl malvalate was more closely investigated, especially an ion at m/e 279, which does not fit the typical series of ions observed in picolinyl esters. It is shown that this ion is caused by cleavage at the picolinyl moiety and contains the fatty acid chain without the picolinyl moiety. This type of cleavage has previously not been observed prominently in picolinyl esters and may therefore be diagnostic for picolinyl esters of cyclopropene fatty acids. The NMR spectra of Thespesia populnea methyl esters are also discussed. Practical applications: The work reports the fatty acid composition of Thespesia seed oil whose derivatives have not yet been extensively utilized for industrial purposes, for example, biodiesel. Knowing this composition is essential for understanding potential uses and, for example, in case of biodiesel the fuel properties. Besides this issue, some data (mass spectrometry and NMR) crucial for obtaining the composition information are analyzed in detail. The biodiesel properties of methyl esters of Thespesia populnea will be reported separately.     

The occurrence of higher fatty acids in corn pollen

Summary The methyl esters of the fatty acids of corn pollen were prepared and fractionated through a Stedman column. Palmitic, stearic, oleic, linoleic, and linolenic acids were identified by the melting points of thep-bromophenacyl esters of the saturated acids and the hydroxy and bromine addition compounds of the unsaturated acids. This investigation was supported by a grant from the National Science Foundation. Journal Article No. 2139 of the Michigan Agricultural Experiment Station.     

Synthesis of deuterated cyclopropene fatty esters structurally related to palmitic and myristic acids

To develop a synthesis of tritiated cyclopropene fatty acids (CPFA), compounds that should prove useful for affinity labeling of desaturases in insect pheromone biosynthetic studies, a series of novel, selectively deuterated CPFA analogues was prepared and characterized. In methyl [16-²H]12,13-methylene-12-hexadecenoate, the incorporation of deuterium was achieved by treatment of the corresponding ω-chloro derivative with sodium borodeuteride in dimethylsulfoxide at 70°C for 24 h (67% yield) following conventional procedures. Alkylation of the tetrahydropyranyl derivative of 13-tridecynol in the presence of lithium diisopropylamide in tetrahydrofuran at −20°C with 1-chloro-3-iodopropane in hexamethylphosphoramide, followed by Jones oxidation of the crude product, yielded 16-chloro-12-hexadecynoic acid (54%), which was esterified to the corresponding methyl ester by treatment with potassium carbonate and methyl iodide in dimethylformamide. Treatment of this acetylenic ester with ethyldiazoacetate in the presence of activated copper-bronze as catalyst followed by hydrolysis in KOH solution at room temperature yielded 16-chloro-12,13-(carboxymethylene)-12-hexadecenoic acid. This diacid was treated with excess oxalyl chloride to give the corresponding diacyl chloride, which was decarbonylated in a diethyl ether solution with zinc chloride, and the cyclopropenium ions thus formed were added at −40°C to a methanolic sodium hydroxide solution of sodium borohydride to give methyl 16-chloro-12,13-methylene-12-hexadecenoate. Analogous procedures were followed to prepare methyl [17-²H]10,11-methylene-10-hexadecenoate, methyl [17-²H]11,12-methylene-11-hexadecenoate and methyl [17-²H]12,13-methylene-12-hexadecenoate from the corresponding diacids using sodium borodeuteride in the reduction of the cyclopropenium ions. Alternatively, methyl [2,2,3,3-²H₄]hexadecynoate, prepared by reaction of methyl 2,11-hexadecadiynoate with magnesium in deuterated methanol at room temperature, was submitted to the above cyclopropenylation and reductive decarbonylation sequence to give methyl [2,2,3,3,17-²H₅]-11,12-methylene-11-hexadecenoate. In summary, complementary methods for the selective incorporation of one to five deuterium atoms into cyclopropene fatty acids, at different sites, in moderate to high yields have been developed. The methods should easily be applicable to the preparation of the corresponding tritiated analogues.     

Composition of triacylglycerols containing cyclopropene fatty acids in seed lipids of Munguba (Bombax munguba Mart.)

The seed lipids of Munguba (Bombax munguba Mart., Bombacaceae) were found to contain substantial proportions (ca. 30%) of cyclopropene acyl moieties (sterculoyl and malvaloyl). In a novel approach to the analysis of glycerolipids containing cyclopropene acyl moieties, the triacylglycerols of Munguba seed were first treated with silver nitrate in acetonitrile-acetone (1:1, v/v), in order to convert the cyclopropene groups to the correspondingα,β-unsaturated ketones. The resulting triacylglycerols were fractionated by thin layer chromatography into molecular species containing none, one and two keto (corresponding to none, one and two cyclopropene) acyl moieties per molecule. Each of these molecular species was further fractionated according to carbon numbers by gas chromatography, and the positional distribution of the acyl moieties in each species was determined. Both sterculoyl and malvaloyl moieties were found predominantly at thesn-2 position of the triacylglycerols. The major triacylglycerols of Munguba seed were found to be 1,3-dipalmitoyl-2-oleoylglycerol, 1,3-dipalmitoyl-2-linoleoylglycerol and 1,3-dipalmitoyl-2-sterculoylglycerol.     

Inhibition by cyclopropene fatty acids of the desaturation of stearic acid in hen liver

The mechanism of the hardening of body fats of animals by dietary lipids which contain cyclopropene fatty acids has been studied. Dietary methyl sterculate increased the stearic acid content of egg yolk lipid and decreased the activity of the stearic acid desaturase system of hen liver. The cyclopropene fatty acids were specific inhibitors of the stearic acid desaturase system of hen livers since other fatty acids, including two possible metabolites of sterculic acid, failed to inhibit the system at equivalent concentrations. Sterculic acid was a more effective inhibitor of the system than malvalic acid. Kinetic studies have shown that the inhibition is irreversible. Apparent kinetic constants were determined for the system. The results support the hypotheses that cyclopropene fatty acids inactivate an essential component of the desaturase system, probably by combination with-SH groups, and that this inhibition causes many of the effects of dietary cyclopropene fatty acids, including permeability disorders of eggs. Based on a paper presented at the AOCS Meeting, Philadelphia, October 1967.     

The common occurrence of furan fatty acids in plants

The observation that F-acids (1) occur in rat chow initiated a search for F-acids in human diet. We observed that the amount of F-acids with a pentyl side chain in α-position taken up with a one-day diet correlates well with the amount of excreted degradation products, the pentyl urofuran acids (2), (3) and (4). Therefore it can be concluded that F-acids with a pentyl side chain are not produced in the human body but are introduced through the diet. The origin of F-acids carrying an α-propyl side chain is less clear. The amount of propyl-urofuran acids (2) and (3) excreted in urine was found in one case out of three to be five times higher than the amount of F-acids carrying a propyl group in α-position taken up by the diet. Therefore, it can presently not be excluded that a portion of the propyl F-acids is produced by the body. F-acids found in human food are mainly introduced into the body by vegetables and fruits. F-acids were found also in birch leaves in considerable amounts, as well as in grasses, dandelion and clover leaves. Thus, we can conclude that F-acids are common constituents of plants.     

Effects of protected cyclopropene fatty acids on the composition of ruminant milk fat

Unsaturated fatty acids can be protected from ruminal hydrogenation, and, when fed to lactating ruminants, the constituent acids are incorporated into milk triacylglycerols. By this means, it has been possible to reduce the melting point of milk triglycerides and to make softer butter fat. This report shows that, by feeding small amounts of protected cyclopropene fatty acids, one is also able to make harder butter fat.Sterculia foetida seed oil, a rich source of cyclopropene fatty acids, was emulsified with casein and spray dried to yield a free flowing dry powder. When this material was treated with formaldehyde and fed to lactating goats (ca. 1 g cyclopropene fatty acids per day), there were substantial increases in the proportions of stearic acid and decreases in the proportions of oleic acid in milk fat. Similar results were obtained when the formaldehyde-treated supplements were fed to lactating cows (ca. 3 g cyclopropene fatty acids per day). The effect was considerably less apparent when theS. foetida seed oilcasein supplement was not treated with formaldehyde, suggesting that cyclopropene fatty acids are hydrogenated in the rumen as are other unsaturated fatty acids. The effect of feeding protected cyclopropene fatty acids on the stearic: oleic ratio in milk fat is probably due to cyclopropene-mediated inhibition of the mammary desaturase enzymes.     

Fatty acid desaturase systems of hen liver and their inhibition by cyclopropene fatty acids

Hen liver preparations which desaturate stearic acid at the 9,10 position to form oleic acid have been found to desaturate other saturated fatty acids of carbon chain length from 12 to 20 and 22. The 9,10-monoenoic fatty acid of the same chain length as the substrate fatty acid is the major product formed. Minor amounts of the 10,11- and 11, 12-monoenoic acids are also formed. Maximum desaturation occurred with the C₁₄ fatty acid substrate and with the fatty acids C₁₇ and C₁₈, suggesting the presence of at least two desaturating systems. The cyclopropene fatty acids, sterculic and malvalic acids, inhibited the desaturation of all thefatty acids at the 9,10 position but desaturation at the 10,11 and 11, 12 positions was affected only slightly. The effect is not due to inhibition of the primary activating enzyme, the long chain acyl CoA synthetase. Sterculic acid is a more effective inhibitor than either malvalic acid or sterculyl alcohol, probably because these cyclopropene compounds do not block the desaturating site of the enzyme as completely as sterculic acid.     

Homogeneously catalyzed hydrogenation and gas-liquid chromatographic analysis of the methyl esters of cyclopropene fatty acids

Various concentrations of cyclopropene fatty acids have been determined down to 0.2% by the use of gas liquid chromatographic (GLC) analysis of the methyl esters of fatty acids that have been quantitatively hydrogenated using a homogeneous transition metal complex catalyst. The effectiveness of the use of bromotris(triphenylphosphine)-rhodium(I), Br(P(C₆H₅)₃)₃Rh, as a homogeneous hydrogenation catalyst to convert the cyclopropene ring to a cyclopropane ring has been evaluated and compared with the analogous chloro- and iodo-complexes. The hydrogenation/GLC method of analysis has been compared with the method of titration with hydrogen bromide in benzene and with the method involving the use of high resolution nuclear magnetic resonance (NMR).     

Fat metabolism in higher plants XLIX fatty acid biosynthesis by subcellular fractions of higher plants

A method is described for the rapid and comprehensive subcellular fraction-ation of plant tissue using a combination of differential and discontinuous Ficoll gradient centrifugation. The procedure has been used to study the synthesis of fatty acids from acetate-1-¹⁴C or malonyl CoA-1,3-¹⁴C, by fractions of germinating pea and lupin seeds and developing avocado fruit, castor bean and safflower seeds. Particle free supernatants of seeds synthesize fatty acids from¹⁴C-malonyl CoA in the presence of added cofactors. Since acetyl CoA carboxylase activity is absent the utilization of¹⁴C-acetate by these fractions is minimal. Other particulate fractions show different activities depending on seed types. Active fractions include the low speed particulate of pea and lupin, the pea microsomes, the avocado mesocarp chloroplasts, and the fat fractions of castor bean and safflower. Individual fractions produce characteristic patterns of acids; especially noteworthy is oleic acid biosynthesis by soluble enzymes of castor bean and safflower from¹⁴C-malonyl CoA. Some characteristics of the avocado supernatant, pea supernatant, and castor bean fat fraction synthesizing systems are compared. As a result of these studies, generalizations derived from work with mammalian or bacterial systems cannot be applied to higher plants. Presented in part at the AOCS Meeting, Houston, May 1971.