HPLC Analysis of cyclopropenoid fatty acids

Cyclopropenoid fatty acid methyl esters have been analyzed by high pressure liquid chromatography (HPLC). The refractive index detector’s linear response to various lipids was studied. Verification of peak identity was by spectroscopy of collected peaks and cochromatography with authentic samples. The HPLC method is simple, convenient and gives precision and absolute, values which are consistent with those from traditional methods.     

Gas-liquid chromatographic analysis of cyclopropenoid fatty acids

A method is described for the analysis of cyclopropenoid fatty acids in oils. The method consists of reacting the methyl esters of the cyclopropenoid fatty acids with silver nitrate in methanol to form ether and ketone derivatives. The derivatives formed from the cyclopropenoid fatty acids are separated from the methyl esters of the normal fatty acids by gas-liquid chromatography on a 15% diethylene glycol succinate column. The method is applicable to oils containing from 0.01% to 100% of cyclopropenoid fatty acids. The derivatives of oils containing lew levels of cyclopropenoids are separated from the normal methyl esters by alumina chromatography prior to gas-liquid chromatography. Studies on the quantitative aspects of the derivative formation, alumina chromatography, and gas-liquid chromatography are reported. Analyses for total cyclopropenoid fatty acid content of cottonseed oil andSterculia foetida oil by the gas-liquid chromatographic and hydrobromic acid titration procedures showed good agreement. Replicate analyses of a sample ofSterculia foetida oil for malvalic and sterculic acid gave coefficients of variation of 6.04% and 1.17%, respectively.     

Variation of cyclopropenoid fatty acids in cottonseed lipids

The proportions of the cyclopropenoid fatty acids (CPA) esters, malvalate and sterculate, varied little in lipids from individual cottonseeds. Coefficients of variation were 10% and 20% for seeds from a lock and 13 varieities, respectively. Within the seed, variations in CPA concentrations were very large. Cyclopropenoid fatty acid concentration in the lipids decreased from 28% in the root tip to 2% in the top of the axis, and to 0.02% in the portion of the cotyledons nearest to the hull. The axial portion was only ca. 5% of the kernel, yet it contained 75% of the CPA. Distribution of dihydrosterculic acid, the precursor of CPA, was similar to that of CPA. High concentrations of CPA were found in immature seeds, root tip and radicle of germinated seeds, and root tips of cotton plants. Presented at the 73rd annual AOCS meeting, Toronto, Ontario, May 1982. One of the facilities of the Southern Region, Agricultural Research Service, U.S. Department of Agriculture. Names of companies or commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Removal of cyclopropenoid fatty acids from cottonseed meals by solvent extraction

Direct solvent-extraction procedures were explored for their effectiveness in removing the residual levels of cyclopropenoid fatty acids from commercial cottonseed meals. Of the seven solvent systems screened, a simple stepwise extraction with an acetone/hexane/water azeotrope was found suitable for the removal of up to 88% of the original CPA content of the meal.     

Hydrogenation of cyclopropenoid fatty acids occurring in cottonseed oil

The hydrogenation of cyclopropenoid acids and their relative reactivities during hydrogenation as compared to linoleic and oleic acids were examined. Pure methyl sterculate and purifiedSterculia foetida oil and its methyl esters, which have a cyclopropene content more than 60 times that of cottonseed oil, were used for the hydrogenation experiments. Nickel, palladium and platinum catalysts were used. The effect of temperature and type of catalyst were demonstrated in a series of hydrogenation experiments of safflower andS. foetida oil mixtures, and methyl oleate and methyl dihydrosterculate mixtures. Partial hydrogenation of methyl sterculate formed as many as twenty compounds in addition to the cyclopropenoid derivatives. Most of these compounds were monounsaturated. The cyclopropene group hydrogenated very readily compared to the 9,12-diene system in linoleate. The cyclopropane group obtained by hydrogenating the cyclopropenoid acids group was quite resistant to further attack by hydrogen and nickel catalyst had little effect. With palladium catalyst, a temperature of 180 C was necessary for the reaction to go to completion. Platinum in acetic acid was a good system for hydrogenolysis of the cyclopropane group at 80 C. Retired.     

Quantitative determination of cyclopropenoid fatty acids in cottonseed meal

A rapid analytical procedure for determining the residual cyclopropenoid fatty acids (CPA) in cottonseed meal has been developed. The procedure involves room-temperature extraction of crude CPA-containing lipids with a hexane-water-acetone azeotrope solvent, followed by a benzenemethanol wash. The crude lipids are then converted to methyl esters by methanolysis with sodium methoxide. Extraction with petroleum ether, followed by washing with aqueous acetone, results in a substance which is free from interfering materials. The purified methyl esters are then analyzed for CPA by a spectrophotometric modification of the Halphen reaction.     

Effects of cyclopropenoid fatty acids on fungal growth and lipid composition

Cyclopropenoid fatty acids (CPE) isolated fromSterculia foetida oil by urea clathration and reverse phase high performance liquid chromatography (HPLC) were introduced into fungal cultures. Stearate levels in phospholipids and triacylglycerols fromUstilago maydis sporidia rose considerably in response to 30 μM CPE. In addition, CPE themselves were incorporated into glycerolipid fractions. Sterol composition was unaffected. Changes in lipid composition were accompanied by inhibition of dry weight accumulation and sporidial number. Treated sporidia showed irregular wall deposition and a branched morphology. Oleate alleviated CPE effects on growth and morphology. Hyphal extension byRhizoctonia solani was inhibited somewhat by 30 μM sterculate, whileFusarium oxysporum showed no appreciable response. Although CPE appeared to inhibit fatty acid desaturation byF. oxysporum, gross increases in the proportion of stearate were limited to the triacylglycerol fraction during 30 μM treatments. The possibility that the CPE synthesized by plants serve as antifungal agents is discussed.     

Participation of cyclopropenoid fatty acids in the tortelli-Jaffe reaction with bromine

The Tortelli-Jaffe reaction of ditertiary double bonds with bromine in the presence of formic acid occurred with cyclopropenoid fatty acid-containing oils after prior bromination of other alkenes in the oil. The blue-colored (650 nm) cyclopropene-bromine complex appeared a basis for colorimetrically assaying cyclopropenoid fatty acids in vegetable oil.     

Determination of cyclopropenoid fatty acids inSterculia seed oils from senegal

Seed oils ofSterculia tomentosa andS. tragacantha (Sterculiaceae) were found to contain malvalic (5.8 and 5.1%), sterculic (11.3 and 30.2%) and dihydrosterculic (0.9 and 0.5%) acids. The total amount of these two cyclopropenoid fatty acids was established by¹H nuclear magnetic resonance and their cooccurrence by gas chromatography. Besides these unusual compounds, the main common fatty acids were palmitic (20 and 24%), oleic (21 and 15%) and linoleic (30 and 16%) acids.     

Chemical inactivation of cyclopropenoid fatty acids in cottonseed meals and their physiological evaluation

Chemical inactivation of cyclopropenoid fatty acids in commercial cottonseed meals was explored with three classes of compounds: anhydrous gases, organic acids and sulfhydryl compounds. Of the reagents screened, sulfur dioxide reduced the cyclopropenoid content by over 90% while free cottonseed fatty acids and thioglycollic acid reduced the cyclopropenoid fatty acid content by over 30%. Large batches of the above three selected meals, as well as a control commercial screw-pressed meal, were then incorporated at 20 wt % levels in the rations of laying hens. A negative control containing 25% soybean meal and a positive control containing a 2% refined cottonseed oil of known CPA content were also employed. During a four-week feeding period, eggs were collected during the third and fourth week and stored at 35 F for periods of 3 and 6 months. Overall egg quality and the fatty acid distribution of the yolk lipids were determined after the 3 and 6 months’ storage periods.     

Cassia siamea seed oil: A minor source of vernolic and cyclopropenoid fatty acids

Cassia siamea oil contains plamitic (19.0%), stearic (7.6%), oleic (11.6%), linoleic (42.7%), malvalic (2.0%), sterculic (3.1%) and vernolic (14.0%) acids.     

Methods for the determination of cyclopropenoid fatty acids. I. Aqueous hydrochloric acid method

An analytical method is described for the estimation of long-chain cyclopropenoid fatty acid derivatives. It is based upon the quantitative addition of a molecule of hydrogen chloride at the cyclopropene ring when the sample is shaken with concentrated hydrochloric acid. The cyclopropenoid content can be calculated, as sterculic acid, from the increase in chlorine content. Epoxy compounds and hydroperoxides interfere and must be removed by one of the accepted pretreatment methods. A laboratory of the So. Utiliz. Res. & Dev. Div., ARS, U.S.D.A.     

Lipoxygenase inhibition by naturally occurring monoenoic fatty acids

Lipoxygenase activity was not detected in preparations from several varieties of rapessed. Erucic acid, one of the main components of rapessed oil, competitively inhibited both soybean and peanut lipoxygenases. Various other long-chain monoenoic fatty acids were assayed for their effects on lipoxygenase activity with linoleic acid as substrate. Fatty acid chain length, from C₁₆, to C₂₄, was not a significant factor, but position of the point of unsaturation did affect enzyme activity. The position of the double bond from the carboxyl group seemed to be more significant than the distance between the point of unsaturation and the methyl terminal group in terms of inhibition.     

Methods for the determination of cyclopropenoid fatty acids. VI. A direct infrared absorption method

A simple rapid method for the estimation of the cyclopropenoid content of glycerides and methyl esters is described based upon the measurement of the characteristic infrared absorptivity of cyclopropenoids at 9.9 μ. Autoxidation products do not interfere, and the sample can be recovered. Equations are given for the calculation of the cyclopropenoid content of both glycerides and methyl esters. One of the laboratories of the So. Utiliz. Res. and Dev. Div. ARS, USDA.     

Effect of subacute toxic levels of dietary cyclopropenoid fatty acids upon membrane function and fatty acid composition in the rat

The effect of subacute toxicity levels of dietary cyclopropenoid fatty acids upon several physiological parameters was determined in the rat. Diets containing 2% corn oil, 2%Sterculia foetida oil or 2% hydrogenatedSterculia foetida oil were fed.Sterculia foetida oil (50% cyclopropenoid fatty acids) fed rats exhibited retarded growth, elevated organ to body wt ratios, increased saturation of tissue lipid, and abnormal histopathology when compared to corn oil and hydrogenatedSterculia foetida oil fed rats. Growth was retarded 50%, liver/body wt doubled, and the percentage of saturated fatty acids in adipose tissue increased 2.5-fold forSterculia foetida oil vs. corn oil comparisons. Three membrane systems were examined in corn oil andSterculia foetida oil fed rats. Erythrocyte hemolysis rate in 0.3 M glycerol was increased by 30%; induction of mitochondrial swelling by reduced glutathione was inhibited completely and microsomal codeine demethylase activity was depressed nearly 50% inSterculia foetida oil fed rats. The ability of cyclopropenoid fatty acids to inhibit fatty acyl desaturase and influence tissue and membrane lipid composition is discussed. Most of the detrimental effects observed in cyclopropenoid fatty acids fed rats may be associated with alteration of normal lipid metabolism and membrane function.     

Methods for the determination of cyclopropenoid fatty acids. VII. The dilution-HBr-titration technique as a general method

The stepwise HBr titration method for the cyclopropenoid analysis of cottonseed oils is subject to serious inaccuracies when applied to samples containing higher cyclopropenoid concentrations, particularly if they contain appreciable amounts of alumina-adsorbable materials. A modification of the method is described which eliminates these sources of error. Its validity has been established by the analysis of a wide range of synthetic compositions including compositions containing massive amounts of interfering HBr-reactive substances and other alumina-adsorbable materials. The method with further modification can be used to analyze glycerides with the same high degree of accuracy. Presented at the AOCS Meeting in Houston, April 1965. So. Utiliz. Res. Dev. Div., ARS, USDA.     

Alteration of liver microsomal proteins from rainbow trout (Salmo gairdneri) fed cyclopropenoid fatty acids

Rainbow trout were fed diets containing cyclopropenoid fatty acids (CPFA) at 50 and 300 ppm with appropriate controls fed CPFA-free diets. Treatment with CPFA altered the overall microsomal protein composition in a manner suggesting a reduction of high molecular weight components. One protein found in low concentration in controls appeared dominant in experimental animals, with the effect more pronounced as dietary levels of CPFA increased. The estimated molecular weight of this component was 41,500 daltons. Membrane fractions from CPFA-fed fish separated on a Bio-Gel P-150 column revealed a significant number of small molecular weight components that suggest degradation of microsomal proteins. These data suggest an alteration by CPFA of membrane protein composition.     

Characterization of cyclopropenoid acids in selected seed oils

Evidence is provided that sterculic and malvalic acids occur together in seed oils ofSterculia foetida, Hibiscus syriacus, andLavatera trimestris. Sterculia foetida oil contains 54.5% sterculic and 6.7% malvalic acids;Hibiscus syriacus oil contains 16.3% malvalic and 3.4% sterculic; andLavatera trimestris oil contains 7.7% malvalic and 0.6% sterculic acids.Hibiscus syriacus oil also contains 1.5% dihydrosterculic acid. The cyclopropenoid acids were characterized by hydrogenation in conjunction with gas-liquid chromatography and by oxidation to β-dioxo acids with subsequent cleavage with peracetic acid. Acetolysis of epoxides in the presence of cyclopropenes was effected by room temperature treatment with acetic acid-10% sulfuric acid (5∶2). This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.