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.     

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.     

Methods for the determination of cyclopropenoid fatty acids: VIII. The HBr titration method applied to small samples

A method is described for the determination of cyclopropenoid fatty acids in small samples of refined and crude glyceride oils and methyl esters. Approximately 0.5 g of cottonseed oil is titrated with HBr-HOAc by a stepwise procedure at 3 C and 55 C after removal of interfering substances by adsorption on activated alumina. Side reactions occurring to the extent of 15% during the titration were uniform and highly reproducible, permitting application of a correction factor. A procedure is given for preparing methyl esters on a small scale to determine the cyclopropenoid content of highly oxidized oils. The So. Utiliz. Res. Dev. Div., ARS, USDA.     

Methods for the determination of cyclopropenoid fatty acids V. A spectrophotometric method for cottonseed oils based upon the Halphen-test reaction

A spectrophotometric method of analysis for the quantitative estimation of cyclopropenoid fatty acids in cottonseed oil based upon the Halphen-test reaction has been described. Various parameters involved in the reaction have been investigated and two pigment fractions responsible for the characteristic Halphen-test cherry-red color have been isolated. The method is applicable to relatively small amts of sample material. The average deviation from the actual cyclopropenoid acid contents as determined by the stepwise HBr titration method was less than ±0.02% in both the refined and crude oil series. S. Utiliz. Res. Dev. Div., ARS, USDA.     

Methods for the determination of cyclopropenoid fatty acids. II. A stepwise hydrogen bromide titration method for cyclopropenid and epoxy derivatives

A rapid titration method is described for the quantitative determination of both cyclopropenoid and epoxy fatty acid derivatives in mixtures. It was found that epoxy compounds can be titrated selectively with Durbetaki reagent at 3C without interference from cyclopropenoid derivatives. Cyclopropenoid derivatives can be titrated much more rapidly to a much sharper end point a 55C that at room temperature. Thus mixtures can be analyzed by first titrating at 3C to determine the epoxy compounds and then continuing the titration at 55C to determine the cyclopropenoid components. A laboratory of the So. Utiliz. Res. & Dev. Div., ARS, U.S.D.A.     

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.     

Methods for the determination of cyclopropenoid fatty acids. IV. Application of the step-wise HBr titration method to the analysis of refined and crude cottonseed oils

A method is described for the determination of cyclopropenoid fatty acids in refined and crude cottonseed oils to within 0.01%. It is based upon a stepwise hydrogen bromide titration at 3C and 55C after removal of interfering substances by adsorption on activated alumina. Highly oxidized cottonseed oils first must be converted to methyl esters. Presented at AOCS Meeting in Minneapolis, 1963. So. Utiliz. Res. and Dev. Div., ARS, USDA.     

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.     

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.     

Studies on the inhibition of the desaturases by cyclopropenoid fatty acids

Unwashed rat liver microsomes were used to study the inhibition of the Δ⁶ and Δ⁹ desaturases by cyclopropenoid fatty acids with the ring structure about the 9,10 or 6,7 carbon atoms. The 9,10 cyclopropenoid acid (sterculic acid) is shown to be an effective inhibitor of only Δ⁹ desaturase and then only in the presence of MgCl₂ and coenzyme A (prepresence of MgCl₂ and coenzyme A (presumably due to the formation of sterculoyl-CoA). Two 6,7 cyclopropenoid acids of different chain lengths showed no marked inhibition of either the Δ⁶ or Δ⁹ desaturase. By the use of [³H]-sterculic acid, it has been shown that under conditions of high inhibition of the Δ⁹ desaturase the inhibitor is not covalently attached to the enzyme at any point. This disproves older ideas on the mechanism of inhibition that assumed reaction between the cyclopropenoid ring and sulphydryl groups on the enzymes. Cyclopropenoid fatty acids are named according to the number of carbon atoms in the chain; thus sterculic acid is a C₁₈ cyclopropenoid fatty acid.     

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.     

An automatized method for determination of free fatty acids

An automated colorimetric method is described for determining free fatty acids (FFA) in vegetable oils using the flow injection analysis (FIA) technique. In this procedure, an almost linear relationship exists between the peak height and the FFA concentration. Liquid samples can be poured directly into the sample cups on the sampler for an automatic analysis of the FFA content. The dynamic range of this method is from 0.01 to almost 5%. Samples with higher FFA content must be diluted before analysis. The sample capacity is 12–20 injections/hr. No evidence of the existence of the earlier proposed cage-like complex (Cu(II)(FFA)₂)₂ in the organic phase was observed in this study.     

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.     

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.     

A crystallization method for the determination of saturated fatty acids in soybean oil

Summary A crystallization method has been developed for the determination of saturated fatty acids in soybean oil. The method is simple and rapid. The results obtained by this method agree quite well with those obtained by the use of the Bertram oxidation method and are about 2 percent higher than those obtained by the use of the modified Twitchell method. The procedure recommended seems to give satisfactory results on soybean and cottonseed oils, but it will probably have to be modified for use with highly unsaturated oils such as perilla, or with oils such as olive which contain large amounts of oleic acid. A cooperative organization participated in by the Bureaus of Agricultural Chemistry and Engineering and Plant Industry of the U. S. Department of Agriculture, and the Agricultural Experiment Stations of the North Central States of Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, Ohio, South Dakota, and Wisconsin.     

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.