Mass spectrometric location of double bonds in unsaturated fatty acids including conjugated acids as their methoxybromo derivatives

Methoxybromo derivatives of unsaturated fatty acids including conjugated acids yield simple mass spectra and can be used to locate the position of double bonds in these acids. The derivatives are prepared under mild conditions by bromination of the unsaturated fatty acids in methanol. The method is illustrated with the methoxybromo derivatives from methyl esters of oleic, petroselenic, erucic, undecenoic, linoleic, linolenic, the conjugated diene acids from dehydrated castor oil, α-eleostearic, punicic and parinaric acids. Unlike other methods using methoxy derivatives, the methoxybromo derivatives yield fewer ions, the diagnostic peaks forming the most intense ions of the spectra. While unambiguous double bond location is possible with monoenoic acids and conjugated fatty acids, only the end carbon atoms of the unsaturation system in nonconjugated polyenoic acids is located. But the characteristic appearance of fragments corresponding to [CH₃(CH₂)_nCH(OMe)CH(Br)CH₂−2H]⁺ and fragments 24 mass units higher than those which locate the end carbons in such fatty acids indicate a methylene-interrupted system of double bonds.     

Unusual gas chromatographic properties of fatty acid pyrrolidides

Fatty acid pyrrolidides have been found to have unusual gas chromatographic properties on a polar stationary phase of the Carbowax type. In particular, exceptional resolution of positional isomers was achieved, and in contrast to methyl esters, unsaturated isomers with double bonds in position 5 or earlier eluted before the corresponding saturated fatty acid derivative.     

Quantitative determination of double bond positions in unsaturated fatty acids after oxidative cleavage

The position and amt of unsaturation in fatty acids have been determined, especially in pure fractions of partially hydrogenated fats. In developing a quantitative method for determination of ethylenic bonds in monounsaturated and polyunsaturated fatty acids several procedures were combined. Key features include oxidative cleavage; recovery of cleaved acids as salts; and their conversion to methyl, ethyl or butyl esters for programmed gas-liquid chromatographic analysis. Monobasic analyses closely agree with the corresponding dibasic analyses, except neither malonic nor propionic acid has been quantitatively estimated. Analyses are shown for cleavage of high purity oleic, linoleic and linolenic acids; forcis andtrans monoenates; and for conjugated and nonconjugated dienoates. Demonstrated are the accuracy, precision and applicability of the procedure to a wide range of pure fractions isolated after both heterogeneous and homogeneous partial catalytic hydrogenation of polyunsaturated fatty acids. Presented at AOCS Meeting in New Orleans, 1964. A laboratory of the No. Utiliz. Res. & Dev. Div., ARS, USDA.     

Mass spectral studies of deuterium-labelled picolinyl fatty esters in the determination of double-bond positions

Deuteration (with Wilkinson’s catalyst) of methyl 12:1(3c), 14:1(5c), 18:1(9c), 18:2(9c,12c), 18:3(9c,12c,15c), 20:4(5c,11c,14c,17c), 20:5(5c,8c,11c,14c,17c) and 22:6(4c,7c,10c,13c,16c,19c) fatty acids gave the corresponding deuterium-labelled saturated fatty esters. The mass spectral analysis of their picolinyl esters gave clear diagnostic ion fragments, which permitted the facile and accurate determination of the positions of the double bonds (up to six) in the alkyl chain of the fatty ester.     

Neonatal polyunsaturated fatty acid metabolism

The importance of n−6 and n−3 polyunsaturated fatty acids (PUFA) in neonatal development, particularly with respect to the developing brain and retina, is well known. This review combines recent information from basic science and clinical studies to highlight recent advances in knowledge on PUFA metabolism and areas where research is still needed on infant n−6 and n−3 fatty acid requirements. Animal, cell culture, and infant studies are consistent in demonstrating that synthesis of 22∶6n−3 involves C24 PUFA and that the amounts of 18∶2n−6 and 18∶3n−3 influence PUFA metabolism. Studies to show that addition of n−6 fatty acids beyond Δ6-desaturase alters n−6 fatty acid metabolism with no marked increase in tissue 20∶4n−6 illustrate the limitations of analyses of tissue fatty acid compositions as an approach to study the effects of diet on fatty acid metabolism. New information to show highly selective pathways for n−6 and n−3 fatty acid uptake in brain, and efficient path-ways for conservation of 22∶6n−3 in retina emphasizes the differences in PUFA metabolism among different tissues and the unique features which allow the brain and retina to accumulate and maintain high concentrations of n−3 fatty acids. Further elucidation of the Δ6-desaturases involved in 24∶5n−6 and 22∶6n−3 synthesis; the regulation of fatty acid movement between the endoplasmic reticulum and peroxisomes; partitioning to acylation, desaturation and oxidation; and the effects of dietary and hormonal factors on these pathways is needed for greater understanding of neonatal PUFA metabolism.     

Determination of the specific positions ofcis andtrans double bonds in polyenes

A method is described for the determination of the positions and geometric configurations of double bonds in polyunsaturated fatty acids. The procedure consists of three steps: 1) Partial reduction of the double bonds with hydrazine under conditions which give high yields of monoenes. 2) Isolation of thecis- and thetrans-monoene fractions by thin-layer chromatography (TLC) directly or in the form of their ozonide derivatives. In the former technique, selective argentation is employed, in the latter, silicic acid adsorption. 3) Determination of the structure of the monoenes via reductive ozonolysis. The position of the double bonds is determined from the structures of the monoenes. Since thecis-monoenes are separated from thetrans-monoenes the geometric configuration of each double bond is determined.The method also provides a direct determination of the spacings of the internal double bonds and it may be employed for the determination of the structures of mixtures of fatty acids in conjunction with direct ozonolysis procedures. The various ramifications of the method are demonstrated on pure fatty acids and model mixtures thereof.     

Isomerization of the double bonds of a conjugated fatty acid during β-oxidation

The β-oxidation of an unsaturated fatty acid containing conjugated double bonds at odd-numbered carbon atoms has not previously been studied. It is, therefore, not clear whether, during the β-oxidation of such an acid, the double bonds will be isomerized by enoyl-CoA isomerase (Δ³-Δ²-enoyl-CoA isomerase) with the loss or retention of its conjugated nature. To investigate the problem, (E,E)-3,5-octadienoyl-CoA was synthesized for use as a model substrate, and enoyl-CoA isomerase was partially purified from bovine liver. The isomerization was followed by spectrophotometric and gas liquid chromatographic methods, and the results suggested that the isomerization of the model substrate proceeded with retention of a conjugated double bond system. It is, therefore, proposed that the β-oxidation intermediate of α-eleostearic acid (Δ^9,11,13 fatty acid) will also isomerize with retention of the conjugated double bond system. Part of doctoral thesis submitted to University of Pretoria, Pretoria, 0001, Republic of South Africa.     

Displacement of the double bonds during hydrogenation of unsaturated fatty acid methyl esters

Summary The displacement of the double bond of several unsaturated fatty acid methyl esters during hydrogenation with a nickel-kieselguhr catalyst at 180°C. was investigated. The analysis of the dicarboxylic acids (obtained by oxidation of the reaction products with KMnO₄ in acetic acid solution) by means of partition chromatography enabled a reliable semiquantitative determination of the position isomers formed. During hydrogenation of methyl esters of oleic, elaidic, petroselinic, and linoleic acid formation of large amounts of position isomers was proved to occur. Migration of the double bonds in both directions took place but was in all cases strongly pronounced in a direction opposite the ester group. The place and configuration (cis or trans) of the double bonds in the starting material apparently were of little importance in this respect. It follows that hydrogenation of fatty acid esters leads to products which are far more complicated, as is generally known. This is especially of importance with respect to the application of hydrogenated fatty oils in the food industries.     

Pyrrolidides for mass spectrometric determination of the position of the double bond in monounsaturated fatty acids

Mass spectra of pyrrolidides of monounsaturated straight chain fatty acids are presented and discussed. The spectra of pyrrolidides contain mainly ions from the polar part of the molecule. This gives simple spectra from which double bond positions can be deduced directly. If an interval of 12 atomic mass units is observed between the most intense peaks of clusters of fragments containing n and n−1 carbon atoms of the acid moiety, the double bond occurs between carbons n and n+1 in the molecule This rule is valid for double bonds occurring at positions Δ⁵-Δ¹⁵ in an 18-carbon chain and has been applied to acids having 10–24 carbon atoms.     

Geometrical isomerisation of double bonds in acid‐catalysed preparation of fatty acid methyl esters

Acid‐catalysed methylation is frequently applied for the preparation of fatty acid methyl esters used for gas chromatographic analysis of fatty acids. A series of artefacts were observed in hydrochloric acid‐catalysed direct methylation of herring (Clupea harengus L.) muscle. The artefacts were identified as trans isomers of eicosapentaenoic and docosahexaenoic acid, and their levels increased with reaction time. The isomers were not found after methylation of a lipid extract of the herring muscle, even after extreme reaction times. In general, the trans isomers are only observed after methylation of certain marine tissues, indicating catalytic activity in these samples. Based on these results, it is recommended that direct methylation procedures are thoroughly validated with each matrix type analysed, and that reaction times should not be longer than necessary to complete the methylation.     

Localization of fatty acid double bonds by gas chromatography of intermediate aldehydes as 1,3-dioxane

A semi-micro quantitative method has been developed for the determination of double bond positions in unsaturated fatty acid methyl esters differing in position, number of double bonds and chain length. This method involves ozonolysis in methylene chloride in the presence of 1,3-propanediol, reduction of the hydroperoxides with dimethyl sulfide and conversion of aldehyde and aldester fragments to stable dioxanes, followed by gas chromatographic analysis under programmed temperature. Propanediol acts as a proton donor for the generation of hydroperoxides as well as a reactant for the formation of dioxanes. All the steps are carried out in a culture tube without any transfers. The absence of unoxidized ester and unreduced hydroperoxide or ozonide has been shown by thin layer chromatography. The method has been tested with pure esters and their binary mixtures. Experimental values for mole percentage composition in the mixtures agreed with the actual values. This method enabled us to isolate and estimate malondialdehyde for the first time as a stable bis-dioxane derivative.     

Comparison of pyrrolidides with other amides for mass spectral determination of structure of unsaturated fatty acids

Amides of unsaturated fatty acids give mass spectra which indicate the locations of the double bonds. A survey of several amides of oleic acid was made to evaluate which amide might be most suitable for routine use in elucidation of structure. Methods of preparation of several amides of oleic acid are presented with mass spectral and gas chromatographic data. Tertiary amides have most easily interpretable mass psectra which indicate the position of the double bond in the fatty acid chain. The pyrrolidide is advantageous for fatty acid gas liquid chromatography and mass spectrometry analyses because it can be prepared easily in high yield on a microscale, it is volatile enough for gas liquid chromatographic separations, and it has a simple and easily interpretable mass spectrum which indicates the structure.     

Reduction with zinc of triple bonds tocis double bonds in long chain conjugated fatty acids

The methyl ester of santalbic acid (trans-11-octadecen-9-ynoic acid) was hydrogenated with powdered zinc in 50% propanol to yield 90% of thecis,trans-diene ester; similarly, 7,9-octadecadiynoic methyl ester was converted tocis,cis-diene ester but yields were no better than 75%. Presented in part at the AOCS Meeting, Chicago, October 1967. East. Market. Nutr. Res. Div., ARS, USDA.     

The cardiac antiarrhythmic effects of polyunsaturated fatty acid

Each year in the United States alone some 250,000 persons die within one hour of an acute myocardial infarction. These deaths are largely due to ischemia-induced ventricular arrhythmias, primarily ventricular fibrillation (VF). Thus a safe, simple means of preventing such arrhythmias has considerable public health benefit potential. We have demonstrated that the intravenous infusion of n-3 polyunsaturated fatty acids (PUFA) from fish oils will prevent ischemia-induced VF in prepared, nonanesthetized, exercising dogs, confirming earlier feeding studies in rats. We show that this protective effect is due to an action of the free acidic form of the PUFA to alter the electrophysiology of individual cardiac myocyte so that the cells are electrically more stable. The electrophysiologic effects, in turn, result from direct and specific effects of the PUFA to block the fast voltage-dependent sodium channels. The binding of the free fatty acids is directly to the protein of the sodium channels and results in prolongation of the inactivated state of these channels. Other ion channels are also affected by the PUFA. Two clinical trials with n-3 PUFA are mentioned which inadvertently support the antiarrhythmic potential of PUFA ingestion.     

Mass spectrometric localization of methyl branching in fatty acids using acylpyrrolidines

Localization of a methyl branch in a fatty acid molecule by mass spectrometry is facilitated by using the pyrrolidide rather than the methyl ester. Branched fatty acid methyl esters are converted to pyrrolidides and are than analyzed by gas chromatography and mass spectrometry. The diagnostic fragments indicate position of the methyl branch.     

Spectrophotometric determination of small proportions of polyunsaturated constituents in fatty materials

Summary Existing ultraviolet spectrophotometric methods have been modified for application primarily to the detection and estimation of low proportions of conjugated and nonconjugated unsaturated constituents in fats, oils, and soaps. The method is applicable also to fatty materials having high proportions of these constituents. Modifications include corrections for absorption by interfering substances, use of alkaline glycerol as an isomerization medium in the analytical procedure, and correction of absorption data on the isomerized product for absorption by conjugated constituents in the material before isomerization. The presence of small proportions of highly unsaturated conjugated and nonconjugated compounds is established in lards, tallows, tallow soaps, and highly purified esters and acids. Tall oil fatty acids are shown to contain approximately 10% of conjugated diene acids and a small amount of linolenic acid. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, United States Department of Agriculture.     

Calcium deficiency modifies polyunsaturated fatty acid metabolism in growing rats

Fatty acid desaturase activities were determined in liver microsomes from calcium-deficient rats and compared to calcium-sufficient ones. The calcium-deprived diet (0.5 g/kg) administered for 60 d caused a 30% inhibition in the Δ5 desaturase activity and a 45–55% decrease in Δ6 and Δ9, respectively, facts that cannot be attributed to a reduction in food intake. In vitro addition of calcium, ethyleneglycol-bis(β-aminoethyl ether)N,N-tetraacetic acid, and/or cytosol fractions from control or calcium-deficient rats to microsomes from both groups of animals indicates that the reduced desaturase capacities would be the consequence of an indirect effect of calcium deprivation. The present work shows that the reduced unsaturated fatty acid biosynthesis might be the result of modifications in the physicochemical properties of microsomal membranes. Such changes could also be derived from the inhibition of phospholipase A₂ activity induced by calcium deficiency.     

Separation of fatty acid methyl esters on the basis of the number of double bonds using alumina plates

The separation of fatty acid methyl esters (FAME) on the basis of the number of double bonds present in the molecule was accom-plished using thin layer chromatography (TLC) on alumina. Hexane was used as the solvent in a continuous development mode using modified tanks. The separations were verified by gas liquid chroma-tography (GLC) and retention of structural integrity was confirmed by infrared analysis fortrans isomerization and by ozonolysis-GLC for double bond position. Presented at the 72nd AOCS annual meeting, New Orleans, 1981.