Bonds Of Unsaturated Fatty Acids Research Articles

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

AbstractMethoxybromo 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.

Hydrazinolysis of lipids and analysis of their constituent fatty acids

Treatment of O-acyl lipids with hydrazine degrades them to hydrazides of the constituent fatty acids. These hydrazides react with acetone to yield less polar and more volatile N'-isopropylidenealkanohydrazides, R-CO-NH-N = C(CH3)2, which strongly absorb ultraviolet light and are amenable to thin-layer and gas-liquid chromatographic analyses. In presence of cyclopentene, reduction of double bonds in unsaturated fatty acids with hydrazine does not occur. The method of hydrazinolysis followed by acetonization has been used for fatty acid analysis of different classes of lipids.

Synthesis and biosynthetic utilization of radioactive photoreactive fatty acids.

A synthesis of a homologous series of highly radioactive photoreactive fatty acids has been developed. Oxidative cleavage of the cis double bonds of unsaturated fatty acids by NaIO4/OsO4 produced the omega-oxo fatty acids; subsequent reduction by [3H]NaBH4 afforded a series of highly radioactive [omega-3H]hydroxy fatty acids to which a variety of photoreactive probes can be readily attached. In this study, the m-diazirinophenoxy group was coupled to the omega-hydroxy groups of the fatty acids, yielding the omega-(m-diazirinophenoxy) derivatives of nonanoic, undecanoic, tridecanoic, and pentadecanoic acid. These photoreactive derivatives were incorporated by L-cells into their major phospholipids; in addition, a number of proteins were shown to be acylated by these fatty acids. The photoreactive group was shown to remain intact as indicated by the cross-linking of the resulting photoreactive phospholipids to other phospholipids as well as to a number of L-cell proteins upon photolysis. This procedure thus identifies integral membrane proteins and directly produces photoaffinity derivatives of those proteins normally acylated by fatty acids.

The quantitative determination of plasmalogen by its reaction with mercuric chloride.

The alk-1-enyl group of 1-alk-1(1)-enyl-2-acyl-glycerophospholipids (plasmalogens) rapidly combines with mercuric chloride. At 0 C, there was a 1:1 stoichiometry for Hg binding to the reactive enol group of plasmalogens. Aldehydes were not released, indicating that the alkenyl ether bond was not cleaved. Hg binding to less reactive double bonds in unsaturated fatty acids was not significant. Quantitative estimation of bound Hg afforded a rapid and sensitive assay for alkenylacyl lipids and gave values similar to those obtained with other methods of analysis. The proportion of plasmalogens in bovine myelin glycerophosphatides and in ethanolamine glycerophosphatide was 35 and 75%, respectively. Plasmalogens account for 23.3% of the total glycerophospholipid of rat erythrocytes.

Taxonomic significance of the position of double bonds of unsaturated fatty acids in Corynebacteria.

The positions of double bonds in cellular monounsaturated fatty acids isolated from six representative strains of corynebacteria were determined by mass fragmentography. Hexadecenoic acid from Corynebacterium glutamicum CNF 016 and Brevibacterium ammoniagenes CNF 096 was principally the Δ7 isomer, while octadecenoic acid from these organisms was the Δ9 isomer. The monounsaturated fatty acids in Corynebacterium diphtheriae CNF 017 and Corynebacterium xerosis CNF 010 consisted mainly of the Δ9 isomers. In Corynebacterium equi CNF 002 and Corynebacterium fascians CNF 006, hexadecenoic acid was a mixture of Δ9 and Δ10 isomers, while octadecenoic acid was composed principally of the Δ9 isomer. The results suggest that the profiles of double bond location in unsaturated fatty acids are very useful for the taxonomic characterization of corynebacteria.

Binding and mutagenicity studies with 3-nitrosofluorene on Salmonella typhimurium TA98 grown at different temperatures

The growth temperature of the histidine auxotroph Salmonella typhimurium TA98 influences the extent of binding of 2-nitrosofluorene (NOF) to this bacterium as well as the mutagenicity of this chemical carcinogen. Gas—liquid chromatography (GLC) of bacteria grown at 37, 27 and 17°C revealed that the unsaturated (hexadecenoic plus octadecenoic) fatty-acid content increased from 15.0% at 37°C to 20.5% at 27°C and to 22.2% at 17°C. It has been shown using both artificial and natural mammalian membranes that NOF adds to carbon-carbon double bonds in unsaturated fatty acids via an “Alder-ene” type reaction to produce a nitroxyl free radical adduct (N-Ȯ-LAF). The same adduct was formed when NOF was incubated with Salmonella typhimurium TA98. There was an increase in N-Ȯ-LAF formed in bacteria grown at 27°C as compared to those grown at 37°C, but very little formed in bacteria grown at 17°C. Therefore the amount of N-ȮLAF formed did not correlate with unsaturated fatty-acid content. 14C-labeled NOF uptake in bacteria grown at different temperatures showed a good correlation with mutagenicity data.

Practical aspects of liquid chromatography-mass spectrometry (LS-MS) of lipids

Techniques for coupling liquid chromatography with mass spectrometry are reviewed and an interface is described for the analysis of lipids by mass spectrometry. The interface for coupling liquid chromatography with mass spectrometry for lipid analysis is based on the moving wire transport principle using an endless stainless steel belt of novel construction. After evaporation of the solvent, the solute remains as a residue on the belt which transports it into a reactor where it is volatilized by evaporation or conversion to hydrocarbons. The volatile compounds are then fed into the source of a chemical ionization mass spectrometer for mass analysis by total or single ion monitoring as well as for structural identification or compositional analysis. The sensitivity of the system was approx. 1 mg per component separated in the eluate of a high efficiency column. The capabilities of the interface were demonstrated by its application to reference compounds representative of triglycerides, sterols, steryl esters, glyceryl ethers, glyceryl ether diesters, glycerophosphatides, sphingolipids, prostaglandins and fatty acid methyl esters. It also was applied to the analysis of methyl ester ozonides to demonstrate the use of LC-MS for the localization of double bonds in unsaturated fatty acids.

Chemical ionization mass spectrometry of polyunsaturated fatty acids of human serum.

Polyunsaturated fatty acids of human serum were analysed by combination gas-liquid chromatography and chemical ionization mass spectrometry, using isobutane as the reagent gas. The method described yielded the molecular ion of long chain unsaturated fatty acids as the base peak; these were weak or unobservable in the corresponding electron impact mass spectra. In the chemical ionization mass spectrometry, the derivatization with trimethylsilyl ether was demonstrated to be useful for the location of the double bonds of unsaturated fatty acids. Applied toteh serum polyunsaturated fatty acids, these methods have given identification of C18:4, C20:4, C20:5, C22:4, C22:5omega-6, C22:5omega-3, and C22:6.

Formation and disappearance of triglyceride droplets in strain L fibroblasts: An electron microscopic study

Formation of lipid droplets in strain L fibroblasts exposed to either oleic or linolenic acid for 5 min to 18 h was studied by ultrastructural morphometry and triglyceride analysis. Esterification of OsO 4 to double bonds of unsaturated fatty acids was used to characterize morphologically the resulting triglyceride droplets. Free cytoplasmic lipid droplets were formed within 5 min of adding exogenous, fatty acid. Of these, trilinolenin droplets were smaller (1.6 × 10 −2 μm 3) than triolein droplets (4.7 × 10 −2 μm 3) but by 18 h both types of droplets had a similar mean volume (46.5 × 10 −2 μm 3). Initial trilinolenin droplets stained dark grey. After 40 min of linolenic acid uptake, trilinolenin droplets stained dark grey. After 40 min of linolenic acid uptake, trilinolenin droplets stained black with OsO 4, corresponding to 2.8 μg trilinolenin per 10 6 cells. Triolein droplets stained pale grey throughout. Lipid droplets were bounded by a thin layer of osmiophilic material which lacked the bilaminar structure and was thinner than a unit membrane. Triolein droplets disappeared more rapidly than trilinolenin droplets after removal of exogenous fatty acids. A disappearance rate more rapid than could be accounted for by cell division, together with a diminution in mean droplet volume indicated the possible presence of a lipase. During the disappearance phase, lipid droplets remained free in the cytoplasm and were not associated with lysomes. By 48 h after removal of exogenous linolenic acid, trilinolenin droplets stained dark grey rather than black, corresponding to a trilinolenin content of 2.4 μg/10 6 cells. Strain L fibroblasts given the two fatty acids in sequence, formed droplets of intermediate staining intensity. All droplets within any given cell stained uniformly and to the same degree, indicating an exchange between droplet triglycerides and intracellular free fatty acids. Only after short sequential exposures to each fatty acid could two distinct populations of droplets be discerned within the same cell. However, this was a rare occurrence and more frequently all droplets were stained with the same intensity.

The position of the double bond in unsaturated fatty acids of a thermophilic [formula omitted] species

The predominant unsaturated fatty acid produced by a thermophilic Bacillus species has been identified as n -Δ 5-hexadecenoic acid. The position of the double bond in the branched-chain, monoenoic fatty acids produced by this organism is also between the fifth and sixth carbon atoms.

Factors governing the extent of overoxidation in permanganate oxidation of unsaturated fatty acids

AbstractThe extent of overoxidation of half‐esters of dicarboxylic acids is governed mainly by the nature and proportions of the oxidizing agent employed and not by reaction temperatures. In procedures which produce overoxidation, this occurs mainly at the time of the scission of the double bonds and no method is known to prevent it. The acetone permanganate procedure overoxidizes monocarboxylic acids (MCA), dicarboxylic acids (DCA) and half esters of DCA. The acetic acid‐acetone permanganate procedure overoxidizes DCA but not MCA half‐esters of DCA or azelaoglycerides. The DCA corresponding to the first double bond in unsaturated fatty acids (UFA) can be isolated quantitatively if the esters or triglycerides are oxidized by the latter procedure.

A simple, rapid micromethod for the determination of the structure of unsaturated fatty acids via ozonolysis

A micromethod for the localization of double bonds in unsaturated fatty acids via ozonolysis employing pyrolytic cleavage of ozonides in the presence of a hydrogenation catalyst is described. Cleavage of the ozonides is carried out in a gasliquid chromatographie instrument in a small glass tube, containing the catalyst, inserted in the top of the column opposite the in input heaters at 225C. Ozonides of methyl esters of straight chain unsaturated fatty acids are cleaved through the action of the catalyst to aldehyde fragments which are swept simultaneously into the column for analysis.The double bond positions are deduced from the chain length of the fragments. The method is demonstrated on methyl oleate, linoleate, linolenate and arachidonate.

A rapid procedure for locating double bonds in unsaturated fatty acids

We have presented a rapid method for determining the locations of the double bonds in unsaturated fatty acids. Fatty acid esters are oxidized with permanganate to form new fatty acids and dicarboxylic acids, and these products are analyzed by GLC. The major components of a mixture of isomers can be quickly identified, and their approximate proportions measured.

Lipolysis in the rumen.

REISER,1, and more recently Shorland et al. 2, reported experiments in which rumen contents of sheep were incubated in vitro with linseed oil and with oleic, linoleic and linolenic acids. These experiments, and others using the living animal3–5, demonstrated that hydrogenation of double bonds of unsaturated fatty acids occurred and that this was effected by the rumen micro-organisms.

Periodate‐permanganate oxidations. IV. Determination of the position of double bonds in unsaturated fatty acids and esters

SummaryThe reaction of the periodate‐permanganate reagent with unsaturated fatty acids, including oleic, elaidic, eicosenoic, 10‐undecenoic, and linoleic acids proceeded smoothly to give the expected end‐products in quantitative yields. The latter were conveniently isolated and determined by partition chromtography on silicic acid columns in 10 to 100 mgm. quantities.Some compounds, which were unreactive due to insolubility in the reagent, gave satisfactory results when the media contained 5 to 10% of pyridine. This was true also for methyl linoleate even though solution was incomplete. However methyl oleate and triolein did not give quantitative results even in media containing 20% or more of pyridine in which the esters were completely dissolved.