Isolation and characterization of the monounsaturated long chain fatty acids ofMycobacterium tuberculosis

The positions of double bond in the monounsaturated C₁₅−C₃₂ fatty acids ofMycobacterium tuberculosis H37Ra were established by gas chromatography/mass spectrometry of the ozonized esters and their pyrrolidide derivatives. The monounsaturated C₁₅−C₂₁ fatty acids had the double bond primarily at the Δ⁹ position while the monounsaturated longer chain fatty acids (C₂₂−C₃₂) had the double bond in several positions. Many of the latter acids, especially the odd-numbered series, were very complex isomeric mixtures. Quantitation showed the most abundant even-numbered long chain fatty acid isomers to be as follow: C₂₂, Δ⁴; C₂₄, Δ⁵; C₂₆, Δ⁷ and Δ⁹; C₂₈, Δ⁹; C₃₀, Δ¹¹ and Δ¹³; C₃₂, Δ¹³ and Δ¹⁵.     

Hydrogenation of a menhaden oil: II. Formation and evolution of the C20 dienoic and trienoic fatty acids as a function of the degree of hydrogenation

To complement studies on monoethylenic fatty acids produced from the major polyunsaturated fatty acid (20:5A5,8,11,14,17) during hydrogenation of a menhaden oil of iodine value (IV) 159, the C₁₀ dienoic and trienoic fatty acid isomers of partially hydro-genated menhaden oils (PHMO) of IV 131.5, 96.5 and 85.5 were isolated by a combination of preparative gas liquid chromatography (GLC), mercuric adduct fractionation, and silver nitrate thin layer chromatography (AgNO₃-TLC). The 20:2 fatty acid methyl esters of the three PHMO samples were transformed to the corresponding alcohols and ozonized in BF₃-MeOH, followed by GLC analysis of the ozonolysis fragments. During the hydrogenation process, re-sidual ethylenic bonds in the 20:2 isomers tend to migrate both towards the carboxyl group and towards the methyl end of the molecule. The hydrazine reaction results revealed that thetrans ethylenic bonds in the 20:2 and 20:3 isomers were distributed all along the the carbon chain, but thecis ethylenic bonds were more localized in the Δ11,Δ14 and Δ17 positions of the preexisting major menhaden oil component 20:5Δ5,8,11,14,17. Iatroscan analyses on AgNO₃-chromarods revealed that, as a result of the hydrogenation process, almost half of the 20:2 isomers were non-methylene-interruptedcis, trans/trans, cis structures. Presented in part at the 73rd annual AOCS meeting, Toronto, 1982.     

The monounsaturated acyl- and alkyl-moieties of wax esters and their distribution in commercial orange roughy (Hoplostethus atlanticus) oil

Wax esters were isolated from commercial orange roughy (Hoplostethus atlanticus) oil by column chromatography and fractionated by argentation thin layer chromatography. Following transesterification, the resultant fatty acid methyl esters and fatty alcohols were analyzed by gas chromatography. both acyl- and alkyl-moieties were mainly of the monoene structure within the 16∶1–22∶1 range. After derivatization, the positions of the double bonds of even numbered fatty acid and fatty alcohol isomers were located by chromatography-mass spectrometry and compared. Results of these positional analyses indicate that the primary desaturation reactions takes place in the Δ9 position of pre-existing (C₁₄ to C₂₄) acyl chains. It is proposed that acyl components from 18∶1 are subjected to chain elongation to form a mixture of 24∶1 isomers as the final product. Apart from the 24∶1 acyl moiety of the wax esters, in which the double bond was almost exclusively in the Δ15 position, de novo biosynthetic reactions on acids and alcohols appear to yield related acyl- and alkyl-moieties of resynthesized wax esters.     

Characterization of unsaturated fatty acids by gas-liquid chromatography

The equivalent chain length (ECL) has been determined on 79 methyl esters of unsaturated fatty acids and on 7 ethyl esters by gas chromatography. Ethylene glycol succinate (EGS), diethylene glycol succinate, β-cyclodextrin acetate and Apiezon L were chosen as the liquid phases to be used. For methyl esters of mono- and polyenoic acids, the differences between ECL on EGS and ECL on Apiezon L approximate 0.84 per double bond. For positional isomers, the ECL on both EGS and Apiezon L are usually greater for the isomer having the longer proximal end of the molecule (smallest ω value). In these terms a triple bond is approximately equal to three double bonds. Esters of nonconjugated dienoic and trienoic acids of the same chain length are not separable on Apiezon L if their proximal structures are the same. This also applies to tetraenoic and pentaenoic acids of the same chain length and the same proximal structure. Conjugation of double bonds, either with the ester carbonyl group or with themselves, yields ECL values on Apiezon L greater than the number of carbon atoms in the acid. Monounsaturated and nonconjugated polyunsaturated esters have ECL values on Apiezon L lower than the number of carbon atoms of the acid. The ECL values of ethyl esters of 18 and 20 carbon acids are greater than the corresponding methyl esters on Apiezon L. Presented at the Chicago meeting of the American Oil Chemists’ Society, Oct. 13, 1964.     

Monoenoic fatty acids in human brain lipids: Isomer identification and distribution

The carbon chain length distribution and the double bond positional isomer composition of the monoenoic fatty acids of the lipids of total human brain tissue have been determined using gas chromatography and gas chromatography/mass spectrometry of the fatty acid methyl and picolinyl esters. The even chain length monoenoic C₁₆ to C₂₈ fatty acids contain predominantly two positional isomer series, the n−7 and n−9cis homologues, whose relative proportion varies significantly with chain length. The odd chain length long-chain fatty acids consist of n−8 and n−10 isomers, whereas the odd chain length very long-chain (more than 22 carbon) fatty acids are n−7 and n−9 isomers.     

Studies on the mechanism of the hydrazine reduction reaction: Applications to selected monoethylenic,diethylenic and triethylenic fatty acids ofcis configurations

The hydrazine reduction ofcis ethylenic bonds has been studied using a number of C₁₆, C₁₈, and C₂₀ monoethylenic, C₁₈ diethylenic, and C₁₈ plus C₂₀ triethylenic fatty acids. Relative reduction rates were determined and correlated with the positions of the ethylenic bonds in the fatty acids. The terminal ethylenic bond was the most reactive and among the others, reactivity was greater nearer the ends of the chain than in the center. Also, the ethylenic bonds closer to the carboxyl group showed greater reactivity than those closer to the methyl end of the chain. Differences in reactivity among the centrally located double bonds were not significant. These reactivity trends are consistent with a mechanism involving protonated diimide as an intermediate in the reduction process. In diethylenic fatty acids, the non-methylene-interrupted dienes showed slightly lower reactivity than the methylene-interrupted counterparts, which indicated that both the position of ethylenic bonds and the relative distance between two ethylenic bonds in the diene influence the rate of reaction. The information obtained from the rate studies should be useful for predicting the product distribution from partial hydrazine reductions of long-chain polyethylenic compounds.     

Column types for the chromatographic analysis of oleochemicals

Chromatography has developed into one of the principle methods of analysis of oleochemicals. Gas chromatography has been used extensively for the analysis of long-chain fatty acids as well as for the analysis of triglycerides and plant sterols. In recent years, high pressure liquid chromatography (HPLC) has been used for the analysis of triglycerides as well as for other related materials. Specialized gas chromatography columns have been developed for the separation of long-chain fatty acids such as the methyl esters. These columns have generally used high polarity stationary phases which separate fatty acids by degree of unsaturation. A specialized use of these high polarity stationary phases is separation ofcis-trans isomers as well ascis-cis andtrans-trans isomers. In this paper, packed and capillary columns are compared for the separation of thecis-trans isomers of fatty acid methyl esters prepared from a hydrogenated vegetable oil. For HPLC separations, the presence of a double bond is approximately equivalent chromatographically to shortening the alkyl chain by two carbons. The long-chain polyenic acids or ethyl esters thus elute near but are resolved from the short-chain saturated fatty acids or esters. HPLC is the method of choice for relatively complex, high molecular weight, or labile esters, such as those of retinyl or cholesterol. Glyceryl esters are particularly well resolved by HPLC in terms of both total chain length and degree of unsaturation. This technique is also useful for lipid class separations and for the analysis of modified fatty acid products, such as prostaglandins and related materials. In general, these analyses are conducted with octadecyl bonded phase column packings.     

Melting points of synthetic wax esters

Saturated, monoenoic and dienoic wax esters, C₂₆−C₄₀, have been synthesized from even-numbered fatty alcohols and acids. In homologous series of saturated esters, the increments of melting points follow a regular trend except for those esters which have an acid moiety two carbon atoms shorter than the alcohol moiety. These wax esters have melting points higher than interpolation would predict. Monoenoic wax esters with the double bond in the alcohol chain have melting points about 10 C higher than their isomers with the double bond in the acid chain.     

Effects ofbis homoallylic and homoallylic hydroxyl substitution on the olefinic13C resonance shifts in fatty acid methyl esters

Substitution of a hydroxyl group at thebis homoallylic position (OH group located three carbons away from the olefinic carbon) in C₁₈ unsaturated fatty acid esters (FAE) induces a 0.73±0.05 ppm upfield and a 0.73±0.06 ppm downfield shift on the δ and ε olefinic¹³C resonances relative to the unsubstituted FAE, respectively. If the hydroxyl group is located on the carboxyl side of the double bond of thebis homoallylic hydroxy fatty acid esters (BHAHFA), the olefinic resonances are uniformly shifted apart by [|1.46+|Δδdb_u‖] where Δδdb_u represents the absolute value of the double bond resonance separation in the unsubstituted FAE and 1.46 ppm is the sum of the absolute values of the δ and ε shift parameters. With hydroxyl substitution on the terminal methyl side of the double bond, the olefinic shift separation is equal to [|1.46−|Δδdb_u‖]. In homoallylic (OH group located two carbons away from the olefinic carbon) substituted FAE the γ and δ induced hydroxyl shifts for thecis double bond resonances are +3.08 and −4.63 ppm, respectively while thetrans double bond parameters are +4.06 and −4.18 ppm, respectively. The double bond resonance separation in homoallylic hydroxy fatty acid esters (HAHFA) can be calculated from the formula [|7.71−|Δδdb_u‖] forcis and [|8.24−|Δδdb_u‖] for thetrans case when the OH substitution is on the carboxyl side of the double bond. Conversely, when the OH resides on the terminal methyl side, the double bond shift separations forcis andtrans isomers are [|7.71+|Δδdb_u‖] and [|8.24+|Δδdb_u‖], respectively. The derived shift parameters can verify the positions of both the double bond and hydroxyl substitution from the olefinic resonance separation in long-chain fatty acid derivatives, obviating the need for destructive analytical methods. Reference to brand or firm name does not constitute endorsement by the United States Department of Agriculture over others of a similar nature not mentioned.     

Separation of the Fatty Acids in Menhaden Oil as Methyl Esters with a Highly Polar Ionic Liquid Gas Chromatographic Column and Identification by Time of Flight Mass spectrometry

The fatty acids contained in marine oils or products are traditionally analyzed by gas chromatography using capillary columns coated with polyethylene glycol phases. Recent reports indicate that 100 % cyanopropyl siloxane phases should also be used when the analyzed samples contain trans fatty acids. We investigated the separation of the fatty acid methyl esters prepared from menhaden oil using the more polar SLB-IL111 (200 m × 0.25 mm) ionic liquid capillary column and the chromatographic conditions previously optimized for the separation of the complex mixture of fatty acid methyl esters prepared from milk fat. Identifications of fatty acids were achieved by applying Ag⁺-HPLC fractionation and GC-TOF/MS analysis in CI⁺ mode with isobutane as the ionization reagent. Calculation of equivalent chain lengths confirmed the assignment of double bond positions. This methodology allowed the identification of 125 fatty acids in menhaden oil, including isoprenoid and furanoid fatty acids, and the novel 7-methyl-6-hexadecenoic and 7-methyl-6-octadecenoic fatty acids. The chromatographic conditions applied in this study showed the potential of separating in a single 90-min analysis, among others, the short chain and trans fatty acids contained in dairy products, and the polyunsaturated fatty acids contained in marine products.     

Fractional vacuum distillation of herring oil methyl esters

Methyl esters of a Canadian Atlantic herring oil containing 62% monoethylenic fatty acids were subjected to batch fractional distillation under vacuum on a pilot plant scale, to study the feasibility of fractionating fatty acid esters of marine oils of low iodine value into monounsaturated fractions with increased commercial value for industrial chemical uses. A total of 64 methyl ester fractions were collected and analyzed by gas liquid chromatography. Recoveries of the major saturated and monounsaturated acids were close to 100%, and some fractions contained over 90% of the desired 22:1 long chain monounsaturated acids. The short chain polyunsaturated acids were recovered in good yields, but the long chain highly unsaturated acids were recovered in yields of 60% or less due to oxidative and thermal decomposition in the particular apparatus employed. If small amounts of unsaturated acids are acceptable, fractional distillation of low iodine value marine oils could inexpensively supply fractions with high concentrations of methyl esters of longer chain (C₂₀ and C₂₂) monounsaturated and shorter chain (C₁₄) saturated acid or (C₁₆) saturated-monounsaturated acid mixture.     

Double-Bond Isomerization of Rapeseed Oil Methyl Esters in a Continuous Flow Reactor Efficiently Catalyzed by H-Mordenite

Unsaturated fatty acids are attractive alternatives to fossil-based materials as a source of hydrocarbons, but potential applications are limited by the preset chain length of 16–20 carbon atoms. However, if the double bond can be randomly moved along the chain, subsequent bond-breaking operations such as ethenolysis or oxidative cleavage will give rise to products with chain lengths ranging from 2 to 18 carbon atoms. A process for the double bond isomerization of rapeseed oil methyl esters in a flow-through reactor using zeolites as the catalyst is herein disclosed. Using H-mordenite as the catalyst at a flow of 0.125 mL min⁻¹ at 290 °C, near equilibrium isomerization is reached with up to 49% recovery of linear monomeric products after 5 h and up to 44% after 48 h. The main side products are oligomers (3%–15%) and skeletal isomerization products (10%–23%). Practical applications: With a suitable follow-up modification addressing the double bond (metathesis, ozonolysis, oxidative cleavage), the product mixtures of double-bond positional isomers can be used in the production of short-chain base chemicals. In repetitive combination with metathesis catalysis, biofuels with customized chain length distributions can be generated.     

The very long chain fatty acids of the green alga,Chlorella kessleri

Fatty acids of the fresh-water green alga,Chlorella kessleri (heterotrophically cultivated), were fractionated (as methyl esters) using high performance liquid chromatography (HPLC). A fraction of fatty acids with chain lengths longer than 28 carbon atoms was analyzed by gas chromatography-mass spectrometry (GC-MS). The very long chain fatty acids, ranging from 31 to 36 carbon atoms, were found for the first time in a green alga.     

Identification of methyl-branched fatty acids from the triacylglycerols of subcutaneous adipose tissue of lambs

A concentrate of branched chain fatty acids (as methyl esters) was prepared from the triacylglycerols of subcutaneous adipose tissue lipids of lambs receiving a carbohydrate-rich (cereal) diet. This was accomplished by procedures which allowed the removal of unsaturated components by peroxidation and straight chain saturated components by urea-adduct formation. The concentrate was analyzed by high resolution gas chromatography in combination with mass spectrometry and was shown to consist of a complex mixture of saturated methyl-substituted fatty acids. Methyl substitution occurred on even-numbered carbon atoms (relative to the carboxyl group) and the chain lengths of the acids ranged from 10 to 18 carbon atoms. Acids with one methyl substituent in the fatty acyl chain were most abundant; di-, tri- and tetramethyl-substituted acids were also present. The biosynthesis of these methyl-substituted acids is discussed briefly.     

Analyse hoch ungesättigter Fettsäuremethylester

Analysis of Highly Unsaturated Methyl Esters of Fatty Acids The conditions for an optimum separation and identification of fatty acids having 0-6 double bonds were investigated. Thin-layer chromatographic separation of methyl esters of fatty acids on silver nitrate containing plates by a special technique in two different solvent systems enables a fractionation according to the degree of saturation as well as a resolution of highly unsaturated ω3 fatty acids from their ω6 positional isomers. Subsequent gas chromatography of individual fractions permits the examination of homologues with the same degree of saturation but different chain length.     

Application of a GC-MS method using deuterated fatty acids for tracingcis-vaccenic acid biosynthesis in kaki pulp

A gas chromatographic-mass spectrometric method using [2,2-²H₂]fatty acids has been developed to trace the biosynthesis ofcis-vaccenic (cis-11-octadecenoic) acid in higher plants. The deuterated fatty acids and other unlabeled fatty acids in the biosynthetic reaction mixture were converted into bis(methylthio) derivatives and analyzed by mass chromatography. The principle of this method was based on the shift of key fragment ions (containing two deuterium atoms) due to the cleavage between the methylthio-substituted carbons. The labeled compounds were detected by the m/z values which shifted 2 mass units from those of the corresponding unlabeled compounds and estimated by a calibration curve based on the peak areas of the key fragment ions. For metabolic experiments, a homogenate fraction was prepared from the pulp part of maturing kaki (Diospyros kaki) fruit and incubated with ammonium [2,2-²H₂]palmitoleate (cis-9-hexadecenoate) or [2,2-²H₂]palmitoleoyl-CoA. The incubation resulted in the formation of detectable amounts of isotopically-labeledcis-vaccenic acid containing two deuterium atoms at the carbon chain between the double bond and the carboxyl group. This experimental evidence proved thatcis-vaccenic acid was formed from palmitoleic acid by chain elongation.     

Fatty acids of unusual double-bond positions and chain lengths found in rat skin surface lipids

The fatty acids of rat skin surface lipids comprise four main skeletal types of chains which occur both as saturates and monoenes and range from C₁₂ to C₃₈: straight even, straight odd, iso and anteiso (the latter two identified by GC retention data only). Two unidentified series of branched monoenes also occur in trace amounts. Reductive ozonolysis of monoenes reveals two characteristic double-bond position patterns, one for the straight even chain series and the other for the straight odd chain series. The straight even chain pattern comprises four series, of which ω7 ≫ω9>ω5>ω11; the straight odd chain series in contrast shows a large number of ω series with irregular distribution. The biosynthesis of the even chain fatty acid monoenes can be thought of as occurring in two stages: synthesis of 14∶Δ9, 16∶Δ9, 18∶Δ9 and 20∶Δ9, with 16∶Δ9 predominating; elongation of these chains mostly by 1, 2, or 3 C₂ units but up to the unusually long lengths by 11 C₂ units. For the formation of the former, two schemes by known pathways are proposed. Iso and anteiso chains which are nearly all saturated comprised 1/3 the total fatty acids. Special terms and abbreviations: Normal even=a straight chain with an even number of carbon atoms, normal odd=a straight chain with an odd number of carbon atoms, ω=terminal carbon atom, iso=a straight chain with a methyl group at the ω−1 position, anteiso=a straight chain with a methyl group at the w−2 position, Δn=a double bond between the n^th and the (n+1)^th carbon atom from the carbonyl group of the fatty acid or ester, ωn=a double bond between the ω∩n^th and the ω-(n−1)^th carbon atom where n is an integer, aldester=aldehyde methyl ester, Me=methyl, GLC=gas-liquid chromatography, TLC=thin-layer chromatography.     

Identification of 3, 6, 9, 12, 15-octadecapentaenoic acid in laboratory-cultured photosynthetic dinoflagellates

Polar and nonpolar chromatograms of fatty acid methyl esters derived from 11 species of photosynthetic, marine dinoflagellates cultured in modified Erd-Schrieber medium contained a component (4-23%) not identifiable by conventional graphic or arithmetic methods. Hydrogenation followed by gas liquid chromatography of the product showed the unknown component to be a straight chained 18 carbon fatty acid methyl ester. Chemical (ch4) ionization mass spectrometry of the isolated ester gave a spectrum characteristic of methyl esters and a mol wt of 288, indicating an 18 carbon molecule with 5 double bonds, or equivalent unsaturation. The IR spectrum showed that the double bonds are nonconjugated, and all are cis in geometry. Electron impact mass spectrometry of the pyrorolidide derivative provided evidence that double bonds are located in the 3, 9, 15 positions and probably also in the 6 and 12 positions of the molecule. these double bond positions were confirmed by NMR spectrometry. Data obtained by quantitation of the algal methyl esters suggest the possibility that these dinoflagellates synthesize 18:5omega3 (shorthand notation for chain length: number of double bond counted from the terminal methyl group) by a 2 carbon chain shortening of 20:5omega3, rather than by the insertion of a delta3 bond into 18:4omega3.     

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.     

Skin lipids. III. Fatty chains in skin lipids. The use ofvernix caseosa to differentiate between endogenous and exogenous components in human skin surface lipid

The literature on the types of fatty chains that occur in skin lipids is reviewed and new data are presented. To ascertain whether certain unusual fatty acids found in human skin surface lipids are truly products of the human skin or are due to some type of external contamination (possibly bacterial), the fatty acids ofvernix caseosa, (the lipoidal material covering the human fetus) were analyzed and compared to those found in human skin surface lipid. The same unusual fatty acids were found invernix caseosa. This indicates that these acids are products of human skin. These acids consist of five classes of saturated branched chain acids and of three classes of monoenes: straight chain,iso andanteiso. All the monoenes are Δ⁶ or derivable from this position by addition of an integral number of 2 carbon units to the carboxyl group. On the polar phase diethylene glycol succinate polyester, the saturated branched chain methyl esters have fractional carbon numbers (by gas chromatography) of 0.15, 0.23, 0.45, 0.63 and 0.75. The series at 0.63 and 0.75 areiso andanteiso, respectively. The series at 0.15 and 0.23 appear to be two newly identified classes of branched chain fatty acids.