Stereospecific analysis of TAG from sunflower seed oil

Stereospecific analysis of TAG from a sunflower seed oil of Tunisian origin was performed. The TAG were first fractionated according to chain length and degree of unsaturation by RP-HPLC. The four major diacid- and triacid-TAG fractions were palmitoyldilinoleoyl-glycerol, dioleoyllinoleoylglycerol, oleoyldilinoleoylglycerol, and palmitoyloleoyl-linoleoyl-glycerol, amounting to 7.2, 16.6, 29.5, and 12 mol%, respectively. The TAG of the four fractions were individually submitted to stereospecific analysis, using a Grignard-based partial deacylation, separation of sn-1,2(2,3)-DAG from sn-1,3-DAG by boric acid-impregnated silica gel TLC plates, conversion of the sn-1,2(2,3)-DAG to their 3,5-dinitrophenylurethane (DNPU) derivatives, fractionation of DNPU derivatives by RP-HPLC, resolution of the DNPU-DAG by HPLC on a chiral column, transmethylation of each sn-DNPU-DAG fraction, and analysis of the resulting FAME by GC. The data obtained were used to determine the triacyl-sn-glycerol composition of the main TAG of the oil. Fifteen triacyl-sn-glycerols were identified and quantified, representing, along with the monoacid-TAG, trilinoleoylglycerol and trioleoylglycerol, more than 90% of the total oil TAG. The two major triacyl-sn-glycerols were trilinoleoyl-glycerol and 1-linoleoyl-2-linoleoyl-3-oleoyl-glycerol (18.6 and 18.5% of the total, respectively). Results clearly identified linoleic acid as the major FA at the sn-2 position, whereas oleic and palmitic acids were the major FA at the sn-3 position. The sn-1 position was occupied to nearly the same extent by linoleic and oleic acids, and to a greater extent by palmitic acid, which was practically absent at the sn-2 position.     

Purification of 2-Monoacylglycerols Using Liquid CO<Subscript>2</Subscript> Extraction

The fatty acid moiety of 2-monoacyl-sn-glycerol (2-MAG) undergoes spontaneous acyl migration to the sn-1(3) position, resulting in a thermodynamic equilibrium of approximately 1:9 of 2-MAG to 1(3)-monoacyl-sn-glycerol (1-MAG). Spontaneous acyl migration is an impediment to synthesizing and isolating specific 2-MAG for use as intermediates in the synthesis of structured triacylglycerols. 2-Monooleoyl-sn-glycerol was synthesized by the enzymatic ethanolysis of triolein and isolated by liquid CO₂ extraction. The resultant MAG, diacylglycerol, and fatty acid ethyl esters were quantified by ¹H NMR and supercritical fluid chromatography. The low polarity of the CO₂ and mild extraction temperature (25 °C) resulted in very low spontaneous acyl migration rates, allowing the MAG to be isolated in 80% yield and in a very high 2-MAG:1-MAG ratios of ≥93 mol%.     

Positional distribution of FA in TAG of enzymatically modified borage and evening primrose oils

Stereospecific analysis was carried out to establish positional distribution of FA in the TAG of DHA, EPA, and (EPA+DHA)-enriched oils. In this study, TAG of enzymatically modified oils were purified using a silicic acid column. The TAG were then subjected to positional distribution analysis using a modified procedure involving reductive cleavage with Grignard reagent. The results showed that in DHA-enriched borage oil (BO), DHA was randomly distributed over the three positions of TAG, whereas γ-linolenic acid (GLA) was mainly esterified at the sn-2 and-3 positions. In DHA-enriched evening primrose oil (EPO), however, DHA and GLA were concentrated in the sn-2 position. In EPA-enriched BO, EPA was randomly distributed over the three positions of TAG, similar to that observed for DHA. In EPA-enriched EPO, however, this FA was mainly located at the primary positions (sn-1 and sn-3) of TAG. In both oils, GLA was preferentially esterified at the sn-2 position. In (EPA+DHA)-enriched BO, EPA and DHA were mainly esterified at the sn-1 and -3 positions of TAG, whereas GLA was mainly located at the sn-2 position. In (EPA+DHA)-enriched EPO, GLA was mainly located at the sn-2 and-3 positions; EPA was preferentially esterified at the sn-1 and-3 positions, and DHA was found mainly at the sn-3 position.     

Stereospecific analyses of seed triacylglycerols from high-erucic acid brassicaceae: Detection of erucic acid at thesn-2 position inBrassica oleracea L. Genotypes

Stereospecific analyses of triacylglycerols from selected high-erucic acid breeding lines or cultivars ofBrassica napus L. andB. oleracea L. have been performed. Initial lipase screening revealed that while allB. napus lines contained little or no erucic acid at thesn-2 position, several of theB. oleracea lines had significant proportions of erucic acid at this position. Detailed stereospecific analyses were performed on the triacylglycerols from these lines by using a Grignard-based deacylation, conversion of thesn-1,sn-2 andsn-3 monoacylglycerols to their di-dinitrophenyl urethane (DNPU) derivatives, resolution of the di-DNPU-monoacylglycerols (MAGs) by high-performance liquid chromatography on a chiral column, transmethylation of eachsn-di-DNPU MAG fraction and analysis of the resulting fatty acid methyl esters by gas chromatography. The findings unequivocally demonstrate for the first time that, within the Brassicaceae, there existsB. oleracea germplasm containing seed oils with substantial erucic acid (30–35 mol%) at thesn-2 position. This has important implications for biotechnology and breeding efforts designed to increase the levels of erucic acid in rapeseed beyond 66 mol% to supply strategic industrial feedstocks. In the first instance, the germplasm will be of direct use in retrieving a gene encoding aBrassica lyso-phosphatidic acid acyltransferase with an affinity for erucoyl-CoA. In a breeding program, the germplasm offers promise for the introduction of this trait intoB. napus by interspecific hybridization and embryo rescue.     

Stereospecific analysis of triacyl-sn-glycerols by chiral high-performance liquid chromatography

A method for the stereospecific analysis of triacyl-sn-glycerols by high-performance liquid chromatography (HPLC) on a chiral column is described. Triacyl-sn-glycerols were partially degraded with ethyl magnesium bromide, and the monoacylglycerols produced were separated as 1- and 2-monoacylglycerol fractions by thin-layer chromatography on boric acid-impregnated silica gel plates. The 1-monoacylglycerols were resolved intosn-1 andsn-3 monoacylglycerol fractions by HPLC on a chiral column (Sumichiral OA-4100) after derivatization with 3,5-dinitrophenyl isocyanate. Fatty acid methyl esters obtained from the original triacyl-sn-glycerols, 1- and 2-monoacylglycerol fractions, andsn-1 andsn-3 monoacylglycerol fractions were analyzed by gas chromatography on open-tubular columns. Stereospecific acyl distributions in triacyl-sn-glycerols were calculated from the data. The acyl distributions of several oils were obtained. The method is rapid, simple and gave reproducible results.     

Lipolysis of different oils using crude enzyme isolate from the intestinal tract of rainbow trout, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

Crude enzyme isolate was prepared from the intestine of rainbow trout. Positional specificity of the crude enzyme isolate was determined from both 1(3)- and 2-MAG products after in vitro lipolysis of radioactive-labeled triolein. The ratio of 2-MAG/1(3)-MAG was 2∶1, suggesting that the overall lipase specificity of the enzyme isolate from rainbow trout tended to be 1,3-specific; however, activity against the sn-2 position also was shown. In vitro lipolysis of four different unlabeled oils was performed with the crude enzyme isolate. The oils were: structured lipid [SL; containing the medium-chain FA (MCFA) 8∶0 in the sn-1,3 positions and long-chain FA (LCFA) in the sn-2 position], DAG oil (mainly 1,3-DAG), fish oil (FO), and triolein (TO). MCFA were rapidly hydrolyzed from the SL oil. LCFA including n−3 PUFA were, however, preserved in the sn-2 position and therefore found in higher amounts in 2-MAG of SL compared with 2-MAG of FO, DAG, and TO. Lipolysis of the DAG oil produced higher amounts of MAG than the TAG oils, and 1(3)-MAG mainly was observed after lipolysis of the DAG oil. The positional specificity determined and the results from the hydrolysis of the different oils suggest that n−3 very long-chain PUFA from structured oils may be used better by aquacultured fish than that from fish oils.     

Enzymatic Analysis of Positional Distribution of Fatty Acids in Solid Fat by 1,3-Selective Transesterification with Candida antarctica Lipase B

A protocol for the analysis of the positional distribution of fatty acids (FA) in solid triacylglycerols (TAG) was developed using sn-1(3) selective alcoholysis catalyzed by immobilized Candida antarctica lipase B (CALB). One part by weight of solid fat and ten parts by weight of ethanol (99.5 %) were warmed to liquefy the fat. After adding 0.44 parts by weight of CALB, the mixture was shaken at 50 °C for 10 min then at 30 °C for 2.8 h. The recovery of 2-MAG after the 3-h transesterification reaction was ca. 85 % of the maximum theoretical yield (33 mol%), with the loss of 15 % attributable to the acyl migration from sn-2 to sn-1(3). The recovery was similar to that of the solvent-free alcoholysis of structured lipids, 1,3-dipalmitoyl, 2-oleoyl glycerol and 1,3-dioleoyl, 2-palmitoyl glycerol, conducted at 30 °C for 3 h. In contrast, the acyl migration from sn-1(3) to sn-2 was hardly observed. Because the detected acyl migration was only in the direction of sn-2 to sn-1(3), and not vice versa, it is proposed to determine the FA composition of the sn-2 position of TAG by the gas chromatographic analysis of 2-MAG fraction recovered from the enzymatic reaction mixture, and the FA composition of sn-1(3) position by a mass balance using the FA composition of TAG and of the sn-2 position as inputs. The procedure was successfully applied to palm oil and shea butter, and docosahexaenoic acid (DHA)-rich single cell oil from Aurantiochytrium sp. KH105 for the first time.     

Regiospecific analysis of triacylglycerols using allyl magnesium bromide

A method for the regiospecific analysis of triacylglycerols (TAG), using the Grignard reagent allyl magnesium bromide (AMB) to partially deacylate TAG, is described. 1,3-Distearoyl-2-oleoyl-glycerol (SOS) and 1,3-didecanoyl-2-palmitoyl-glycerol (CPC) were reacted with AMB. From the resulting mixture, the four different classes of partial acylglycerols and TAG were isolated, and the mole ratios between stearic acid and oleic acid, or decanoic acid and palmitic acid, respectively, were determined in each fraction. Different approaches of calculating the composition of the fatty acids in positionssn-1(3) andsn-2 of the original TAG were compared. For thesn-2 position, the best estimate was the direct determination of the fatty acid composition of 2-monoacylglycerol (MAG). Mole percentages of stearic acid and decanoic acid in thesn-1(sn-3) positions of SOS and CPC, respectively, were most accurately estimated from the fatty acid compositions of TAG and 2-MAG according to the formula: 1.5×TAG−0.5×2-MAG. Using AMB and the present method of calculation, the results obtained were more accurate and showed smaller standard derivations than those obtained using other common deacylating agents, such as ethyl magnesium bromide or pancreatic lipase.     

Micro method for stereospecific analysis of triacyl-sn-glycerols by chiral-phase high-performance liquid chromatography

A procedure for micro stereospecific analysis of triacyl-sn-glycerols (TGs) by high-performance liquid chromatography (HPLC) on a chiral column is presented. TGs were partially hydrolyzed with ethyl magnesium bromide, and total products were immediately converted to 3,5-dinitro-phenylurethane derivatives. Each of the 1- and 2-monoacylglycerol (MG) derivatives was isolated by HPLC on a silica column. The 1-MGs were resolved intosn-1 andsn-3 MG fractions by HPLC on a Sumichiral OA-4100 column (Sumitomo Chemical, Osaka, Japan). Fatty acid methyl esters obtained from thesn-1,sn-2 andsn-3 MG fractions were analyzed by gas-liquid chromatography on a capillary column. Analyses of standard TGs showed that, even with 1 mg of sample, accuracy was comparable to that obtained with 100-mg samples. Applying this procedure to the stereospecific analysis of 5 mg of jujube pulp, TGs revealed the positional distribution of the (n-5) series of monounsaturated fatty acids they contained. Honored Student Award Address presented at the 83rd AOCS Annual Meeting held in Toronto, Canada, May 10–14, 1992.     

Identification of natural diacylglycerols as the 3,5-dinitrophenylurethanes by chiral phase liquid chromatography with mass spectrometry

This study reports a facile identification of the molecular species of enantiomeric diacylglycerols by combining chiral phase high-performance liquid chromatography with positive chemical ionization mass spectrometry. For this purpose the 3,5-dinitrophenylurethane (DNPU) derivatives ofsn-1,2(2,3)-diacylglycerols are separated on an (R+)-naphthylethylamine polymer column (25 cm × 0.46 cm ID) using an isocratic solvent system made up of hexane/dichloroethane/acetonitrile (85∶10∶5, by vol) or isooctane/tert-butyl methyl ether/acetonitrile/isopropanol (80∶10∶5∶5, by vol). About 1% of the column effluent (1 mL/min) was admitted to a quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA)via direct liquid inlet interface, and positive chemical ionization spectra were recorded over the range of 200–900 mass units. The DNPU derivatives of diacylglycerols yield characteristic [M-DNPU]⁺ and [RCO+74]⁺ ions for each diacylglycerol species from which the molecular weight and exact pairing of fatty acids can be unequivocally obtained. The characteristic ions appear to be generated in nearly correct mass proportions as indicated by preliminary quantitative comparisons. The abbreviations 14∶0, 16∶1, 18∶2, etc. represent normal chain fatty acids of 14, 16, 18, etc. acyl carbons and 0, 1, 2, etc. double bonds, respectively; 16∶0–18∶1, etc. represent diacylglycerols containing 16∶0 and 18∶1 fatty acids of unspecified positional distribution;sn indicates stereospecific numbering of glycerol carbons;sn-1,2-diacylglycerols andsn-2,3-diacylglycerols are enantiomeric diacylglycerols of unspecified fatty acid composition;rac-1,2-diacylglycerols are racemic diacylglycerols representing equal amounts ofsn-1,2-andsn-2,3-enantiomers;sn-1,2(2,3)-diacylglycerols are a mixture ofsn-1,2-andsn-2,3-diacylglycerols of unspecified proportion of enantiomers and unspecified fatty acid compisition and positional distribution; X-1,3-diacylglycerols are diacylglycerols of unspecified fatty acid composition and reverse isomer content.     

Positional distribution of highly unsaturated fatty acids in triacyl-sn-glycerols of Artemia nauplii enriched with docosahexaenoic acid ethyl ester

This paper presents the positional distribution of fatty acids in triacyl-sn-glycerols (TAG) of Artemia nauplii used in aquaculture as a live food for marine fish larvae. The nauplii were enriched with docosahexaenoic acid (DHA) ethyl ester (EE) in the form of gelatin-acacia microcapsules for 4, 18, and 24 h. TAG of the initial, enriched, and unenriched Artemia nauplii were subjected to stereospecific analysis. A remarkable increase of DHA content in the enriched Artemia TAG confirmed the view that DHA-EE is effectively assimilated and incorporated into the TAG fraction of Artemia nauplii. TAG of the nauplii enriched with 25 mg/L of DHA-EE contained DHA at concentrations of 5.9–6.8, 4.3–6.0, and 14.3–22.3 mol% in the sn-1, sn-2, and sn-3 positions, respectively. When the nauplii were enriched with 100 mg/L of DHA-EE, proportions of DHA in the sn-1, sn-2, and sn-3 positions were 5.2–8.6, 3.9–6.0, and 12.2–25.4 mol%, respectively. In all of the enriched Artemia, DHA was preferentially located in the sn-3 position followed in sequence by the sn-1 and sn-2 positions. The lower content of DHA in the sn-1 and sn-2 positions was consistent with low content of this acid in 1,2-diacyl-sn-glycerophospholipids. When fish larvae are reared on Artemia nauplii enriched with LL-type DHA oil, the larvae feed on DHA esterified in TAG with a positional distribution pattern similar to that of marine mammals (sn-3≫sn-1>sn-2) rather than that of fish or marine invertebrates (sn-2≫sn-3>sn-1).     

Positional distribution of Δ5-olefinic acids in triacylglycerols from conifer seed oils: General and specific enrichment in the sn-3 position

Triacylglycerols (TAG) were purified from the storage lipids extracted from the seeds of several conifer species (Taxus baccata, Larix decidua, Sciadopytis verticillata, and Juniperus communis), each species belonging to one of the four families Taxaceae, Pinaceae, Taxodiaceae, and Cupressaceae, respectively. Each species was characterized by a high content of 5,9-18:2, 5,9,12-18:3, 5,11,14-20:3, or 5,11,14,17-20:4 acids, respectively. TAG were partially deacylated with ethylmagnesium bromide, and the resulting 1,2-, 2,3-diacylglycerols (DAG), and 2-monoacylglycerols (MAG) were purified by thin-layer chromatography. 1,2- and 2,3-DAG were further fractionated by chiral column high-performance liquid chromatography of the 3,5-dinitrophenylurethane derivatives. Alternately, TAG were subjected to porcine pancreatic lipase, and the resulting 2-MAG were purified for further analysis. Gas-liquid chromatography of fatty acid methyl esters prepared from the separated DAG and MAG, coupled with appropriate calculations, indicated that the Δ5-olefinic acids, irrespective of the species, chainlengths and number of ethylenic bonds, were considerably enriched in the sn-3 position of TAG where they accounted for ca. 35 to 74 mole% of fatty acids esterified to this position (depending on the initial level of total Δ5-olefinic acids in TAG), which corresponded to 79–94% of Δ5-olefinic acids esterified to the three positions. On the other hand, Δ5-olefinic acids were less than 10% in the sn-2 position and less than 6% in the sn-1 position of TAG. This specific enrichment of Δ5-olefinic acids in the sn-3 position thus appears to be a general characteristic of conifer seed TAG. These results were extended to TAG from the seeds of two pine species (Pinus koraiensis and P. pinaster) that are rich in Δ5-olefinic acids and available commercially on a ton-scale.     

Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids

Six oils of marine, algal, and microbial origin were analyzed for stereospecific distribution of component fatty acids. The general procedure involved preparation ofsn-1,2-(2,3)-diacylglycerols by partial deacylation with ethylmagnesium bromide or pancreatic lipase, separation of X-1,3- andsn-1,2(2,3)-diacylglycerols by borate thin-layer chromatography, resolution of thesn-1,2- andsn-2,3-enantiomers by chiral phase high-performance liquid chromatography following preparation of dinitrophenylurethane derivatives, and determination of the fatty acid composition by gas chromatography. Unexpected complications arose during a stereospecific analysis of triacylglycerols containing over 33% of either 20∶4 or 22∶6 fatty acids. Thesn-1,2(2,3)-diacylglycerols made up of two long-chain polyunsaturated acids migrated with the X-1,3-diacylglycerols and required separate chiral phase resolution. Furthermore, the enzymatic method yieldedsn-1,2(2,3)-diacylglycerols, overrepresenting the polyenoic species due to their relative resistance to lipolysis, but prolonged digestion yielded correct composition for the 2-monoacylglycerols. The final positional distribution of the fatty acids was established by pooling and normalizing the data from subfractions obtained by norman- and chiral-phase separation of diacylglycerols. The molecular species of X-1,3-,sn-1,2- andsn-2,3-diacylglycerol dinitrophenylurethanes were identified by chiral-phase liquid chromatography/mass spectrometry with electrospray ionization, which demonstrated a preferential association of the paired long-chain acids with thesn-1,2- andsn-2,3-diacylglycerol isomers.     

Triacylglycerol composition and structure in genetically modified sunflower and soybean oils

Changes in composition were examined in oils extracted from genetically modified sunflower and soybean seeds. Improvements were made to the analytical methods to accomplish these analyses successfully. Triacylglycerols (TAG) were separated on two 300 mm × 3.9 mm 4μ Novapak C18 high-performance liquid chromatography (HPLC) columns and detected with a Varex MKIII evaporative light-scattering detector. Peaks were identified by coelution with known standards or by determining fatty acid composition of eluted TAG by capillary gas chromatography (GC). Stereospecific analysis (fatty acid position) was accomplished by partially hydrolyzing TAG with ethyl magnesium bromide and immediately derivatizing the resulting diacylglycerols (DAG) with (S)-(+)-1-(1-naphthyl)ethyl isocyanate. The derivatized sn-1,2-DAG were completely resolved from the sn-2,3-DAG on two 25 mm × 4.6 mm 3 μ silica HPLC columns. The columns were chilled to −20°C to obtain baseline resolution of collected peaks. The distribution of fatty acids on each position of the glycerol backbone was derived from the fatty acid compositions of the two DAG groups and the unhydrolyzed oil. Results for the sn-2 position were verified by hydrolyzing oils with porcine pancreatic lipase, isolating the resulting sn-2 monoacylglycerols by TLC, and determining the fatty acid compositions by GC. Results demonstrated that alterations in the total fatty acid composition of these seed oils are determined by the concentration of TAG species that contain at least one of the modified acyl groups. As expected, no differences were found in TAG with fatty acid quantities unaffected by the specific mutation. In lieu of direct metabolic or enzymatic assay evidence, the authors’ positional data are nevertheless consistent with TAG biosynthesis in these lines being driven by the mass action of available acyl groups and not by altered specificity of the acyltransferases, the compounds responsible for incorporating fatty acids into TAG.     

Docosahexaenoic Acid is More Stable to Oxidation when Located at the sn-2 Position of Triacylglycerol Compared to sn-1(3)

Regio-isomeric effects on the oxidative stability of triacylglycerols (TAG) containing docosahexaenoic acid (DHA) were investigated using two pairs of regio-isomerically pure TAG, namely 1,3-dihexadecanoyl-2-(4,7,10,13,16,19-docosahexaenoyl)glycerol (PDP)/1,2-dihexadecanoyl-3-(4,7,10,13,16,19-docosahexaenoyl)glycerol (PPD) and 1,3-dioctadecenoyl-2-(4,7,10,13,16,19-docosahexaenoyl)glycerol (ODO)/1,2-dioctadecenoyl-3-(4,7,10,13,16,19-docosahexaenoyl)glycerol (OOD) where P, O, and D represent palmitic acid, oleic acid, and DHA respectively. Each pair of regio-isomers was subjected to accelerated auto-oxidation (at 40 or 50 °C inside a dark oven). In each case, the TAG oxidized more slowly when DHA was located at the sn-2 position (PDP and ODO) compared to the sn-1(3) position (PPD and OOD), as evidenced by slower development of peroxide value, slower depletion of DHA, and slower generation of secondary oxidation products propanal and trans, trans-2,4-heptadienal. The positional effect on auto-oxidation was more pronounced when DHA occurred in combination with oleic acid than with palmitic acid.     

Dibutyrate derivatization of monoacylglycerols for the resolution of regioisomers of oleic, petroselinic, and cis-vaccenic acids

Dibutyrate derivatives of monoacylglycerols of oleic, petroselinic, and cis-vaccenic acids were prepared by diesterification of monoacylglycerols with n-butyryl chloride. The resulting triacylglycerols were analyzed by gas chromatography (GC) with a 65% phenyl methyl silicone capillary column and separated on the basis of both fatty acid composition and regiospecific position. The petroselinic acid derivatives eluted first, followed sequentially by the oleic and cis-vaccenic acid derivatives, with the sn−2 positional isomer eluting before the sn−1(3) isomer in each case. Separation of the peaks was almost baseline between petroselinic and oleic acids as well as between oleic and cis-vaccenic acids. To assess the accuracy of the method, mixtures of triolein, tripetroselinin, and tri-cis-vaccenin in various known proportions were partially deacylated with the use of ethyl magnesium bromide and derivatized and analyzed as above. The results showed that this method compares favorably to the existing methods for analysis of oleic, petroselinic, and cis-vaccenic fatty acids by GC with respect to peak separation and accuracy, and it also provides information on the regiospecific distribution of the fatty acids. The method was applied to basil (Ocimum basilicum) and coriander (Coriandrum sativum) seed oils. cis-Vaccenic, oleic, and linoleic acids were mainly distributed at the sn−2 position in basil seed oil, and higher proportions of linolenic, palmitic, and stearic acids were distributed at the sn−1(3) position than at the sn−2 position. In coriander seed oil, petroselinic acid was mainly distributed at the sn−1(3) position, and both oleic and linoleic acids were mostly located at the sn−2 position, whereas palmitic, stearic, and cis-vaccenic acids were located only at the sn−1(3) position.     

<Superscript>13</Superscript>C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates

The regio-isomeric distribution of the omega-3 polyunsaturated fatty acids (PUFA) cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in the triacylglycerols (TAG) of anchovy/sardine fish oil was determined by ¹³C nuclear magnetic resonance (NMR) analysis under quantitative conditions. From the measurements of sn-1,3 and sn-2 carbonyl peak areas it was established that EPA was mainly located in the sn-1,3 positions, whereas DHA primarily occupied the sn-2 position. Reconstituted TAG prepared by Candida antarctica lipase-B (CALB) glycerolysis of the ethyl ester (EE) or the free fatty acid (FFA) forms of anchovy/sardine fish oil, displayed a different pattern: EPA was equally distributed, while DHA was preferentially attached to the sn-1,3 positions. TAG concentrates of varying EPA and DHA molar fractions were prepared by CALB-catalyzed glycerolysis of the corresponding EE and FFA. ¹³C-NMR analysis of the purified products revealed a lack of CALB regioselectivity for EPA and a slight sn-1,3 regioselectivity for DHA. Since this pattern was observed in all cases of this study, it was concluded that the lipase regioselectivity during TAG synthesis is independent of both the acyl donor type (carboxylic acid or ester) and the fatty acid content of the oil substrate.     

Determination of positional distribution of short-chain fatty acids in bovine milk fat on chiral columns

The positional distribution of acetic and butyric acids in bovine milk fat triacylglycerols was determined by chiral-phase high-performance liquid chromatography (HPLC) of the derived diacylglycerols. Enriched fractions of acetic and butyric acid-containing triacylglycerols were isolated by normal-phase thin-layer chromatography (TLC) from a molecular distillate of butter oil, and they were fully hydrogenated. Mixedsn-1,2(2,3)- andX-1,3-diacylglycerols of short- and long-chainlength, which were generated by partial Grignard degradation of the hydrogenated triacylglycerols, were isolated by borate-TLC. The enantiomericsn-1,2-andsn-2,3-diacylglycerols and theX-1,3-diacylglycerols as their 3,5-dinitrophenylurethanes were resolved by HPLC on chiral columns. Both acetic and butyric acids were exclusively associated with thesn- 2,3- andX-1,3-diacylglycerols of short and long chainlength. These results establish the presence of acetic and butyric acids in thesn-3-position of bovine milk fat triacylglycerols. Other short-and medium-chainlength acids were found in progressively increasing proportions also in thesn-1- andsn-2-positions.     

Synthesis of acetyl, docosahexaenoyl-glycerophosphocholine and its characterization using nuclear magnetic resonance

Docosahexaenoic acid (DHA) circulates in mammals in lipoproteins and bound to serum albumin as a nonesterified fatty acid as well as esterified in lysophosphatidylcholine (lysoPC). 1-Lyso,2-DHA-glycerophosphocholine (GPC) is an unstable isomer because of a primary alcohol at the sn-1 position. To keep DHA at the sn-2 position of lysoPC, its usual position for the corresponding lysoPC to be acylated into PC in tissues, we synthesized 1-acetyl,2-DHA-GPC and confirmed its structure by use of nuclear magnetic resonance (NMR) spectroscopy in comparison with its positional isomer, 1-DHA,2-acetyl-GPC. 1-Lyso,2-DHA-GPC was prepared from 1-stearoyl,2-DHA-GPC by enzymatic hydrolysis and purified by high-performance liquid chromatography. The isomerization of 1-lyso,2-DHA-GPC into 1-DHA,2-lyso-GPC was obtained by keeping the former overnight at room temperature under nitrogen. Both lysoPC isomers were acetylated by acetic anhydride into 1-acetyl,2-DHA-GPC and 1-DHA,2-acetyl-GPC, respectively, and the resulting phospholipids were fully characterized by NMR. In particular, the 1,2 substitution pattern of the acetyl and DHA chains could be easily detected by 2D heteronuclear multibond correlation. We conclude that 1-acetyl,2-DHA-GPC might be considered as a stable form of 1-lyso,2-DHA-GPC for its delivery to tissues, if the latter exhibits acetyl hydrolase activity.     

A simple method for regiospecific analysis of triacylglycerols by gas chromatography

A simple method for regiospecific analysis of triglycerides was developed. It consists of partial deacylation of triglycerides by ethylmagnesium bromide followed by derivatization of monoglycerides with n-butyryl chloride, and direct analysis of dibutyrate derivatives of monoglycerides by gas chromatography. The chromatographic conditions were carried out with monoglycerides of C₁₂ to C₂₀ fatty acids and resulted in separation of dibutyrate derivatives between those bearing the medium- or long-chain fatty acid in the sn-1(3) and sn-2 positions of glycerol. Beef tallow and grapeseed and cotton seed oils were analyzed using this new method, and their regiospecific distributions were compared with literature data. The method does not require separation of products by thin-layer chromatography or special analytical equipment other than a standard gas chromatograph, and it can thus be used for routine regiospecific analysis of triglycerides.