Analysis of diastereomeric DAG naphthylethylurethanes by normal-phase HPLC with on-line electrospray MS

Normal-phase HPLC resolution of sn-1,2(2,3)- and x-1,3-DAG generated by partial Grignard degradation from natural TAG was carried out with both (R)-(−) and (S)-(+)-1-(1-naphthyl)ethylurethane derivatives. The diastereomeric sn-1,2- and sn-2,3-DAG derivatives were resolved using two Supelcosil LC-Si (5 μm, 25 cm × 4.6 mm i.d.) columns in series and an isocratic elution with 0.37% isopropanol in hexane at a flow rate of 0.7 mL/min. The DAG were detected by UV absorption at 280 nm and were identified by electrospray ionization MS in the positive ion mode following postcolumn addition of chloroform/methanol/30% ammonium hydroxide (75∶24.5∶0.5, by vol) at 0.6 mL/min. Application of the method to a stereospecific analysis of the molecular species of TAG of rat VLDL showed that the TAG composition of VLDL circulating under basal conditions differs markedly from that of VLDL secreted by the liver during inhibition of serum lipases. The inhibition of serum lipases resulted in a significant proportional decrease in 16∶0 and PUFA and an increase in 18∶0 and oligoenoic FA in the sn-1-position, whereas the FA compositions in the sn-2- and sn-3-positions were much less affected.     

Study of TAG ethanolysis to 2-MAG by immobilized Candida antarctica lipase and synthesis of symmetrically structured TAG

Regiospecific ethanolysis of homogenous TAG with immobilized Candida antarctica lipase (Novozym 435) was studied using trioleoylglycerol (TO) as a model substrate. Optimization of the reactant weight ratio revealed that the 2-MAG reaction yield increased when a larger amount of ethanol was used. These results suggested that Novozym 435 showed strict regiospecificity in an excess amount of ethanol. The process optimization (reaction temperature and reactant molar ratio) and a study of lipase specificity for various substrates were performed. Under the optimized conditions (ethanol/TO molar ratio=77∶1 and 25°C), 2-monooleoylglycerol (2-MO) was obtained in more than 98% content among glycerides of the reaction mixture and approximately 88% reaction yield in 4 h. The above reaction conditions were applied for ethanolysis of tridocosahexaenoylglycerol, trieicosapentaenoylglycerol, triarachidonoylglycerol, tri-α-linolenoylglycerol, and trilinoleoylglycerol. Reaction yields ranging from 71.9 to 93.7% were obtained in short reaction times (2.5 to 8 h). Purified (>98%) 2-MO and 2-monodocosahexaenoylglycerol (2-MD) were reesterified with caprylic acid by immobilized Rhizomucor miehei lipase (Lipozyme IM) to afford symmetrical structured TAG. At a stoichiometric ratio of 2-MAG/caprylic acid, 25°C and 2–5 mm Hg vacuum, the glyceride composition of the esterification mixture was approximately 95% 1,3-dicapryloyl-2-oleoylglycerol (COC) at 4 h, and 96% 1,3-dicapryloyl-2-docosahexaenoylglycerol (CDC) at 4 h, and 96% 1,3-dicapryloyl-2-docosahexaenoylglycerol (CDC) at 8 h. The regioisomeric purity of both COC and CDC was 100%.     

Synthesis and Structural Analysis of Structured Triacylglycerols with CLA Isomers in the <Emphasis Type="Italic">sn</Emphasis>-2- Position

The present research deals with the chemical esterification of the sn-2- position of sn-1,3-diacylglycerol (sn-1,3-DAG) with 9cis,11trans (c9,t11) and 10trans,12cis (t10,c12) conjugated linoleic acid (CLA) isomers to obtain structured triacylglycerols (TAG); the sn-1,3-DAG substrates were produced from extra virgin olive oil by means of enzymatic reactions while CLA isomers were obtained using a three-step procedure based on alkaline hydrolysis of sunflower oil, urea purification of linoleic acid (LA) and alkaline isomerization of LA. The results showed good levels of CLA incorporation in structured TAG at the tested temperatures: 37.5% at 4 °C and 39.1% at 14 °C. To evaluate the incorporation of CLA isomers in sn-2- position of sn-1,3-DAG structural analysis of the newly synthesized TAG was carried out using an enzymatic and a chemical method. The results of the structural analysis also showed up the occurrence of acyl migration. The pancreatic lipase method allowed the direct determination of the fatty acid composition of TAG sn-2- position but this enzymatic method showed different results (p < 0.05) in respect to the chemical one; this occurrence could be due to an acylic specificity of the lipase. High incorporation of CLA isomers in sn-2- position of TAG was observed, 77.0% at 4 °C and 81.5% at 14 °C, considering the results of the chemical procedure.     

The regiospecific position of 18∶1 cis and trans monoenoic fatty acids in milk fat triacylglycerols

TAG of butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver-ion HPLC. The fractions containing TAG with either cis-or trans-monoenoic FA were collected and fractionated further by reversed-phase HPLC to obtain fractions containing cis TAG of ACN:DB (acyl carbon number:double bonds) 48∶1, 50∶1, and 52∶1 as well as trans 48∶1, 50∶1, and 52∶1. The FA compositions of these fractions were elucidated by GC. The MW distribution of each fraction was determined by ammonia negative-ion CI-MS. Each of the [M-H]⁻ parent ions was fractionated further by collision-induced dissociation with argon, which gave information on the location of cis-and trans-FA between the primary and secondary positions of TAG. The results suggest that the sn-positions of the monoenoic cis-and trans-FA depend on the two other FA present in the molecule. With 14∶0 FA in the TAG molecule, the 18∶1 FA in the sn-2 position are mostly present as cis-isomers. When there is no 14∶0 in the TAG molecule, the trans-18∶1 isomers seem to be more common in the sn-2 position. Also when other long-chain FA are present, the trans-isomers are more likely to be located in the secondary (sn-2) position.     

Enzymatic Synthesis of Structured Triacylglycerols Containing CLA Isomers Starting from <Emphasis Type="Italic">sn</Emphasis>-1,3-Diacylglycerols

The synthesis of structured triacylglycerols (TAG) by the enzymatic reaction between sn-1,3-diacylglycerols (sn-1,3-DAG) and conjugated linoleic acid (CLA) isomers was studied. Both the substrates of the reaction were produced from vegetable oils, the sn-1,3-DAG from extra virgin olive oil and the CLA isomers from sunflower oil. The enzymatic reactions between these substrates were catalyzed for 96 h by an immobilized lipase from Rhizomucor miehei (Lipozyme IM) and the reactions carried out in solvent were monitored every 24 h by using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The enzymatic reactions were carried out in different reaction media (hexane, isooctane and solvent free) and with different CLA/sn-1,3-DAG ratios. Total % acidic composition and structural analysis data were evaluated to verify the presence of CLA isomers in sn-2- position of synthesized TAG. The results showed good levels of CLA incorporation in sn-1,3-DAG, from 19.2% of TAG synthesized in solvent free conditions with a 0.5:1 substrate ratio, to 47.5% of TAG synthesized in isooctane with a 2:1 substrate ratio. It was observed that for all the reaction media, the best sn-2- acylic specificity was obtained with a 0.5:1 substrate ratio.     

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.     

Chylomicron and VLDL TAG structures and postprandial lipid response induced by lard and modified lard

Alterations in chylomicron and VLDL TAG and the magnitude of postprandial lipemia were studied in healthy volunteers after two meals of equal FA composition but different TAG-FA positional distribution. Molecular level information of individual lipoprotein TAG regioisomers was obtained with a tandem MS method. The incremental area under the response curve of VLDL TAG was large (P=0.021) after modified lard than after lard. In plasma TAG, the difference did not quite reach statistical significance (P=0.086). In general, there were less TAG with palmitic acid in the sn-2 position and more TAG with oleic acid in the sn-2 position in chylomicrons than in fat ingested. From 1.5 to 8 h postprandially, the proportion of individual chylomicron TAG was constant or influenced by TAG M.W. VLDL TAG regioisomerism was similar regardless of the positional distribution of fat ingested. Significant alterations were seen in VLDL TAG FA, in M.W. fractions, and in individual regioisomers with respect to time. The TAG sn-14∶0-18∶1-18∶1+sn-18∶1-18∶1-14∶0, sn-16∶0-16∶1-18∶1+sn-18∶1-16∶1-16∶0, and sn-16∶1-18∶1-18∶1+sn-18∶1-18∶1-16∶1 decreased (P<0.05); and sn-16∶0-16∶0-18∶2+sn-18∶2-16∶0-16∶0, sn-16∶0-16∶0-18∶1+sn-18∶1-16∶0-16∶0, sn-16∶0-18∶1-16∶0, and sn-16∶0-18∶1-18∶2+sn-18∶2-18∶1-16∶0 increased (P<0.05) after both meals. In conclusion, positional distribution of TAG FA was found to affect postprandial lipid metabolism in healthy normolipidemic subjects.     

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.     

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.     

Evaluation of ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of triacylglycerols containing highly unsaturated fatty acids

The suitability of a recently proposed method based on ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of oils containing long-chain PUFA such as [PA and DHA has been evaluated using selected marine oils and regio-isomerically enriched synthetic TAG substrates. 1,3-Regios-electivity of the lipase was enhanced when the ethanolysis was conducted in a high excess of ethanol, typically 10–50 times by weight of the oil. This enabled the reaction to be conducted on a milligram scale. However, irrespective of the ethanol-to-oil ratio, C. antarctica lipase released FA from TAG at different rates depending on the degree of unsaturation and/or chain length of the FA. Differences in lipolysis rates were particularly significant for EPA and DHA, with EPA released faster than DHA. Although DHA can be measured with reasonable accuracy by ethanolysis with C. antarctica, the method requires further optimization before it can be adopted for reliable regiospecific analyses that are as accurate as those obtainable by ¹³C NMR analysis for all major FA occurring in oils rich in long-chain PUFA.     

Distribution of the major fatty acids of human milk betweensn-2 andsn-1,3 positions of triacylglycerols

Human milk triacylgycerols (TAG) were analyzed by tandem mass spectrometry. The SIMPLEX method and a simple linear model were used to interpret the distribution of fatty acids between thesn-2 andsn-1,3 positions in 24 major molecular weight groups of TAG. The number of regio-isomeric pairs of TAG varied between 3 and 18 in each of these groups. Hexadecanoic (16∶0), tetradecanoic (14∶0) and dodecanoic acids (12∶0) typically occupied thesn-2 position in TAG containing less than 54 acyl carbons, whereas long-chain C18 and C20 acids were predominantly located at the primary positions. The positions of the three fatty acids within a TAG molecule were shown to depend on the fatty acid combination. The maximum of 12∶0 in thesn-2 position appeared at acyl carbon number (ACN) 48, the maxima of 14∶0 were at ACN 44 and ACN 50, and for 16∶0 at ACN 46 and 52.     

Effect of triacylglycerol structure on absorption and metabolism of isotope-labeled palmitic and linoleic acids by humans

The effect of dietary TAG structure and fatty acid acyl TAG position on palmitic and linoleic acid metabolism was investigated in four middle-aged male subjects. The study design consisted of feeding diets containing 61 g/d of native lard (NL) or randomized lard (RL) for 28 d. Subjects then received an oral dose of either 1,3-tetradeuteriopalmitoyl-2-dideuteriolinoleoyl-rac-glycerol or a mixture of 1,3-dideuteriolinoleoyl-2-tetradeuteriopalmitoyl-rac-glycerol and 1,3-hexadeuteriopalmitoyl-2-tetradeuteriolinoleoyl-rac-glycerol. Methyl esters of plasma lipids isolated from blood samples drawn over a 2-d period were analyzed by GC-MS. Results showed that absorption of the ²H-fatty acids (²H-FA) was not influenced by TAG position. The ²H-FA at the 2-acyl TAG position were 85±4.6% retained after absorption. Substantial migration of ²H-16∶0 (31.2±8.6%) from the sn-2 TAG position to the sn-1,3 position and ²H-18∶2n−6 (52.8±6.4%) from the sn-1,3 position to the sn-2 position of chylomicron TAG occurred after initial absorption and indicates the presence of a previously unrecognized isomerization mechanism. Incorporation and turnover of the ²H-FA in chylomicron TAG, plasma TAG, and plasma cholesterol esters were not influenced by TAG acyl position. Accretion of ²H-16∶0 from the sn-2 TAG position in 1-acylphosphatidylcholine was 1.7 times higher than ²H-16∶0 from the sn-1,3 TAG positions. Acyl TAG position did not influence ²H-18∶2n−6 incorporation in PC. The concentration of ²H-18∶2n−6-derived ²H-20∶4n−6 in plasma PC from subjects fed, the RL diet was 1.5 times higher than for subjects fed the NL diet, and this result suggests that diets containing 16∶0 located at the sn-2 TAG position may inhibit 20∶4n−6 synthesis. The overall conclusion is that selective rearrangement of chylomicron TAG structures diminishes but does not totally eliminate the metabolic and physiological effects of dietary TAG structure.     

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.     

Utilization of reaction medium-dependent regiospecificity of Candida antarctica lipase (Novozym 435) for the synthesis of 1,3-dicapryloyl-2-docosahexaenoyl (or eicosapentaenoyl) glycerol

A highly efficient enzymatic method for the synthesis of regioisomerically pure 1,3-dicapryloyl-2-docosahexaenoyl glycerol (CDC) in two steps was established. 2-Monoglyceride (2-MG) formation by ethanolysis of tridocosahexaenoylglycerol (DDD) with immobilized Candida antarctica lipase (Novozym 435) as catalyst was the key step of the synthesis. CDC was finally obtained by reesterification of 2-MG with ethylcaprylate (EtC) catalyzed by Rhizomucor miehei lipase (Lipozyme IM). The regiospecificity of Novozym 435 depended on the type of reaction and the initial composition of the reaction medium. It displayed strict 1,3-regiospecificity for ethanolysis at a high excess of ethanol in the reaction mixture although it displayed no regiospecificity in transesterification and esterification reactions. The highest yield of CDC (85.4%) was obtained by ethanolysis at a 4∶1 weight ratio of ethanol/DDD for 6 h followed by reesterification at a 20∶1 molar ratio of EtC/initial DDD for 1.5 h. The regioisomeric purity of CDC was 100%. Good results were obtained also for the synthesis of 1,3-dicapryloyl-eicosapentaenoylglycerol (CEC) by the same method: 84.2% yield and 99.8% regioisomeric purity at the same reactant ratios as above. The yield of the reesterification step and the regioisomeric purity of the product were influenced by the molar ratio of the reactants for both CDC and CEC syntheses: higher excess of EtC favored higher yields and regioisomeric purity of the products.     

Generation and remodeling of highly polyunsaturated molecular species of rat hepatocyte phospholipids

Freshly isolated rat hepatocytes were incubated for 20 min with [U-¹⁴C]glycerol in the presence or absence of unlabeled linoleic (18∶2n-6), arachidonic (20∶4n-6), or docosahexaenoic (22∶6n-3) acid, added as albumin complex in 10% ethanol. Most of the radioactivity (≈95%) recovered in hepatocyte lipids was present in phosphatidylcholine (PC), phosphatidylethanolamine (PF), and triacylglycerol (TAG). The presence of exogenous fatty acids resulted in (i) higher incorporation of [U-¹⁴C]glycerol, (ii) higher percentage of label in TAG, and (iii) enhanced formation of PC and PE molecular species bearing the exogenous fatty acid at both the sn-1 and sn-2 positions of glycerol. In each case, these molecular species contained 60 to 70% of the label in that lipid class. Further incubation of the cells for 40 and 80 min in the absence of labeled substrate and exogenous fatty acids resulted in a redistribution of label among PC and PE molecular species due to deacylation-reacylation at the sn-1 position of glycerol.     

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.     

Distribution of medium-chain FA in different lipid classes after administration of specific structured TAG in rats

Structured TAG (STAG) containing medium-chain FA (MCFA) in the sn-1,3 positions and essential FA in the sn-2 position were synthesized by lipase-catalyzed acidolysis. In our previous studies we found that part of the MCFA from STAG could be absorbed in the small intestine; however, it was unclear how they were absorbed. In order to get a better understanding of the metabolism of STAG to improve future design and application of STAG, in the present study lymph lipids collected after feeding STAG were fractionated into different classes and the FA composition of each lipid class was studied by GC after methylation to FAME. Caprylic acid was detected in the fraction of TAG only after administration of 1,3-dioctanoyl-2-linoleyl-sn-glycerol (8∶0/18∶2/8∶0), whereas lauric acid was detected in TAG, DAG, and FFA as well as phospholipids after administration of 1,3-didodecanoyl-2-linoleyl-sn-glycerol (12∶0/18∶2/12∶0). We conclude that the enterocyte has the ability to reacylate the MCFA into TAG and that the intestinal absorption of MCFA from STAG mainly occurs by resynthesis of TAG. Caprylic acid from STAG is not incorporated into phospholipids, whereas lauric acid from STAG can be incorporated into phospholipids.     

Lipase-catalyzed synthesis of structured triacylglycerides from 1,3-diacylglycerides

A new method for the lipase-catalyzed synthesis of structured TAG (ST) is described. First, sn1,3-dilaurin or-dicaprylin were enzymatically synthesized using different published methods. Next, these were esterified at the sn2-position with oleic acid or its vinyl ester using different lipases. Key to successful enzymatic synthesis of ST was the choice of a lipase with appropriate FA specificity, i.e., one that does not act on the FA already present in the sn1,3-DAG, but that at the same time exhibits high selectivity and activity toward the FA to be introduced. Reactions were performed in the presence of organic solvents or in solvent-free systems under reduced pressure. With this strategy, mixed ST containing the desired compounds 1,3-dicaprylol-2-oleyl-glycerol or 1,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at 87 and 78 mol% yield, respectively, using immobilized lipases from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C. However, regiospecific analysis with porcine pancreatic lipase indicated that in CyOCy, 25.7% caprylic acid and in LaOLa 11.1% lauric acid were located at the sn2-position. Oleic acid vinyl ester was a better acyl donor than oleic acid. Esterification of sn1,3-DAG and free oleic acid gave very low yield (<20%) of ST in a solvent system and moderate yield (>50%) in a solvent-free system under reduced pressure.     

Regiospecific analysis by ethanolysis of oil with immobilized <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

A mixture of oil/ethanol (1∶3, w/w) was shaken at 30°C with 4% immobilized Candida antarctica lipase by weight of the reaction mixture. The reaction regiospecifically converted FA at the 1- and 3-positions to FA ethyl esters, and the lipase acted on C₁₄−C₂₄ FA to a similar degree. The content of 2-MAG reached a maximum after 4 h; the content was 28–29 mol% based on the total amount of FA in the reaction mixture at 59–69% ethanolysis. Only 2-MAG were present in the reaction mixture during the first 4 h, and 1(3)-MAG were detected after 7 h. After removal of ethanol from the 4-h reaction mixture by evaporation, 2-MAG were fractionated by silica gel column chromatography. The contents of FA in the 2-MAG obtained by ethanolysis of several oils coincided well with FA compositions at the 2-position, which was analyzed by Grignard degradation. It was shown that ethanolysis of oil with C. antarctica lipase can be applied to analysis of FA composition at the 2-position in TAG.     

Molecular weight distribution and regioisomeric structure of triacylglycerols in some common human milk substitutes

Triacylglycerol (TAG) molecular weight distribution and regioisomeric structure of selected molecular weight species in human milk and in 32 human milk substitutes was determined. Negative ion chemical ionization mass spectrometry was used to determine the molecular weight distribution and collisionally induced dissociation tandem mass spectrometry applied to identify the sn-2 and sn-1/3 positions of fatty acids in TAG. The main molecular weight species of human milk TAG in decreasing order of abundance were 52∶2, 52∶3, 52∶1, 54∶3, 50∶2, 50∶1, 54∶4, 48∶1, 54∶2, 48∶2, 46∶1, 52∶4, and 50∶3 (acyl carbon number/number of double bonds), constituting 83 mol% of total TAG molecular species. In human milk substitutes, the proportion of the corresponding molecular weight species varied from 33 to 87 mol%. The main TAG regioisomers within the molecular weight species 52∶2, 52∶3, and 50∶1 in human milk were 18∶1-16∶0-18∶1 (83 mol%), 18∶1-16∶0-18∶2 (83 mol%), and 18∶1-16∶0-16∶0 (80 mol%), respectively. In human milk substitutes, the corresponding proportions varied in a wide range of 0–82 mol%, 0–100 mol%, and 0–73 mol%, respectively. Although TAG structures in some human milk substitutes closely resembled those in human milk, the great variation among samples leads to the conclusion that it is still possible to improve the TAG composition in human milk substitutes by applying novel methods to synthesize structured TAG.