Synthetic Triacylglycerols Research Articles

Separation of regioisomers and enantiomers of triacylglycerols containing branched fatty acids (iso and/or anteiso).

A combination of two chromatographic and one enzymatic methods was used for identification of the molecular species of triacylglycerols (TAGs) from Streptomyces avermitilis. Streptomyces avermitliswas cultured on various carbon sources and the ratio of iso- (i-FAs), anteiso- (ai-FAs), and straight-chain- (n-FAs) fatty acids was modified by precursor-directed biosynthesis. Saturated TAGs were separated from other lipids (including TAGs containing unsaturated FAs) using Ag+ ion cartridges. Analysis of TAGs wereperformed by RP-HPLC/ESI+ tandem mass spectrometry. Both the synthetically prepared sn-TAGs and the natural mixture of TAGmolecular species of wereseparated and identified by tandem MS. The structures of synthetic TAGs werefurther confirmed by pancreatic lipase, which cleaves sn-TAGs into sn-2-monoacylglycerols. The retention times (tR) of the individual regioisomers and enantiomers were found to be depend on the structure of the TAGs. If one branched acyl (iso or anteiso) is present in the TAG molecule, then the elution order is enantiomer (n/n/br), opposite enantiomer (br/n/n), regioisomer (n/br/n). In the case where two branched acyls are in the TAG molecule, the order of the elution is different, that is, br/n/br, n/br/br, br/br/n. In all cases, it was further demonstrated that tandem MS of either synthetically prepared TAGs or TAGs obtained from natural material, that is, n-16:0/ai-15:0/n-16:0 and i-16:0/n-15:0/i-16:0 are identical. Unfortunately, it is not possible to distinguish by ESI+ tandem MS such TAGs, which differ only in the branching of the acyls. The results of our analyses of TAGs are in good agreement with previously published data in other streptomycetes.

Biocatalytic synthesis and characterization of sn-1/3 and sn-2 monoacylglycerols.

To investigate the lipase-catalyzed synthesis of high purity sn-1/3 and sn-2 monoacylglycerols (1/3-MAG and 2-MAG) of different fatty acids (FAs). The 1/3-MAGs of three FAs (16:0, 17:0, 16:1) were synthesized using lipase-catalyzed esterification of glycerol with FAs. The 2-MAGs were obtained from the ethanolysis of synthetic triacylglycerols using sn-1,3 regiospecific lipase. The effects of lipase types, substrate ratio, temperature, reaction time and lipase load on the MAG conversion were studied. Under the optimal conditions, high purities (96.74%, 95.44%, 92.96%) with acceptable isolated yields (51.00%, 54.28%, 46.00%) were obtained for 1/3-16:0-MAG, 1/3-17:0-MAG, and 1/3-16:1-MAG, respectively. For 2-16:0-MAG, 2-17:0-MAG, and 2-16:1-MAG, the purities were 92.64, 95.04, and 96.48%, with isolated yields of 50.64, 52.16, and 26.12%, respectively. The molecular structures of the synthetic compounds were confirmed by 1H NMR, and MS and the melting points were characterized by DSC. High purity MAG isomers can be synthesized via lipase-catalyzed reactions to be building blocks for production of functional lipids, the melting points of which are largely governed by the hydrophobic interactions among the alkanyl chains.

Polarized THG Microscopy Identifies Compositionally Different Lipid Droplets in Mammalian Cells

Cells store excess lipids as two major compounds, triacylglycerols (TAGs) and cholesteryl esters (CEs), inside lipid droplets (LDs). The degree of lipid ordering is considered to play a major role in the mobility and enzymatic processing of lipids in LDs. Here, we provide evidence that polarized third-harmonic generation (THG) microscopy distinguishes between native TAG- and CE-enriched LDs in cells due to the different ordering of the two lipid species. We first demonstrate that the responses from synthetic TAG- and CE-enriched LDs using THG microscopy with linear and circular polarizations differ according to their different intrinsic ordering. We then employ simulations to dissect how polarization effects influence the THG from an isotropic LD. Finally, we induce TAG- and CE-enriched LDs in murine macrophages and demonstrate that polarized THG responses increase in a nonlinear fashion with increasing CE/TAG ratio. This suggests that with an increasing CE content, there is a rather sharp transition toward increased LD ordering. Our results demonstrate that polarized THG microscopy enables label-free quantitative analysis of LD ordering and discriminates between compositionally different LDs in intact mammalian cells.

High resolution 13CNMR detection of short‐ and medium‐chain synthetic triacylglycerols used in butterfat adulteration

Recently in European countries, control laboratories have revealed real cases of deceptive practices in which synthetic triacylglycerols (TAGs) were used for adulterating butterfat. The complex fat blends are difficult to detect with the classical methods of analysis, especially when the adulterating TAGs contain fatty acids (FAs) in the proportions similar to that of genuine butter. Nevertheless, because butyric acid (C4) exclusively occurs at the sn‐3 position in milk fat TAGs, the determination of its distribution on the glycerol backbone provides a diagnostic indicator to differentiate genuine butter from mixtures with synthetic TAGs. Herein, we have demonstrated the straightforward application of mono‐dimensional 13C NMR spectroscopy for detecting synthetic TAGs in mixtures with authentic butterfat. The method is based on the 13C NMR determination of the regioisomeric distribution of C4 between the sn‐1,3‐ and sn‐2‐positions and boasts several analytical advantages, such as specificity, robustness and minimal sample handling. The limit of detection (LOD) and limit of quantification (LOQ) of the synthetic fats, estimated to be 1 and 2.5% w/w, respectively, easily surpass the limit of 10% at which a fraudulent practice might be economically attractive.Practical applications We have developed a robust and efficient method, based on high resolution 13C NMR spectroscopy, for detecting the adulteration of butterfat with synthetic low‐ and medium‐carbon number TAG mixtures. In milk fat TAGs, butyric acid is esterified exclusively at the sn‐3 position of the glycerol backbone. Thus, the method relies on 13C NMR monitoring of the carbonyl resonance of butyric acid at the sn‐2 glycerol position, which is diagnostic of synthetic or interesterified TAGs. The 13C NMR‐based analysis overcomes the pitfalls of traditional pattern recognition methodologies for the assessment of butter authenticity, which can be ineffective when butter is adulterated with synthetic TAGs obtained from FA in proportions similar to those of natural milk fat. The method achieves reliable quantification up to 2.5% w/w of adulterating fats (LOQ) and can detect quantities as low as 1% w/w of synthetic TAGs blended with butter (LOD).

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases.

Oxidation progress in an emulsion made from metmyoglobin and different triacylglycerols

Emulsions made using metmyoglobin as emulsifier and different synthetic and natural triacylglycerols (TAG), have been studied with respect to oxidation progress during 48h at ambient temperature. The investigated model systems contained polyunsaturated TAGs of linolein (C18:2n-6, tri-LIN), arachidonin (C20:4n-6, tri-ARA), docosahexanoin (C22:6n-3, tri-DHA), salmon oil (SO) and cod liver oil (CLO). The synthetic TAGs were added in ratios of one part to nine parts trioctanoin (C8:0), yielding model composite oil systems. The total amount of volatiles, determined using dynamic headspace GC/MS, were relatively larger for tri-LIN and tri-ARA than for the marine systems during the first 6h of observation. After 48h of incubation, the marine oils had more total volatiles than had tri-LIN and tri-ARA. Hexanal and propanal were observed as the marker degradation compounds for n-6 and n-3 fatty acids, respectively. Myoglobin tended to aggregate in these model systems, and only for trioctanoin and tri-LIN could the protein monomer be recovered unmodified and subsequently observed using MALDI-TOF MS after 48h of incubation. Myoglobin was only recovered after 4h of incubation in the tri-DHA system, and it then appeared adducted with lipid fragments up to ∼1000Da. Final oxidation (after 48h) products from the marine lipids were trienes, most likely as alcohols or acids. The natural oils revealed a reaction pattern close to that of tri-DHA.

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape(TM) from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.

RP‐HPLC/MS‐APCI Analysis of Branched Chain TAG Prepared by Precursor‐Directed Biosynthesis with Rhodococcuserythropolis

Reversed phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/MS-APCI) was used to analyze both synthetic triacylglycerols (TAG) having 1-3 branched fatty acids (FA) in the molecule, and natural TAG prepared by precursor directed biosynthesis from valine, leucine and isoleucine and the corresponding branched short-chain acids in cultivations of Rhodococcus erythropolis. The technique made it possible to identify and quantify TAG differing in a single branched-chain FA. Altogether 11 TAG were synthesized, out of which 8 were synthesized stereospecifically. Branched- and straight-chain-TAG were separated and identified while TAG differing only in iso or anteiso FA could not be separated. The APCI mass spectra of iso-, anteiso- and straight-chain TAG were completely identical. The natural material was found to contain 19 TAG having at least one branched FA. Cultivation on six different substrates showed, apart from the presumed and common incorporation of precursors to iso-even, iso-odd and anteiso FA, also some unusual features such as an increase in the content of odd-FA after the addition of Val (attributed to catabolism of Val to propionate) or the appearance of branched monounsaturated FA. The two-sample paired t test, when applied to the TAG, showed that only the pair Val and isobutyrate differ in incorporation into FA--see, e.g. proportions of M/M/O and brM/brM/O (1.2:1.2 and 1.9:1.2, respectively). Also, incorporation of Val (isobutyrate) yielded only TAG having two branched FA in the molecule, whereas Leu and Ile (isovalerate and 2-methyl-butyrate) gave only TAG with a single branched FA in the molecule.

EFFECTS OF DOCOSAHEXAENOIC ACID POSITIONAL DISTRIBUTION ON THE OXIDATIVE STABILITY OF MODEL TRIACYLGLYCEROL IN WATER EMULSION

ABSTRACT The effect of the positional distribution of docosahexaenoic acid (DHA) within triacylglycerol (TAG) molecules on the oxidative stability of oil‐in‐water emulsions was investigated by using a synthetic TAG regio‐isomer pair SDS (1,3‐dioctadecanoyl‐2‐[4,7,10,13,16,19‐docosahexaenoyl] glycerol) and SSD (1,2–dioctadecanoyl‐3‐[4,7,10,13,16,19‐docosahexaenoyl] glycerol), where S and D represent stearic acid and DHA, respectively. Oil‐in‐water emulsions (10%, w/w) of each isomer were subjected to accelerated autoxidation by continuous exposure to air at 50C in the absence of light. Oxidation during the exposure (storage) was monitored by measuring a series of volatile compounds characteristic of DHA oxidation. SSD emulsion oxidized faster than SDS emulsion, showing that DHA is more stable to oxidation when located at the sn‐2 position of the TAG compared with the sn‐1(3) position. This regio‐isomeric effect is similar to that previously reported for bulk oil oxidation.PRACTICAL APPLICATIONSMany of the food products that have been selected for fortification with omega‐3 oils such as milk, yoghurt, salad dressings and juices are oil‐in‐water emulsions. This study, for the first time, demonstrated that the regio‐isomeric effects on oxidative stability of docosahexaenoic acid observed for bulk oil also apply to oil‐in‐water emulsion. Thus, potential exists for enhancing the oxidative stability of omega‐3‐fortified emulsion foods through modification of triacylglycerol structure.

The Renaissance of High-Energy CID for Structural Elucidation of Complex Lipids: MALDI-TOF/RTOF-MS of Alkali Cationized Triacylglycerols

Triacylglycerols were analyzed as cationized species (Li(+), Na(+), K(+)) by high-energy CID at 20 keV collisions utilizing MALDI-TOF/RTOF mass spectrometry. Precursor ions, based on [M + Li](+)-adduct ions exhibited incomplete fragmentation in the high and low m/z region whereas [M + K](+)-adducts did not show useful fragmentation. Only sodiated precursor ions yielded product ion spectra with structurally diagnostic product ions across the whole m/z range. The high m/z region of the CID spectra is dominated by abundant charge-remote fragmentation of the fatty acid substituents. In favorable cases also positions of double bonds or of hydroxy groups of the fatty acid alkyl chains could be determined. A-type product ions represent the end products of these charge-remote fragmentations. B- and C-type product ions yield the fatty acid composition of individual triacylglycerol species based on loss of either one neutral fatty acid or one sodium carboxylate residue, respectively. Product ions allowing fatty acid substituent positional determination were present in the low m/z range enabling identification of either the sn-1/sn-3 substituents (E-, F-, and G-type ions) or the sn-2 substituent (J-type ion). These findings were demonstrated with synthetic triacylglycerols and plant oils such as cocoa butter, olive oil, and castor bean oil. Typical features of 20 keV CID spectra of sodiated triacylglycerols obtained by MALDI-TOF/RTOF MS were an even distribution of product ions over the entire m/z range and a mass accuracy of +/-0.1 to 0.2 u. One limitation of the application of this technique is mainly the insufficient precursor ion gating after MS1 (gating window at 4 u) of species separated by 2 u.

Hydrolysis of Acyl‐Homogeneous and Fish Oil Triacylglycerols Using Desalted Midgut Extract from Atlantic Salmon, Salmo salar

Despite several studies aimed at evaluating the positional and fatty acid specificity of fish triacylglycerol (TAG) digestive lipases, there is still much uncertainty regarding these issues. The aim of the present study was therefore to address these questions in Atlantic salmon (Salmo salar L.). Crude luminal midgut extracts were collected from fed salmon and the hydrolysis studied for various substrates including triolein (Tri-18:1), trilinolein (Tri-18:2), trilinolenin (Tri-18:3), trieicosapentaenoin (Tri-20:5), tridocosahexaenoin (Tri-22:6) and natural fish oil TAG. Using Tri-18:1, in a time-curve model showed an initial high degree of sn-1 or sn-3 specificity where sn-1,2(2,3)-diacylglycerol (1,2(2,3)-DAG) and free fatty acid (FFA) were the main hydrolytic products up to 15 min. Lack of initial sn-2 specificity was confirmed by negligible sn-1,3-diacylglycerol (1,3-DAG) being produced. During the further hydrolysis of DAG, all positions appeared susceptible to attack causing a concomitantly small increase in sn-1(3)-monoacylglycerol (1(3)-MAG) and 2-MAG, but not at the level expected for an exclusively sn-1,3-specific lipase. Oleic acid (18:1n-9) and eicosapentaenoic acid (20:5n-3) were preferred substrates for hydrolysis using both fish oil and acyl-homogeneous TAGs with FFA as the main product of lipolysis. Hydrolysis of the natural fish oil TAG appeared slower yet produced proportionally more MAG and DAG after 5 min, and similar specificities, as for synthetic TAG substrates, were exhibited with 18:1n-9 and 20:5n-3 accumulating in the FFA fraction after 30 min. Notably, 16:0 was particularly conserved in MAG. As TAG resynthesis of absorbed lipid in salmon enterocytes proceeds preferably with 2-MAG as templates, the absorption of 2-MAG, produced during initial stages of TAG hydrolysis, would need to occur rapidly to be effectively utilised via the MAG pathway.

The Position of Rumenic Acid on Triacylglycerols Alters Its Bioavailability in Rats

The metabolic fate of rumenic acid (9cis,11trans-octadecenoic acid) related to its position on the glycerol moiety has not yet been studied. In the present work, synthetic triacylglycerols (TAG) esterified with oleic and rumenic acids were prepared. Rats were force-fed synthetic dioleyl monorumenyl glycerol with (14)C labeled rumenic acid in the internal (sn-2) or in the external position (sn-1 or sn-3). Rats were then placed in metabolic cages for 16 h. At the end of the experiment, the radioactivity in tissues, carcass and expired CO(2) was measured. Rumenic acid that was esterified at the external positions on the TAG was better absorbed and oxidized to a greater extent than when esterified at the internal position. The fatty acid from the 2-TAG form was also better incorporated into the rat carcass. In the liver, rumenic acid appeared mainly in TAG (50%) and to a lesser extent in phospholipids (33%) whatever its dietary form. Moreover, analyses of lipids from Camembert cheese and butter revealed that rumenic acid was located mainly on the sn-1 or sn-3 positions (74%). Taken together, these data suggest that rumenic acid from dairy fat may be well absorbed and used extensively for energy production.

Reversed-phase high-performance liquid chromatographic separation of tert.-butyl hydroperoxide oxidation products of unsaturated triacylglycerols

Triacylglycerols containing monounsaturated fatty acids are known to be relatively resistant to autoxidation and require long periods of exposure to dilute oxidants. Use of concentrated solutions of synthetic hydroperoxides, however, yields in addition to the hydroperoxides also unidentified oxidation by-products. In the present study we have employed synthetic triacylglycerols containing one (18:0/18:1/18:0 and 18:1/16:0/16:0) and two (18:0/18:0/18:2 and 18:1/18:1/18:0) double bonds per molecule to reinvestigate the formation of oxotriacylglycerols using tert.-butyl hydroperoxide as an oxidant. Reversed-phase HPLC was used to separate and tentatively identify the oxidation products based on relative retention times of standards and the estimated elution factors for functional groups and their positional distribution. Hydroperoxides, diepoxides and hydroxides were the major components of the oxidation mixtures (50–95% of total). Previously unidentified peroxide-bridged tert.-butyl adducts were present in significant amounts (5–50% of total oxidation products) in all preparations. In several instances more than one functional group was present on a single fatty chain. The tentative reversed-phase chromatographic identification of the adducts was confirmed by determination of the molecular mass of each component by on-line LC with electrospray MS. The oxidation products were quantified by HPLC with light scattering detection.

Effect of medium-chain fatty acid positional distribution in dietary triacylglycerol on lymphatic lipid transport and chylomicron composition in rats.

The present study was carried out to examine if the positional distribution of medium-chain fatty acid (MCF) in dietary synthetic fat influences lymphatic transport of dietary fat and the chemical composition of chylomicrons in rats with permanent cannulation of thoracic duct. Four types of synthetic triacylglycerol were prepared: (i) sn-1 (3) MCF-sn 2 linoleic acid, (ii) interesterified sn-1(3) MCF-sn 2 linoleic acid, (iii) sn-2 MCF-sn-1(3) linoleic acid, and (iv) interesterified sn-2 MCF-sn-1(3) linoleic acid. A purified diet composed of equal amounts of the synthetic fat and cocoa butter was given to rats with permanent lymph duct cannulation. The positional distribution of MCF in the dietary fat had no significant effect on the lymph flow, triacylglycerol output, phospholipid output, lipid composition of chylomicrons, or the particle size. The positional distribution of MCF in the synthetic triacylglycerol was maintained in the chylomicron triacylglycerol. These results showed that MCF in the dietary triacylglycerol is transported into lymphatics and the positional distribution is well preserved in chylomicron triacylglycerol.

Double-blind, randomized trial of a synthetic triacylglycerol in formula-fed term infants: effects on stool biochemistry, stool characteristics, and bone mineralization

The low sn-2 palmitate content of infant formulas results in formation of fatty acid calcium soaps in the stools and reduced calcium absorption. Our objective was to test the hypotheses that increasing the proportion of sn-2 palmitate in formula for term infants would result in greater skeletal mineral deposition and reduced stool hardness. Healthy term neonates were randomly assigned to receive standard formula (n = 103) or formula containing 50% sn-2 palmitate (high-sn-2 formula; n = 100) for 12 wk. One hundred twenty breast-fed infants were also studied. The main outcome measures were 1) radial (single-photon absorptiometry) and whole-body (dual-energy X-ray absorptiometry) bone mineral content (WBBMC) at 12 wk and 2) stool frequency, volume, and consistency at 6 and 12 wk. Secondary outcome measures included stool fatty acid content. Infants receiving high-sn-2 formula had higher WBBMC (128.1 +/- 9.7 compared with 122.7 +/- 10.1 g, adjusted for size and sex), softer stools at 6 and 12 wk, and a lower proportion of stool soap fatty acids than did infants receiving the control formula. Breast-fed infants had adjusted WBBMC values (128.3 +/- 9.1 g) similar to those of infants fed high-sn-2 formula and significantly higher than those of infants fed the control formula. Changing the stereoisomeric structure of palmitate in infant formula resulted in higher WBBMC, reduced stool soap fatty acids, and softer stools more like those of breast-fed infants. The greater bone mass measured could be important if it persists beyond the trial period; this merits further investigation.

Production and characteristics of the lipase from Yarrowia lipolytica 681

The production of lipase by Yarrowia lipolytica 681 was enhanced significantly ( P < 0.05) by olive or corn oil when used as both carbon and inducer sources. Urea was the best nitrogen source for lipase production. Low levels of aeration increased lipolytic activity and mycelia formation in the culture medium. Concentrations of Tween 80 between 0.5 and 2 g/l increased extracellular lipase activity without changes in the concentration of biomass. A response surface analysis predicted a temperature of 29.5°C, an initial pH of 4.7 and an incubation time of 54 h as the optimum conditions for lipase production. Lipase production was strongly correlated to biomass during 52 h of incubation. The enzyme had a maximum activity at pH 6 and 37°C. The lipase did not lose significant amounts of activity during incubation at pH 6 and 5°C for 72 h. The enzyme was highly active towards synthetic triacylglycerols and olive oil. However, the lipase was strongly inhibited by oleic acid.

Elution factors of synthetic oxotriacylglycerols as an aid in identification of peroxidized natural triacylglycerols by reverse-phase high-performance liquid chromatography with electrospray mass spectrometry.

Selected elution factors were determined for model oxotriacylglycerols as an aid in identification of the peroxidation products of natural triacylglycerols by reverse-phase high-performance liquid chromatography (HPLC) with electrospray mass spectrometry (LC/ES/MS). For this purpose synthetic triacylglycerols of known structure were converted to hydroperoxides, hydroxides, epoxides, and core aldehydes and their dinitrophenylhydrazones by published procedures. The oxotriacylglycerols were resolved by normal-phase thin-layer chromatography and reverse-phase HPLC, and the identities of the oxotriacylglycerols confirmed by LC/ES/MS. Elution factors of oxotriacylglycerols were determined in relation to a homologous series of saturated triacylglycerols, ranging from 24 to 54 acyl carbons, and analyzed by reverse-phase HPLC, using a gradient of 20-80% isopropanol in methanol as eluting solvent and an evaporative light-scattering detector. It was shown that the elution times varied with the nature of the functional group and its regiolocation in the triacylglycerol molecule. A total of 31 incremental elution factors were calculated from chromatography of 33 oxygenated and nonoxygenated triacylglycerol species, ranging in carbon number from 36 to 54 and in double-bond number from 0 to 6.

Non-aqueous reversed-phase high-performance liquid chromatography of synthetic triacylglycerols and diacylglycerols

Reversed-phase high-performance liquid chromatography (HPLC) methods were developed for the separation of molecular species of 45 synthetic triacylglycerols and diacylglycerols. These methods used linear gradients of methanol-isopropanol and UV detection at 205 nm as well as radioactive flow detection, which we add for metabolic studies. The elution orders of triacylglycerols and diacylglycerols depend on the polarity of their fatty acid constituents with elution time increasing as polarity decreases. The elution orders of triacylglycerols depending on their fatty acid constituents were as follows: ricinoliec acid<linolenic acid<palmitoleic acid<myristic acid<palmitelaidic acid<linoleic acid<linolelaidic acid<oleic acid<palmitic acid<elaidic acid<petroselinic acid<petroselaidic acid<stearic acid, while the elution orders of diacylglycerols were: palmitic acid<oleic acid<stearic acid. For both classes of glycerides, elution corresponded closely with chain length, degree of unsaturation and presence of polar groups and they were similar to the elution orders of fatty acids on an aqueous C 18 HPLC which we reported recently: Other structural features also affect eltion order, as triacylglycerol containing a cis-fatty acid elutes slightly earlier than its isomer containing a trans-fatty acid. Additionally, higher polarity in the sn-2 position causes earlier elution; Diacylglycerol with a hydroxy group at sn-2 position of the glycerol backbone and triacylglycerol with a polar group on the fatty acid chain at sn-2 position elute slightly earlier than their respective sn-1(3) isomers.

Oxidation of synthetic triacylglycerols containing eicosapentaenoic and docosahexaenoic acids: Effect of oxidation system and triacylglycerol structure

AbstractThirteen synthetic triacylglycerols (TAG) containing eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) were oxidized in the presence of 2,2′‐azobis(2,4‐dimethyl‐valeronitrile) (AMVN) and 2,2′‐azobis(2‐amidinopropane)dihydrochloride (AAPH) as aqueous and nonaqueous radical initiators to investigate the influence of TAG structure and oxidation system on the oxidative stability of TAG that contain highly unsaturated fatty acids (HUFA). A 2:1 (mol/mol) mixture of trieicosapentaenoylglycerol and tripalmitoylglycerol was most susceptible to the AMVN‐initiated oxidation among three types of TAG that contained EPA and palmitic acid (2:1, mol/mol). Compared with 1,2 (or 2,3)‐dieicosapentaenoyl‐3(or 1)‐palmitoylglycerol (EEP) and 1,3‐dieicosapentaenoyl‐2‐palmitoylglycerol (EPE), the oxidative rate of EEP was somewhat higher. A similar result was obtained for DHA‐containing TAG. The oxidative rate of TAG that contained EPA and palmitic acid (1:2, mol/mol) showed a positive correlation with the amount of EPA in a single TAG molecule. Moreover, in the nonaqueous system, the oxidative rate of EPA‐containing TAG was affected by unsaturation and carbon chainlength of constituent fatty acids. In the AAPH‐initiated oxidation in the aqueous system, the oxidative rate of TAG with EPA and palmitic acid was higher with the increased quantity of EPA in a single TAG molecule. Also, constituent fatty acids modified the oxidative rate of EPA‐containing TAG in an aqueous system. The glycerol position of EPA and DHA also affected the oxidative rate of the TAG. EPA and DHA located at the 1,2 (or 2,3)‐position of glycerol were more oxidizable than those at the 1,3‐position during AAPH‐initiated oxidation. Thus, 1,2(or2,3)‐dipalmitoyl‐3(or 1)‐eicosapentaenoylglycerol was oxidized faster than 1,3‐dipalmitoyl‐2‐eicosapentaenoylglycerol. These observations suggest that the oxidative stability of TAG that contain HUFA could be modulated by the oxidation system and TAG structure.

Kinetics for the Autoxidation of Triacylglycerols Containing Eicosapentaenoic Acid

The oxidative rate of three kinds of synthetic triacylglycerols (TAGs) consisting of eicosapentaenoic acid (EPA) and palmitic acid was measured by the induction period method to understand the mechanism for the autoxidation of TAGs containing highly unsaturated fatty acids (HUFAs) such as marine oils. The propagation rate, oxidizability, and kinetic chain length increased with the number of moles of EPA in a single TAG molecule, although the initiation rate was almost the same for all the TAG samples. These results demonstrate that the oxidative rate of EPA in TAGs was affected by the TAG structure and that EPA highly concentrated in a TAG molecule was very susceptible to free-radical oxidation.