Stereospecific analysis of triacyl-sn-glycerols in Docosahexaenoic acid-rich fish oils

This paper presents the positional distribution of fatty acids in docosahexaenoic acid (22∶6n-3)-rich fish oil triacyl-sn-glycerols (TG). Stereospecific analysis of TG was carried out by a nonenzymatic method. The TG of bonito head oil, obtained after a winterization process, contained 22∶6n-3 at concentrations of 28,7, and 49 mole % in thesn-1,sn-2, andsn-3 positions, respectively. In the TG of oil before the winterization process, 22∶6n-3 was concentrated in thesn-3 position, followed evenly by thesn-1 andsn-2 positions. Tuna orbital oil, obtained after winterization, showed the preferential association of 22∶6n-3 to thesn-3 position, followed by thesn-1 position. This distribution pattern was similar to that observed for seal oil TG rather than sardine oil TG. The bonito head and tuna orbital oils are useful as fish oils with characteristics different from those of common fish oils, such as menhaden, sardine, and herring oils.     

In vitro hydrolysis of natural and synthetic γ-linolenic acid-containing triacylglycerols by pancreatic lipase

The present study compared thein vitro hydrolysis of two 18:3n-6-rich oils—evening primrose oil (EPO) and borage oil (BO)—and different synthetic 18:3n-6-containing triacylglycerols (TG). Incubation of EPO and BO with pancreatic lipase lipolyzed 18:3n-6 from the TG species. The rate of lipolysis of TG species containing two or three molecules of 18:3n-6, which comprised 36% of total 18:3n-6 in BO and only 7% in EPO, was significantly slower than those containing only one molecule of 18:3n-6. This was found especially in those with two molecules of linoleic acid, which constituted 20% of total 18:3n-6 in BO, whereas over 80% were present in EPO. In a separate study, various synthetic 18:3n-6-containing TG were also subjected toin vitro hydrolysis by pancreatic lipase. Results showed that release of 18:3n-6 from thesn-1/sn-3 positions was significantly slower when two other stereospecific positions in the same TG molecule were occupied by either palmitic acid (16:0) or monounsaturated (18:1 and 20:1) fatty acids than when occupied by 18:2n-6. The rate of hydrolysis ofsn-2-γ-linolenyl-sn-1(3)-diacylglycerol to formsn-2-mono-γ-linolenyl glycerol was also significantly slower when both thesn-1 andsn-3 positions in TG molecules were occupied by either saturated fatty acids (16:0 and 18:0) or long-chain monounsaturated fatty acids than when occupied by 18:2n-6. These findings suggest that the stereospecific position of 18:3n-6 in TG molecules and the constituent of its neighboring fatty acids modulated availability of 18:3n-6 from 18:3n-6-containing TG or 18:3n-6-rich oils.     

Positional distribution of 24∶6(n−3) in triacyl-sn-glycerols from flathead flounder liver and flesh

This paper presents the positional distribution of very long-chain fatty acids, 24∶6(n−3), in triacyl-sn-glycerols (TG) of flathead flounder (Hippoglossoides dubius). Each of the liver and flesh TGs was subjected to the stereospecific analysis. The liver TGs contained 24∶6(n−3) at concentrations of 1.5, 1.2 and 1.7 mole % in thesn-1,sn-2 andsn-3 positions, respectively, and the flesh TGs had 9.0, 7.8 and 7.1 mole % in thesn-1,sn-2 andsn-3 positions, respectively. This fatty acid was distributed almost evenly among the three positions of the TGs. No preference for thesn-2 position was observed in contrast to the general tendency for the distribution of longer-chain polyunsaturated fatty acids, such as 22∶6(n−3), 22∶5(n−3) and 20∶5(n−3). There was essentially no difference in the positional distributions of the liver and flesh TGs. The results obtained in this study give new fundamental information to the investigation of very long-chain fatty acids.     

Stereospecific analysis of triacylglycerols from vegetable oils by two procedures—II: Normal and high-oleic sunflower oils

Triacylglycerol stereospecific analysis of normal (NOS) and high-oleic sunflower (HOS) oils was carried out by two procedures to study the influence of variety and growing conditions. Four cultural varieties, two NOS and two HOS, were grown in seven different places of Italy. Three of the four varieties were grown both in dry conditions and with irrigation. Concerning the triacylglycerol fatty acid compositions, the results showed no significant differences between irrigated and nonirrigated samples (P>0.05), between the two NOS, and between the two HOS varieties. Between NOS and HOS varieties, only stearic acid showed no significant differences (P>0.05). The fatty acid compositions of the sn-2 position of NOS and HOS samples showed different percentage abundances (P<0.01), especially for oleic and linoleic acids. Fatty acid distributions in the sn-1 and sn-3 positions indicated a certain asymmetry. The relationships between the percentage intrapositional content of each acid (one sn-position at a time) and its percentage content in the original triacylglycerol matrix were studied. A general regression model was used to verify if the content of each acid at the three stereospecific positions changed at the same rate as the content in the intact triacylglycerols. The interpositional compositions of all varieties of NOS and HOS oils showed analogous trends for each acid.     

Regioselective analysis of the fatty acid composition of triacylglycerols with conventional high-performance liquid chromatography

A new method for regioselective analysis of triacyglycerols via conventional high-performance liquid chromatography (HPLC) has been developed. The method is simple and avoids the time-consuming purification processes normally characteristics of regioselective analyses. The procedure utilizes an sn-1,3-specific lipase from Rhizopus arrhizus to deacylate the fatty acid residues located at the sn-1 and sn-3 positions of triacylglycerols. The fatty acid residues esterified at the sn-2 position are determined by subtraction of the results of the sn-1,3 analysis from an overall composition analysis based on complete saponification of the original sample. The fatty acid mixtures are converted to p-bromophenacyl esters and analyzed using conventional HPLC techniques. The analytical procedure has been verified using a standard structured triacylglycerol. The analytical results for three edible vegetable oils are compared with those obtained via the method proposed by P.J. Williams and co-workers.     

Preparation of Infant Formula Fat Analog Containing Capric Acid and Enriched with DHA and ARA at the sn-2 Position

An infant formula fat analog with capric acid mostly esterified at the sn‐1,3 positions, and substantial amounts of palmitic, docosahexaenoic (DHA), and arachidonic (ARA) acids at the sn‐2 position, was prepared by physically blending enzymatically synthesized structured lipids (SL) with vegetable oils. The components of the blend included high sn‐2 palmitic acid SL enriched with capric acid (SL_CA), canola oil (CAO), corn oil (CO), high sn‐2 DHA (DHAO_m), and high sn‐2 ARA (ARAO_m) enzymatically modified oils. Each component was proportionally blended to match the fatty acid profile of commercial fat blends used for infant formula. The infant formula fat analog (IFFA₁) was characterized for total and positional fatty acids (FA), triacylglycerol (TAG) molecular species, thermal behavior, and tocopherol content. IFFA₁ contained 17.37 mol% total palmitic acid of which nearly 35 % was located at the sn‐2 position. The total capric acid content was 13.93 mol%. The content of DHA and ARA were 0.49 mol% (48.18 % at sn‐2) and 0.57 mol% (35.80 % at sn‐2), respectively. The predominant TAG were OPO (24.09 %), POP (15.70 %), OOO (11.53 %), and CLC (7.79 %). The melting completion and crystallization onset temperatures were 18.65 and ?2.19 °C, respectively. The total tocopherol content was 566.45 μg/g. This product might be suitable for commercial production of infant formulas.     

Stereospecific analysis of glycerolipids of egg yolk of Japanese quail (Coturnix coturnix japonica)

Phosphatidylcholine, phosphatidylethanolamine and triacylglycerol were isolated from egg yolk of the Japanese quail. Fatty acid compositions at the two and three positions of glycerol in the glycerolipids were determined by stereospecific analysis employing phospholipase A₂. The distribution of the total number of carbon atoms in the fatty acid moieties of triacylglycerol was also quantitated by high temperature gas liquid chromatography. The distribution of acyl groups in each of the positions of the phosphatidylcholine, phosphatidylethanolamine and triacylglycerol was not random, and each position has a characteristic composition. The phosphatidylcholine and phosphatidylethanolamine had distinctive fatty acid distributions for positionsn-2 of the triacylglycerol had a predominance of unsaturated fatty acids of which 18∶1 (69.9%) was the major component. Positionsn-3 contained 49.3% saturated fatty acids and was more saturated than positionsn-1 by 8.1%. The experimentally determined distribution of the carbon numbers in triacyl glycerol deviated significantly from the distribution predicted by 1-random-2-random-3-random association of the fatty acids. The data suggest that in Japanese quail there is marked preferencial synthesis of some triacylglycerols.     

Triacylglycerols of Apiaceae seed oils: Composition and regiodistribution of fatty acids

Oils from the seeds of caraway (Carum carvi), carrot (Daucus carota), celery (Apium graveolens) and parsley (Petroselinum crispum), all from the Apiaceae family, were analyzed by gas chromatography for their triacylglycerol (TAG) composition and fatty acid (FA) distribution between the sn‐1(3) and sn‐2 positions of TAG. Twenty‐two TAG species were quantified. Glyceryl tripetroselinate was the major TAG species in seed oils of carrot, celery and parsley, with levels ranging from 38.7 to 55.3%. In caraway seed oil, dipetroselinoyllinoleoylglycerol was the major TAG species at 21.2%, while the glyceryl tripetroselinate content was 11.4%. Other TAG species were linoleoyloleoylpetroselinoylglycerol and dipetroselinoyloleoylglycerol. Predominantly, TAG were triunsaturated (72.2–84.0%) with diunsaturates at 14.4–25.9%, and small amounts of monounsaturated TAG. Results for regiospecific analysis showed a non‐random FA distribution in Apiaceae for palmitic, petroselinic, linoleic and oleic acids. Petroselinic acid was predominantly located at the sn‐1(3) position in carrot, celery and parsley seed oils, while it was mainly at the sn‐2 position in caraway seed oil. The distribution of linoleic acid was opposite to that of petroselinic acid. Oleic acid was mostly located at the sn‐2 position, except for caraway, where it was evenly distributed between the sn‐1(3) and sn‐2 positions. Both the saturated FA, palmitic and stearic acid, were located mainly at the sn‐1(3) position. The presence of a high level of tripetroselinin in parsley seed oil (55.3%) makes it a potential source for the production of petroselinic acid.     

Acylglycerol structure of genetic varieties of peanut oils of varying atherogenic potential

Detailed investigation was made of the triacylglycerol structure of three varieties of peanut oils of varying atherogenic activity. By means of chromatographic and stereospecific analyses, it was shown that all the oils had markedly nonrandom enantiomeric structures with the long chain saturated fatty acids (C₂₀−C₂₄) confined exclusively to thesn-3-position, whereas the palmitic and oleic acids were distributed about equally between thesn-1-andsn-3-positions, with the linoleic acid being found preferentially in thesn-2-position. On the basis of detailed studies of the molecular species of the separatesn-1,2-,sn-2,3- andsn-1,3-diacylglycerol moieties, it was concluded that the fatty acids in the three positions of the glycerol molecule are combined with each other solely on the basis of their relative molar concentrations. As a result, it was possible to calculate the composition of the molecular species of the peanut oil triacylglycerols (including the content of the enantiomers and the reverse isomers) by means of the 1-random 2-random 3-random distribution. In general, the three peanut oils possessed triacylglycerol structures which where closely similar to that derived earlier for a commercial peanut oil of North American origin. Since their oil has exhibited a high degree of atherogenic potential, it was anticipated that the present oils would likewise be atherogenic, which has been confirmed by biological testing. However, there are certain differences in the triacylglycerol structures among these oils, which can be correlated with the variations in their atherogenic activity. The major differences reside in the linoleic/oleic acid ratios in the triacylglycerols, especially in thesn-2-position, and in the proportions in which these acids are combined with the long chain fatty acids. On the basis of the characteristic structures identified in the earlier analyzed atherogenic peanut oil, the peanut oil of South American origin would be judged to possess the greatest atherogenic potential and this has been borne out by biological testing.     

Phospholipid fatty acid composition and stereospecific distribution of soybeans with a wide range of fatty acid composition

Phospholipid (PL) fatty acid composition and stereospecific distribution of 25 genetically modified soybean lines with a wide range of compositions were determined by gas chromatography and phospholipase A₂ hydrolysis. Pl contained an average of 55.3% phosphatidylcholine, 26.3% phosphatidylethanolamine, and 18.4% phosphatidylinositol. PL class proportions were affected by changes in overall fatty acid composition. PL fatty acid composition changed with oil fatty acid modification, especially for palmitate, stearate, and linolenate. Stereospecific analysis showed that saturated fatty acids were primarily located at the sn-1 position of all PL, and changes of the saturates in PL were largely reflected on this position. Oleate was distributed relatively equally between the sn-1 and sn-2 positions. Linoleate was much more concentrated on sn-2 than on sn-1 position for all PL. Linolenate was distributed relatively equally at low concentration but preferred sn-2 position at high concentration.     

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.     

The triacylglycerol structure of olive oil determined by silver ion high-performance liquid chromatography in combination with stereospecific analysis

The compositions of positionssn-1,sn-2 andsn-3 of triacylglycerols from “extra-virgin” olive oil (Olea europaea) were determined. The procedure involved preparation of diacyl-rac-glycerols by partial hydrolysis with ethyl magnesium bromide; 1,3-, 1,2- and 2,3-diacyl-sn-glycerols as (S)-(+)-1-(1-naphthyl)ethyl urethanes were isolated by highperformance liquid chromatography (HPLC) on silica, and their fatty acid compositions were determined. The same procedure was also carried out on the five main triacylglycerol fractions of olive oil after separation according to the degree of unsaturation by HPLC in the silver ion mode. Although stereospecific analysis of the intact triacyl-sn-glycerols indicated that the compositions of positionssn-1 andsn-3 were similar, the analyses of the molecular species demonstrated marked asymmetry. The data indicate that the “1-random, 2-random, 3-random” distribution theory is not always applicable to vegetable oils.     

Positional distribution of fatty acids in triacylglycerols and phospholipids from adzuki beans (Vigna angularis)

The fatty acid distributions of triacylglycerols (TAG) and major phospholipids (PL) obtained from adzuki beans (Vigna angularis) were investigated. The total lipids extracted from the beans were separated by thin‐layer chromatography (TLC) into eight fractions. The major lipid components were PL (63.5 wt‐%), TAG (21.2 wt‐%), steryl esters (7.5 wt‐%) and hydrocarbons (5.1 wt‐%), while free fatty acids, diacylglycerols (1,3‐DAG and 1,2‐DAG) and monoacylglycerols were also present in minor proportions (0.2–1.1 wt‐%). The major PL components isolated from the beans were phosphatidylcholine (45.3 wt‐%), phosphatidylethanolamine (25.8 wt‐%) and phosphatidylinositol (21.5 wt‐%). Phosphatidylinositol was unique in that it had the highest saturated fatty acid content among the three PL. With a few exceptions, however, the principal characteristics of the fatty acid distribution in the TAG and three PL were evident in the beans: Unsaturated fatty acids were predominantly concentrated in the sn‐2 position while saturated fatty acids primary occupied the sn‐1 or sn‐3 position in the oils of the adzuki beans. In general, these results could be useful to both consumers and producers for the manufacture of traditional adzuki foods in Japan.     

Acylglycerol structure of peanut oils of different atherogenic potential

Detailed investigation was made of the triacylglycerol structure of native, simulated, and interesterified peanut oils, which had previously been shown to differ markedly in their atherogenic potential. By means of chromatographic and stereospecific analyses, it was shown that the more atherogenic native oil contains a significantly greater proportion of triacylglycerols with linoleic insn-2-position and arachidic, behenic, and lignoceric acids insn-3-position than the synthetic oils. It is suggested that the atherogenicity may arise from a relative metabolic unavailability of the linoleic acid from the native oil, which may be due in part to the presence of long chain saturated acids in the outer position. This might render the oil metabolically more saturated than the interesterified oils of the same total fatty acid composition, which contain a much greater proportion of the linoleic acid in the primary positions of the triacylglycerol molecule. The identification of specific triacylglycerols may allow the experimental testing of this hypothesis by feeding synthetic triacylglycerols incorporating the potentially atherogenic features.     

Stereospecific analysis ofOnosmodium hispidissimum Mack. seed oil triglycerides

The objective of the study was to determine the fatty acid composition ofOnosmodium hispidissimum Mack. seed oil and the stereospecific distribution of γ-linolenic and stearidonic acids in the seed oil triglycerides. The seed oil contained about 20% γ-linolenic acid and about 8% stearidonic acid. About 90% of both γ-linolenic acid and stearidonic acids were esterified to thesn-2 andsn-3 positions. This paper is NRCC No. 36484.     

Reinvestigation of positional distribution of fatty acids in docosahexaenoic acid-rich fish oil triacyl-sn-glycerols

Positional distribution of fatty acids in triacyl-sn-glycerols of docosahexaenoic acid (DHA)-rich tuna orbital and bonito head oils has been reanalyzed by a method based on chromatographic separation of isomeric and enantiomeric monoacyl-sn-glycerol (MAG) derivatives. When boric acid thinlayer chromatography (TLC) was used for separation of 1(3)- and 2-MAG analytical intermediates, the stereospecific analysis showed the preferential association of DHA to the sn-2 position followed by the sn-3 position. This distribution pattern differed from that obtained by silicic acid LTC of their bis-3,5-dinitrophenylurethane (DNPU) derivatives. Reversed-phase high-performance liquid chromatography elution profiles of 1(3)- and 2-MAG intermediates revealed that 1(3)- and 2-MAG made up of both short- and long-chain lengths cannot be clearly resolved by TLC after preparation of the DNPU derivatives. The 1(3)- and 2-MAG must be resolved by boric acid TLC prior to derivatization.     

Triacylglycerols and regiospecific fatty acid analyses of philippine seed oils

Three Philippine seed oils, namely coconut (Cocos nucifera Linn.), pilinut (Canarium ovatum Engl.), and cashew (Anacardium occidentale Linn.), which were selected for their local abundance and availability, were examined for their triacylglycerol profiles and fatty acid compositions. Triacylglycerol molecular species in terms of carbon number and partition number were determined by gas chromatography and liquid chromatography, respectively. The distribution of fatty acids in the primary and secondary positions of the glycerol backbones for the three oils were examined by regiospecific analysis by using pancreatic lipase. Coconut oil had high concentrations of lauric and myristic acids, while the other two oils did not have such fatty acids. Lauric acid in coconut oil and linoleic acid in pilinut oil were distributed mainly in the primary positions (sn-1,3) of the glycerol backbone. Trilaurin and dioleylpalmitoylglycerol were the major triglycerides in coconut and pilinut oils, respectively.     

Studies on effects of dietary fatty acids as related to their position on triglycerides

This article reviews published literature on how the stereospecific structure of dietary triglycerides may affect lipid metabolism in humans. Animal studies have shown enhanced absorption of fatty acids in the sn-2 position of dietary triglycerides. Increasing the level of the saturated fatty acid palmitic acid in the sn-2 position (e.g., by interesterification of the fat to randomize the positions of the fatty acids along the glycerol backbone) has been shown in rabbits to increase the atherogenic potential of the fat without impacting levels of blood lipids and lipoproteins. In contrast, enhancing the level of stearic acid in the sn-2 position has not been found to affect either atherogenic potential or levels of blood lipids and lipoproteins in rabbit. Fatty acids other than palmitic and stearic have not been studied systematically with respect to possible positional effects. A limited number of human studies have shown no significant effects of interesterified fats on blood lipid parameters. However, it is unknown whether modifying the stereospecific structure of dietary triglycerides would affect atherogenicity or other long-term health conditions in humans. It is possible that incorporation of palmitic acid into the sn-2 position of milk fat is beneficial to the human infant (as a source of energy for growth and development) but not to human adults. Additional research is needed to determine whether processes like interesterification, which can be used to alter physical parameters of dietary fats (e.g., melting characteristics), may result in favorable or unfavorable long-term effects in humans.     

Positional analyses of triacylglycerol fatty acids in the milk fat of the antarctic fur seal (Arctocephalus gazella)

The positional distribution of fatty acids has been determined for the milk triacylglycerols of the Antarctic fur seal,Arctocephalus gazella. Of particular interest was the positional distribution of the polyunsaturated n−3 fatty acids in milk triacylglycerols (TG). In adipocytes of pinnipeds, TG are synthesized with the n−3 fatty acids primarily in thesn-1,3 positions. To determine the positional distribution, extracts of enzymatically digested lipids were separated by thin-layer chromatography, and the constituent fatty acids were separated and quantified by gas-liquid chromatography. Monoenoic and saturated fatty acids comprised over 75% of the total, the ratio of monoenoic to saturated fatty acids being 2∶1. The percent content of the long-chain n−3 fatty acids, 20∶5, 22∶5 and 22∶6, ranged between 15–20%. The positional analyses revealed that at thesn-2 position of milk TG, saturated fatty acids were in excess (57%), and the content of n−3 fatty acids was less than 5%. More than 80% of the n−3 fatty acids in milk were located in thesn-1,3 positions. The data indicate that in pinnipeds TG are synthesized in the mammary gland and adipose tissue with fatty acids having similar positional distributions.     

The development and application of novel vegetable oils tailor‐made for specific human dietary needs

Conventional edible oils, such as sunflower, safflower, soya bean, rapeseed (canola) oils, were modified to obtain high‐oleic, low‐linoleic or even low‐linolenic oils. The aim was to develop salad, cooking and frying oils, that are very stable against lipid peroxidation. They are also suitable for margarine blends, as additives to cheeses and sausages, or even as feed components. Oils containing higher amounts of medium‐chain length or long‐chain polyunsaturated fish oil fatty acids are suitable as special dietetic oils or as nutraceuticals. High‐stearic oils are designed as trans‐fatty acid‐free substitutes for hydrogenated oils. New tailor‐made (designer) oils are thus a new series of vegetable oils suitable for edible purposes, where conventional oils are not suitable.