Glycerides ofLimnanthes douglasii seed oil

Brockerhoff-type procedures were used to determine the amounts of each acyl group at each glyceride position ofLimnanthes douglasii seed oil. During the course of the analyses, small quantities of three acids isomeric with those previously found in the oil were identified by their ozonolysis products and their gas-liquid chromatographic (GLC) behavior. The newly discovered constituents of the oil were 3-octadecenoic acid (0.1%), 5-octadecenoic acid (0.9%) and 11-eicosenoic acid (3%). The saturated acids and those with ω-9-unsaturation are esterified most often to β-glyceride positions inLimnanthes seed, while the acids with Δ5-unsaturation occur generally at the outer glyceride positions. Although the Δ5-unsaturated acids as a group exhibited no obvious preference for one outer position over the other, individual acids were unequally distributed between the 1- and 3-sn-glycerol positions. The probabilities of occurrence of the various triglycerides were calculated from the stereospecific analysis data by assuming a 1-random, 2-random, 3-random distribution of the acyl groups. The calculations are in agreement with the composition of the whole oil, as determined by GLC. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Positional analysis and determination of triacylglycerol structure ofArgania spinosa seed oil

The distribution of fatty acids between the sn-1, sn-2 and sn-3 positions of triacylglycerols fromArgania spinosa seed oil of Morocco has been determined. Saturated fatty acids showed a preference for external positions. The sn-1 position contained slightly more palmitic acid than the sn-3 position, whereas stearic acid was preferentially esterified at the sn-3 position. Linoleic acid occurred predominantly in the sn-2 position with lesser amount evenly distributed between the sn-1 and the sn-3 positions, as generally found in vegetable oils. Oleic acid was distributed with a slight preference shown for the internal position, whereas the distribution between the external positions revealed a slight preference for the sn-1 position. The distribution of the triacylglycerols determined from high-performance liquid chromatography (HPLC) is at variance with that calculated from the 1-random 2-random 3-random distribution theory. This is particularly true for trioleoyl and trilinoleoylglycerols. In contrast, the agreement between theory and experiment is good for triacylglycerols containing two oleoyl and one linoleoyl chains, one oleoyl, one linoleoyl and one palmitoyl chains or one oleoyl, one palmitoyl, and one stearoyl chains.     

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.     

Composition of fatty acids and structure of triglycerides in medium and low erucic acid rapeseed

Total triglycerides in medium (MEAR) and low (LEAR) erucic acid cultivars of rapeseed were fractionated by argentation chromatography into twelve and ten fractions, respectively. Gas liquid chromatography of the fatty acids in the triglyceride fractions and their 2-monoglycerides was used to evaluate the structural characteristics of the individual fractions. Fractionation occurred on the basis of degree of unsaturation, molecular weight and positional characteristics. The most mobile fractions contained 34–50% of saturated fatty acids while the less mobile had 59–65% of polyunsaturated fatty acids. In the medium erucic acid oil, long chain fatty acids (C20–C22) were found in all fractions, but four fractions of low erucic acid oil were essentially free of long chain acids. Two of these fractions in the latter oil, which represented 44% of the total triglycerides, were glycerol trioleate and dioleoyllinoleoylglycerol. The majority of the 2-positions were occupied by unsaturated C18 fatty acids, generally in the order of linoleic ≥linolenic> oleic acids. The saturated and long chain fatty acids occurred predominantly in the 1-and 3-positions. The various fractions of medium and low erucic acid oils were similar in structural composition except that eicosenoic and erucic acids substituted for oleic acid in some external positions. Erucic acid did not appear to substitute directly for oleic acid in the 2-position.     

Evaluation of mathematical distribution methods for the determination of triglyceride composition

The triglyceride composition of a number of animal and vegetable fats was determined directly by means of selective argentation and lipase hydrolysis, and compared to that given by the 1,3-random,2-random method of analysis described by Vander Wal [JAOCS37, 18 (1960)]. Exceptions to the basic assumption of the 1,3-random,2-random method that the fatty acids in the 2-position are distributed randomly are reported. The analyses of some fats determined by the 1,3-random,2-random method agreed closely with those determined by the direct method, but the overall results indicated that methods based on mathematical distribution patterns generally are not as precise as direct methods. Presented at AOCS meeting, Houston, 1965.     

Lipids of cultured hepatoma cells: VII. Structural analyses of glycerolipids in minimal deviation hepatoma 7288C

Phosphatidylcholine. phosphatidylethanolamine, and triglycerides were isolated from minimal deviation hepatoma 7288C cells cultured as monolayers to confluency in roller flasks containing Swim's 77 medium supplemented with 5% fetal calf serum, plus 20%, 10%, or 5% bovine serum. Fatty acid distribution at each position of glycerol was determined for the 3 glycerolipid classes, and carbon number distributions of triglycerides and diglycerides derived from phosphatidylcholine and phosphatidylethanolamine were quantitated by high temperature gas liquid chromatography. Fatty acid composition was only marginally affected by the level of bovine serum in the culture medium. Percentage composition of fatty acids esterified at each position of the 3 glycerolipids was different, indicating a nonrandom distribution of acyl groups in triglycerides and the 2 diacyl phosphatides. The carbon number distribution of diglycerides derived from phosphatidylcholine and phosphatidylethanolamine was different, and neither carbon number distribution agreed with the calculated 1-random, 2-random diacyl distribution, thus indicating pairing of certain acids in the diglycerides derived from these phospholipd classes. The determined triglyceride carbon number distributions did not show complete agreement with those calculated, assuming a 1-random, 2-random, 3-random type of fatty acyl distribution, suggesting preferential pairing of some acids in this lipid class. The 1-, 2-diglycerides derived from phosphatidylcholine, phosphatidylethanolamine, and triglycerides differed, indicating either selectivity in utilization of diglyceride species in biosynthesis of these glycerolipids, or modification of glycerolipids after their initial synthesis.     

The Triglyceride Composition of Mango (Mangifera indica) Kernel Fat

The triglyceride composition of the kernel fat of 9 different mango varieties has been determined. Stearic and oleic acids represent respectively from 32.7 to 44.0% and from 43.7 to 53.4% of the total fatty acids. The remaining fatty acids were palmitic (6.7-9.7%), linoleic (3.6-6.9%), arachidic (1.1-2.5%) and linolenic (0.3–1.0%) acids. The triglyceride components were determined by separating the triglycerides according their degree of unsaturation by means of thin-layer chromatography on silica gel impregnated with silver nitrate. The fatty acid composition of the different triglyceride fractions and of the fatty acids incorporated at the sn-2-position of each triglyceride fraction was determined. Moreover, the triglycerides were separated according to their carbon number by gas liquid chromatography using an open-tubular glass column, wall-coated with CP-Sil 5. The triglyceride compositions obtained by thin-layer chromatography on silica gel impregnated with silver nitrate were in agreement with the compositions predicted by the 1,3-random-2-random distribution hypothesis.     

Milk fat structure of a patient with type 1 hyperlipidemia

Structural analyses were performed on milk fat samples obtained 3–10 days postpartum from a lactating patient with primary Type 1 hyperlipidemia. The milk triacylglycerols contained 3–7% C₁₀, 14–21% C₁₂, 20–30% C₁₄, 22–26% C₁₆ and 20–30% C₁₈ (largely oleic) acids. Gas liquid chromatographic (GLC) analyses of the X-1,3- and X-1,2-diacylglycerols on polar siloxane columns showed a markedly non-random association of acyl chains. Stereospecific analyses indicated that the short chain length fatty acids were confined essentially to the sn-3-position of the triacylglycerol molecule. Furthermore, these acids were largely absent from the phosphatidylcholines and the endogenous sn-1,2-diacylglycerols of the milk fat. It is concluded that the short chain fatty acids are incorporated into the milk triacylglycerols during the final stage of biosynthesis via the phosphatidic acid pathway, and that the overall fatty acid distribution is consistent with the 1-random 2-random 3-random hypothesis.     

Triacylglycerol structure of an african peanut oil

Triacylglycerols were isolated from an African peanut oil, then fractionated by unsaturation into classes, and the triacylglycerol structure was determined on these classes using pancreatic lipase hydrolysis. Fatty acid analysis of monoacylglycerols and, in some cases, of 1 or 2 classes of diacylglycerols, allowed the proportions of 84 isomers to be calculated. The oil had a high oleic acid content (60.3%) and contained nearly 25% of trioleoylglycerol, the major triacylglycerol. The 4 most abundant isomers together represented more than one-half of the total triacylglycerols. In 30 isomers, the 2-position was occupied by linoleic acid, and in 39 isomers, by oleic acid. The very long-chain saturated fatty acids (20:0, 22:0, 24:0) that formed 5.1% of the fatty acid content of the oil, were not found in the 2-position. In most cases, each was associated with 2 molecules of an unsaturated fatty acid. The placement of fatty acids, respectively, at the 1,3-position and the 2-position was relatively close to a l,3-random-2-random distribution, except for trioleoylglycerol (24.7% instead of 21.7% by the random hypothe-sis). To whom requests should be sent.     

The Saturated sn-2-Triglycerides of Palm-Kernel Oil

The saturated sn-2-triglycerides (the fatty acid esterified at the 2-position is known) of palm-kernel oil, representing 78%, was isolated by argentation thin-layer chromatography. The nine groups of this fraction (triglycerides with the same total acyl carbon atoms) were fractionated by gas-liquid chromatography and their component fatty acids determined. From the fatty acid composition of each group it was possible to determine mathematically the component triglyceride types of the group (the 3 fatty acids are known, but their positional distribution is not). The proportion of 46 types were calculated in this way. The major fractionate groups (6 out of 9) were hydrolyzed by pancreatic lipase. From the component 2-mono-glycerides it was possible to determine the proportion of 44 sn-2-triglycerides which account for 75% of the oil triglycerides. Trilaurin (21%) is the major component, followed by sn-2-lauro-1,3-lauromyristin (12%). These two triglycerides together form one third of the glyceride content of the oil. Sn-2-lauro-1,3-caprylolaurin (7%), 5 sn-2-triglycerides (2 to 5%), 9 (1 to 2%) were also found, and 27 triglycerides present at less than 1%. The calculated 1,3-random-2-random distribution of fatty acids on glycerol molecules exhibited great differences from the experimentally determined distribution. Consequently, calculations of this kind cannot replace a complete analytical analysis of palm-kernel oil such as the one reported in this work.     

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.     

Quantitative analysis of polymerized fatty acids using gel permeation chromatography

The acid clay catalyzed dimerization of tall oil fatty acids and oleic acid yields commercial products which are complex mixtures of monomers, dimers, trimers, and higher polymers of various structures. Analyses for monomer, dimer, and trimer concentrations are important to ensure good quality control and reproducible end use performance. Techniques for direct determination of monomer, dimer, and trimer acids by gel permeation chromatography are presented. The components are separated using Bio Beads SX-2 gel. Heptanoic acid is used as an internal standard. The standard deviations for determination of dimer, trimer, and monomer are 1.0, 0.4, and 0.2% respectively. Calibration was accomplished using dimer and trimer fractions isolated from a preparative scale chromatographic system. It is suggested that a measure of higher polymeric acids can be obtained by difference after correction for neutral materials.     

Studies of triacyglycerol structure of very low density lipoproteins of normolipemic subjects and patients with type III and type IV hyperlipoproteinemia

The triacylglycerols of very low density lipoproteins (VLDL-TG) were analyzed in samples from normal subjects and patients with Frederickson’s Type III and Type IV hyperlipoproteinemia. VLDL were obtained by conventional ultracentrifugation, and the triacylglycerols were isolated by thin-layer chromatography (TLC). Representative sn-1,2(2,3)- and sn-1,3-diacylglycerols were generated by Grignard degradation of the triacylglycerols, and were resolved by TLC on borate-treated silica gel. The molecular association of the fatty acids in the diacylglycerol moieties was determined by gasliquid chromatography with mass spectrometry (GC/MS) of the tertiary-butyldimethylsilyl ethers. The positional distribution of the fatty acids was established by the Brockerhoff stereospecific analysis. The results showed a marked asymmetry in the distribution of the fatty acids in all samples, with the saturated acids predominantly in the sn-1-position and the unsaturated fatty acids distributed about equally between the sn-2- and sn-3-positions. In all instances, the molecular species composition of the sn-1,2-, sn-2,3- and sn-1,3-diacylglycerols was found to be similar to that calculated for 1-random 2-random 3-random distribution of triacylglycerols. There were marked differences in the quantitative composition of the molecular species of the VLDL-TG between normal subjects and patients, but these discrepancies were attributed to differences in the fatty acid composition of the samples.     

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.     

An investigation of rice bran oil tank settling

A wax-like settling is observed in tanks in which rice bran oil is stored. “Soft” and “hard” wax fractions have been isolated from this settling by solvent Crystallization. Previous investigation has shown that the settling consists mainly of wax esters of long chain alcohols and long chain fatty acids. The present work describes the column chromatographic analysis of unhydrolyzed tank settling. The presence of an aromatic moiety is indicated in the infra red spectrum. Comparison of data obtained by analysis of the tank settling before and after hydrolysis shows that it contains only 33% of monomeric esters; the remainder may be present as a polymeric ester, as found in carnauba wax. Investigation of different samples of rice bran oil has shown that the ratio of monomeric to polymeric fraction varies with the history of the bran.     

Analyses of lipids and oxidation products by partition chromatography. Dimeric and polymeric products

A liquid-partition chromatographic method was developed to determine dimers in fats. Silicie acid treated with 20% methanol in benzene served as the immobile phase. A mixture of 2% methanol in benzene was the mobile solvent. Chromatographic separation of free fatty acids from oxidized-deodorized oils gave three well-isolated fractions composed of unoxidized acids, dimeric or polymeric fatty acids, and polar fraction (ethyl ether eluate). Recovery of acidic materials from the column was essentially quantitative (96–100%), reproducibility was good, and the standard error of regression was ±0.26. A linear relationship exists between the dimer content of deodorized soybean oil and the peroxide value of the oil before deodorization. An increase of 1% in dimer concentration corresponds to an increase in peroxide value of approximately 40. Dimer content of different vegetable oils varied from 1 to 3%. The chromatographic method can be used to estimate the degree of oxidation that an oil has received before deodorization and to follow various phases of fat oxidation, polymerization, and processing. Presented at spring meeting, American Oil Chemists' Society, Dallas, Tex., April 4–6, 1960. This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

Anatomical variation in fatty acid composition and triglyceride distribution in animal depot fats

The fatty acid composition and glyceride distribution of fatty acids in pork, beef and lamb depot fats from different localities within the same animal were determined by a combination of gas liquid chromatography and lipase hydrolysis techniques. The glyceride distribution was calculated according to the method of Vander Wal, based on the 1, 3-random and 2-random distribution pattern. Both fatty acid composition and glyceride structure were found to vary depending on the position within the animal from which the depot fat was obtained. Presented at the AOCS meeting in Chicago, October, 1964.     

Comparative studies of triacylglycerol structure of very low density lipoproteins and chylomicrons of normolipemic subjects and patients with type II hyperlipoproteinemia

The triacylglycerols of very low density lipoproteins (VLDL) and of chylomicrons were analyzed in the fasting and postabsorptive states from normolipemic subjects and patients with Frederickson's Type II hyperlipoproteinemia, who subsisted on free choice diets, standard diets excluding lard, or were given a breakfast enriched in lard. The VLDL and chylomicrons were obtained by conventional ultracentrifugation, and the triacylglycerols were isolated by thin-layer chromatography (TLC). Representative sn-1,2, 2n-2-3- and sn-1,3-diacylglycerols were generated by partial Grignard degradation of the triacylglycerols and a stereospecific hydrolysis by phospholipase C of the mixed sn-1,2(2,3)-diacyl phosphatidylcholines prepared as intermediates. Representative sn-2-acylglycerols were obtained by hydrolysis with pancreatic lipase. Positional distribution of the fatty acids was established by subtracting in turn the fatty acid composition of the sn-2-position from the fatty acid composition of the sn-1,2- and sn-2,3-diacylglycerols. The molecular association of the fatty acids in the diacylglycerol moieties was determined by gas-liquid chromatography with mass spectrometry (GC/MS) of the tertiary-butyldimethylsilyl (t-BDMS) ethers. The molecular association of the fatty acids in the triacylglycerols was determined by 1-random 2-random 3-random calculation following experimental validation of the distribution. The results confirm a marked asymmetry in the positional distribution of the fatty acids in all triacylglycerol samples, with the palmitic acid predominantly in the sn-1-position, the unsaturated acids about equally divided between the sn-2-and sn-3-positions, and the stearic acid divided about equally between the sn-1- and sn-3-positions. The overall structure of the VLDL and chylomicron triacylglycerols from patients and control subjects was characterized by a noncorrelative distribution of fatty acids under all dietary conditions.     

Acylglycerols Containing Trihydroxy Fatty Acids in Castor Oil and the Regiospecific Quantification of Triacylglycerols

Ricinoleate, a monohydroxy fatty acid in castor oil, has many industrial uses. Dihydroxy and trihydroxy fatty acids can also be used in industry. We report here the identification of diacylglycerols (DAG) and triacylglycerols (TAG) containing trihydroxy fatty acids in castor oil. The C₁₈ HPLC fractions of castor oil were used for mass spectrometry of the lithium adducts of acylglycerols to identify trihydroxy fatty acids and the acylglycerols containing trihydroxy fatty acids. Two DAG identified were triOH18:1–diOH18:1 and triOH18:0–OH18:1. Four TAG identified were triOH18:1–OH18:1–OH18:1, triOH18:0–OH18:1–OH18:1, triOH18:1–OH18:1–diOH18:1 and triOH18:0–OH18:1–diOH18:1. The structures of these two newly identified trihydroxy fatty acids were proposed as 11,12,13-trihydroxy-9-octadecenoic acid and 11,12,13-trihydroxyoctadecanoic acid. The locations of these trihydroxy fatty acids on the glycerol backbone were almost 100% at the sn-1,3 positions or at trace levels at the sn-2 position. The content of these acylglycerols containing trihydroxy fatty acids was at the level of about 1% or less in castor oil.     

The component triacylglycerols of avocado fruit-coat

The component triacylglycerols of avocado fruit-coat fat have been determined by thin layer argentation chromatography and by pancreatic lipolysis. The fat was found to contain a total of 33 triacylglycerols (10 major and 23 minor) belonging to triunsaturated (43.0%), diunsaturated-monosaturated (44.1%), disaturated-monounsaturated (12.4%) and trisaturated (0.5%) types of acylglycerols. The results of the distribution of individual fatty acids show a 1,3-random-2-random distribution pattern. Among the unsaturated fatty acids, linoleic acid was esterified at the 2 position with greater preference than either oleic or palmitoleic acid. Presented at the AOCS meeting in Houston, Texas, May 1971.