Positional distribution of fatty acids in depot triglycerides of aquatic animals

Stereospecific triglyceride analyses were performed on fats of the following animals: five aquatic invertebrates, five freshwater fish, six marine fish, three marine birds, two amphibia, two seals, a whale, and a marine turtle. The distribution of faty acids was asymmetrical in most cases. A formula is presented which describes the general tendencies of fatty acid distribution in many animal fats, and some special rules which modify this formula are stated. Visit sponsored by a grant from the National Institutes of Health (AM-06011).     

Positional distribution of fatty acids in the triglycerides of bovine milk fat with elevated levels of linoleic acid

A stereospecific distribution of fatty acids in bovine milk fat containing 15.5% linoleic acid has been compared with the distribution in bovine milk fat containing a normal level (1.8%) of linoleic acid. The positional distribution was obtained by the separate analysis of milk fat triglycerides of high, medium, and low molecular weight. The order of preference for linoleic acid in the high molecular weight triglycerides was position 3>position 2 >position 1. There was an accompanying altered distribution of myristic acid and palmitic acid in favor of position 1 at the expense of position 3. However, the proportions of myristic acid and palmitic acid in position 2, relative to positions 1 and 3 were identifical in the high molecular weight fractions of the two milk fats. The distribution of linoleic acid in the medium molecular weight triglycrides of linoleic-rich milk fat was position 1=position 2>position 3. This resulted in a change in the distribution of 18 carbon monounsaturated fatty acids in favor of position 2 at the expense of position 1, but the distribution of myristic acid and palmitic acid was virtually unaltered. The distribution of linoleic acid in the low molecular weight triglycerides was position 2>position 1>position 3. The amounts of myristic acid and palmitic acid in position 2 and of palmitic acid in position 1 decreased in the low molecular weight triglycerides of the milk fat containing elevated levels of linoleic acid. Changes in the distribution of fatty acids which were observed in the separate analysis of the high, medium, and low molecular weight triglycerides were not apparent when comparing the distribution in the total milk fats. For example, the distribution of myristic acid and palmitic acid appeared to be unchanged, while the distribution of 18 carbon monounsaturated fatty acids was slightly altered in favor of positions 2 and 3. Moreover, linoleic acid showed an almost equal preference for the three positions of the glycerol moiety in milk fat containing elevated levels of this fatty acid with some concentration at position 2.     

Positional specificity of gastric hydrolysis of long-chain n−3 polyunsaturated fatty acids of seal milk triglycerides

Long-chain n−3 polyunsaturated fatty acids (n−3 PUFA) of marine oils are important dietary components for both infants and adults, and are incorporated into milks following maternal dietary intake. However, little is known about the hydrolysis of these PUFA from milk triglycerides (TG) by lipases in suckling young. Seals, like humans, possess gastric lipase; however, the milk lipids of seals and sea lions are almost devoid of the readily hydrolyzable medium-chain fatty acids, and are characterized by a large percentage (10–30%) of n−3 PUFA. Gastric hydrolysis of milk lipids was studiedin vivo in suckling pups of three species (the California sea lion, the harp seal and the hooded seal) in order to elucidate the actions and specificity of gastric lipases on milk TG in relation to fatty acid composition and TG structure. Regardless of milk fat content (31–61% fat) or extent of gastric hydrolysis (10–56%), the same fatty acids were preferentially released in all three species, as determined by their relative enrichment in the free fatty acid (FFA) fraction. In addition to 16∶1 and 18∶0, these were the PUFA of 18 carbons and longer, except for 22∶6n−3. Levels of 20∶5n−3 were most notably enriched in FFA, at up to five times that found in the TG. Although 22∶6n−3 was apparently also released from the TG (reduced in the diglyceride), it was also notably reduced in FFA. Positional analysis of milk TG based on the products of Grignard hydrolysis revealed that these PUFA, including 22∶6n−3, were preferentially esterified at the α-position of the TG, and that the fatty acids not released during gastric hydrolysis were located at thesn-2 position. The extreme reduction of 22∶6n−3 and enrichment of 20∶5n−3 in FFA is discussed. Results from this study are consistent with reports that gastric lipase acts stereo-specifically to release fatty acids at the α-positions (sn−3,sn−1). We conclude that the n−3 PUFA in milk are efficiently hydrolyzed by gastric lipase and that this has important implications for digestion of milks enriched in PUFA by neonates in general. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990; part of this work is from the doctoral dissertation by S.J.I., University of Maryland, 1988.     

Specific distribution of fatty acids in the milk fat triglycerides of goat and sheep

The triglycerides of the fat globules of sheep and goat milk were isolated and separated into short and long chain lengths by silicic acid column chromatography. The short chain lengths comprised major triglycerides with 34–44 acyl carbon atoms and accounted for nearly 50% of the total milk fat. The long chain lengths contained major triglycerides with 40–54 acyl carbons. Stereospecific analyses of the short chain triglyceride fraction showed that of the 20–23 moles per cent of C₄−C₈ fatty acids present, at least 95% were specifically attached to the glycerol molecule in the position corresponding to carbon 3 ofsn-glycerol. The distribution of the other fatty acids (C₁₀ or greater) did not show such marked specificity for either the 1 or the 2 position. Although individual triglycerides were not identified, the specific placement of the fatty acids could best the accounted for by assuming a common pool of long chain 1,2-diglycerides which served as precursors of the bulk of both short and long chain triglycerides during milk fat synthesis. Presented in part at the AOCS Meeting, New York, October 1968.     

Positional distribution of the fatty acids in the triglycerides of the oil of some african peanut varieties

The distribution of fatty acids between the sn-1-, sn-2- and sn-3-positions of the triglycerides from the oils of eight African peanut varieties has been determined. The saturated fatty acids and eicosenoic acid occur almost exclusively at the sn-1- and sn-3-positions. The sn-1-position contained slightly more palmitic acid than the sn-3-position. The fatty acids with a chain length exceeding 18 carbons were preferentially incorporated in the sn-3-position. Linoleic acid was preferentially esterified at the sn-2-position, whereas oleic acid was equally distributed among the three positions. The amount of the saturated fatty acids, i.e., palmitic and stearic acid, and of oleic acid and linoleic acid incorporated in the sn-1-, sn-2-and sn-3-position, were each linearly related to their respective content in the triglycerides.     

Positional distribution of the fatty acids in the triglycerides of mango (Mangifera indica) kernel fat

Triglycerides of mango seed kernel fat contain, depending on the variety, 32.4–44.0% of stearic acid and 43.7–54.5% of oleic acid. Palmitic and linoleic acids represent, respectively, 5.9–9.1% and 3.6–6.7% of the fatty acids. The triglycerides also contain minor amounts of arachidic and linolenic acids. Palmitic, stearic and arachidic acids were almost exclusively distributed among thesn-1-andsn-3-positions. Oleic acid represented 85–89% of the fatty acids at thesn-2-position. Oleic acid at thesn-1- andsn-3-positions showed a preference for thesn-1-position. Linoleic acid was mainly esterified at thesn-2-position. The amounts of saturated fatty acids, i.e., palmitic and stearic acids, and of oleic acid, at thesn-1- and sn-3-positions, were linearly related to their respective contents in the total triglycerides.     

Positional analysis of triglycerides and phospholipids rich in long-chain polyunsaturated fatty acids

Four sources of long-chain polyunsaturated fatty acids (LCP) differing in their chemical structure (triglycerides or phospholipids) and in their origin (tuna triglycerides, fungal triglycerides, egg phospholipids, and pig brain phospholipids) were analyzed to determine the distribution of the component fatty acids within the molecule. Lipase and phospholipase A₂ hydrolysis was performed to obtain 2-monoacylglycerols and lysophospholipids, respectively, which allowed us to determine the distribution of fatty acids between the sn-2 and sn-1,3 positions of triglycerides or between the sn-1 and sn-2 position of phospholipids. Fatty acids in the LCP sources analyzed were not randomly distributed. In tuna triglycerides, half of the total amount of 22∶6n−3 was located at the sn-2 position (49.52%). In fungal triglycerides, 16∶0 and 18∶0 were esterified to the sn-1,3 (92.22% and 91.91%, respectively) 18∶1 and 18∶2 to the sn-2 position (59.77% and 62.62%, respectively), and 45% of 20∶3n−6 and only 21.64% of 20∶4n−6 were found at the sn-2 position. In the lipid sources containing phospholipids, LCP were mainly esterified to the phosphatidylethanolamine fraction. In egg phospholipids, most of 20∶4n−6 (5.50%, sn-2 vs. 0.91%, sn-1) and 22∶6n−3 (2.89 vs. 0.28%) were located at the sn-2 position. In pig brain phospholipids, 22∶6n−3 was also esterified to the sn-2 (13.20 vs. 0.27%), whereas 20∶4n−6 was distributed between the two positions (12.35 vs. 5.86%). These results show a different fatty acid composition and distribution of dietary LCP sources, which may affect the absorption, distribution, and tissue uptake of LCP, and should be taken into account when supplementing infant formulas.     

Intramolecular fatty acid distribution in milk fat triglycerides of monkeys

Pancreatic lipase hydrolysis was used to determine the distribution of fatty acids in the milk triglycerides of four species of monkeys and of human milk. The patterns of the major fatty acids were generally similar in all species examined, but there were some differences in the relative concentrations of individual fatty acids esterified at either the 2 or 1,3 positions. Caprylic, stearic, oleic, and linoleic acids were found predominantly at the 1,3 positions; in contrast, lauric, myristic, palmitic, and palmitoleic were concentrated at the 2 position. Monkey milk fats had greater proportions of these acids at the respective positions than did bovine milk fat. Also, the monkey fats were relatively uniform both in total unsaturated fatty acids (41–48%) and in the proportion of these esterified at the 2 position (19–26%). In general, both the fatty acid composition and the specific distribution of fatty acids in the monkey milk fats more closely resembled the patterns in human milk fat than did those in ruminant milk fats.     

Positional distribution of fatty acids in cardiolipin of mitochondria from 21-day-old rats

Pure cardiolipins (1,3-diphosphatidylglycerol) were prepared from mitochondria of heart, liver and kidney from 21-day-old male Wistar rats and submitted toNaja naja venom phospholipase A₂ (EC 3.1.1.4) action. Incubation conditions were controlled carefully, and a complete hydrolysis of cardiolipin to lysocardiolipin {di [1 (1″) acylsn-glycero-3-phosphoryl] 1′, 3′-sn-glycerol} and fatty acids from positions 2 (2″) was obtained in less than two hr practically without side reactions. Cardiolipins from the three organs contained low levels of saturated fatty acids; stearic acid accounted for 0.4–0.7% and palmitic acid for 1.4–3.5% of total fatty acids. These percentages apparently depended on the organ. In all three cases, linoleic acid was the major component, but its percentage varied from 62–78% of total fatty acids. Acyl chains linked to positions 1 (1″) of all three cardiolipin preparations exhibited a similar pattern; they were composed of linoleic acid for 85–89%. This fatty acid also was the main component esterified at position 2 (2″), but its percentage was much more variable: from 39.8% in heart to 51.2% in kidney and 67.8% in liver mitochondria. The remaining acids comprised octadecenoic and polyunsaturated fatty acids with more than 18 carbon atoms in different proportions. As opposed to other phospholipids,cis-vaccenic acid, and not oleic acid, was the main octadecenoic acid present in cardiolipins. Octadecenoic acids were nine- to 10-fold more concentrated at positions 2 (2″) than at positions 1 (1″). The percentage ofcis-vaccenic acid was four- to five-fold higher than that of oleic acid at positions 2 (2″), whereas oleic acid dominated at positions 1 (1″). From results presented in this study and selected literature data, it may be concluded that fatty acids are asymmetrically distributed in cardiolipins of different origins, with linoleic acid showing a definite preference for position 1 (1″).     

Synthesis of triglycerides from fish oil fatty acids

The generally accepted methods for the synthesis of triglycerides are unsatisfactory when they are applied to highly unsaturated systems such as those present in fish oil. The methods either fail to g ive sufficiently high yields, or they are prohibitive in cost when applied to large scale production. Of the numerous reactions studied, the most feasible was the zinc-catalyzed esterification of fish oil fatty acids with glycerol. Thinlayer chromatography (TLC) showed that this reaction gave yields of 75–78% triglycerides. Gas-liquid chromatography (GLC) demonstrated the composition of the triglycerides was essentially the same as that of the original fatty acids.     

Determination of milk fat in chocolates by gas-liquid chromatography of triglycerides and fatty acids

The combination of two routine methods is proposed to determine the content of milk fat (MF) in chocolates, which is applicable even in the presence of lauric fats or others. The content of MF is obtained from the sum of C₄₀, C₄₂, and C₄₄ medium-chain triglycerides, determined by capillary gas-liquid chromatography (GLC). A new method, based on methyl esters of lauric acid and on minor acids situated between myristic and palmitic, is proposed. It enables detection and estimation of potential lauric fats, as well as the determination of the actual content of MF. The influence of other vegetable and animal fats is discussed. We analyzed 45 MF samples extracted from industrial milk powders and from pure or fractionated MF for chocolate manufacturing or pastry by GLC of triglycerides. We also analyzed by capillary GLC the methyl esters from 22 of those fats. Mixtures of these 22 MF samples with a cocoa butter also were used for chromatographic analyses of methyl esters and triglyceride. Results from the various analytical methods have been presented.     

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.     

Positional distribution of fatty acids in the triacylglycerols of developing oil palm fruit

The pattern of accumulation of triacylglycerols, their fatty acid compositions and the positional distribution of the fatty acids at thesn-2- andsn-1,3-positions of the triacylglycerol molecules at progressive stages of oil palm fruit development were determined. There was an exponential rate of increase of triacylglycerols and their fatty acids toward the end of fruit development. The fatty acid composition of the triacylglycerols in the early stages of development, prior to active accumulation, was more or less similar, but differed appreciably from the later stages, and the transition of fatty acid composition toward that of normal palm oil occurred at around 16 wk after anthesis (WAA) and stabilized at 20 WAA. All fatty acids increased in terms of absolute quantity. There was an overall consistency in fatty acid positional distribution, irrespective of development stage. More saturated fatty acids were found to be esterified at thesn-1,3-positions and more unsaturated fatty acids at thesn-2-position of triacylglycerol. Higher rate of incorporation of 16:0 at the 1,3-positions during the active phase of triacylglycerol synthesis was observed, while 18:1 acid exhibited a reverse trend.     

The metabolic fate of fatty acids derived from dietary triglycerides

The metabolic fates of dietary tricaprylin, trimyristin, tripalmitin, triolein, and trilinolein at the 15% level were followed with tracer doses of the corresponding C¹⁴-labeled acids. Distribution of the label in respiratory C¹⁴O₂ and in fatty acids of adipose tissue and liver lipids as well as the fatty acid composition of these unfractionated tissue lipids led to the following conclusions: Tissue fatty acid compositional homeostasis is limited mainly by the degrees to which dietary fatty acids can be converted to endogenous fatty acids. Other factors, such as their effects on lipogenesis and the relative degrees to which they are catabolized and stored, also play roles.     

Surface tension of fatty acids and triglycerides

Surface tension as a function of temperature was measured for four fatty acids (lauric, myristic, palmitic, and oleic) and two triglycerides (tricaprylin and tripalmitin). These surface tension measurements were performed using a K12 vers. 3.1 (Krüss GmbH) tensiometer at temperatures from 20 (or the melting point of each substance) to 90°C. The constants for a van der Waals-type correlation as well as for a linear equation are presented. Both equations are quite accurate, presenting mean deviations not exceeding 0.570%. Such correlation constants are valuable in the design or evaluation of processing equipment, especially that involving gas-liquid contact such as distillation and stripping columns, deodorizers, reactors, and equipment for physical refining.     

Adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides

Rat adipose hormone-sensitive lipase-mediated release of fatty acids from triglycerides was studied in three model systems: i) cultured preadipocytes containing polyunsaturated fatty acid-enriched triglyceride; ii) perfused epididymal fat pads; and iii)in vitro incubations of crude preparations of hormone-sensitive lipase with synthetic triglyceride-analogues as substrates. We found that cultured preadipocytes challenged with 10μM norepinephrine tended to release more ω6 and ω3 polyunsaturated fatty acids than saturated fatty acids. Fat pads perfused with 10 μM norepinephrine preferentially released arachidonate and α-linolenate but tended to retain oleate and linoleate. Finally, crude preparations of hormonesensitive lipase released from the triglyceride-analogue substrates α-linolenate twice as fast as oleate. We conclude that rat adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides. We suggest that this may be a mechanism by which these fatty acids are kept from being trapped in fat depots and maintained in the circulation.     

Positional specificity oftrans fatty acids in fetal lecithin

Differences in the positional incorporation of 9-trans[1-¹⁴C] octadecenoic (elaidic) and 9-trans,12-trans[1-¹⁴C] octadecadienoic (linoelaidic) acids in fetal lecithin of rats were demonstrated. On the 20th day of gestation, a¹⁴C-labeled albumin complex of elaidic or linoelaidic acid was injected into the jugular vein of pregnant rats. For comparative purposes, 9-cis[1-¹⁴C] octadecenoic (oleic) or 9-cis,12-cis[1-¹⁴C] octadecadienoic (linoleic acid) was injected into the maternal circulation of rats. Animals were killed 6 hr later. Distribution of label in total lipids and phospholipids (PL) of fetal tissue was measured by TLC. Irrespective of the label, the highest percentage of total radioactivity was associated with PL-59 to 67%. Within PL, the major portion of radioactivity was found in choline phosphoglycerides (CPG)-53 to 67%, and in ethanolamine phosphoglycerides (EPG)-18 to 33%. While linoelaidic acid was predominantly esterified in the 2-position of CPG, elaidic acid was nearly equally distributed between positions 1 and 2 of lecithin. Distribution of radioactivity within fatty acid methyl esters (FAME) of CPG measured by radio-GLC suggested that oleic and possibly linoleic acids may be converted to nervonic and arachidonic acid, respectively, in the rat by the 20th day of gestation. Following injection of elaidate, radioactivity of FAME was distributed between palmitate and elaidic acid indicating that rat fetal tissue may metabolize elaidic acid via β-oxidation. In contrast, following injection of linoelaidate, radioactivity of FAME was primarily associated withtt-18∶2, suggesting little biotransformation to other fatty acids by fetal tissues.     

Positional distribution on n−3 fatty acids in triacylglycerols from rat adipose tissue during fish oil feeding

The present study was designed to investigate the metabolism of the n−3 olyunsaturated fatty acids (PUFA) in adipose tissue and its dependence upon dietary factors. Changes in the positional distribution of the fatty acids in triacylglycerols from retroperitoneal adipose tissue were studied as a function of time on rats fed for 4 wk a diet enriched with fish oil. The stereospecific analysis of triacylglycerols was based on random formation ofrac-1,2-diacylglycerols by Grignard degradation. This was followed by synthesis ofrac-phosphatidic acids and treatment with phospholipase A₂. In the triacylglycerols of the fish oil diet, 57% of the total n−3 fatty acids were in position 3,i.e., two-thirds of 22∶5n−3 and 22∶5n−3 were esterified insn-3 position, whereas 22∶6n−3 was equally distributed in positions 2 and 3. After 4 wk of feeding fish oil, the fatty acid composition of adipose tissue triacylglycerols reached a steady state. Half of the n−3 fatty acids were found in position 3, namely 75% of 22∶5n−3, 50% of 20∶5n−3 and 18∶4n−3 and 45% of 22∶6n−3, the latter being equally distributed in positions 2 and 3. This pattern of distribution resembled that found in triacylglycerols of the fish oil diet, except for a higher proportion of 20∶5n−3 in adipose tissue in position 1 at the expense of position 3. Throughout the 4-wk period of fish oil feeding, the distribution pattern of minor n−3 fatty acids (18∶4n−3 and 22∶5n−3) in adipose tissue triacylglycerols remained unchanged. On the other hand, at the onset of fish oil feeding, 20∶5n−3 and 22∶6n−3 became concentrated in position 3, but thereafter 20∶5n−3 was progressively incorporated into position 1 and 22∶6n−3 into position 2. We thus conclude that n−3 fatty acids are differentially esterified in triacylglycerols of white adipose tissue. Despite the complex sequence of hydrolysis and acylation steps involved, the positional distribution of n−3 fatty acids was found to be similar in both the fish oil diet and the stored fat, in contrast to what was observed for nonessential fatty acids.     

Positional distribution of octadecadienoic acids in sponge phosphatidylethanolamines

The distribution of C14–C22 fatty acids in the phosphatidylethanolamines isolated from the spongesAgelas sp. andSpongia tampa was investigated. Selective changes with phospholipase A₂ (fromAgkistrodon halys blomhoffii) followed by thin-layer chromatographic separation of the resulting lysophosphatidylethanolamines and free fatty acids and subsequent methylation with HCl/MeOH and diazomethane, respectively, revealed that the 5,9-octadecadienoic acid and the 9,12-octadecadienoic acid present showed no preference for either positionsn-1 orsn-2 in these phosphatidylethanolamines. The other saturated and unsaturated fatty acids with chains between 14 and 22 carbons long were also found to be equally distributed between positionssn-1 andsn-2 in the phosphatidylethanolamines in these sponges. The results contrast with what is known about the distribution in most mammalian phospholipids, such as the phosphatidylcholines from human erythrocytes, where octadecadienoic acid typically occupies thesn-2 position.