Two-step enzymatic reaction for the synthesis of pure structured triacylglycerides

Structured triacylglycerides with medium-chain fatty acids (caprylic acid) in sn1- and sn3-positions and a long-chain unsaturated fatty acid (oleic or linoleic acid) in the sn2-position of glycerol (MLM) were synthesized by lipase catalysis in a two-step process. First, pure 2-monoacylglycerides (2-MG) were synthesized by alcoholysis of triacylglycerides (triolein, trilinolein, or peanut oil) in organic solvents with 1,3-regiospecific lipases (from Rhizomucor miehei, Rhizopus delemar, and Rhizopus javanicus). The 2-MG were purified by crystallization and obtained in up to 71.8% yield. These 2-MG were esterified in a second reaction with caprylic acid in n-hexane to form almost pure MLM. For 2-MG obtained from peanut oil, the final product contained more than 90% caprylic acid in the sn1- and sn3-positions, whereas the sn2-position was composed of 98.5% unsaturated long-chain fatty acids. Reaction conditions for both steps were optimized with respect to source and immobilization of lipase, water activity, and solvent.     

Varietal difference in lipid content and fatty acid composition of highbush blueberries

Lipids from five cultivars of highbush blueberries (Vaccinium corymbosum L.) were extracted and fractionated into neutral lipids (60–66%), glycolipids (20–22%) and phospholipids (14–18%). The major fatty acids in all fractions were palmitic (16∶0), oleic (18∶1), linoleic (18∶2), and linolenic (18∶3) acids. All lipid classes had a large concentration of C₁₈ polyunsaturated acids (84–92%), indicating that blueberries are a rich source of linoleic and linolenic acids. Changes in the fatty acid composition of neutral lipids and phospholipids were not significantly different among the five cultivars, but significant differences were noted in the ratios of linoleic and linolenic acids in the glycolipids fraction.     

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.     

Characterization of oil exhibiting high γ-linolenic acid from a genetically transformed canola strain

The seed oil from a genetically transformed canola (Brassica napus) containing 43% (w/w) of γ-linolenic acid (G, 18∶3n−6), 22% linoleic acid (L, 18∶2n−6), and 16% oleic acid (O, 18∶1n−9) was evaluated. In this high γ-linolenic acid canola oil (HGCO), the predominant 18∶3n−6-containing triacylglycerol (TG) molecular species were GGL (23%), GLO (20%), and GGG (11%). In the total TG, approximately 75% of the 18∶3n−6 was located at the sn-1,3 positions, while only 34% of linoleic acid was at the sn-1,3 positions. The GGL molecular species of HGCO contained approximately equal amounts of GLG and GGL positional isomers, while the GLO molecular species had 95% GOL and 5% GLO isomers. The general characteristics and the tocopherol and phytosterol contents were mostly similar between HGCO and nontransformed canola oil. No detectable amounts of amino acids and nucleotides were observed in the HGCO.     

Effects of different medium-chain fatty acids on intestinal absorption of structured triacylglycerols

To study the effect of the chain length of medium-chain fatty acids on the intestinal absorption of long-chain fatty acids, we examined the lymphatic transport of fat following administration of five purified structured triacylglycerols (STAG) containing different medium-chain fatty acids in the sn-1, 3 positions and long-chain fatty acids in the sn-2 position in a rat model. Significant amounts of medium-chain fatty acids were found in lymph samples after intragastric administration of 1,3-dioctanoyl-2-linoleyl-sn-glycerol (8∶0/18∶2/8∶0), 1,3-didecanoyl-2-linoleyl-sn-glycerol, and 1,3-didodecanoyl-2-linoleyl-sn-glycerol. The accumulated lymphatic transport of medium-chain fatty acids increased with increasing carbon chain length. The recoveries of caprylic acid (8∶0), capric acid (10∶0), and lauric acid (12∶0) were 7.3±0.9, 26.3±2.4, and 81.7±6.9%, respectively. No significant differences were observed for the maximal intestinal absorption of linoleic acid (18∶2n−6) when the chain length of medium-chain fatty acids at the primary positions was varied, and the absorption of 18∶2 and oleic acid (18∶1) from 8∶0/18∶2/8∶0 and 1,3-dioctanoyl-2-oleyl-sn-glycerol was similar. We conclude that the chain length of the medium-chain fatty acids in the primary positions of STAG does not affect the maximal intestinal absorption of long-chain fatty acids in the sn-2 position in the applied rat model, whereas the distribution of fatty acids between the lymphatics and the portal vein reflects the chain length of the fatty acids. Presented in part at the 3rd ISSFAL Conference, Lyon, France, June 1–5, 1998.     

Fatty acid and positional selectivities of gastric lipase from premature human infants:in vitro studies

Gastric lipase activity in aspirates from premature human infants was tested for fatty acid and positional selectivity using racemic diacid triacylglycerols (TG) as substrates. The resulting free fatty acids and monoacylglycerols (MG) were recovered and analyzed. Octanoic acid (8∶0) and decanoic acid (10∶0) were hydrolyzed with a preference of 61.5∶1 and 2.4∶1 compared to palmitic acid (16∶0) fromrac-16∶0–8∶8∶0 andrac-16∶0–10∶0–10∶0, respectively. The ratio of lauric acid (12∶0) to oleic acid (18∶1) hydrolyzed fromrac-18∶1–12∶0 was 13∶1. Myristic acid (14∶0), 18∶1 and linoleic acid (18∶2) were released at similar rates. These data and the composition of the MG suggest that,in vitro, the lipase is selective for shorter chain fatty acids and for fatty acids on the primary positions of the TG backbone.     

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.     

Divergent incorporation of dietary trans fatty acids in different serum lipid fractions

Trans fatty acids may be involved in atherosclerotic vascular diseases. We investigated the incorporation of dietary trans fatty acids and oleic acid into the serum triglycerides (TG), cholesterol esters (CE), and phospholipids (PL). Fourteen healthy female volunteers, aged 23.2±3.1 yr (mean±SD), body mass index 20.8±2.1 kg/m² participated in this study. All subjects consumed both a trans fatty acid-enriched diet (TRANS diet) and an oleic acid-enriched diet (OLEIC diet) for 4 wk according to a randomized crossover design. Both experimental diet periods were preceded by consumption of a baseline diet for 2 wk which supplied 37% of total energy (E%) as fat: 18 E% from saturated fatty acids (SFA), 12 E% from monounsaturated fatty acids, and 6 E% from polyunsaturated fatty acids. Five E% of the SFA in the baseline diet was replaced by trans fatty acids (18∶1 t and 18∶2 c,t+18∶2t,t, where c is cis and t is trans) in the TRANS diet and by oleic acid (18∶1n-9) in the OLEIC diet. After the TRANS diet, the proportions of 18∶1t and 18∶2t increased (P <0.001) in all serum lipid fractions analyzed. The increase of 18∶1 t in TG and PL (1.80±0.28 vs. 5.26±1.40; 1.07±0.34 vs. 3.39±0.76 mol% of total fatty acids, respectively) was markedly higher than that in CE (0.44±0.07 vs. 0.92±0.26), whereas that of 18∶2t was nearly the same in all three fractions. The proportions of palmitic, stearic, arachidonic, and eicosapentaenoic acids in TG, CE, and PL and that of oleic acid in TG and CE were decreased when compared with the baseline value. In contrast, the proportion of palmitoleic acid in TG and PL and that of linoleic acid in PL increased on the TRANS diet. After consumption of the OLEIC diet, the proportion of oleic acid increased in all three lipid fractions analyzed, and the percentage increase was nearly the same in all fractions. In contrast, the proportions of 18∶1 t in TG and PL and 18∶2 t in TG and CE decreased when compared with the baseline value. In conclusion, a moderate increase in dietary trans fatty acids resulted in a marked incorporation into serum lipids and decreased the conversion of linoleic acid to its more unsaturated long-chain metabolites. Analysis of 18∶1 t from serum TG and PL seems to reflect reliably the dietary intake of this fatty acid.     

Lipase-catalyzed acidolysis of tristearin with oleic or caprylic acids to produce structured lipids

Two different structured lipids (SL) were synthesized by transesterifying tristearin with caprylic acid (C8∶0) or oleic acid (C18∶1). The objective was to synthesize SL containing stearic acid (C18∶0) at the sn-2 position as possible nutritional and low-calorie fats. The reaction was catalyzed by IM60 lipase from Rhizomucor miehei in the presence of n-hexane. The effects of reaction parameters affecting the incorporation of caprylic acid into tristearin were compared with those for incorporating oleic acid into tristearin. For all parameters studied, oleic acid incorporation was higher than caprylic acid. The range of conditions favorable for synthesizing high yields of C8∶0-containing SL was narrower than for oleic acid. An incubation time of 12–24 h and an enzyme content of 5% (w/w total substrates) favored C8∶0 incorporation. The mole percentage of incorporated C18∶1 did not increase further at enzyme additions greater than 10%. C18∶1 incorporation decreased with the addition of more than 10% water (w/w total substrates) to the tristearin-oleic acid reaction mixture. Increasing the mole ratio of fatty acid (FA) to triacylglycerol increased oleic acid incorporation. The highest C8∶0 incorporation was obtained at a 1∶6 mole ratio of tristearin to FA. Positional analysis confirmed that C18∶0 remained at the sn-2 position of the synthesized SL. The melting profiles of tristearin-caprylic acid and tristearin-oleic acid SL displayed peaks between −20 to 30°C and −20 to 40°C, respectively. Their solid fat contents (∼25%) at 25°C suggest possible use in spreads or for inclusion with other fats in specialized blends.     

Cis-trans isomerization of oleic,linoleic and linolenic acids

The equilibrium composition ofcis andtrans isomers obtained by isomerizing oleic, linoleic, and linolenic acids with selenium or nitrous acid has been studied using gas chromatography and infrared spectroscopy. The oleic/elaidic equilibrium mixture was found to contain 75–80% elaidic acid instead of the generally accepted 66% value. It is felt that the greater accuracy of gas chromatography and infrared analyses over older methods allows this equilibrium to be defined with greater precision. Similar studies on thecis-trans isomerization of linoleic and linolenic acids indicated that their equilibrium mixtures also contained 75–80%trans double bonds. With linoleic acid, thesetrans bonds were shown to be randomly distributed among the double bonds present. Cis-trans isomerization of linoleic or linolenic acids with selenium produced by-products having elution times equivalent to 18∶2, 18∶1, and 18∶0 on a gas chromatograph. No such by-products were observed when oleic acid was isomerized. Apparently some type of hydrogen-transfer reaction accompanies thecis-trans isomerization of polyunsaturated acids with selenium. Presented at the AOCS meeting in Toronto, Canada, 1962.     

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

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

Prevention of cholesterol cholelithiasis by dietary unsaturated fats in hormone-treated female hamsters

We examined the effect of diet on gallstone incidence and the composition of biliary phosphatidylcholines in methyltestosterone-treated female hamsters. These hamsters were fed a nutritionally adequate purified lithogenic diet containing 2% corn oil, 4% butterfat, 0.3% cholesterol, and 0.05% methyltestosterone, resulting in a cholesterol gallstone incidence of 86%. This incidence was lowered when mono-and polyunsaturated fats or fatty acids were added to the diet: 2.5% oleic acid resulted in total prevention of cholesterol cholelithiasis, 2.5% linoleic acid, and 4% safflower oil (78% linoleic acid content) reduced gallstone incidence to 26 and 8%, respectively. An additional 4% butterfat (29% oleic acid content) produced gallstones in 50% of the animals. At the end of the 6-wk feeding period, the bile of all hamsters was supersaturated with cholesterol. The major biliary phosphatidylcholine species in all groups were (sn-1-sn-2): 16:0–18:2, 16:0–18:1, 18:0–18:2, 16:0–20:4, and 18:2–18:2. The safflower oil-and linoleic acidfed hamsters exhibited an enrichment of 16:0–18:2 (16–18%); added butterfat or oleic acid increased the proportion of 16:0–18:1 (9 and 25%, respectively). We conclude that the phosphatidylcholine molecular species in female hamster bile can be altered by dietary fats/fatty acids and that mono-and polyunsaturated fatty acids play a role in suppressing the induced cholelithiasis.     

Incorporation of n−3 fatty acids of fish oil into tissue and serum lipids of ruminants

This study examines the biohydrogenation and utilization of the C₂₀ and C₂₂ polyenoic fatty acids in ruminants. Eicosapentaenoic (20∶5n−3) and docosahexaenoic (22∶6n−3) acids were not biohydrogenated to any significant extent by rumen microorganisms, whereas C₁₈ polyenoic fatty acids were extensively hydrogenated. The feeding of protected fish oil increased the proportion of 20∶5 from 1% to 13–18% and 22∶6 from 2% to 7–9% in serum lipids and there were reductions in the proportion of stearic (18∶0) and linoleic (18∶2) acids. The proportion of 20∶5 in muscle phospholipids (PL) increased from 1.5% to 14.7% and 22∶6 from 1.0% to 4.2%; these acids were not incorporated into muscle or adipose tissue triacylglycerols (TAG). In the total PL of muscle, the incorporated 20∶5 and 22∶6 substituted primarily for oleic (18∶1) and/or linoleic (18∶2) acid, and there was no consistent change in the porportion of arachidonic (20∶4) acid.     

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″).     

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.     

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.     

General characteristics of Pinus spp. Seed fatty acid compositions, and importance of Δ5-olefinic acids in the taxonomy and phylogeny of the genus

The Δ5-unsaturated polymethylene-interrupted fatty acid (Δ5-UPIFA) contents and profiles of gymnosperm seeds are useful chemometric data for the taxonomy and phylogeny of that division, and these acids may also have some biomedical or nutritional applications. We recapitulate here all data available on pine (Pinus; the largest genus in the family Pinaceae) seed fatty acid (SFA) compositions, including 28 unpublished compositions. This overview encompasses 76 species, subspecies, and varieties, which is approximately one-half of all extant pines officially recognized at these taxon levels. Qualitatively, the SFA from all pine species analyzed so far are identical. The genus Pinus is coherently united—but this qualitative feature can be extended to the whole family Pinaceae—by the presence of Δ5-UPIFA with C₁₈ [taxoleic (5,9–18∶2) and pinolenic (5,9,12–18∶3) acids] and C₂₀ chains [5,11–20∶2, and sciadonic (5,11,14–20∶3) acids]. Not a single pine species was found so far with any of these acids missing. Linoleic acid is almost always, except in a few cases, the prominent SFA, in the range 40–60% of total fatty acids. The second habitual SFA is oleic acid, from 12 to 30%. Exceptions, however, occur, particularly in the Cembroides subsection, where oleic acid reaches ca. 45%, a value higher than that of linoleic acid. α-Linolenic acid, on the other hand, is a minor constituent of pine SFA, almost always less than 1%, but that would reach 2.7% in one species (P. merkusii). The sum of saturated acids [16∶0 (major) and 18∶0 (minor) acids principally] is most often less than 10% of total SFA, and anteiso-17∶0 acid is present in all species in amounts up to 0.3%. Regarding C₁₈ Δ5-UPIFA, taxoleic acid reaches a maximum of 4.5% of total SFA, whereas pinolenic acid varies from 0.1 to 25.3%. The very minor coniferonic (5,9,12,15–18∶4) acid is less than 0.2% in all species. The C₂₀ elongation product of pinolenic acid, bishomo-pinolenic (7,11,14–20∶3) acid, is a frequent though minor SFA constituent (maximum, 0.7%). When considering C₂₀ Δ5-UPIFA, a difference is noted between the subgenera Strobus and Pinus. In the former subgenus, 5,11–20∶2 and sciadonic acids are ≤0.3 and ≤1.9%, respectively, whereas in the latter subgenus, they are most often ≥0.3 and ≥2.0%, respectively. The highest values for 5,11–20∶2 and sciadonic acids are 0.5% (many species) and 7.0% (P. pinaster). The 5,11,14,17–20∶4 (juniperonic) acid is present occasionally in trace amounts. The highest level of total Δ5-UPIFA is 30–31% (P. sylvestris), and the lowest level is 0.6% (P. monophylla). Uniting as well as discriminating features that may complement the knowledge about the taxonomy and phylogeny of pines are emphasized.     

<Emphasis Type="Italic">sn</Emphasis>-Position determination of phospholipid-linked fatty acids derived from erythrocytes by liquid chromatography electrospray ionization ion-trap mass spectrometry

The sn-position of FA in membrane lipids has an influence on the physiological function of cells, is predictive for diseases, and therefore is useful for diagnostics. The current study compares the compositions of acyl chain substituents in the sn-1 and sn-2 positions of the glycerol backbones of phospholipids derived from human erythrocytes by using RP-HPLC coupled with on-line electrospray ionization ion trap MS. Preferential loss of the acyl group in the sn-1 position was used to determine the degree of regiospecific preference exhibited by the phospholipid molecules. The identities of the molecular species and the positions of the acyl substituents were identified using product-ion spectra of major precursor ions selected from the mass spectra averaged across peaks in the total ion chromatogram. Saturated FA were found to be located mainly in the sn-1 position of the glycerol backbones of erythrocyte phospholipids, whereas PUFA were found primarily in the sn-2 position. All measured phospholipids revealed palmitic acid (16∶0) at the sn-1 position. Linoleic acid (18∶2n−6) and arachidonic acid (20∶4n−6) were found to be attached exclusively to the sn-2 position of the backbone, whereas eicosadienoic (20∶2n−6) and eicosatrienoic acid (20∶3n−9) occurred in both positions of the backbone of PC. Oleic (18∶1n−9), linoleic (18∶2n−6), and octadecatrienoic (18∶3) acids of PE and PS were linked to both positions. Lignoceric acid (24∶1n−9) was found to be strictly localized at the sn-2 position, whereas nervonic (24∶1n−9) acid of PS was associated with both positions of the backbone. A detailed analysis of the blood cell membrane lipids by MS might be helpful to characterize postprandial kinetics of pharmacological or dietary lipid applications, as well as environmental influences on cell membranes.     

Incorporation into lipid classes of products from microsomal desaturation of isomerictrans-octadecenoic acids

The microsomal desaturation of positional isomers oftrans-octadecenoic acids is effected by the Δ9-desaturase and, with concomitant geometric isomerization,cis,trans- andcis,cis-octadecadienoic acids of unusual structure are formed. Incorporation of the substrates and their products into lipids varied from 50.5% for incubations with 14–18∶1 to 81.0% for 6–18∶1. A detailed study of the composition of each of the major lipid classes, i.e., phospholipids, triacylglycerol and cholesteryl esters, as well as the composition of the free fatty acid fraction, revealed a complex picture. Generally, thec,c-18∶2 products were enriched in the phospholipid fraction, whereas thec,t-18∶2 appeared preferentially in cholesteryl esters. The 18∶1 substrates themselves did not show marked preferences for any of the lipid classes. Phospholipase A₂ action on phosphatidylcholine and phosphatidylethanolamine demonstrated enrichment of thec,c- and thec,t-18∶2 products in the 2-position, whereas the 18∶1 substrates were preferentially inserted into the 1-positions. Thec,c- andc,t-18∶2 formed by desaturation oft11–18∶1 varied from this pattern, probably due to their conjugated double bond structures. Linoleic acid,c9,c12–18∶2, formed during desaturation oft12–18∶1, surprisingly showed enrichment in the 1-position of phosphatidylcholine. Incubation experiments witht5- andt6-isomers using liver microsomes from rats fed a corn-oil-supplemented diet showed conversion and incorporation rates similar to the rates obtained with microsomes from EFA-deficient rats. The fatty acid composition of lipid classes and the distributions of products and substrate between the 1- and 2-positions of phosphatidylcholine also agreed with results obtained using microsomes from EFA-deficient rats.     

Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C₂₂ residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.