Analysis of triglycerides using atmospheric pressure chemical ionization mass spectrometry

Atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated as a new method for analysis of a mixture of triglycerides separated by reverse-phase high-performance liquid chromatography. A mixture of homogeneous (monoacid) triglyceride standards containing fatty acids with zero to three double bonds was analyzed to demonstrate the quality of mass spectra obtained by using the APCI interface. The mass spectra showed that minimal fragmentation occurs, resulting primarily in diglyceride [M−RCOO]⁺ ions and [M+1]⁺ protonated molecular ions. The degree of unsaturation within the acyl chains had a marked effect on the proportion of diglyceride ions vs. the [M+1]⁺ ions formed in the APCI source. The mass spectra of triglycerides containing fatty acids with two or three double bonds showed predominantly protonated triglyceride ions, with diglyceride peaks representing 13 to 25% of the base peak. The triglycerides containing singly unsaturated fatty acids gave diglyceride ions as the base peak, and [M+1]⁺ ions with an intensity 20 to 28% that of the base peak. Only diglyceride ions were observable in the spectra of triglycerides containing saturated fatty acids.     

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.     

The incorporation of14C-glycerol into different species of diglycerides and triglycerides in rat liver slices

The relative rates of de novo synthesis of species of diglycerides and triglycerides from¹⁴C-glycerol were examined in rat liver slices. Diglycerides containing one or two double bonds per molecule and triglycerides containing four or more double bonds per molecule represented 70% and 60% respectively of the newly synthesized diglycerides and triglycerides. The newly synthesized triglycerides were more unsaturated than the endogenous triglycerides. Our results suggest that a nonrandom synthesis of species of diglycerides occurred followed by an almost random utilization of the various diglyceride species for the biosynthesis of triglycerides.     

Structure of bovine milk fat triglycerides: II. Long chain lengths

The long chain triglycerides of bovine milk fat were isolated by thin layer chromatography, and their chemical structure determined by combined thin layer and gas liquid chromatography, and a stereospecific analysis of a molecular distillate of butteroil of comparable composition. The milk fat fraction (39% of total) contained C₈–C₂₀ fatty acids which were distributed among the glycerides of 40–56 acyl carbon atoms in a manner not unlike that found for the same acids in the short chain triglycerides. Although individual triglycerides were not identified, the specific distribution of the fatty acids could best be accounted for by assuming a common pool of long chain 1,2-diglyceride precursors from which the bulk of both short and long chain triglycerides are synthesized by a stereospecific introduction of C₄–C₁₈ fatty acids in position 3 of sn-glycerol. This hypothesis is compatible with the results of stereospecific analyses of the short and long chain fractions and of the total butteroil. It is supported by the nonrandom distributions demonstrated for the molecular weights of the milk fat triglycerides of different degrees of saturation. Presented in part at the AOCS meeting, Philadelphia, October, 1966.     

Studies on biosynthesis of waxes by developing jojoba seed tissue

Slices of developing jojoba cotyledons incorporated a variety of precursors in to wax, free alcohols, and polar lipids.¹⁴C-Decanoic and¹⁴C-lauric acids were elongated and desaturated, whereas¹⁴C-myristic and¹⁴C-longer chain fatty acids, although incorporated into wax, were insignificantly modified. Exogenously added¹⁴C-acetate contributed mainly to chain elongation of endogenous oleic acid, whereas¹⁴C from added glucose was uniformly distributed throughout the acyl chain of the fatty acids. These data suggest the existence of metabolically separate pools of acetate and/or sites for de novo synthesis and elongation of acyl chains.     

Incorporation of radioactive polyunsaturated fatty acids into liver and brain of developing rat

The incorporation of radioactivity from orally administered linoleic acid-1-¹⁴C, linolenic acid-1-¹⁴C, arachidonic acid-³Hg, and docosahexaenoic acid-¹⁴C into the liver and brain lipids of suckling rats was studied. In both tissues, 22 hr after dosing, 2 distinct levels of incorporation were observed: a low uptake (from 18∶2-1-¹⁴C and 18∶3-1-¹⁴C) and a high uptake (from 20∶4-³H₈ and 22∶6-¹⁴C). In adult rats, the incorporation of radioactivity into brain lipids from 18∶2-1-¹⁴C and 20∶4-³H was considerably lower than the incorporation into the brains of the young rats. In the livers of the suckling rats, the activity from the 18 carbon acids was associated mostly with the triglyceride fraction, whereas the activity from the 20∶4-³H₈ and 22∶6-¹⁴C was concentrated in the phospholipid fraction. In the brain lipids, the activity from the different fatty acids was associated predominantly with the phospholipids. In the liver and brain phospholipid fatty acids, some of the activity in the 18∶2-1-¹⁴C and 18∶3-1-¹⁴C experiments was associated with 20 and 22 carbon polyunsaturated fatty acids; however, radioactivity from orally administered 20∶4-³H₈ and 22∶6-¹⁴C was incorporated intact into the tissue phospholipid to a much greater extent compared with the incorporation of radioactivity into 20∶4 and 22∶6 in the experiments where 18∶2-1-¹⁴C and 18∶3-1-¹⁴C, respectively, were administered. Possible reasons for these differences are discussed. Rat milk contains a wide spectrum of polyunsaturated fatty acids, including linoleate, linolenate, arachidonate, and docosahexaenoate. During the suckling period in the rat, there is a rapid deposition of 20∶4 and 22∶6 in the brain. The results of the present experiments suggested that dietary 20∶4 and 22∶6 were important sources of brain 20∶4 and 22∶6 in the developing rat.     

Fractionation of anhydrous milk fat by short-path distillation

Anhydrous milk fat was fractionated by short-path distillation into four fractions at temperatures of 245 and 265 C and pressures of 220 and 100 μm Hg. Two fractions (LF1 and LF2) were liquid, one fraction (IF) was semi-solid and one fraction (SF) was solid at room temperature. The fractions were characterized by melting temperature profile, solid fat index and triglyceride and fatty acid compositions. The peak melting temperature progressively increased (8.8 to 38.7 C) from liquid to solid fractions. The solid fat content ranged from 0 to 27.5% at 20 C, while native milk fat was 15.4%. The short chain (C24–C34) triglycerides were enriched in the LF1 fraction, long chain (C42–C54) triglycerides were concentrated in the SF fraction, and medium chain (C36–C40) triglycerides in the IF fraction; in the LF2 fraction, though, both short and medium chain triglycerides were enriched. Short chain (C4–C8) fatty acids gradually decreased from liquid to solid fractions and the trend was reverse for long chain (C14–C18) fatty acids, both saturated and unsaturated. The weight average molecular weights and geometric mean-carbon number of milk fat fractions were in the range of 590.7–782.8 and 31.9–46.3, respectively, compared to 729.3 and 41.0, respectively, for native milk fat, suggesting short-path distillation effects a very high degree of molecular weight separation.     

Properties of lysophosphatidylcholine acyltransferase from <Emphasis Type="Italic">Brassica napus</Emphasis> cultures

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT; EC 2.3.1.23) catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine (LPC) to produce PC and CoA. LPCAT activity may affect the incorporation of fatty acyl moieties at the sn-2 position of PC where PUFA are formed and may indirectly influence seed TAG composition. LPCAT activity in microsomes prepared from microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf) was assayed using [1-¹⁴C]acyl-CoA as the fatty acyl donor. LPCAT activity was optimal at neutral pH and 35°C, and was inhibited by 50% at a BSA concentration of 3 mg mL⁻¹. At acyl-CoA concentrations above 20 μM, LPCAT activity was more specific for oleoyl (18∶1)-CoA than stearoyl (18∶0)- and palmitoyl (16∶0)-CoA. Lauroyl (12∶0)-CoA, however, was not an effective acyl donor. LPC species containing 12∶0, 16∶0, 18∶0, or 18∶1 as the fatty acyl moiety all served as effective acyl acceptors for LPCAT, although 12∶0-LPC was somewhat less effective as a substrate at lower concentrations. The failure of LPCAT to catalyze the incorporation of a 12∶0 moiety from acyl-CoA into PC is consistent with the tendency of acyltransferases to discriminate against incorporation of this fatty acyl moiety at the sn-2 position of TAG from the seed oil of transgenic B. napus expressing a medium-chain thioesterase.     

Structural comparison between triglycerides and phospholipids from pig kidney

The compositional specificity of the major diacyl phosphatides, plasmalogens and triglycerides of pig kidney has been determined. The triglycerides have been shown to be esterified predominantly with saturated fatty acids in the 2 position while the phosphatides have predominantly unsaturated fatty acids in this position. Such complete “inversion” of the structure of triglycerides and phospholipids is not normally seen in mammalian tissue and suggests that the synthesis of structural polar lipids involves other steps than the reaction of a diglyceride with CDP-ethanolamine or CDP-choline. This marked difference in the fatty acid distribution of the polar lipids and triglycerides of the pig kidney may render this tissue especially suitable for studies on the turnover and synthesis of structural lipids.     

Digestion of butyrate glycerides by pancreatic lipase

The racemic triglycerides, glyceryl-1-palmitate-2,3-dibutyrate (PBB), glyceryl-1-butyrate-2,3-dipalmitate (PPB), glyceryl-2-butyrate-1,3-dipalmitate (PBP), and the diglyceride, racemic glyceryl-1-palmitate-3-butyrate (P-B) were synthesized and digested with pancreatic lipase. Each triglyceride was mixed with equimolar amounts of triolein (OOO) prior to incubation. The following order of digestion rates was observed: PBB>PPB>PBP>P-B. There was no evidence for short-chain fatty acid specificity; however the triglycerides containing butyric acid were hydrolyzed more rapidly than OOO. Based upon the fatty acid composition of partial glycerides, digestion of butyrate glycerides was not a simple phenomenon. For example, in the digestion of PBB, butyric acid accumulated faster than palmitic acid in the diglycerides, and monobutyrin was found to accumulate when the diglyceride, P-B, was digested. As evidenced by the fatty acid composition of the monoglycerides, positional specificity of pancreatic lipase was always maintained. Scientific contribution No. 245, Agricultural Experiment Station, University of Connecticut, Storrs. Presented in part at the AOCS Meeting, Philadelphia, October 1967.     

Stereospecificity of monoacylglycerol and diacylglycerol acyltransferases from rat intestine as determined by chiral phase high-performance liquid chromatography

Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol systhetase of rat intestinal mucosa. With 2-oleoyl[-³H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97–98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was thesn-1,2-and 10% thesn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was thesn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-³H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.     

Triacylglycerols of the olive fruit (Olea europaea L.): characterization of mesocarp and seed triacylglycerols in different cultivars by liquid chromatography and 13C NMR spectroscopy

Triacylglycerols of mesocarp and seed oils extracted from olive fruits of different cultivars were characterized. The analysis of the profiles of the triglyceride isomers which were determined by HPLC, evidenced that ECN 48 (OOO-OOP) and ECN 46 (OLO) triglycerides were the major components of mesocarp and seed oils, whereas triglycerides acylated with the linoleoyl chain appeared to be more abundant in seed oil, i.e. LLL (ECN 42), OLL-PLL (ECN 44), and POL-OLO (ECN 46). Mesocarp and seed oils, likewise the cultivars differed significantly (P<0.05) with regard to all the triglyceride components.¹³C NMR spectroscopy based on carbonyl carbon resonances of triglyceride acyl chains was applied to carry out the regiospecific analysis of triacylglycerols. Acyl chain composition, chain distribution among 1(3)- and 2-glycerol positions, and chain 2-positional specificity, were determined. The results confirmed that two different pools of fatty acids esterified at 1(3)- and 2-positions in triacylglycerols of mesocarp and seed oils exist, the saturated chains being by almost 100% at 1(3)-positions. 2-positional specificity evidenced that the oleoyl chain moved away from a pure random model less than the linoleoyl chain. 2-Distribution and 2-specificity data of oleoyl and linoleoyl chains along with linoleoyl 1(3)-distribution values, appeared to discriminate significantly (P<0.05) mesocarp and seed oils, but no cultivar discrimination was achieved. The effect of fatty acid concentration on their distribution between triacylglycerol positions and the low variability of 2-positonal specificity values of unsaturated chains over a wide range of vegetable oils were highlighted.     

Fatty acid metabolism in the chloroplast lipids of green and blue-green algae

The pattern of uptake of radioactivity into chloroplast lipids when a green alga (Chlorella vulgaris) was incubated with sodium 2-¹⁴C-acetate differed appreciably from that obtained when two blue-green algae (Anabaena cylindrica andAnacystis nidulans) were incubated under similar conditions. The fatty acids of the digalactosyl diglyceride and sulphoquinovosyl diglyceride fractions from the blue-green algae were labeled more rapidly than were those of the corresponding fractions fromC. vulgaris, whereas the activity in the acids of the phosphatidyl glycerol fraction fromA. cylindrica andA. nidulans was relatively lower than that in the green alga. The results indicate that the metabolic behavior of chloroplast lipids may vary considerably according to the class of alga concerned. In all three alga, the evidence points to an intermediary function for the chloroplast lipids in fatty acid synthesis. Only limited exchange of acyl groups between the different chloroplast lipids seemed to occur during photoautotrophic growth.     

Triglyceride structure of milk fats

The enantiomeric nature of the triglycerides of bovine milk fat was reinvestigated by determining the stereospecific distribution of fatty acids in rearranged butterfat, following partial hydrolysis with pancreatic lipase, and in certain molecular distillates of native butterfat, following Grignard degradation. The results with rearranged butterfat confirmed the validity of pancreatic lipase hydrolysis as a means of generating representative diglycerides from milk fat triglycerides. The Grignard degradation and lipolysis gave identical distributions for fatty acids when included as part of the assay system in the stereospecific analysis. Characteristically, butyric acid and the other short chain acids occupied the 3 position in the native butterfat, while in the rearranged oil they were distributed more or less randomly. Gas chromatographic analysis of the short chain glycerides on polyester columns allowed an effective resolution of butyryl, caproyl and caprylyl glycerides of identical numbers of total acyl carbons and double bonds. The method was especially well suited for resolution of the 2,3-diglycerides, which were recovered either as the more polar fraction from thin layer chromatography of the X-1,2-diacylglycerols, or by acetolysis of the residual phenolphosphatides resulting from phospholipase A digestion. It was shown that butyric acid in the 3 position was preferentially paired with myristic, palmitic and oleic acid in the 2 position, and palmitic and oleic acid in the 1 position, which was also characteristic of the other short chain acids. One of eight papers presented in the symposium “Milk Lipids,” AOCS Meeting, Ottawa, September 1972.     

The structure of rat liver triglycerides

The fatty acid compositions at the 1-, 2-, and 3-positions³ of rat liver triglycerides were determined by using pancreatic lipase and diglyceride kinase. The distribution of acids between the 1- and 3-positions is not random; rather each position has a characteristic composition. The relative abundance of species and positional isomers in the triglyceride mixture was predicted by using values from the stereospecific analysis and assuming that the composition of each position is independent of the other two. The total triglyceride was resolved into species by using TLC with silver nitrate and Silica Gel G, and the relative amounts corresponded closely with those predicted on the basis of this assumption. The major species were isolated, and the distribution of their fatty acids among the three glyceride positions was determined. From these data the relative amount of each positional isomer was calculated. The results indicate that the esterification of fatty acids at each position proceeds with a specificity that is not correlated with the composition of the other positions with the molecule. The relative abundance of the different liver triglyceride species is also found to be related in part to the composition of the 1,2-diglyceride units found in the lecithins of this tissue. Presented at the AOCS Meeting, Philadelphia, October 1966.     

Fatty acid composition of liver lipids during development of rat

The fatty acid composition of triglycerides and phospholipids from rat liver was determined throughout the period of growth in the rat. Major changes in the triglyceride fatty acid composition were observed during the period studied. The triglycerides from fetal and newborn rats contained only a small percentage of polyunsaturated acids compared with suckling and weanling rats. During the suckling phase the liver triglycerides were rich in long chain polyunsaturated acids such as 20∶4, 20∶5, 22∶5, and 22∶6; once the pups were weaned, the percentage of these acids in the liver triglycerides fell. In these experiments, 18∶2 and 18∶3 were the only polyunsaturated acids in the maternal diet. However, the stomach contents of the suckling pups contained 18∶2 and 18∶3, as well as the long chain polyunsaturated acids. Radioactive 18∶3 and 22∶6 were fed to suckling pups, and the results suggested that the LCP in the rat liver triglycerides during the suckling period were derived from the long chain polyunsaturated acids in the dam's milk, rather than by synthesis from either 18∶2 or 18∶3 within the pups.     

Lipid metabolism in plasma,liver, and adipose tissue of rats with experimental chronic nephrotic syndrome

Plasma, liver, and adipose tissue lipid composition and synthesis from [1-¹⁴C] acetate were studied three months following induction of nephrotic syndrome in rats by injection of antiglomerular basement membrane protein. Plasma triglyceride concentrations and specific radioactivities were elevated, and the triglycerides contained increased proportions of oleic acid. Plasma cholesterol and phospholipid concentrations were also increased, but free fatty acid levels were not. Liver triglyceride concentrations were decreased and incorporation of [1-¹⁴] acetate into liver triglycerides was also depressed below that of normal controls. Nephrotic rat liver triglycerides contained a higher proportion of oleic acid and lower arachidonic acid than did controls. Incorporation of [1-¹⁴C] acetate into adipose tissue lipids of the nephrotic rats was increased, and the proportion of palmitic acid was decreased. In the chronic nephrotic rat, the major source of the increased plasma triglycerides may be fatty acids mobilized from adipose tissue stores.     

The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase

The importance of Glu87 and Trp89 in the lid of Humicola lanuginosalipase for the hydrolytic activity at the water/lipid interfacewas investigated by site-directed mutagenesis. It was foundthat the effect on the hydrolytic activity upon the replacementof Trp89 with Phe, Leu, Gly or Glu was substrate dependent TheTrp89 mutants displayed an altered chain length specificitytowards triglycerides, with a higher relative activity towardstriacetin and trioctanoin compared with tributyrin. Trp89 wasshown to be lessimportant in the hydrolysis of vinyl esterscompared with ethylesters and triglycerides. An exclusive effecton the acylation reaction rate by the mutation of Trp89 wasconsistent with the data. It is suggested that Trp89 is importantin the process of binding the acyl chain of thesubstrate intothe activesite for optimal acylation reaction rate. The Trp89Phemutation resulted in an increased hydrolytic activity towards2-alkylalkanoic acid esters. This is suggested to be due toreduction of unfavourable van der Waals contacts between Trp89and the 2-substituent of the substrate. Thus, in contrast tonatural substrates, Trp89 has a negative impact on the catalyticefficiencywhen substrates with bulky acyl chains are used. Incontrast to the Trp89 mutations, the effect on the hydrolyticactivity of the Glu87Ala mutation was almost substrate independent,35–70% activity of wild-type lipase. Areduction of boththe acylation and deacylation reaction was consistent with thedata.     

Lipid specificity and location of the sterol carrier protein-2 fatty acid-binding site: A fluorescence displacement and energy transfer study

Although it was recently recognized that sterol carrier protein-2 (SCP-2) interacts with fatty acids, little is known regarding the specificity of SCP-2 for long-chain fatty acids or branched-chain fatty-acid-like molecules. Likewise the location of the fatty-acid binding site within SCP-2 is unresolved. A fluorescent cis-parinaric acid displacement assay was used to show that SCP-2 optimally interacted with 14–22 carbon chain lipidic molecules: polyunsaturated fatty acids > monounsaturated, saturated > branched-chain isoprenoids > branched-chain phytol-derived fatty acids. In contrast, the other major fatty-acid binding protein in liver, fatty-acid binding protein (L-FABP), displayed a much narrower carbon chain preference in general: polyunsaturated fatty acids > branched-chain phytol-derived fatty acids > 14- and 16-carbon saturated > branched-chain isoprenoids. However, both SCP-2 and l-FABP displayed a very similar unsaturated fatty-acid specificity profile. The presence and location of the SCP-2 lipid binding site were investigated by fluorescence energy transfer. The distance between the SCP-2 Trp⁵⁰ and bound cis-parinaric acid was determined to be 40 Å. Thus, the SCP-2 fatty-acid binding site appeared to be located on the opposite side of the SCP-2 Trp⁵⁰. These findings not only contribute to our understanding of the SCP-2 ligand binding site but also provide evidence suggesting a potential role for SCP-2 and/or L-FABP in metabolism of branched-chain fatty acids and isoprenoids.     

Effect of dietary fat supplementation on the composition and positional distribution of fatty acids in ruminant and porcine glycerides

Dietary fats which were protected from ruminal metabolism were fed to ruminants, and the constituent fatty acids subsequently appeared in the glycerides of tissues and secretory products. These dietary fat induced alterations in tissue lipid composition were particularly apparent when the fat source was enriched with linoleic acid. Similarly, when pigs were fed linoleic-enriched fats, the linoleic acid was incorporated into the adipose tissue triglycerides. Stereospecific analyses were carried out on triglycerides from various tissues and secretory products obtained from animals fed control or linoleate-enriched diets. The analysis of adipose tissue triglycerides showed that linoleate and oleate were preferentially esterified to positions 2 and 3 (cattle and sheep), and positions 1 and 3 (pigs). Of the other major adipose tissue fatty acids, palmitate was preferentially esterified at position 1 (ruminants) and position 2 (pigs), and stearate was preferentially esterified at positions 1 and 3 (ruminants), and position 1 (pigs). Stereospecific analysis of high mol wt milk triglycerides showed that linoleate was either evenly distributed on all three positions (goats), or predominantly on position 3 (cows). Furthermore, the incorporation of this linoleate did not markedly alter the positional specificity of the other major milk triglyceride fatty acids. Of these fatty acids, the short and medium chain length acids (butyratelaurate) were mainly on position 3, myristate and palmitate on positions 1 and 2, and stearate and oleate evenly distributed. Thoracic duct lymph triglycerides from sheep tended to show preferential incorporation of linoleate at position 3, palmitate at position 2, and stearate at position 1 and 3; oleate, on the other hand, tended to be evenly distributed on all three positions of the lymph triglyceride. The stereospecific arrangement of fatty acids in sheep liver triglycerides was similar to that of lymph triglycerides, and this may reflect the uptake of intact or partially hydrolysed chylomicron and/or very low density lipoprotein triglycerides by the liver. There were also some analogies in the stereospecific arrangement of fatty acids on ruminant lymph and milk triglycerides and this may reflect an incomplete hydrolysis of chylomicron and/or very low density lipoprotein triglycerides prior to uptake by the mammary gland. An unusual feature of lymph from sheep fed linoleate was the presence of phospholipids which contained large amounts of linoleate in ca. equal proportion at both positions 1 and 2 of the phospholipid molecule.