Fatty acid composition of rat hearts as influenced by age and dietary fatty acids

Fatty acid composition of neutral and polar lipid fractions from rat hearts was determined in rats of different ages as their diet source changed. Piebald rats were weaned at 21 days and were fed standard lab chow. Lipids from rat hearts, mothers milk and lab chow were purified on a Sephadex G-25 fine column and separated into neutral and polar lipid fractions by silicic acid column chromatography. These lipid fractions were then hydrolyzed and methylated with BF₃ in methanol, prior to gas liquid chromatographic separation on a 1/8 in. × 10 ft aluminum column of 15% EGS on 80–100 mesh acid-washed Chromosorb W. Three major fatty acids in the neutral lipid fraction comprised 72% of total neutral lipid fatty acids from young hearts. At sexual maturity (at least 74 days old) C_18∶1 was the major fatty acid, followed by C_16∶0 and C_18∶0. The same three fatty acids comprised 83% of total polar lipid fatty acids, but C_18∶0 was the major fatty acid, followed by C_16∶0 and C_18∶1. The fatty acid composition of dietary lipids influenced the total neutral lipid fatty acid composition of the rat heart, but had little influence on the fatty acid composition of the polar lipid fraction. Presented in part at the AOCS Meeting, New Orleans, April 1970.     

Isovaleric acid as a precursor of odd numbered iso fatty acids in Tetrahymena

Tris isovalerate-supplementedTetrahymena pyriformis W showed no qualitative change in fatty acid composition; however, an increase in polar lipids that contain odd numbered iso acids (C₁₃, C₁₅, C₁₇, C₁₉) occurred. This change was accompanied by a decrease in the proportional amount of even numbered normal acids (C₁₄, C₁₆, C₁₈). The neutral and polar lipids from cells incubated with [1-¹⁴C] isovaleric acid were found to contain radioactivity. The methyl esters of the saturated fatty acids obtained from the polar lipids by alkaline methanolysis were separated by reversed phase chromatography, the identities confirmed by gas chromatography-mass spectrometry, and the specific activities determined. Iso acids were found to be the most heavily labeled materials. In addition to ceramide, two sphingolipid components were detected. One yielded saturated fatty acids after acidic methanolysis, while the other contained >93% α-hydroxy fatty acids. Radioactivity was noted in the long chain base fraction derived from the sphingolipids. Progressive growth inhibition occurred as the isovalerate concentration was increased in the culture medium; however, the ciliates were morphologically indistinguishable from unsupplemented cells.     

Fatty acids of bovine milk glycolipids and phospholipids and their specific distribution in the diacylglycerophospholipids

Milk lipids were fractionated by silicic acid column chromatography and preparative thinlayer chromatography (TLC). Ceramide monohexoside (CMH), ceramide dihexoside (CDH), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), phosphatidyl serine (PS), and sphingomyelin (Sph) were isolated, and the purity of each was checked by infrared spectroscopy and TLC. The diacylphospholipids were hydrolyzed with phospholipase A and the products separated by TLC. Fatty acid methyl esters were prepared from the various fractions and analyzed by gas chromatography. The glycolipids, CMH and CDH, and Sph contained large amounts of long-chain saturated fatty acids, especially C_22:0, C_23:0, and C_24:0, PE, PS, and PC contained C₁₀-C₂₂ normal and branched-chain saturated fatty acids, and C₁₅-C₂₀ unsaturated fatty acids (mainly monoenes). The distributions of saturated acids between the α′- and β-positions were respectively: PE, 46 and 11%; PS, 65 and 19%; and PC, 72 and 53%. PC was exceptional in that there was 10.8% myristic acid in the β-position and only 5.6% in the α′-position. PE and PS were similar in composition except that in PE oleic acid was evenly distributed, and in PS was largely in the β-position. In general, PC was much more saturated than PE or PS, and there was no overall pattern governing the specific distribution of the fatty acids in the three diacylphospholipids. Comparison with PC from other bovine tissues and from egg lecithin showed that fatty acids are located much less specifically in milk phospholipids than in PC from other sources. Presented at the AOCS Meeting, Houston, Texas, April, 1965.     

Characterization of skin-surface lipids from the monkey (Macaca fascicularis)

Skin-surface lipids from the monkeyMacaca fascicularis are composed of sterol esters (38%), cholesterol (4%) and two types of wax diesters, identified as Type II (IIa and IIb, 17% and 40%, respectively). Type IIa contained diesters of 1,2-alkanediols esterified with two molecules of long-chain (C₁₄−C₃₄) fatty acids having straight and branched chains. In the diesters IIa, fatty acids shorter than C₁₉ predominated in position 1, and fatty acids longer than C₂₀ predominated in position 2. Type IIb contained diesters of 1,2-alkanediols esterified with C₄ and C₅ branched-chain fatty acids (predominantly isovaleric acid) at position 1 and long-chain (C₁₄−C₂₇) acids, having straight and branched chains, at position 2. The shortchain acids were converted to 2-nitrophenylhydrazides and analyzed by high-performance liquid chromatography (HPLC). Ammonia chemical ionization (CI)-gas chromatography (GC)-mass spectrometry (MS) resolved the intact diesters IIb into 12 peaks corresponding to molecular weights ranging from 597 to 748, and showed that the molecular species, such as C₂₁−C₁₆−C₅ (diol, fatty acid in position 2, fatty acid in position 1), C₂₂−C₁₆−C₅ and C₂₃−C₁₆−C₅, were prevalent. The fatty acids from both diesters were mostly (>98%) saturated. The 1,2-alkanediols from both diesters consisted of C₁₆−C₂₆ saturated straight- and branched-chain components. The acyl groups of sterol esters contained 86% C₁₄−C₃₄ branched-chain acids. The unsaturated fatty acids (5.4%) belonged to a straight-chain monoenoic series having extremely long chains (C₁₈−C₃₄). The branched-chain structures in the fatty acids and diols were iso and anteiso. These results show the species-specific profile for the skin-surface lipid synthesis.     

Occurrence ofcis-5,cis-9-Hexacosadienoic andcis-5,cis-9,cis-19-Hexacosatrienoic acids in the marine spongeMicrociona prolifera

Fatty acid analysis of the total lipids from the marine spongeMicrociona prolifera by gas liquid chromatography on an EGSS-X column revealed two major peaks with equivalent chain length values of 27.08 and 27.74. Each of these components was isolated as a separate band by thin layer chromatography on AgNO₃-silicic acid. Characterization of the two unknowns by IR spectroscopy, NMR, hydrogenation, and gas liquid chromatography revealed that the unknown acids weren-26∶2 andn-26∶3 containing only nonmethylene interruptedcis-double bonds. Reductive ozonolysis identified the 26∶2 ascis-5,cis-9-hexacosadienoic acid and the 26∶3 ascis-5,cis-9,cis-19-hexacosatrienoic acid. Analysis of the fatty acid composition ofMicrociona total lipids showed 14% 26∶2 and 31% 36∶3. The neutral lipids, phosphatidylethanomaline, and phosphatidylserine all contained >41% C₂₆ acids; but only 4% C₂₆ was present in the phosphatidylcholine.     

Changes in lipids of pigeon “milk” in the first week of its secretion

Pigeon “milk” (PM) collected from the crop of 1- to 5-day-old squabs was analyzed to examine whether there were changes in lipid composition during the first week of secretion. The high PM fat content (9–11%) remained fairly constant in the first 5 days of secretion. The mean percentage of neutral lipids, glycolipids and phospholipids was 80, 12 and 8%, respectively. Unlike the content of neutral lipids, glycolipid and phospholipid levels increased significantly between day 1 and day 5 of secretion. Triglycerides, the major neutral lipids, decreased by 24% between day 1 and day 5, while free sterols, monoglycerides and hydrocarbons increased by 8%, 11% and 2.5%, respectively, during the same period; diglycerides and sterol esters, however, remained unchanged. The ratio of saturated to unsaturated fatty acids was 0.27 and it remained unchanged. Medium-chain (C₁₀, C₁₂ and C₁₄) and oddchain (C₁₅ and C₁₇) fatty acid contents were low. Fatty acids longer than C₂₀ were absent. Palmitic acid, the major saturated fatty acid, increased by 42% from day 1 to day 5, whereas stearic acid decreased by 48% during the same period. Oleic acid, the predominant unsaturated fatty acid, also decreased from 51 to 45% between the first and fifth day of PM secretion. Polyunsaturated acids (18∶2, 18∶3 and 20∶4) accounted for 26% and 30% of the total fatty acids on day 1 and day 5, respectively. Although lipid changes in the crop of squabs prior to collection of samples cannot totally be ruled out, the nature of lipid changes is likely to reflect cellular breakdown that precedes PM secretion by parent pigeons.     

Trans-6-hexadecenoic acid and the corresponding alcohol in lipids in the sea anemoneMetridium dianthus

Trans-6-hexadecenoic acid was found in polar lipids, triglycerides, was esters and diacylglyceryl ethers of the sea anemoneMetridium dianthus from Passamaquoddy Bay. The corresponding alcomaquoddy Bay. The corresponding alcohol also apparently occurs in the wax esters of this species. The long-chain (C₂₀, C₂₂) monoethylenic alcohols reported for other species of sea anemones from neighboring waters were absent and the major alcohol and glyceryl ether chain both had 16∶0 structures. The isomers of C₁₈ and C₂₀ monoethylenic fatty acids in polar lipids and triglycerides were unusual in their high proportion of theω ₇ isomer. These two lipids also contained higher proportion of the polyunsaturated fatty acids than the others.     

The high content of monoene fatty acids in the lipids of some midwater fishes: Family myctophidae

The total lipids of eleven species of Myctophids caught at depths between 20 and 700 m in the northern Pacific Ocean were analyzed using silicic acid column chromatography (lipid classes) and capillary gas chromatography (fatty acid and fatty alcohol composition). The major components in the lipid classes were triacylglycerols or wax esters; triacylglycerols were the dominant acyl neutral lipids (68.1–96.1%) in eight species, and wax esters were found as the dominant lipid (85.5–87.9%) in three species. The major fatty acids and alcohols contained in the was esters of the three fishes were 18:1n–9, 20:1n–9, 20:1n–11, and 22:1n–11 for fatty acids, and 16:0, 18:1, 20:1, and 22:1 for fatty alcohols. Fatty acids in the triacylglycerols ranging from C₁₄ to C₂₂ were predominantly of even chain length. The major components were 16:0, 16:1n–7, 18:1n–9, 20:1n–11, 22:1n–11, 20:5n–3 (icosapentaenoic acid), and 22:6n–3 (docosahexaenoic acid). In both the triacylglycerols and the wax esters, the major fatty components were monoenoic acids and alcohols. It is suggested from the lipid chemistry of the Myctophids that they may prey on the same organisms as the certain pelagic fishes such as saury and herring, because the large quantities of monoenoic fatty acids are similar to those of saury, herring, and sprats whose lipids originate from their prey organisms such as zooplanktons which are rich in monoenoic wax esters.     

Skin surface lipids of the dog

The skin surface lipid of the dog has been reported to contain a high proportion of diol diesters having a lower mobility on thin layer chromatography than diesters from other species in spite of containing similar fatty acid and diol components. In the present study, dog skin surface lipid was separated by preparative thin layer chromatography into sterol esters (42%), wax diesters (32%), free sterols (9%), polar lipids (7%), and unidentified components (10%). The diesters contained 1,2-diols, each esterified with one long chain fatty acid and one isovaleric acid moiety. The diols were principally branched chain C₂₁ and C₂₂ compounds while the long chain fatty acids esterified with them were mainly C₂₀ and C₂₁ branched compounds. The fatty acids from the sterol esters were mostly saturated, branched chain C₁₉ to C₂₃, together with 7% of straight chain monoenoic acids, principally C₂₁ and C₂₂. There were only trace amounts of free sterols other than cholesterol, while the esterified sterols contained 96% cholesterol and 4% lathosterol.     

The effect of lyophilization on the solvent extraction of lipid classes,fatty acids and sterols from the oysterCrassostrea gigas

The lipid class compositions of adult Pacific oysters [Crassostrea gigas (Thunberg)] were examined using latroscan thin-layer chromatography/flame-ionization detection (TLC/FID), and fatty acid compositions determined by capillary gas chromatography and gas chromatography/mass spectrometry (GC/MS). The fatty acid methyl esters were separated using argentation TLC and also analyzed as their 4,4-dimethyloxazoline derivatives using GC/MS. Major esterified fatty acids inC. gigas were 16∶0, 20∶5n−3, and 22∶6n−3. C₂₀ and C₂₂ nonmethylene interrupted (NMI) fatty acids comprised 4.5 to 5.9% of the total fatty acids. The NMI trienoic fatty acid 22∶3(7,13,16) was also identified. Very little difference was found in the proportions of the various lipid classes, fatty acids or sterols between samples of adult oysters of two different sizes. However, significant differences in some of the lipid components were evident according to the method of sample preparation used prior to lipid extraction with solvents. Lyophilization (freeze drying) of samples led to a significant reduction in the amounts of triacylglycerols (TG) extracted by solvents in two separate experiments (7.0 and 52.5% extracted). Extracts from lyophilized samples had less 16∶0, C₁₈ unsaturated fatty acids, and 24-ethylcholest-5-en-3β-ol, while C₂₀ and C₂₂ unsaturated fatty acids comprised a higher proportion of the total fatty acids. There was no significant change in the amounts of polar lipids, total sterols, free fatty acids or hydrocarbons observed in extracts from lyophilized samples relative to extracts from nonlyophilized samples. Addition of water to the freezedried samples prior to lipid extraction greatly improved lipid yields and resulted in most of the TG being extracted.     

Changes in flax (Linum usitatissimum L.) seed lipids during germination

Flax (Linum usitatissimum L.) seeds were germinated for 8 d under laboratory conditions, and changes in their lipid fraction were studied by various chemical and chromatographic methods. Total lipid content of the seeds was reduced fourfold at the end of the 8-d germination period as compared to ungerminated seeds on a fresh weight basis. The neutral lipids comprised the major fraction of seed lipids, and triacylglycerols predominated over all other lipid components even during the germination period. Both the spectrophotometric and thin-layer chromatography-flame-ionization detection methods of quantification showed a considerable increase in the content of free fatty acids. The glycolipid fraction of lipids increased, but the phospholipid fraction exhibited only minor changes. Lipase activity of flaxseed increased at the beginning of germination and then remained constant until the fifth day. Phosphatidylcholine was the major phospholipid of flaxseed lipids, and its content was reduced during the germination. The contents of lysophosphatidylcholine and phosphatidic acid increased from negligible amounts to 46% of the total phospholipids. Linolenic, linoleic, and oleic acids, respectively, were the predominant fatty acids of all the lipid fractions of flaxseed, and remained unchanged during the germination period. The glycolipid fraction had the lowest content of polyunsaturated fatty acids. Fatty acids C_14:0, C_20:0, C_24:0, C_20:1, C_22:1, and C_20:5 appeared after d 2 of germination in neutral, glyco- and phospholipid fractions.     

Occurrence of wax esters and 1-O-alkyl-2,3-diacylglycerols in goat epididymal sperm plasma membrane

Two unusual lipid classes were detected by thin-layer chromatography in the neutral lipids derived from goat cauda-epididymal sperm plasma membrane. The lipids were identified as wax esters and 1-O-alkyl-2,3-diacylglycerols based on chromatographic properties, identity of their hydrolysis products, and infrared/¹H nuclear magnetic resonance spectral evidence. The membrane containedca. 3 and 5 μg/mg protein of wax esters and alkyldiacylglycerols, respectively. The relative proportions of wax esters and alkyldiacylglycerols in the total neutral lipids were 1.5% and 2.4%, respectively. The lipids contained fatty acids with chain lengths of C₁₄ to C₂₂. The major fatty acids of the wax esters were 14∶0, 16∶0, 16∶1ω7, 18∶0 and 18∶1ω9. The fatty acids in alkyldiacylglycerol were 16∶0, 18∶0, 22∶5ω3 and 22∶6ω3. Alkyldiacylglycerol was particularly rich in docosahexaenoic acid 22∶6ω3) representing 30% of the total fatty acids. The alcohols of wax ester were all saturated with C₂₀–C₂₉ carbon chains. The deacylated products derived from alkyldiacylglycerols were identified as hexadecyl, octadecyl and octadec-9′-enyl glycerol ethers.     

cis-5-olefinic unusual fatty acids in seed lipids of gymnospermae and their distribution in triacylglycerols

Open-tubular gas chromatographic analysis of fatty acids in the lipids from the seeds of 20 species of Gymnospermae showed that they all contained nonmethylene-interrupted polyenoic (NMIP) acids as minor components and palmitic, oleic, linoleic and α-linolenic acids as major components. The NMIP acids have an additional 5,6-ethylenic bond in ordinary plant unsaturated fatty acids and the following C₂ elongation acids:cis-5,cis-9-octadecadienoic acid (5,9–18∶2) (I); 5,9,12–18∶3 (II); 5,9,12,15–18∶4, 5,11–20∶2, 5,11,14–20∶3 (III); and 5,11,14,17–20∶4 (IV). The main NMIP acids found in neutral lipids are I in two species ofTaxus, II in seven species of Pinaceae, III in two species of Podocarpaceae,Torreya nucifera, Cycas revoluta, andGinkgo biloba, and III and IV in each of three species of Taxodiaceae, and Cupressaceae. The polar lipids constitute the minor fraction of seed lipids in general. The content and composition of NMIP acids in these lipids differe considerably from those in neutral lipids. Analysis of the partial cleavage products of triacylglycerols showed that the NMIP acids distribute mainly in the 1,3-position.     

Mass spectrometric analysis of the fatty acids and nonsaponifiable lipids ofEimeria tenella oocysts

The fatty acids and nonsaponifiable lipids ofEimeria tenella oocysts were analyzed by gas liquid chromatography and combined gas liquid chromatographymass spectrometry. The fatty acids detected were identified as C_14∶0, C_16∶0, C_16∶1, C_18∶0, C_18∶1, and C_18∶2. Though the wt of the fatty acid fraction decreased during sporulation from 91 μg per 10⁶ oocysts to 47 μg per 10⁶ oocysts, the relative amounts of these fatty acids did not change appreciably. The nonsaponifiable lipids ofE. tenella consisted of cholesterol and unbranched primary alcohols of 22, 24, 26, 28, 30, and 32 carbons. Mass fragmentography demonstrated that each species of alcohol consisted of saturated and monounsaturated derivatives. Trimethylsilyl ethers of fatty alcohols were found to offer several important advantages over free alcohols for mass spectrometric characterization. Before sporulation, most fatty alcohols were in the oocyst wall. During sporulation, the wt of the nonsaponifiable lipids increased from 16 μg per 10⁶ oocysts of 44 μg per 10⁶ oocysts due largely to synthesis of C₂₄ and C₂₆ alcohols. The newly synthesized fatty alcohols were not deposited in the oocyst wall.     

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.     

Characterization of feline omentum lipids

Feline omental lipid extracts, previously reported to be angiogenic in the cornea of rabbits, were fractionated and the major lipid components characterized. Approximately 97% of the chloroform/methanol extract consisted of triglycerides containing primarily 16∶0, 18∶0, 18∶1 and 18∶2 fatty acids. Trace quantities of free fatty acids, cholesterol, di- and monoglycerides were also detected. The phospholipid fraction, obtained by solvent partition and Unisil column chromatography and characterized by high performance liquid chromatography (HPLC)-mass spectrometry, was found to consist of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine. The neutral glycolipids, isolated by solvent partition and Unisil column chromatography and identified by high performance thin layer chromatography and HPLC of their perbenzoylated derivatives, were found to consist of glucosyl- and galactosylceramides, galabiosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The complex glycolipid fraction, obtained from Folch upper phase solvent partition, was found to consist primarily of Forssman glycolipid and gangliosides GM₃ and GD₃. Smaller amounts of GM₁ and other unidentified gangliosides were also present. The ganglioside nomenclature is according to the system of Svennerholm (J. Neurochem. 10, 613–623, 1963)     

Thermophilic fungi: IV. The lipid composition of six species

The lipid composition of six thermophilic fungi (Myriococcum albomyces, Mucor miehei, Papulaspora thermophila, Rhizopus sp.,Thielavia thermophila (+)Thielavia thermophila (−), andTorula thermophila) was examined. The relative per cent total lipids (4.9–26.3%), neutral lipids (55.5–88.3%), polar lipids (11.7–44.6%) and the fatty acid profile of each lipid fraction was determined. The predominant fatty acids were 16∶0, 18∶0 and 18∶2, and lesser amounts of 12∶0, 14∶0, 15∶0, 16∶1, 16∶2, 17∶0 and 18∶3 were present. The total lipids contained an average of 0.96 double bonds per mole fatty acid (unsaturation index [USI]) the neutral lipids 0.86 USI and the polar lipids 0.84 USI, excluding the values forTorula thermophila. These data show a high degree of saturation and are consistent with data reported for other fungal thermophiles.Torula thermophila possessed abnormally high USI values (1.15–1.50) and was cultured at three different temperatures (25, 45 and 51 C). As the culture temperature ofTorula thermophila increased, the USI decreased. The USI of the polar lipids ofTorula thermophila at 25, 45 and 51 C were 1.50, 1.28 and 1.11, respectively. Thus the membrane lipids of this fungus appear unusual for a thermophile.     

Lipids of six cultivated barley (Hordeum vulgare L.) varieties

The lipids of representative varieties of 2-row spring, 6-row spring, and 6-row winter-type barleys were studied. Total barley lipids were classified by silicic acid gel column chromatography and separated by thin layer chromatography, and the fatty acid composition was determined by gas liquid chromatography. Total lipid content of the 6 barley varieties ranged from 3.12%–3.56% (dry wt basis). The average values for neutral lipids, glycolipids, and phospholipids were 71, 9, and 20%, respectively. The fatty acid composition of barley was rather typical of plant tissue. The neutral lipids and glycolipids from all the varieties contained a higher percent of linoleic and linolenic (C 18∶2 and C 18∶3) acids than the phospholipid fraction. South Dakota Experiment Station Paper 1248.     

Production of odd chain polyunsaturated fatty acids byMortierella fungi

A soil isolate,Mortierella alpina 1S-4, was found to show high production of odd chain polyunsaturated fatty acids (PUFAs) among various arachidonic acid-producingMortierella strains tested. The fungus mainly accumulated 5,8,11,M-cis-nonadecatetraenoic acid. With 5%n-hepta-decane and 1% yeast extract as growth substrates, the amount of C_19:4:4 acid accumulated reached 44.4 mg/g dry mycelia (0.68 mg/mL of culture broth). This value accounted for 11.2% of the total fatty acids in the extracted lipids from mycelia, and odd chain fatty acids comprised over 95% of the total mycelial fatty acids. The addition of sesamin, a specific inhibitor of A5 desaturation, caused an increase in C_19:3 acid and an accompanying decrease in C_19:4 acid. On the other hand, species ofMortierella that could not produce C-20 PUFAs accumulated C-17 acids, but no C-19 PUFAs, when grown with fatty substrates with an odd chain skeleton. The odd chain PUFAs were distributed in both neutral and polar lipids. The biosynthetic route to C_19:4 acid was presumed to mimic the n-6 route to arachidonic acid as follows: C_17:0 → C_17:1→ C_17:2→ C_17:3 → C_19:3 → C_19:4 acids. On leave from Suntory Ltd.     

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.