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

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

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

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

Lipid composition of beef and human pituitary glands

The lipid composition of beef and human pituitary was determined by chromatographic and spectrophotometric methods. Beef pituitary lipid contained about 25% nonpolar lipids and 75% phospholipids whereas nonpolar lipids made up approximately 60% of the total in human pituitaries. The main nonpolar (i.e., low polarity) lipids in human pituitary were triglycerides, cholesterol, free fatty acids and an unidentified component in the triglyceride fraction. Cholesterol was the major nonpolar lipid component in freshly collected beef anterior and posterior pituitary, but the amount of free fatty acids appeared to increase during storage. Preliminary investigation of the unknown nonpolar lipid in human pituitaries suggested that it was an unsaturated hydroxy compound with no carbonyl functions. Thin layer chromatography indicated that it was also present in smaller amounts in freshly collected beef pituitaries. The main phospholipids of beef anterior, posterior and human pituitary were phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl inositol, phosphatidyl serine and sphingomyelin. The fatty acid composition of total nonpolar lipids, free fatty acids, total phospholipids, phosphatidyl ethanolamine and phosphatidyl choline of beef anterior and posterior pituitary was determined by gas liquid chromatography. Mixtures of saturated and unsaturated fatty acids ranging from C₁₂ to C₂₂ were present; the main fatty acids were palmitic, stearic, oleic, linoleic and arachidonic.     

Lipid composition ofNeurospora crassa

The lipids ofNeurospora crassa, isolated in pure form from freeze-dried mycelium, were found to contain squalene, sterol esters, triglycerides, free fatty acids, geranylgeraniol, free sterols, carotenoids, cardiolipin, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, and phosphatidic acid. The above compounds were isolated in pure form by column and thin layer chromatography and were characterized by infrared spectroscopy and chromatographic mobilities. Fatty acid moieties were characterized by gas liquid chromatographic retention times of their methyl esters relative to those of authentic standards. The fatty acid composition of the triglycerides was found to be similar to that of phosphatidic acid, cardiolipin, and lecithin.     

Distribution of cholesteryl esters and other lipid in subcellular fractions of the adrenal gland of the pig

Total lipids from whole pig adrenal glands as well as from their mitochondria, microsomes, liposomes, and cell sap were extracted and fractionated first into neutral lipids and phospholipids. The highest percentage of neutral lipids was found in the cell sap, and the lowest in the microsomal fraction. Neutral lipids were subfractionated into cholesteryl esters, free cholesterol, triglycerides, and free fatty acids. Cholesteryl esters were distributed throughout the liposomes. Free fatty acids represented a substantial part of cell sap lipids, but were present also in the mitochondria, microsomes, and liposomes. Fatty acids of all fractions were analyzed by gas liquid chromatography. Free fatty acids and cholesteryl ester fatty acids from all cellular fractions were similar in composition and were characterized by considerable quantities of linoleic and arachidonic acid. Triglycerides were characterized by an increased percentage of palmitic and a low content of arachidonic acid. Phosphatidyl choline, phosphatidyl ethanolamine, diphosphatidyl glycerol, and sphingomyelin plus phosphatidyl inositol were isolated from the lipids by preparative thin layer chromatography, and their fatty acids analyzed by gas liquid chromatography. Phosphatidyl choline and phosphatidyl ethanolamine from mitochondria, microsomes, and cell sap were very similar in respect of their fatty acid composition. Sphingomyelin plus phosphatidyl inositol was characterized by a high content of C22:2omega6. Diphosphatidyl glycerol was present in mitochondria and in the cell sap.     

Fatty acid composition of milk phospholipids. III. Camel,ass, and pig milks

Phospholipids were isolated from camel, ass, and pig milks, and their fatty acid compositions were determined by gasliquid chromatography. The specific distributions of fatty acids in phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) were determined. The results are compared with previous results for bovine, sheep, Indian buffalo, and human milks. The milk phospholipids which were studied can be grouped, on the basis of their fatty acid compositions, into those from ruminant herbivores, nonruminant herbivores, and nonherbivores. The phospholipids of camel milk however have features typical of all groups as well as 15% plasmalogen in the PE fraction. For Parts I and II, see References 1 and 2.     

Phospholipids of human serum

Phospholipids extracted from normal human serum were fractionated into lecithin, lysolecithin, sphingomyelin, phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl inositol. Identification of each was established by thin-layer chromatography and infrared spectrophotometry. The content of plasmalogen was determined in both lecithin and phosphatidyl ethanolamine fractions. The composition of fatty acids and fatty aldehydes in isolated phospholipids is presented. The degree of unsaturation as reflected in the average content of double bonds per molecule of the fatty acids in phospholipids was: lecithin 1.2, choline plasmalogen 2.1, lysolecithin 0.6, sphingomyelin 0.2, phosphatidyl ethanolamine 2.8, lysophosphatidyl ethanolamine 1.0, phosphatidyl serine 1.0, and phosphatidyl inositol 1.8. Both chlline and ethanolamine plasmalogen aldehydes were predominantly saturated. Molecular weight of each phospholipid was calculated from determined fatty acid and fatty aldehyde compositions; the phosphorus factor for each phospholipid was computed. On a weight percent basis, lecithin, sphingomyelin, and lysolecithin accounted for 95% of the total phospholipids. The ethanolamine-containing phospholipids accounted for 2.5%, and the remainder was divided among phosphatidyl inositol, choline plasmalogen and phosphatidyl serine. Presented in part at the AOCS Meeting, Houston, April, 1965. Dept. of Health, Education and Welfare, USPHS.     

Lipid composition and endogenous respiration of pig heart mitochondria

Lipid composition and endogenous respiration of pig heart mitochondria were studied in parallel, since the level of endogenous respiration affects the oxidation of added substrates and therefore the regulation of oxidative phosphorylation; mitochondrial lipids can interfere either as substrates or as partner in the energy conservation mechanism. O₂ uptake kinetics were measured in presence of different additives: ATP, ADP, NAD⁺ and hexokinase + glucose. The lipid composition of pig heart mitochondria was determined by chromatographic and spectrophotometric methods. Total lipids were 90% phospholipids; the main phosphatides were cardiolipin, phosphatidyl choline and phosphatidyl ethanolamine; the two latter were rich in plasmalogens. The main nonpolar lipids were triglycerides and free fatty acids. The fatty acid composition of total lipids, phospholipids, free fatty acids and triglycerides was determined by gas liquid chromatography. Mitochondrial lipids were characterized by a high content of unsaturation. Part of this work is included in “Thèse de Doctorat de Spècialitè en Biochimie” de J. Comte, Lyon, June 26, 1970.     

Animal endogenous triglycerides: III. Swine,rat and chicken liver: Comparison with adipose tissue

The endogenous triglycerides of swine, rat and chicken livers were fractionated by silver ion thin layer chromatography and the resulting fractions were analyzed for their fatty acid composition and distribution. Whereas the endogenous triglycerides of swine adipose tissue differ markedly from those of rat and chicken adipose tissue in the location of the major fatty acids, the liver triglycerides of the three species are quite similar. They also resemble rat and chicken adipose triglycerides.     

Postnatal changes in the phospholipid composition of livers from young lambs

The total lipids were extracted from the livers of newborn lambs, from the livers of lambs during the first week after birth and from the livers of adult sheep. After separation from the nonphospholipids on columns of silicic acid the phospholipids were analyzed by thin layer chromatography and quantitative gas liquid chromatography. In all samples phosphatidyl choline and phosphatidyl ethanolamine together accounted for about 80% of the total liver phospholipids. The phosphatidyl choline-phosphatidyl ethanolamine ratio in the livers of the newborn lambs was markedly less than the ratio in the livers of the adult sheep. Moreover there was a pronounced increase in the phosphatidyl cholinephosphatidyl ethanolamine ratio in the livers of the lambs during the first week after birth. In the liver phospholipids of the lambs the concentration of phosphatidyl inositol was lower and the concentrations of phosphatidyl serine and sphingomyelin were greater than the corresponding concentrations in the liver phospholipids of the adult sheep. It is proposed that the change in the phosphatidyl choline-phosphatidyl ethanolamine ratio in the livers of the lambs during the first week after birth is due, at least in part, to the marked change that occurs in the linoleic acid-arachidonic acid ratio in the tissues of the lamb during this period.     

Lipid Composition of Garlic

Neutral, glyco- and phospholipids of garlic were resolved into their component fractions by thin layer chromatography. Neutral lipids contained considerable quantities of monoglycerides (18.5%), diglycerides (14.2%), sterols (16.3%) and triglycerides (41.5%) respectively. The phospholipid fraction was rich in phosphatidyl choline (23.5%), phosphatidyl ethanolamine (17.9%), lysophosphatidyl choline (11.8%) and lysophosphatidyl ethanolamine (8.2%). Digalactosyl diglyceride (10.1%), sterol glycoside (15.6%), cerebrosides (8.1%), acylsterol glycoside (38.6%) and monogalactosyl diglyceride (22.5%) were the major components of the glycolipids of garlic. Lauric, myristic, palmitic and linoleic acids constituted the major fatty acids of monoglycerides, diglycerides and free fatty acid fractions whereas palmitic, linoleic and linolenic acids were the major fatty acids of triglycerides. Palmitic and linoleic acids were the major fatty acids of garlic phospholipids. Except the acylsterol glycoside fraction glycolipids were rich in lauric, palmitic, linoleic and linolenic acids; palmitic acid was the only major fatty acid of acylsterol glycosides.     

Some structural investigations on phospholipids from membranes

A complete chemical characterization of phophatidyl glycerol from green leaves and algae demonstrated that 1-linoleneoyl-trans-Δ³ represented the major molecular species and its occurrence appeared to be related to photosynthesis. Beef-heart cardiolipin was demonstrated to be identical to synthetic diphosphatidyl glycerol. Chemical structures of synthetic amino acid esters of phosphatidyl glycerol were compared with those of amino acid and glucosamine containing phospholipids from bacterial cell membranes. The molecular species of lecithin from animal tissues were recognized and the influence of dietary fats on their composition was determined. Physical characteristics of natural and synthetic phospholipids indicate that nature is eloquent to preserve the properties offered by particular fatty acid combinations in the phospholipid molecule. Mammalian tissues were found to contain phospholipase A activity which produces two structurally isomeric monoacyl-phosphoglycerides. Utilizing five isomeric lysolecithins of known structure micro methods involving enzymic hydrolysis were developed to distinguish among these isomers. Lysolecithins from different natural sources were demonstrated to consist of both 1-acyl-glycero-3-phosphorylcholine and 2-acyl-glycero-3-phosphorylcholine. In connection with the nonrandom distribution of fatty acids of different apolarity at the two positions of phosphoglycerides, the various metabolic pathways of lysolecithin enantiomers in red cell ghosts, yeast and liver were investigated using different doubly-labeled substrates.     

Quantitative analysis of the phospholipids of some marine bioluminescent bacteria

Quantitative analyses of the phospholipids of three strains of marine bioluminescent bacteria were carried out after separation by two-dimensional thin layer chromatography. The phospholipids of all three species consisted of about 75% phosphatidyl ethanolamine, 13% phosphatidyl glycerol and 7% cardiolipin. The composition was only slightly affected by drastic changes in the growth conditions. One of the species contained poly-β-hydroxybutyrate. The fatty acids of another species contained principally straight and branched-chain 16 and 18 carbon fatty acids. No clue as to the nature of the elusive “aldehyde factor” of bacterial bioluminescence was found by analysis of aldehyde deficient mutants, indicating that possibly the factor is not a major phospholipid of these bacteria.     

Quantitation of phosphatidyl N-methyl- and N,N-Dimethylaminoethanol in rat liver

The contents of phosphatidyl N-methyl-and N,N-dimethylaminoethanol were determined in the liver of rats injected with (Me-C¹⁴) methionine. Total phospholipids were extracted from aliquots of the liver and fractionated by two-dimensional thin layer chromatography after addition of carrier phosphatidyl-N-methyl-and N,N-dimethylaminoethanol. The radioactivity present in the two phosphatide spots was determined and used to calculated total disintegrations per min/100 g body wt. The remainder of the livers was pooled, and total phospholipids were isolated and subjected to acid hydrolysis. N-methyl- and N,N-dimethylaminoethanol were purified by thin layer chromatography, and their specific activity was determined after quantitation by gas liquid chromatography and radioactivity measurement. The liver contents of phosphatidyl N-methyl- and N,N-dimethylaminoethanol were determined by dividing disintegrations per min/100 g body wt by the specific activity of N-methyl- or N,N-dimethylaminoethanol.     

The Nature of Cottonseed Phospholipids

Degumming of crude cottonseed oil yielded 1% of phospholipids. Repeated acetone precipitations still left 18% of triglycerides and 3.6% of gossypol. The UV-spectrum of the total phospholipids showed a λ_max at 391 mμ which was characteristic of the bound form of gossypol, possibly with the free amino group of the phosphatidyl ethanolamine present. The phospholipids were separated into a number of fractions by silicic acid column chromatography. Gossypol occurred in free form in the eluted fractions indicating cleavage during column chromatography. Analysis of the fractions by thinlayer chromatography as such and after different types of hydrolysis revealed the composition of the total phospholipids to be: phosphatidyl ethanolamine 22%, inositol phospholipids 37%, phosphatidyl choline 33% and unidentified 8%.     

Lipid metabolism ofAgaricus bisporus (Lange) sing.: I. Analysis of sporophore and mycelial lipids

The lipid components of four strains ofAgricus bisporus (Lange) Sing., the cultivated mushroom, were analyzed. Both sporophore and mycelial samples were obtained from beds in normal production. A method for obtaining mycelium free of compost was developed. Neutral lipids were separated from polar lipids by silicic acid column chromatography. Each fraction was separated by thin layer chromatography. Fatty acid methyl esters were analyzed by gas liquid chromatography and mass spectrometry. Sporophore extracts contained free sterol, free fatty acid, triglycerides, phosphatidyl choline and phosphatidyl ethanolamine. High amounts of linoleic acid were found in both neutral and polar lipid fractions. Mycelial extracts contained free fatty acids, triglycerides, phosphatidylcholine and phosphatidyl ethanolamine. No free sterol could be detected. Linoleic acid was also present in large amounts. Paper 3798 in the Journal Series of The Pennsylvania Agricultural Experiment Station.     

The Triglyceride Composition of Mango (Mangifera indica) Kernel Fat

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

Effect of heat on triglycerides of corn oil

Results of a study on the effect of heating corn oil in air to a 200C temp are reported. Heated oil was separated on a silicic acid column into 8 fractions. The first four fractions, constituting about 62% original oil, were found to be unchanged triglycerides. The remaining 4 fractions constituted polymeric and degraded products of high molecular wt. Percentage losses from the respective positions in the oleo- and linoleoglyceride fractions suggest that fatty acids in primary positions are slightly more susceptible to heat than those in the 2-position. Assuming a 1,3-random 2-random distribution, triglyceride fraction in the heated oil contained 6.7% trilinolein as compared to 17.7% in fresh oil. Evidence is presented which shows presence of branching in short chain unsaturated acids and of hydroxy acids in the saponified polymeric fractions. Presented in part at the AOCS Meeting, New Orleans, 1962     

Positional distribution of fatty acids in triglycerides from milk of several species of mammals

Milk triglycerides from the echidna, koala, Tammar wallaby, guinea pig, dog, cat, Weddell seal, horse, pig and cow were subjected to fatty acid and stereospecific analysis to determine the positional distribution of the fatty acids in the triglycerides. The samples presented a wide range of fatty acids, most of which varied in content among species. The compositions of the acids at the 3 positions also varied among species, reflecting the content of these acids in the triglycerides. However, there was a general similarity in fatty acid positional distribution patterns for all the species with the exception of the echidna. The echidna exhibited a completely different fatty acid positional distribution pattern. The saturated acids were preferentially esterified at thesn-1-position whereas the unsaturated acids were selectively esterified at thesn-2-position. The triglyceride carbon number distribution of milk from the above species (with the exception of the Weddell seal) was determined by gas liquid chromatography and compared to that predicted by the 1-random-2-random-3-random fatty acid distribution hypothesis. Agreement was excellent between observed and predicted composition for echidna, koala, Tammar wallaby, guinea pig and pig milk, and agreement was reasonable for dog, cat, horse and cow milk. Results are discussed in relation to biochemical mechanisms.     

The triglyceride composition ofMyrica carolinensis fruit coat fat (bayberry tallow)

The triglycerides ofMyrica carolinensis fruit coat fat (bayberry tallow) contain only three fatty acids: myristic (21.5 mole %), palmitic (77.5%), and stearic (1.0%). The component triglycerides of this simple fat were determined by gas-liquid chromatography and pancreatic lipase hydrolysis. Triglycerides with carbon numbers 42, 44, 46, 48, and 50 were found. Lipase hydrolysis showed a preferential but not exclusive esterification of myristic acid at the 2-position. The triglyceride composition calculated from the combined experimental results did not conform to a random or 1,3-random-2-random distribution pattern. Regional differences in fatty acid and triglyceride composition within the fruit coat were also observed. Presented at the AOCS meeting in Houston, Texas, 1965.