Lipid composition of beef brain,beef liver,and the sea anemone: Two approaches to quantitative fractionation of complex lipid mixtures

Two new schemes for fractionation of complex lipid mixtures are presented. Their use for the study of lipids of beef brain, beef liver, and the sea anemone are described. Apparatus and techniques for working in an inert atmosphere, evaporation of solutions in the cold under nitrogen, use of infrared spectroscopy for examination of lipids and their hydrolysis products, preparation and clution of diethylaminoethyl (DEAE) cellulose and silicic acid-silicate columns and general column combinations that can be used to fractionate complex lipid mixtures are considered in detail. The first scheme, employing DEAE cellulose columns followed by thin layer and paper chromatographic examination of the fractions, was applied to liver lipids. The many components, some of them new lipids not previously detected, are clearly seen with this technique but are not seen when paper or thin layer chromatography alone or silicic acid chromatography are used. The second scheme employing DEAE for initial fractionation, followed by complete separation on silicic acid and silicic acid-silicate columns, was applied to lipids of the sea anemone and beef brain. Typical lecithin and phosphatidyl ethanolamine were isolated, but sphingomyelin was not found. A new sphingolipid, ceramide aminoethylphosphonate, with a free amino group and a direct carbon to phosphorus bond was isolated and characterized. The methods used for quantitative isolation, the infrared spectra, and the amounts of cholesterol, ceramide, cerebroside, galactosylglyceride, sulfatide, sphingomyelin, lecithin, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, triphosphoinositide, phosphatidic acid, cardiolipin, and ganglioside of beef brain are presented. Finally, the types of lipid-nonlipid interactions disclosed by column chromatography and their potential application to biological problems are discussed.     

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.     

Phospholipid Composition of <Emphasis Type="Italic">Jatropha curcus</Emphasis> Seed Lipids

Jatropha curcus L. oil has emerged as one of the most important raw materials for biodiesel production. However, no detailed study has been reported on characterizing the lipid constituents of jatropha oil. The present study revealed that the total oil content of jatropha seeds was 32% with a composition of 97.6% neutral lipids, 0.95% glycolipids and 1.45% phospholipids. The fatty acid composition of total lipids, neutral lipids, phospholipids and glycolipids was also determined and found to contain oleic acid (18:1) and linoleic acids (18:2) as major fatty acids. The phospholipids fraction was further characterized and quantified and found to contain phosphatidyl choline (PC) 60.5%, phosphatidyl inositol (PI) 24% and phosphatidyl ethanolamine (PE) 15.5%. The fatty acid composition and the positional distribution of the fatty acids of individual phospholipids were also reported.     

Thermophilic fungi: III. The lipids ofHumicola grisea var.thermoidea

The lipids of the thermophilic fungusHumicola grisea var.thermoidea were qualitatively and quantitatively determined. The polar lipids consisted of 38.4–42.3% of the total lipids. The relative per cent phospholipids based upon the total phospholipids were as follows: phosphatidyl choline, 32.3–33.7%; phosphatidic acid, 24.5–31.7%; phosphatidyl ethanolamine, 15.8–20.9%; phosphatidyl inositol, 12.5–13.0%; phosphatidyl serine, 2.3–5.4%; and diphosphatidyl glycerol, 3.9–4.0%. The relatively high concentration of phosphatidic acid may be characteristic of fungi grown at elevated temperatures. Several sterol glycosides (3.1–6.0%) were present in the polar lipids. The neutral lipids consist of triglycerides, 28.6–36.0%; free fatty acids, 5.3–13.5%; sterols, 11.4–13.9%; sterol esters, 1.8–3.0%; and diglycerides, 2.2–3.4%. The sterols and derivatives comprise an unusually large fraction of the total lipids (16.3–22.9%) suggesting a role in thermostability.     

Study of free and bound lipids ofBrassica campestris,var yellow sarson

Mature seeds ofBrassica campestris var. yellow sarson were extracted with hexane to yield free lipid. The residue then was extracted with chloroform-methanol to release bound lipid. Free and bound lipids were separated into polar and nonpolar fractions chromatographically. The nonpolar fraction of both free and bound lipid consisted mainly of triglycerides with small amounts of steryl esters, free sterols, mono- and di-glycerides, and free fatty acids. The principal components of polar bound lipid were phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and steryl glycoside. In the free polar lipid, there was more phosphatidyl inositol and less phosphatidyl choline and phosphatidyl ethanolamine. Erucic acid content was much greater in the nonpolar fractions and in the polar free lipid than in the polar bound lipid.     

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.     

Phosphatidyl choline: Donor of 18-carbon unsaturated fatty acids for glycerolipid biosynthesis

Kinetics of radiocarbon inforporation into the phyosphatidyl choline of pumpkin leaf fed 1(-14)C-acetate at low light intensity were strongly suggestive of lipid bound fatty acids acting as substrates for desaturase enzymes. After pulse labeling in direct sunlight with the same precursor, phosphatidyl choline and phosphatidyl glycerol contained up to 90% of total glycerolipid radiocarbon at the shortest sampling times. Subsequent loss of radiocarbon from phyosphatidyl choline and a corresponding gain in other glycerolipids is taken to indicate a net flow of long chain fatty acids through phosphatidyl choline and into other lipids. It is proposed that there may be 2 separate synthetases in leaf tissue, one producing oleic and the other palmitic acids as their end products. Oleic acid is transferred almost exclusively to phosphatidyl choline where it is further desaturated to linoleic and linolenic acids before being made available for the biosynthesis of other lipids. Palmitic acid is transferred mainly to phosphatidyl glycerol, where it is desaturated to trans-delta3-hexadecenoic acid.     

Genetic variability of human plasma and erythrocyte lipids

Genetic variation of fasting plasma lipids, lipoproteins and erythrocyte membrane lipids was studied in 67 sets of like-sexed twins and 3 sets of triplets. All of the plasma lipids were more variable in dizygotic twins than monozygotic twins with the exception of phosphatidyl ethanolamine, but only cholesteryl esters, lecithin, phosphatidyl inositol and β-lipoprotein showed significant genetic variation. In contrast, no significant genetic variability was found in any of the erythrocyte membrane lipids and erythrocyte phosphatidyl ethanolamine had significantly greater variation in monozygotic twins. Two sets of twins had an extra lipoprotein band (slow α1); in one family the variant appeared to be segregating as a dominant trait. Presented in part at the ISF-AOCS World Congress, Chicago, September 1970.     

Cyclic acetal as coinitiator for bimolecular photoinitiating systems

Cyclic acetals were used to replace the conventional amines in bimolecular photoinitiating systems. The mixtures of benzophenone derivatives and cyclic acetals were used to initiate the UV photopolymerization of 1,6-hexanedioldiacrylate (HDDA). Camphorquinone (CQ)/1,3-benzodioxole (BDO) combinations were used to initiate the visible light photopolymerization of 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane (BisGMA)/triethylene glycol dimethacrylate (TEGDMA) (70/30 wt%) for dental application. The kinetics was recorded by real-time infrared spectroscopy (RTIR). Ethyl 4-N,N-dimethylaminobenzoate (EDMAB) was used as control in the same photocuring condition. The results showed that the addition of cyclic acetals greatly increased the rate of polymerization (R_p) and final double bond (DC) of HDDA. Combination of p-chlorobenzophenone (CBP)/BDO had the highest initiating reactivity. BDO also showed an effective coinitiator for camphorquinone-based initiator system. Comparing with EDMAB, CBP/BDO and CQ/BDO indicated comparable initiating reactivity. Moreover, the natural component characteristics of BDO made it a promising alternative to commercial amine in biomolecular photoinitiating system.     

Methodology for the separation o plant lipids and application to spinach leaf and chloroplast lamellae

Procedures for separation of complex plant lipids and results obtained are reviewed. Procedures based on DEAE cellulose and silicic acid chromatography, which may be preceded by countercurrent distribution, are presented for separation of the individual glyceroland sphingolipid classes of spinach leaf and chloroplast lamellae. These procedures appear to be generally applicable to photosynthetic tissue of plants and algae. The separation and infrared spectra of mono-and digalactosyl diglycerides, lecithin, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl inositol, plant sulfolipid, cerebroside, and sterol glycosides from spinach are recorded. Chloroplast lamellae lipids are in the molar ratio monogalactosyl diglyceride (14.0), digalactosyl diglyceride (8.0), phosphatidyl glycerol (5.5), sulfolipid (3.9), lecithin (2.0), phosphatidyl inositol (1.0). Phosphatidyl ethanolamine, cerebrosides, and sterol glycosides were not detected in chloroplast lamellae. Fatty acid composition of individual lamellae lipids have been determined: The galactosyl lipids contain more than 90% trienoic acids.Trans-3- hexadecenoic acid is restricted almost exclusively to phosphatidyl glycerol. In this report techniques which have been applied to the isolation of plant glycero- and sphingolipids are reviewed and a new scheme presented for the separation of several of the plant lipid classes. Results obtained with spinach leaf and its photosynthetic apparatus are presented and discussed. Paper III in the series Plant and Chloroplast Lipids. Presented at the 15th Annual Summer Program, “Symposium on Quantitative Methodology in Lipid Research”, Aug. 3–7, 1964. Literature is reviewed to July 1964.     

Lipid Studies of Pimpinella acuminata

The seed oil of Pimpinella acuminata species of the family Umbelli-ferae was extracted with chloroform : methanol (2:1) for the separation of different classes of lipids as hydrocarbons (1.7%), sterol esters (traces), triglycerides (74.1%), free fatty acids (6.6%), 1,3-diglycerides (1.6%), 1,2-diglycerides (1.6%), sterols (1.8%), mono-glycerides (2.0%), phosphatidyl ethanolamine (0.8%), phosphatidyl choline (1.2 %), lyso phosphatidyl ethanolamine (0.4%), phosphatidyl inositol (1.2%) and unknown (7.0%). The fatty acids as determined by application of gas liquid chromatography is C₁₀-C₂₂, whereas C_16:0, C_18:0-C_18:2 and C_18:2 are predominantly present in all polar and non-polar lipid classes.     

Liver lipids during development

The fatty acid composition of the major liver microsomal phospholipids has been studied during pre- and postnatal development of the rabbit. The fatty acid composition of the total lipids, phosphatidyl choline, and phosphatidyl ethanolamine from animals −6, −3, 0, +3, +6, +9, +16, and +112 days of age was determined. Fatty acid composition is similar in phosphatidyl choline and phosphatidyl ethanolamine for oleic acid at +3, +6, +9, and +16 day old animals; palmitoleic acid at +9 day old animals and linoleic acid at −6, −3, and 0 day old animals. Palmitoleic acid demonstrated a uniform decrease during early development in the total lipids and in both phosphatidyl choline and phosphatidyl ethanolamine; however, in the 112 day animal, the amount was just slightly lower than that observed for the earliest prenatal animal studied. Oleic acid decreased considerably during early postnatal development in the total lipids, phosphatidyl choline and phosphatidyl ethanolamine, but an increase in the 112 day animal was observed. Linoleic acid fluctuated considerably throughout postnatal development in the total lipids as well as in the two major phosphatides. Lecithin biosynthesis has been studied by two pathways during development of rabbit liver from −6 days to +110 days. The two pathways of lecithin biosynthesis were evaluated by assaying the activities of the liver enzymes choline phosphotransferase and phosphatidylmethyltransferase at different time intervals during development. The greater enzymatic activity was observed in the cholinephosphotransferase during development.     

The lipid composition of wheat flours varying widely in bread-making potentialities

Lipids were extracted with petroleum ether (average 0.93%) and with water-saturated n-butanol (average 1.20%) from flours milled from composite 8 hard red winter, 5 hard red spring, and each from red soft, durum, and club wheat varieties. The butanol-extracted lipids were fractionated into nonpolar and polar lipids by silicic acid column chromatography, and the two major fractions were subfractionated by thin-layer chromatography. The extracted, washed, lipids contained about 52% nonpolar, and 48% polar lipids. Flours milled from durum wheat contained substantially less polar lipids, than flours milled from hard red winter or hard red spring wheats. The triglycerides constituted about 50% of the nonpolar lipids. Among the polar lipids, digalactosyl glyceride was the major component (about 40%); an unidentified compound, and a mixture of monogalactosyl glyceride with phosphatidic acid were about 20% each; and phosphatidyl ethanol amine, phosphatidyl choline and phosphatidyl serine comprised about 4, 7, and 4.5% of the polar lipids, respectively. Contribution No. 514. Kansas Agricultural Experiment Station, Manhattan. A report of work done in part under contract with the US Department of Agriculture and authorized by the Research and Marketing Act 1946. This contract was supervised by the W. Res. and Dev. Div., ARS, USDA.     

Polar lipids ofMacrophomina phaseolina

Macrophomina phaseolina was grown on a defined medium at three different carbon/nitrogen ratios. The lipids of the mycelia and the sclerotia were extracted; fractionated into polarity groups; and separated by thin layer, column, and gas liquid chromatographies. Sclerotia contained higher levels of neutral lipids and lower amounts of polar lipids than mycelia. The neutral lipid content of sclerotia increased, up to 77% of total lipids, and phospholipids decreased as carbon/nitrogen ratio increased from 10 to 320. The glycolipid content was not altered significantly by changes in carbon/nitrogen ratios. Although cardiolipin could not be detected in sclerotial polar lipids, both sclerotia and mycelia contained similar phospholipid profiles with major quantitative differences. Phosphatidic acid and phosphatidyl glycerol were major components of sclerotia, whereas phosphatidyl ethanolamine and phosphatidyl inositol were the major phosphatides of mycelia. Phosphatidyl choline was present in both mycelia and sclerotia. The fatty acid distribution did not show any particular pattern of saturation or unsaturation due to differences in carbon/nitrogen ratio. However, mycelial lipids tended to contain C24∶1, C18∶3, and C22∶1 as major fatty acids, whereas the major fatty acids in sclerotial lipids were C18∶2, C18∶1, C22∶1, C20∶0, and C16∶1. Saturated fatty acids were present in lesser concentrations.     

Lipids of maturing grain of corn (Zea mays L.): I. Changes in lipid classes and fatty acid composition

The total lipids of the grain from three strains of corn were compared throughout the growing season. The Illinois High Oil stock and the two inbreds, H51 and K6, represented high, intermediate and low oil-producing lines. In all three strains lipid synthesis was most active between 15 and 45 days after pollination. The lipids were extracted from the grain with a mixture of chloroform, methanol and water and were separated into classes by silicic acid and thin layer chromatography. Triglycerides constituted 10–17% of the total lipids at 10 days after pollination and increased to 75–92% at 75 days. Polar lipids at 10 days represented 70–72% and at 75 days 4–21%. Fatty acid compositions of the triglycerides and polar lipids changed as the grain matured, but the fatty acids of the polar lipids were more saturated than those of the triglycerides throughout the sampling periods. The major polar lipids were digalactosyl diglyceride, monogalactosyl diglyceride, phosphatidyl choline, phosphatidyl inositol and phosphatidyl ethanolamine. Presented in part at the AOCS-AACC Joint Meeting, Washington, D.C., April 1968.     

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 lipid components of white potato tubers (Solanum tuberosum)

Four Canadian varieties of potatoes were examined for their lipid composition. Lipids, extracted with chloroformmethanol, were shown by TLC and column chromatography to consist of 16.5% neutral lipids, 45.5% phospholipids and 38.1% glycolipids. Among the phospholipids and glycolipids, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, the galactolipids and the sterol glucosides were the major lipids. The predominant fatty acids were palmitic (19.5%), linoleic (44.8%) and linolenic (30.4%, in Kennebec). Analyses of the fatty acids of stored potatoes showed a marked decrease in linoleic acid and an increase in linolenic acid, in the Irish Cobbler and Sebago potatoes. β-sitosterol comprised 85.0% of total sterols. Nearly half of the carotenoids was lutein (xanthophyll), the others being α-carotene, β-carotene, an unidentified pigment and lutein epoxide. Contribution No. 101 of the Food Research Institute, Canada Department of Agriculture.     

Total,polar and neutral lipids ofRhizopus arrhizus fischer

The changes in total lipid content, neutral and polar lipids, total fatty acids, and free fatty acids were investigated over a 4 day period in the zygomycete,Rhizopus arrhizus Fischer. The highest concentration of lipids occurred at the 72 hr period. The degree of unsaturation in the total fatty acid fraction increased during the growth period, whereas the degree of unsaturation decreased in the free fatty acid fraction during the same time period. The ratios of neutral to polar lipids over the 4 day period were: 0.75, 0.22, 1.94 and 0.94. The major components of polar lipids were phosphatidyl ethanolamine, lecithin, lysolecithin, and fatty acids. The fatty acids in the mono- and diglycerides were predominately saturated (67–96%). The fatty acids in the triglycerides shifted from a predominately unaturated (69%, 24 hr) to a more saturated pattern (62%, 96 hr).     

Upper critical solution temperature—type cononsolvency of poly(N,N-dimethylacrylamide) in water—organic solvent mixtures

The behaviour of linear poly(N,N-dimethylacrylamide) (PDMAM) chains was studied by turbidimetry and viscometry in mixtures of water with the polar organic solvents methanol, dioxane and acetone. The swelling-deswelling behaviour of PDMAM gels in the same solvent mixtures was also investigated. Contrary to the behaviour in water-methanol mixtures, in water-dioxane and water-acetone mixtures a significant shrinkage of polymer chains and deswelling of polymer gels, followed by phase separation, was observed for high organic solvent fractions. Cononsolvency phenomena were found to be temperature-dependent, as demixing occurred upon decreasing temperature. This upper critical solution temperature (UCST) phase separation behaviour in mixed solvents was studied by turbidimetry and compared to the well-known lower critical solution temperature (LCST) behaviour of poly(N-isopropylacrylamide) (PNIPAM) in similar solvents mixtures.