Lipid changes in maturing oil-bearing plants

Seeds ofCrambe abyssinica C.D. 6619 and theBrassica napus varieties Golden and Zero-erucic were collected at different stages of maturity and the free lipid extracted with hexane. The lipid thus obtained was separated into lipid classes by silicic acid column chromatography. The lipid classes were further examined by thin-layer chromatography and the component fatty acids and sterols by gas-liquid chromatography. The relative amounts of the lipid classes in crambe and both rape varieties varied as the seed matured and a period of great change occurred about 10 days after fertilization. The greatest change was in triglycerides and phospholipids plus glycolipids. Free fatty acids, present in immature seeds, has almost disappeared at maturity. The lipid classes of crambe and both types of rape were in similar proportion at maturity. Differences in phospholipid and glycolipid composition were found between crambe and rape and between immature and mature rape. The fatty acid composition differred between lipid classes and changed with maturity. Changes in 18-carbon acids of Zero-erucic rape were concurrent with the development of erucic and eicosenoic acids in Golden rape. Contribution No. 36 of the Food Research Institute. Presented at the AOCS Meeting, Cincinnati, October 1965.     

The effect of temperature on the fatty acid and phospholipid composition ofCephalosporium falciforme andCephalosporium kiliense

The effect of temperature on the lipid composition ofCephalosporium falciforme andCephalosporium kiliense, causative agents of maduromycosis, was investigated. The fungi were grown at 28.5 C and 37 C in a chemically defined medium. The lipids were solvent extracted, purified on Sephadex, and separated into their component classes by silicic acid column chromatography. Five lipid classes were found: (a) sterol esters, (b) triacylglycerides, (c) free fatty acids, (d) sterols, and (e) phospholipids. Fatty acids were analyzed by gas liquid chromatography and phospholipids by thin layer chromatography. Temperature induced changes of varying degrees occurred in both the fatty acid phospholipid fractions of each organism.     

Lipid changes in maturing oil-bearing plants

Flax and safflower blossoms were tagged at the onset of fertilization. At intervals of approximately 10 days, samples of leaves, seeds, bolls, and bracts were collected, frozen in liquid nitrogen, and kept in deep-freeze until analyzed. A comparison of the behavior during maturation of the seeds of Raja and Rocket flax and Indian safflower showed the following points of similarity and difference. Flax and safflower seed had similar patterns for changes in total extractable matter and true free and true bound lipid, dry matter, iodine absorbed by the seed oil, and isotopic carbon incorporation. The phosphorus and nitrogen contents of the free and bound lipid of flax and safflower seed had different patterns of variation. At a given stage of development, fresh flax seed tissue insafflower tissue. The effect of adverse growing conditions was reflected more clearly by the behavior of safflower than by that of flax. Raja and Rocket flax developed similarly and differed only in regard to response to climatic conditions. Presented at the Fall Meeting, American Oil Chemists’ Society, Los Angeles, Calif., September 28–30, 1959. Contribution No. 43 from the genetics and Plant Breeding Research Institute, Research Branch, Canada Department of Agriculture, Ottawa.     

The endosperm lipids of three Canadian wheats

Flour samples were prepared from intact and degermed kernels of Hard Red Spring, Soft White Spring, and Amber Durum wheats. The “free” (hexane soluble) and “bound” (hexane resistant, water-saturatedn-butanol extractable) lipids were extracted from the six flours and separated quantitatively by silicic acid column chromatography. Thin-layer chromatography (TLC) was used to monitor the column and to resolve the lipid classes into components. Gas-liquid chromatography (GLC) was used to obtain the fatty acid composition of the triglyceride, sterol ester, and phospholipid fractions, and also to determine the nature of the sterol components of the unesterified sterol and sterol ester classes. Similar patterns of lipid classes were shown by all three varieties; the differences were in the degree of dominance. In fatty acid composition some varietal differences were found but the greatest difference was between lipid classes. Contribution No. 5 of the Food Research Institute. Presented at the AOCS Meeting, Toronto, 1962.     

Lipids in wheat from various classes and varieties

Lipids were extracted with petroleum ether and with water-saturated n-butanol from 8 hard red winter, 5 hard red spring, and one each from soft red, durum, and club wheat varieties from 2 harvests. 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. Durum wheats contained the highest lipid contents, and the highest concentration of nonpolar lipids. The breadmaking wheat varieties had a lipid content which was consistent for the 2 years examined. The total and nonpolar lipid contents of hard red spring wheats were higher than of hard red winter wheats. The polar lipid contents of wheats from the two classes were essentially equal. Total lipid contents were substantially higher in wheats than in flours milled from the wheats. Nonpolar lipids constituted about one-half of the flour lipids and two-thirds of the wheat lipids. Concentrations of triglycerides were higher in wheat than in flour nonpolar lipids. Glycolipids were present in comparable concentrations in wheat and in flour polar lipids; concentration in polar lipids of phosphatidyl choline was higher and of other phospholipids was lower in wheat than in flour polar lipids. No. 547, Kansas Agrieultural Experiment Station, Manhattan. Done in part under contract with the U.S. Department of Agriculture and authorized by the Research and Marketing Act of 1946. Supervised by the Western Utilization Research and Development Div., ARS.     

Lipid changes in maturing oil-bearing plants. II. Changes in fatty acid composition of flax and safflower seed oils

Changes in the fatty acids composition of the oil in flax and safflower seed that occur during the seed-ripening period have been measured. Concentrations of lipid or of specific fatty acid, expressed on a weight-per-seed basis, have been plotted as functions of days after fertilization and of percentage of oil development. Relations between these two independent variables have been established, and limitations to the unsefulness of the latter variable have been pointed out. Days after fertilization proved to be the more useful abscissa. Nonpolar solvents were used to remove free lipid from the tissue, and the total extractable matter was separated into true lipid and nonlipid components. With both flax and safflower, weight of true free lipid per seed and total unsaturation increased during the same period of growth. Nonlipid extractable matter was an inverse function of the extent of development. In developing flax seed, oleic, linoleic, and linolenic acids all increased continuously; oil in immature seed however was more saturated than oil in more mature seed. Nevertheless the ratio of linolenic acid to linoleic acid that characterizes linseed oil was established by the 20th day after fertilization during a normal growing season. In developing safflower seed, oleic acid concentration increased slowly during the first 30 days after fertilization and then appeared to level off in some cases as maturity was approached. Initially linoleic acid was present in almost the same amount as oleic acid, but by the 20th day after fertilization its concentration was three times that of oleic acid. This ratio of linoleic to oleic acid tended to increase steadily during the latter part of seed development.     

Lipids of seven cereal grains

Grain samples of representative varieties of barley, corn, oats, rye, sorghum, triticale, and wheat grown commercially in the north central US were analyzed. Chemical constituents of the varieties studied are presented to provide an overview of their characteristics. Lipids of the milled grain samples were solvent extracted, classified by silicic acid column chromatography, and separated by thin layer chromatography. Fatty acid composition of the total lipid was determined by gas liquid chromatography and the fatty acid content was determined by saponification and extraction. Total lipid content of the grains ranged from 2.3% for ‘Polk’ wheat to 6.6% for ‘Chief’ oats. Lipid composition varied considerably. The row crops, corn and sorghum, have a high neutral lipid and low glycolipid content. The small grain varieties have a more balanced distribution among neutral lipids, glycolipids, and phospholipids. Fatty acid composition of the total lipid was similar for all grains. Minor qualitative differences were noted among the lipid classes of the 7 cereals.     

Lipid composition of oats (Avena sativa L.)

Compositions of lipids extracted from a sample of Hinoat oat by seven solvent systems and that extracted with chloroform/methanol (2:1 v/v) from six selected cultivars representing high and low lipid contents are reported. Lipid components (steryl esters, triglycerides, partial glycerides, free fatty acids, glycolipids and phospholipids) were separated by silicic acid column chromatography and thin layer chromatography and quantitated by GLC analysis of fatty acids and phosphorus determinations. Twelve oat cultivars were examined for the fatty acid composition of lipid extracted with n-hexane. Lipids extracted from Hinoat by different solvent systems ranged from 5.6 to 8.8%. Quantitative distribution of lipid components extracted with chloroform/methanol from six cultivars containing 4.6 to 11.6% lipid showed a significant correlation (γ=0.99) between the total lipid and the neutral lipid content. Phospholipid content was similar in all cultivars, but glycolipids showed a two-fold increase in high lipid oats. Triglycerides contained less palmitic and more oleic acid than the glycolipids or phospholipids. Nine glycolipids and 11 phospholipids have been identified, and the polar lipid composition of Hinoat oat is presented.     

Quantitative analysis of brain and spinach leaf lipids employing silicic acid column chromatography and acetone for elution of glycolipids

Quantitative elution of acidic and neutral glycolipids of brain and spinach leaves from silicic acid columns with acetone was demonstrated. Cerebrosides and sulfatides of brain and sulfolipid and glycosyl diglycerides of spinach leaves were eluted quantitatively with acetone while prospholipids remained on the column. The observations provide the basis for an analytical procedure employing column and quantitative thin-layer chromatography (TLC). Sephadex column chromatography is utilized for separation of lipids from nonlipids; silicic acid column chromatography for separation into neutral lipid, glycolipid and phospholipid fractions; and quantitative TLC for analysis of lipid classes of each column fraction.     

Lipids in sea water

Acidified and filtered sea water samples which were extracted with petroleum ether and ethyl acetate have been shown to contain a variety of lipid compounds in trace amounts. Concentrations of these solvent-soluble substances ranged from 0.5 to 6.0 mg/liter, the lower concentrations being found in offshore waters. The solvent extracts of the sea water were separated into eight lipid classes by column chromatography on silicic acid. The fractions eluted with solvents of increasing polarity were characterized by thin-layer chromatography, infrared and ultraviolet absorption and gas chromatography. These techniques revealed a complex mixture of alkanes, alkenes, fatty acids, steroids, phospholipids and many as yet unidentified components. Twenty to thirty alkanes were present as indicated by gas chromatography. No aromatic hydrocarbons were detected. Chromatography of the methyl esters of the fatty acids indicated the presence of acids with chain lengths varying from 14 to 22 carbons, both saturated and unsaturated. In many samples the unsaturated fatty acids containing 18 to 22 carbons predominated. The lipid components varied somewhat in composition as well as concentration from location to location and with season and depth.     

Dietary modulation of phospholipid fatty acid composition and lipoxygenase products in mouse lung homogenates

This study investigated the potential of dietary fats to modulate the arachidonic acid content of mouse lung phospholipids and the formation of lipoxygenase products from arachidonic and eicosapentaenoic acids. Prior to breeding, female mice were fed for five months diets with 10 wt% of either olive oil, safflower oil, fish oil, or linseed oil. The same diets were fed to the females during gestation and to the pups from day 18 to day 42 postpartum. On day 42, the phospholipids were extracted from fresh lung tissue and separated into classes [phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylinositol (Pl)] by thin-layer chromatography. Methyl esters of phospholipid fatty acids and unesterified fatty acids were analyzed by gas chromatography. At comparable dietary n-3/n-6 ratios, arachidonic acid was reduced 85 and 75% in lungs from mice fed linseed oil and fish oil, respectively, compared to lungs of safflower oil-fed mice. Dietary fats affected the proportion of arachidonic acid in phospholipids in the order: PE>PS>PS>Pl. Following incubation of homogenized lung tissue, the total amount of 12-lipoxygenase products was lowest in lungs from mice fed olive oil, and 12-hydroxyeicosatetraenoic acid was lowest in incubated lungs from mice fed linseed oil. Comparison of the amounts of lipoxygenase substrate fatty acids in the individual phospholipids with the lipoxygenase products suggested that the major substrate pool for the 12-lipoxygenase pathway in mouse lung homogenates was PC.     

Lecithins and lysolecithins of wheat flour

Lecithin and lysolecithin from the bound lipid of Thatcher wheat endosperm were separated and purified by column and thin-layer silicic acid chromatography. Lecithin was hydrolyzed with phospholipaseA (Crotalus adamanteus) and the products isolated and purified by silicic acid chromatography. The fatty acid composition of the original lecithin and lysolecithin and of the hydrolysis products was determined by gas-liquid chromatography. The fatty acids in the beta position of the lecithin were found to be almost entirely unsaturated, whereas those in the alpha position were saturated and unsaturated in nearly equal amounts. The differences between the fatty acid composition of the original lysolecithin and that of the lysolecithin obtained by hydrolysis of lecithin with phospholipaseA suggested the presence of both alpha and beta acyl species in the naturally occurring lysolecithin. Contribution No. 48, Food Research Institute, Research Branch, Canada Department of Agriculture, Ottawa, Canada.     

Fatty acid composition of garlic (Allium sativum Linnaeus) lipids

Chloroform-methanol extracted lipids of garlic (Allium sativum Linnaeus) amounted to 0.6% on a dry weight basis. Fractionation by silicic acid column chromatography showed that garlic lipids comprise 62.6% neutral lipids 14.0% glycolipids and 23.4% phospholipids. Fatty acid compositions of total lipids and component lipid fractions were determined.     

Lipid Profile and Fatty Acid Composition of Moth Bean (Vigna aconitefolia) Seeds

The seeds of moth bean (Vigna aconitefolia) were found to contain 4.5 % of lipid. Fractionation of this lipid by silicic acid column chromatography yielded 44.5 % neutral lipids (NL), 23.4 % glycolipids (GL) and 32.1 % phospholipids (PL). Fatty acid composition of the total lipid and lipid fractions showed that palmitic acid (37.3-54.7 %), stearic acid (7.8-8.0%) oleic acid (6.8-13.9 %) linoleic acid (23.1-35.6 %) and linolenic acid (3.0-10.0%) are the major fatty acids. The phospholipid fraction was found to be different from the rest in containing higher palmitic acid (54.7%) and lower unsaturated fatty acids.     

Analysis of complex lipid mixtures by thin-layer chromatography and complementary methods

Conclusion Further work has been done on the application of thin-layer adsorption chromatography to the fractionation of complex lipid mixtures into classes. New methods, the use of siliconized silicic acid plates and the application of thin-layer adsorption chromatography combined with the complementary techniques of gas-liquid chromatography and paper chromatography, are presented for the resolution of classes of lipids into their constituents. In contrast to such elaborate conventional techniques as column chromatography, analyses using the methods reported in this paper can be performed rapidly in large numbers on a routine basis. Supported in part by the U. S. Public Health Service National Institutes of Health Research Grant 5817, and the National Science Foundation.     

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.     

Lipids ofCrassostrea virginica. I. Preliminary investigations of aldehyde and phosphorous containing lipids in oyster tissue

Oyster tissue contained 2.4% lipid, 0.14 μmole aldehyde per milligram lipid and at least 10 μg phosphorous per milligram lipid. The neutral lipid represented 58%, the glycolipid 6%, and the polar lipid 36% of the total lipid recovered after silicic acid column chromatography. Aldehydes were found in all fractions, but the presence of plasmalogen was verified in only the neutral and polar lipid fractions. At least 68% of the plasmalogen in oyster lipid was found in the polar lipid fraction. At least 13% of the phosphorous in oyster lipids was present as phosphonolipid. The distribution of phosphate and phosphonate lipids was: diacyl phospholipid 38.1%, plasmalogen phospholipid 21.8%, sphingophosphonolipid 13.5%, glyceryl ether phospholipid 8.3%, sphingophospholipid 6.9%, plasmalogen phosphonolipid 6.4%, diacyl phosphonolipid 2.6%, and glyceryl ether phosphonolipid 2.4%. When the per cent of phosphorous as phosphonolipid within the plasmalogen and glyceryl ether classes was calculated, similar values were obtained. These results support the hypothesis that there is a product precursor relationship between these two classes of lipids. Some of the data taken from a thesis to be submitted to the Graduate School, University of Maryland, by Leslye Johnson in partial fulfillment of the requirements for the M.S. degree in biochemistry.     

Fatty acids of cows' milk. B. Composition by gas-liquid chromatography aided by other methods of fractionation

A fresh sample of cows' milk was converted directly to the methyl esters by methanolysis and the esters were fractionated into eleven distilled fractions and an undistilled portion. The latter, which contained the bulk of the polyunsaturated and long chain esters, was further fractionated by adsorption chromatography on a silicic acid column. Each fraction from the distillation and adsorption chromatography was analyzed by gas-liquid chromatography on columns containing polyester stationary phases of different polarity. Twenty-seven minor components, including some not previously reported, were present each in less than 0.1%. The fatty acid distribution of the major components fell within the range generally reported. Presented at the 53rd Meeting of the American Oil Chemists' Society, October 17–19, 1960, New York. Eastern Utilization Research and Development Division, Agricultural Research Service, United States Department of Agriculture.     

Studies of the phospholipids,glycolipids and sterols of wheat endosperm

The phospholipid-glycolipid mixture, isolated chromatographically from the “free” (hexane soluble) and “bound” (hexane insoluble, water-saturated butanol extractable) lipid of wheat endosperm, was fractionated by column and thinlayer silicic acid chromatography. The components were identified by hydrolysis followed by thin-layer or paper chromatography of the products. They included a sterol-containing glycolipid, hitherto unreported in wheat. The fatty acid and sterol compositions of the phospholipidglycolipid components were determined by gasliquid chromatography. Differences were found between varieties and between components. Contribution No. 14 of the Food Research Institute, Canada Dept. of Agriculture, Ottawa, Can. Presented at the AOCS Fall Meeting, Minneapolis, 1963.     

Chlorinated fatty acids in lipid class fractions from cardiac and skeletal muscle of Chinook salmon

Analysis of chlorinated fatty acids (CFA) in tissues can be difficult because of their low concentrations. This task becomes even more difficult when samples are from organisms living in remote locations with very little exposure to environmental contamination. Therefore, enrichment of CFA is necessary prior to analysis. In this study, CFA were enriched from fractionated lipid classes of cardiac and skeletal muscle of Chinook salmon (Oncorhynchus tshawytscha) to determine CFA distribution among lipid classes and tissue types and to demonstrate the sensitivity of this method to detect CFA at trace concentrations. The lipids extracted from cardiac and skeletal muscle of O. tshawytscha were separated into fractions containing TAG, FFA, and phospholipids. After transesterification, the FAME derivatives from each lipid class were analyzed by GC with a halogen-selective detector (XSD) to determine the concentrations of dichlorostearic acid and dichloropalmitic acid. Other chlorinated compounds detected byGC-XSD were analyzed by GC-MS. CFA were observed in all lipid classes in both cardiac and skeletal muscle tissues. However, the highest concentrations of CFA were found in the phospholipids of both tissue types, about 1–2 mg/g lipid. It was also shown that dichloropalmitic acid concentrated in cardiac phospholipids whereas dichlorostearic acid was found primarily in the phospholipids of skeletal tissue. CFA concentrations in TAG and FFA fractions were below 150 mg/g lipid. This study demonstrates a small-scale approach to the study of CFA at trace concentrations and their distribution among lipid classes.