Effect of the conditions of analysis and obtaining of the sample on the level of free fatty acids in the brain of the rat

Free fatty acids (FFA) have been determined in the rat brain by gas-liquid chromatography after isolation by thin-layer chromatography on silica gel. The brains were removed under three experimental conditions, 1) after freezing in situ with liquid nitrogen, 2) after immersion of the animal in liquid nitrogen, 3) after decapitation, the brain being frozen 3 minutes later. The total FFA level was found to be equal respectively to 20.1, 33.1 and 168 micrograms/g. In any case, the main fatty acids were palmitic, stearic and oleic acid but there were marked increases in arachidonic and docosahexaenoic acids following decapitation. A cause of error in the FFA determination originated in the use of commercial silica gel which contained significant amounts of fatty acids.     

Purification and spectroscopic properties of pyrene fatty acids

Pyrene fatty acids are routinely purified by silica based column chromatography and analyzed on thin-layer silica plates (H.-J. Galla et al., Chem. Phys. Lipids, 23 (1979) 239-251). Although pyrene decanoic acid runs as a single spot on thin-layer chromatography (TLC), gas-liquid chromatography (GC) of the methyl ester derivatives of a representative sample revealed four separate peaks with the major component only 92% of the total. High performance reverse phase liquid chromatography (HPLC) was used to purify pyrene decanoic acid and separate the contaminants. After two passes on a C18 reverse phase HPLC column, pyrene decanoic acid is 99.98% pure by GC analysis. Absorption, fluorescence, and NMR spectra were recorded for pyrene decanoic acid and the major impurities. The results indicate that one impurity is a C10 fatty acid with an altered aromatic moiety. Two other impurities are pyrene derivatives but their acyl chains probably are not decanoic acid.     

Isolation of a novel sphingoglycolipid containing glucuronic acid and 2-hydroxy fatty acid from Flavobacterium devorans ATCC 10829

A new acidic sphingoglycolipid has been isolated from a Gram-negative, glucose-non-fermentative (obligatory aerobic) bacterium, Flavobacterium devorans ATCC 10829, by thin-layer chromatography on silica gel after mild alkaline hydrolysis of the cellular lipids. Chemical degradation studies, thin-layer chromatographic behavior, IR and mass-spectrometric analysis of the original and reduced glycolipid with LiA1H4 revealed that the lipid contained glucuronic acid, long-chain bases, and fatty acids in a molar ratio of approximately 1:1:1. The major long-chain bases were identified by gas chromatography-mass spectrometry as dihydrosphingosine (d-18 :0) and longer homologues, while the N-acyl group was exclusively 2-hydroxy myristic acid. The most probable structure of this glycolipid appeared to be a ceramide glucuronic acid (N-acyl dihydrosphingosine 1-glucuronic acid).     

Phospholipids from the hepatopancreas of Indian horseshoe crab Carcinoscropius rotundicauda

Total phospholipids were extracted from the heart, hepatopancreas, and hemolymph of the Indian horseshoe crab Carcinoscorpius rotundicauda by the conventional method. Characteristic group reaction and 2-dimensional thin-layer chromatography on silica gel were used for identification of different phospholipids. The phospholipid profile obtained from hemolymph and 2 major organs are comparable and show phosphatidyl choline (PC) and phosphatidyl ethanolamine to be the major phospholipids. A phospholipid has been consistently detected migrating immediately below the PC in the thin-layer chromatogram of lipids extracted from the hepatopancreas. When mixed methyl esters of this slower moving PC are resolved on a silica gel plate ran in hexane ether:acetic acid 80:20:1, with appropriate controls, an additional spot is seen just below the normal methyl ester, indicating a difference between the fatty acid compositions of 2 PC (e.g., regular and slower). The slower mixed methyl esters were found to comprise mainly the 4 saturated fatty acids: lauric, myristic, palmitic, and stearic. The slow moving PC seems to consist mainly of molecular species with the above-mentioned saturated fatty acids at both Sn 1 and Sn 2 positions.     

A rapid and sensitive method for the determination of the lithogenic index of bile by thin-layer chromatography and densitometry using a dual-wavelength chromatoscanner

A rapid and sensitive method for quantitative determination of three bile lipid components, cholesterol, bile acids, and lecithin, is described. These components were separated by thin-layer chromatography on a silica gel plate, spots were visualized with a phosphomolybdate reagent, and their color intensities were estimated by direct densitometry using a dualwavelength chromatoscanner. The lithogenic index was estimated by the molar ratio of the three lipids. This method can be applied to the routine analysis of bile in patients with gallstones. It has been evaluated by comparison with the method using standard gas-liquid chromatography and spectrophotometry.     

A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.     

Studies on the Lipid Components of Endotoxin II. Chemical Analyses and Biological Properties of Neutral,Polar-I and Polar-II Subfractions

Lipid components obtained from Salmonella typhosa O-901 endotoxin by acid hydrolysis were separated into neutral, polar-I and polar-II lipid fractions by silica gel column chromatography. These lipids were further separated by silica gel column and/or thin-layer chromatography. The subfractions were analyzed by thin-layer chromatography, gas chromatography and infrared spectrophotometry. Seven subfractions obtained from the neutral lipid fraction contained lauric, myristic, palmitic, 3-OH-myristic acid, artificial products of 3-OH-myristic acid, or a small amount of two unidentified fatty acids. These fatty acids and glucosamine were commonly detected in six subfractions obtained from the polar-I lipid fraction. Fatty acids, glucosamine, and O-phosphorylethanolamine were detected in all of the 13 subfractions obtained from the polar-II lipid fraction. Chick embryo lethal activity, rabbit pyrogenicity and in vitro interferon inducing activity were found in three polar-I lipid subfractions and five polar-II lipid subfractions, but not in neutral lipids. The activities were highest in a polar-II lipid subfraction, which contained smaller amounts of O-phosphorylethanolamine and glucosamine than the other subfractions. However, no particular chemical constituent (s) related to the biological activities could be found. Prolonged acid hydrolysis of the polar-II lipids gave rise to neutral and polar-I lipids. Chemical and biological aspects of the lipid constituents of endotoxin are discussed.     

Phospholipids from the hepatopancreas of Indian horseshoe crab Carcinoscorpius rotundicauda

Total phospholipids were extracted from the heart, hepatopancreas, and hemolymph of the Indian horseshoe crab Carcinoscorpius rotundicauda by the conventional method. Characteristic group reaction and 2-dimensional thin-layer chromatography on silica gel were used for identification of different phospholipids. The phospholipid profile obtained from hemolymph and 2 major organs are comparable and show phosphatidyl choline (PC) and phosphatidyl ethanolamine to be the major phospholipids. A phospholipid has been consistently detected migrating immediately below the PC in the thin-layer chromatogram of lipids extracted from the hepatopancreas. When mixed methyl esters of this slower moving PC are resolved on a silica gel plate ran in hexane ether:acetic acid 80:20:1, with appropriate controls, an additional spot is seen just below the normal methyl ester, indicating a difference between the fatty acid compositions of 2 PC (e.g., regular and slower). The slower mixed methyl esters were found to comprise mainly the 4 saturated fatty acids: lauric, myristic, palmitic, and stearic. The slow moving PC seems to consist mainly of molecular species with the above-mentioned saturated fatty acids at both Sn 1 and Sn 2 positions.     

Determination of content and specific activity of amino acids in central nervous system tissue utilizing tritium and carbon-14 dual labeling

A method is reported for measuring content and specific activity of amino acids in central nervous system tissue of the rat. Tritiated dinitrofluorobenzene is used to produce dinitro-[³H]phenyl amino acid derivatives which are separated by two-dimensional thin-layer chromatography on silica gel. Both content and specific activity can be determined simultaneously by pulse-labeling with an appropriate ¹⁴C precursor and measuring radioactivity in the individual spots of dinitrophenyl amino acids scraped from the chromatogram. This method is sensitive to at least 100 pmol and is shown to be more rapid than previously described procedures.     

Synthesis of lipids from acetate by human preputial and abdominal skin in vitro

Lipogenesis in vitro from acetate-1-(14)C was studied in human preputial skin and abdominal skin. Radioactive lipids were separated by column chromatography on Florisil and by thin-layer chromatography on silica gel. Radioactivity was incorporated chiefly into the triglyceride, sterol, and polar lipid fractions, while lesser amounts of (14)C were found in the hydrocarbon, wax, diglyceride, monoglyceride, and fatty acid fractions; labeling of steryl esters was minimal. On thin-layer chromatography, the radioactive polar lipids had mobilities similar to lysolecithin, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidic acid. The radioactive fatty acids of the different lipid fractions were separated by gas-liquid chromatography. The major (14)C-labeled acids were 16:0 and 18:0. Radioactivity was also detected in acids 14:0, 15:0, 16:1, 18:1, 18:2, 20:0, 20:1, 22:0, 24:0, 24:1, and 26:0. No radioactivity could be detected in arachidonic acid, although this fatty acid comprises 9% of the chromatographed fatty acids. The pattern of incorporated (14)C was different from the percentage mass composition of the fatty acids. Skin is therefore active in the biosynthesis of a wider variety of lipids than previously demonstrated.     

Separation of brain phosphatidylserines according to degree of unsaturation by thin-layer chromatography

Phosphatidylserines (PS) have been prepared from bovine brain using DEAE column chromatography. A method involving AgNO₃-impragnated silica gel H thin-layer chromatography is presented for separating intact PS according to the degree of unsaturation of their fatty acids. A detailed analysis was made of the fatty acid composition of the various fractions using gas chromatography. Some data are presented on the composition of molecular species of PS in bovine brain. The two main molecular species found in cerebral cortex are tentatively assigned the structures of 1-octadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphorylserine.     

Free ceramide, sphingomyelin, and glucosylceramide of isolated rat intestinal cells.

Free ceramide, glucosylceramide, and sphingomyelin were isolated from mature cells of adult rat small intestine. Free ceramide and ceramide cleaved from sphingomyelin by enzymatic hydrolysis were fractionated by thin-layer chromatography on borate-impregnated silica gel plates. Sphingoid bases were characterized by gas-liquid chromatography of aldehydes formed upon periodate oxidation. Fatty acids were quantified as methyl esters. Ceramide structures were confirmed by direct-inlet mass spectrometry. Free ceramide was found to contain two major long-chain bases in nearly equal quantity: sphingosine, mainly linked to palmitic acid, and 4D-hydroxysphinganine associated with C20 to C24 fatty acids, 22% being hydroxylated. Sphinganine occurred as a minor component linked to nonhydroxy fatty acids. Sphingomyelin contained the three long-chain bases and 63% of its ceramide was N-palmitoyl-sphingosine. Mass spectrometry of glucosylceramide confirmed 4D-hydroxyshingamine as the major sphingoid base associated preferentially with longer chain hydroxy fatty acids.     

Isolation and characterization of gangliosides from pig lymphocytes

Two major gangliosides from pig spleen lymphocytes, accounting for 57% of the total lipid-bound sialic acids, were isolated and purified to homogeneity by column chromatography on DEAE-Sephadex and silica gel. They were identified as GM3 (II3Neu5GcLacCer), and GD3 (II3(Neu5Gc)2LacCer), by thin-layer chromatography in comparison with standards and by analysis of the constituent sugars. The major fatty acids of these gangliosides were stearic acid and myristic acid, respectively. In addition to these gangliosides, GD2 and bands comigrating on thin-layer chromatography with authentic GM2, GM1, GD1a and GD1b were found. These compounds also occur in pig peripheral blood lymphocytes, where, however, GD3 represents about 70% of the total lipid-bound sialic acid.     

Separation of individual sulfated bile acid conjugates as calcium complexes using reversed-phase partition thin-layer chromatography.

A method for separating individual monosulfated primary bile acid conjugates by reversed-phase partition thin-layer chromatography on octadecyl-bonded silica gel is described. The solvent system is acetonitrile containing calcium, probably as calcium carbamate. Excellent resolution of the 3- and 7-monosulfated glycine conjugates, as well as 3- and 7-monosulfated taurine conjugates of cholic and chenodeoxycholic acids is reported. A convenient class separation of sulfated from nonsulfated primary bile acid conjugates by adsorption thin-layer chromatography on low-polarity silica gel is also described.     

我国健康成人红细胞膜脂类的脂酸组成

 本文采用双向簿层层析分离红细胞膜脂类,继以毛细管气相色谱法分析其脂酸含量,检测了15名我国健康成人红细胞膜脂类的脂酸摩尔百分组成。结果表明:各脂类中,脂酸的类别基本相同,但其含量组成相差甚远。如磷脂酰乙醇胺(PE)富含C_(20:4);磷脂酰胆碱(PC)富含C_(18:2);神经鞘磷脂(SM)主要含C_(16:0);磷脂酰丝氨酸(PS)主要含C_(18:0);而以红细胞糖苷脂(GL)中脂酸含量最少。膜总脂中饱和脂酸与不饱和脂酸的含量大致相等,胆碱磷脂(PC+SM)的脂酸饱和度则明显高于氨基磷脂(PE+PS)。     

Dry-column chromatographic isolation of fatty acid esters of phorbol from croton oil

The family of mixed fatty acid esters of 4,9,12,13,20-pentahydroxy-tiglia-1,6-dien-3-one (phorbol) has been isolated from croton oil by column chromatography on silica gel and preparative dry-column chromatography on silica gel HF/254 packed in a cellophane membrane. The tumorpromoting agents are obtained rapidly and with minimum difficulty.     

Rapid profiling of bacterial quinones by two-dimensional thin-layer chromatography

Bacterial quinones were analysed by two-dimensional thin-layer chromatography with ready-made, multi-phase silica gel plates which allowed good separation of complicated quinone mixtures. A combination of this method and silver-ion-modified thin-layer chromatography made it possible to identify partially hydrogenated quinones.     

Resolution and chromatographic configuration analysis of 2-hydroxy fatty acids

2-Hydroxyoctadecanoic acid was resolved into D and L isomers as salts of 1-phenylethylamine enantiomers The diastereomers of phenylethylamides of 2-hydroxy fatty acids and the corresponding derivatives with protected hydroxy group (acetyl, methyl, trifluoro-acetyl, trimethylsilyl) are well separated by thin-layer or gas-liquid chromatography. This allows a simple microanalysis of configuration and optical purity of 2-hydroxy fatty acids. With this method 2-hydroxy fatty acids from sphingomyelin of the honey-bee were shown to belong exclusively to the D series.     

Assessment of fatty acids in cardiac tissue as 9-anthryldiazomethane esters by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the rapid assessment of fatty acids in cardiac tissue is described. A level of 50.4 ± 14.9 nmol fatty acids per g wet weight of rat myocardial tissue could be monitored. The content of the individual fatty acids C_14:0, C_16:0, C_16:1, C_18:0, C_18:1, C_18:2 and C_20:4 amounted to 1.9, 13.5, 0.6, 14.4, 6.1, 6.5 and 7.2 nmol/g wet weight, respectively. A comparison of this method with a well established gas chromatographic technique yielded good agreement. In contrast with time-consuming gas chromatographic techniques, there is no need to isolate (unesterified) fatty acids from the other lipid classes with column chromatography or thin-layer chromatography, because the derivatizing reagent 9-anthryldiazomethane reacts highly specifically with fatty acids.     

Effects of Phosphatidylcholine or Ergosteryl Oleate on Physiological Properties of Saccharomyces sake

Chromatographically homogeneous phosphatidylcholine identified by thin-layer chromatography on silica gel G was isolated from mycelia of Aspergillus oryzae or egg yolk by a combination of alumina and silicic acid column chromatography. Although the 2-position in both Asp. oryzae- and egg yolk-phosphatidylcholine was occupied nearly exclusively by unsaturated fatty acids, significant proportions of unsaturated fatty acids were also found in the 1 -position of Asp. oryzae-phosphatidylcholine. In nitrogen gas-sparging anaerobic culture of Saccharomvces sake Kyokai No. 7, supplementing the basal synthetic medium with phosphatidylcholine enhanced the yeast growth and fermentative activity, whereas adding ergosteryl oleate enhanced alcohol-endurability. Supplementation with both phosphatidylcholine and ergosteryl oleate promoted the yeast growth, fermentative activity and alcohol-endurability of cells.