Gradient-thickness thin-layer chromatography for the isolation and analysis of trace amounts of free fatty acids in large lipid samples

A thin-layer chromatographic method for quantitative isolation of free fatty acids is described. This method appears to be more satisfactory than existing methods in offering the combination of advantages of specificity, simplicity, rapidity, reproducibility, accuracy, high sensitivity, and applicability as a preparative technique. The method involves chromatography on a thin-layer plate on which the layer of Silica Gel G decreases linearly in thickness from 1000 micro at the base to 125 micro at the upper end. This gradient-thickness design allows the separation and densitometric quantitation of very small traces of free fatty acids from relatively large and complex lipid samples in a single chromatographic step. The method has been shown to be applicable directly to the crude total lipid extracts of several mammalian tissues. It appears to generate little if any artifactual free fatty acids from the breakdown of complex lipids, in contrast to the undesirable behavior of silicic acid columns in this respect. Gradient-thickness thin-layer chromatography promises to be useful for the quantitative isolation of trace amounts not only of other types of lipids but also of classes of compounds other than lipids.     

Isolation and characterization of fractions of Mycoplasma pneumoniae. II. Antigenicity and immunogenicity

Sobeslavsky, O. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), B. Prescott, W. D. James, and R. M. Chanock. Isolation and characterization of fractions of Mycoplasma pneumoniae. II. Antigenicity and immunogenicity. J. Bacteriol. 91:2126-2138. 1966.-Chemical and chromatographic fractions of disrupted Mycoplasma pneumoniae organisms were examined for serological and immunogenic activity. Complement-fixing activity was associated with lipid components, whereas precipitin activity was chiefly associated with polysaccharide components. When chemically extracted lipids were separated by thin-layer silica gel chromatography, only three of the nine fractions exhibited complement-fixing activity. Although lipids were highly active serologically, they were only weakly immunogenic. However, lipids combined with protein in lipoprotein complexes were highly immunogenic, stimulating high levels of complement-fixing, indirect-hemagglutinating, and growth-inhibiting antibodies. The specificity of these antibodies was directed chiefly against the serologically active lipid constituents of the organism. It was suggested that these serologically active lipids are present at the sites on the limiting membrane of M. pneumoniae at which antibody acts to inhibit growth of the organism. Only protein fractions adsorbed to tanned erythrocytes. The main function of protein in the indirect-hemagglutination reaction appeared to be that of serving as a carrier for the serologically active lipids.     

Occurrence of an endogenous inhibitor of platelet-activating factor in rat liver

Hepatocytes obtained from livers derived from fed rats perfused with a collagenase-containing mixture were found to contain significant levels of platelet-activating factor activity as isolated by Silica Gel G thin layer chromatography. However, when soybean trypsin inhibitor was included in the collagenase-containing perfusion medium for hepatocyte preparation, platelet-activating factor activity could not be detected on Silica Gel G chromatograms. Examination of the lipids extracted from freeze clamped perfused rat livers revealed low, but detectable, levels of platelet-activating factor. Further investigation of these observations indicated that a lipid-like inhibitor was present in freeze-clamped perfused livers as well as in hepatocytes isolated in the presence of soybean trypsin inhibitor. In each instance platelet-activating factor and this newly discovered inhibitor, which comigrated at the same RF value on Silica Gel G thin layer chromatography plates, could be separated by further chromatography on high performance thin layer plates. The present study shows that platelet-activating factor is present in unstimulated liver and that its detection is masked by an endogenous lipid-like inhibitor.     

Preparative isolation of monogalactosyl and digalactosyl diglycerides by thin-layer chromatography

Monogalactosyl and digalactosyl diglycerides were separated from leaf and Chlorella vulgaris lipid extracts by thin-layer chromatography with Silica Gel G as the stationary phase and acetone-acetic acid-water as the mobile phase. Phospholipids are completely removed and with two-dimensional development can themselves be fractionated.     

Origin of some derivatives of retinoic acid found in rat bile

After the intraportal injection of retinoic acid-15-(14)C into rats, all-trans methyl retinoate, a cis isomer of methyl retinoate, retinoyl beta-glucurono-gamma-lactone, retinoic acid, and retinoyl beta-glucuronide were isolated from methanol extracts of rat bile by chromatography on anion-exchange resin and silicic acid columns and characterized on thin-layer plates of Silica Gel G. On the other hand, when bile was extracted with n-butanol or analyzed directly by thin-layer chromatography, only retinoyl beta-glucuronide and a very small amount of retinoic acid could be detected. Butanol extracts of the liver and the intestine, however, still contained a small radioactive nonpolar fraction. When retinoyl beta-glucuronide was incubated with an anion-exchange resin in the presence of methanol, several nonpolar products appeared. Apparently the methyl retinoate, retinoyl beta-glucurono-gamma-lactone, and most of the retinoic acid previously found in bile after retinoic acid administration are produced from retinoyl beta-glucuronide during the isolation procedure.     

Chromatographic separation of plasmalogenic, alkyl-acyl, and diacyl forms of ethanolamine glycerophosphatides

The plasmalogenic, alkyl-acyl, and diacyl forms of ethanolamine glycerophosphatides were completely separated from each other as methylated dinitrophenyl derivatives by thin-layer chromatography on Silica Gel G. The relatively high resolving power needed was obtained by multiple unidimensional development with solvents that give very low mobility to the lipids. Under these conditions the plasmalogens moved fastest, the alkyl-acyl lipids were intermediate, and the diacyl lipids were the slowest. The presence of all these forms of lipids in the ethanolamine phosphatides of hen's eggs, ox brain, and human blood plasma could be directly demonstrated with the new method.     

Isolation and Properties of Carbohydrate Rich Fraction of Wheat Gluten

Two carbohydrate rich fractions A and B were isolated from wheat gluten. Fraction B contained more lipid than fraction A. Lipid portion of fraction B consisted mainly of glycolipid and was fractionated into five fractions by thin-layer chromatography. The two main fractions were extracted and determined to be galactolipid and glucolipid, respectively, by the analyses of fatty acid and sugar components by gas chromatography. Defatted fraction A was assumed to consist of glycoprotein. After complete pronase digestion of defatted fraction A, the remaining glycopeptide moiety was isolated by column chromatography on DEAE-cellulose followed by gel filtration through Sephadex G–25. The amino acid and sugar components of the glycopeptide were investigated.     

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.     

Four-directional-development thin-layer chromatography of lipids using trimethyl borate

Solvent mixtures containing trimethyl borate virtually eliminated the pronounced interconversion of 1,2- and 1,3-dipalmitins during their resolution by thin-layer chromatography on Silica Gel G. With trimethyl borate, an average of 1-2% of 1,2-dipalmitin was converted to 1,3-dipalmitin. A four-directional-development TLC procedure incorporating trimethyl borate resolves cholesteryl glucoside, ceramides, monogalactosyl diglyceride, 1- and 2-monopalmitin, palmitic acid, cholesterol, 1,2- and 1,3-dipalmitin, tripalmitin, methyl palmitate, cholesteryl palmitate, beta-carotene and some of its degradation products, squalene, and tetracosane. Digalactosyl diglyceride, phosphatidic acid, phosphatidylglucose, cerebrosides, and other phospholipids remain near the origin. A mixture containing triolein, 1,2- and 1,3-diolein, 1- and 2- monoolein, oleic acid, and cholesterol was resolved in one dimension. A similar series of palmitic-containing neutral lipids was also resolvable in one dimension. These procedures were applied to the TLC of human sera lipids.     

Separation of gluco- and galactocerebrosides by means of borate thin-layer chromatography

Gluco- and galactocerebrosides can be separated by thin-layer chromatography on Silica Gel G prepared with sodium borate solution instead of water. The most successful developing system was chloroform-methanol-water-15 M NH(4)OH 280:70:6:1.     

New sialic acid-containing sulfolipid: "ungulic acid"

Human epidermis, hair, nails, and kidney as well as bovine and horses' hooves were found to contain a lipid fraction, which on thin-layer chromatography migrated slightly ahead of the cerebroside sulfate esters and gave the color reaction specific for sialic acid. This fraction was isolated from horse hoof, in which it constituted nearly half of the total lipids. The purified fraction contained sulfur, but no phosphorus. The IR spectrum revealed the presence of a sulfate group, which was also determined by the benzidine method. Thin-layer and gas-liquid chromatography of the products of acid hydrolysis revealed the presence of sphingosine, galactose, galactosamine, and sialic acid. Fatty acid analysis showed that stearic acid was the major component, with minor amounts of palmitic and arachidic acids. The fraction isolated contained ceramide, sialic acid, galactose, galactosamine, and sulfate in equimolar amounts. We conclude that the new lipid is a ganglioside sulfate, which we have called "ungulic acid" because it was first separated and identified from a horse's hoof (Latin, ungula).     

Analysis of ear sebum of the Syrian hamster (Mesocricetus auratus) reveals pronounced sexual dimorphism

External ear of male and female Syrian hamsters (Mesocricetus auratus) were extracted with hexane and separated by class on thin-layer chromatography (TLC). Separated lipid classes were eluted and saponified, and non-saponifiable lipids further characterized by TLC, gas-liquid chromatography (GLC), UV and IR spectroscopy and functional group analyses. Many sex differences were observed, most notably the presence of sex-specific sterols of males and females. Mature animals were found to have greater quantities of ear sebum, but the characteristic qualitative lipid profiles of each sex were apparent in immature animals.     

Chromatographic behaviour of rat-liver monophosphoinositide

1. Chromatography of rat-liver lipids on a column of silicic acid or a mixture of silicic acid and Hyflo Super-Cel, with chloroform–methanol mixtures, gave monophosphoinositide-containing fractions which were invariably contaminated by the presence of nitrogen-containing phospholipids. The behaviour of the inositide was extremely sensitive to column loading and the results with different batches of silicic acid were not reproducible. 2. However, when chromatography on an alumina column was used, the solvent system chloroform–methanol–water (23:23:4, by vol.) completely eluted the neutral lipids, choline-containing phospholipids and phosphatidylethanolamine. An increase of the water content of the solvent to 14% (by vol.) then led to the elution of the monophosphoinositide component, now free from nitrogen-containing phospholipids, but still contaminated by the presence of a phospholipid, which from its properties was taken to be polyglycerophosphatide. 3. Most of the polyglycerophosphatide could be removed from a rat-liver lipid extract by silicic acid chromatography with chloroform–methanol (19:1, v/v). The other phospholipids were then eluted and applied to an alumina column, whereby a monophosphoinositide fraction of much greater purity was obtained. 4. Further purification of the monophosphoinositide was achieved by chromatography on a mixture of silicic acid and cellulose powder. The final product was virtually pure by thin-layer chromatography and gave the expected analysis for monophosphoinositide.     

The skin-reactive antigens of Mycoplasma pneumoniae.

Guinea pigs experimentally infected with Mycoplasma pneumoniae or immunized with the orgnaism in combination with Freund's complete adjuvant developed a delayed hypersensitive skin reaction following on intradermal injection of the M. pneumoniae antigen. The amount of protein necessary to produce the delayed skin reaction was as low as 0.01 mug. When the sonicated whole cells were extracted with aqueous acetone, the delayed skin reactivity was found mostly in the acetone insoluble (lipid-depleted) fraction. On the other hand, the lipid fraction which was isolated by a chloroform-methanol extraction of the acetone-soluble fraction and had a high titer of complement-fixing activity, exhibited little delayed skin reactivity. The lipid-depleted antigens as the whole cell antigens produced delayed skin reactivities in human patients.     

Lipids of Thermoplasma acidophilum

Cells of Thermoplasma acidophilum contain about 3% total lipid on a dry weight basis. Total lipid was found to contain 17.5% neutral lipid, 25.1% glycolipid, and 56.6% phospholipid by chromatography on silicic acid. The lipids contain almost no fatty acid ester groups but appear to have long-chain alkyl groups in ether linkages to glycerol. The phospholipid fraction includes a major component which represents about 80% of the lipid phosphorus and 46% of the total lipids. We believe this component to be a long-chain isopranol glycerol diether analogue of glycerolphosphoryl monoglycosyl diglyceride. The glycolipids appear to contain isopranol diether analogues. Several components of the complex, neutral lipid fraction have been identified as hydrocarbons, vitamin K(2)-7, and isopranol glycerol diether analogues. Sterols are present in the neutral lipids but do not appear to be synthesized by the organism.     

Partial characterization of lipid A of intraperiplasmically grown Bdellovibrio bacteriovorus.

The lipid A components of substrate cell origin incorporated by Bdellovibrio bacteriovorus during intraperiplasmic growth (D. R. Nelson and S. C. Rittenberg, J. Bacteriol. 147:860-868, 1981) were shown to be integrated into its lipopolysaccharide structure. Lipid A isolated from bdellovibrios grown on Escherichia coli was resolved into two fractions by thin-layer chromatography. Fraction 2 had the same Rf as the single lipid A fraction of axenicaly grown bdellovibrios, and both stained identically with aniline-diphenylamine reagent. Fraction 1 resembled, in Rf and staining reaction, the slower migrating of two lipid A fractions obtained from the E.coli used as the substrate cell. Both fractions 1 and 2 contained glucosamine, a substrate cell-derived compound. Greater than 65% of the fatty acids in fraction 1 were derived from the substrate cell, whereas more than 60% of the fatty acids of fraction 2 were synthesized by the bdellovibrio. Nevertheless, each fraction contained significant amounts of fatty acid of both origins. The substrate cell-derived fatty acids had the same distribution of N-acyl and O-acyl linkages as in E. coli lipid A. The data indicate that the two lipid A moieties in lipopolysaccharide of intraperiplasmically grown bdellovibrios are hybrids of substrate cell-derived and bdellovibrio-synthesized components. The data also suggest that disaccharide units and N- and O-acyl linkages preexisting in the substrate cell lipid A may be conserved. A possible explanation for the unequal distribution of substrate cell-derived material in the two lipid A fractions of the bdellovibrio is suggested.     

Association of a lipoprotein-like particle with bovine fetuin

Fetuin is a major protein of fetal bovine serum that exhibits heterogeneity and has been found to be associated with some biological active growth factors. Preliminary studies indicated that commercial fetuin preparations contain lipids. We investigated in detail the nature of lipids associated with fetuin by using ultracentrifugation and agarose gel chromatography followed by lipid analysis. Fetuin was associated with a variety of lipids, predominantly cholesterol, cholesteryl ester with smaller amounts of phospholipids, triglycerides, and free fatty acids. Adjustment of fetuin preparation for various densities followed by ultracentrifugation resulted in a fraction with a density 1.063-1.21 g/ml (1-2% of total protein) that contained the bulk of the lipids. This fraction eluted as a single peak upon high pressure liquid chromatography and agarose gel chromatography. Delipidation of the lipoprotein-like particle followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a major band in the range of fetuin itself. These studies suggest that a fraction of fetuin (or isoform) binds lipids forming a particle with floating characteristics similar to high density lipoproteins.     

Covalent interaction of chloroacetic and acetic acids with cholesterol

The covalent interaction of chloroacetic acid with rat liver lipids was studied in vivo. Rats were given a single oral dose (8.75 mg/kg, 50 microCi) of 1-[14C]chloroacetic acid and sacrificed after 24 hours. Lipids extracted from the livers were separated into neutral lipids and phospholipids by solid-phase extraction using sep-pak silica cartridges. The neutral lipid fraction was further fractionated by preparative thin-layer chromatography followed by reverse-phase high-performance liquid chromatography. The fraction corresponding to the retention time of standard cholesteryl chloroacetate gave a pseudomolecular ion peak at m/z 480/482 ratio: (3:1) on ammonia chemical ionization mass spectrometry, and the fragmentation pattern was found to be similar to that of the standard sample. Under similar conditions, acetic acid resulted in the formation of cholesteryl acetate. The effect of such conjugation reactions on the cell membrane and their contribution to toxicity is presently unknown.     

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell II. Distribution of the alterations in the various lipid fractions

The distribution of carbon tetrachloride-induced alterations of membrane lipids in various fractions of liver microsomal lipids was studied. The chromatographic spot (referred to as the “D” spot in the previous paper [1]) which has been shown to contain the compounds responsible for the diene conjugation absorption [1], was found in the fatty acid methyl esters prepared from the fraction containing phosphatidylethanolamine (PE) and also in those obtained from the fraction containing phosphatidylserine (PS) and phosphatidylinositol (PI). The absorption of conjugated dienes was very marked in PE and less intense in PS and PI. The fatty acid methyl esters prepared from the fraction containing phosphatidylcholine (PC) showed no presence of the “D” spot and minimal absorption of conjugated dienes.A decrease in arachidonic acid content was found in the fraction containing PE, while no change in content of this fatty acid was found in the fraction containing PC. Results similar to those observed for PC were also found for neutral lipids (NL).Analysis of the fatty acid methyl esters of the various lipid fractions by gas-liquid chromatography (GLC) with an electron capture detector (ECD) gave a qualitative index of the free radical attack by CCl₄ metabolites. Quantitative estimation was attained by study of the irreversible binding of ¹⁴C from ¹⁴CCl₄ to the various lipid fractions. It was found that the fraction containing PS had the highest specific activity, while the fraction containing PC had the lowest specific activity of all the phospholipids. Thin layer chromatography (TLC) of the fraction containing PS revealed that only 11% of the radioactivity was associated with the pure PS moiety, while the remainder was associated with uncharacterized lipids (probably oxidation products).The possible relevance of the alterations induced by carbon tetrachloride in the various phospholipid fractions of liver microsomes to functional changes is discussed.     

Isolation and characterization of fractions of Mycoplasma pneumoniae. I. Chemical and chromatographic separation

Prescott, B. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), O. Sobeslavsky, G. Caldes, and R. M. Chanock. Isolation and characterization of fractions of Mycoplasma pneumoniae. I. Chemical and chromatographic separation. J. Bacteriol. 91:2117-2125. 1966.-Fractionation of Mycoplasma pneumoniae, cultured on a beef heart infusion-horse serum-yeast extract medium, was carried out by chemical and chromatographic procedures. The chemical method yielded eight fractions consisting of lipid, carbohydrates, and proteins. Four protein-rich fractions were isolated by chromatographing a supernatant fluid of sonically treated organisms on Sephadex G-25. The 12 fractions were tested for serological and antigenic activity in vitro and in vivo. The lipid fraction was serologically active and the relative order of activity of the protein fractions appeared to depend on the amount of lipid present in the molecule. The highly serologically active Sephadex G-25 protein fraction 1 prepared chromatographically contained 15% lipid in the molecule, whereas the less serologically active protein fraction 2 prepared by chemical means contained 2% lipid. The acetone-extracted lipid fraction was chromatographed on thin-layer chromatography plates and found to consist of nine fractions. Serological activity was associated with only the first three spots above the origin. Lipid extracted from the protein fractions seemed to be similar to the acetone-extracted lipid from the sediment of the sonically treated organisms.