George Rouser

Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots

Separation of polar lipids by two-dimensional thin layer chromatography providing resolution of all the lipid classes commonly encountered in animal cells and a sensitive, rapid, reproducible procedure for determination of phospholipids by phosphorus analysis of spots are described. Values obtained for brain and mitochondrial inner membrane phospholipids are presented.

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.

Analytical separation of nonlipid water soluble substances and gangliosides from other lipids by dextran gel column chromatography.

A column chromatographic procedure is reported utilizing a dextran gel (Sephadex) for the complete separation of the major lipid classes from water-soluble nonlipids. Lipids other than gangliosides are eluted first with chloroform/methanol 19/1 saturated with water, gangliosides with chloroform/methanol/water containing acetic acid, and water-soluble nonlipids with methanol/water 1/1. Results for adult human whole brain, grey and white matter, and normal infant whole brain lipids are presented. With beef brain lipid as sample the ganglioside fraction is essentially pure, but with human brain lipid samples only about 70% of the second fraction is ganglioside. All ganglioside and water soluble nonlipid of a human spleen chloroform/methanol extract was separated from lipids with the procedure. Control studies with P32O4≡ and C14 labelled glucose showed that all counts were present in fraction 3. Similar studies with C14 labelled amino acids (glycine, serine, alanine, phenylalanine) showed that only phenylalanine counts were eluted in fraction 2 along with the gangliosides. The procedure was applied for removal of large amounts of ammonium acetate from DEAE cellulose column fractions and for complete removal of adsorbent and salts from lipids eluted from thin-layer chromatograms. After passage through the dextran gel columns, lipids eluted from thin-layer chromatograms were found to give infrared spectra identical to those of pure samples obtained by other procedures.

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

AbstractsQuantitative 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.

Laboratory contaminants in lipid chemistry: Detection by thin-layer chromatography and infrared spectrophotometry and some procedures minimizing their occurrence.

Many sources of contamination for lipid preparations exist in the laboratory. These contaminants can be detected using thin-layer chromatography (TLC) and infrared spectroscopy. Numerous components that are potential contaminants and can lead to false analyses were demonstrated by TLC in laboratory soaps, cleaners, hand creams and lotions, hair tonics, laboratory greases, floor waxes, oil vapors, tobacco smoke, hydrocarbon phases for gas-liquid chromatography, etc. Procedures preventing introduction of contaminants are presented including descriptions of equipment and precautions to eliminate or minimize contamination. These are useful in isolation of pure polar and nonpolar lipids.

LIPIDS OF SUBCELLULAR PARTICLES.

Methods for isolation and characterization of subcellular particles as well as procedures for analysis of lipid class composition are discussed. The literature on distribution of lipids in subcellular particles is then reviewed. Pertinent new data from our laboratories are presented as well. The isolated particles are related to the organelles to which they correspond in the cell and are discussed with regard to heterogeneity and morphological integrity. Confusion can arise with regard to subcellular particles unless it is appreciated that: 1) preparation of particles of high purity generally requires more than the classical differential centrifugation scheme (both differential and gradient centrifugation may be required); 2) it is hazardous to apply exactly the same procedure for all tissues; 3) all subcellular fractions must be thoroughly characterized.The more recently devised DEAE cellulose column and thin-layer chromatographic procedures for analysis of lipid class composition are more reliable than the older hydrolytic or silicie acid column or paper chromatographic techniques.The chief lipid components of mitochondria from all organs and species are lecithin, phosphatidyl ethanolamine, and cardiolipin (diphosphatidyl glycerol). Despite the fact that reports in the literature are in agreement that phosphatidyl inositol is a major component of mitochondria, it is concluded on the basis of new data obtained from highly purified mitochondria and improved analytical methods that phosphatidyl inositol is not a major component of mitochondria. The presence of a relatively large amount of phosphatidyl inositol in mitochondrial preparations is probably related in part to contamination with other particles. Some analytical procedures are demonstrated to give erroneous values for this lipid class. It is also concluded that phosphatidyl serine, phosphatidic acid, sphingomyelin, cerebrosides, and lysophosphatides, reported to occur in mitochondria, are not characteristic mitochondrial components and furthermore that the large amount of uncharacterized mitochondrial phospholipid reported is actually an analytical artifact. Microsomes appear to be similar to mitochondria except that cardiolipin is either low in or absent from microsomes. Available data indicate nuclei to be rather similar to mitochondria and microsomes, at least in some organs.Studies of the fatty acids of subcellular particles indicate that different particles from one organ have very similar fatty acid compositions. It is clear that there are marked variations in fatty acid composition of particles from different organs and from different species. Differences in dietary fat may be associated with marked changes in fatty acid composition, although brain mitochondrial lipids are largely unchanged. Each lipid class from mitochondria of most organs appears to have a fairly characteristic fatty acid composition. Cardiolipin from some organs contains primarily linoleic acid, phosphatidyl ethanolamine contains large amounts of linoleic and higher polyunsaturates, and lecithin is similar to phosphatidyl ethanolamine except that it does not contain as much arachidonic acid and/or other highly unsaturated fatty acids. New data, the first to be reported, are presented for heart mitochondrial cardiolipin, phosphatidyl ethanolamine, and lecithin.It is concluded that there are two basically different types of membranous structures. Myelin is the chief representative of the metabolically stable type of membrane structure while mitochondria represent the more labile type. The two types of membranes have very different in vivo properties and very different lipid compositions. Myelin is characterized by a high content of cholesterol and sphingolipids with more long chain saturated or monoenoic fatty acids while mitochondria are characterized by a low cholesterol content, little or no sphingolipid, and highly unsaturated fatty acids. It is clear that formulations of the myelin type membrane structure such as that of Vandenheuvel cannot apply to mitochondria. It is postulated that membrane structures intermediate between the extremes represented by myelin and mitochondria exist.

Analytical fractionation of complex lipid mixtures: DEAE cellulose column chromatography combined with quantitative thin layer chromatography

A quantitative chromatographic procedure for the fractionation of complex lipid mixtures is described. The method utilizes diethylaminoethyl (DEAE) cellulose column chromatography followed by thin layer chromatography (TLC). Spots produced in TLC are charred with sulfuric acid-potassium dichromate and heat and are then measured by quantitative densitometry. Results obtained with beef brain and beef heart mitochondrial lipids are presented, and the close correspondence between column isolation procedures and the new procedure is demonstrated. Methods utilizing only column chromatography, column chromatography and TLC, and one- and two-dimensional TLC without column chromatography are compared.

Species variations in phospholipid class distribution of organs: I. Kidney, liver and spleen

Improved procedures for preparation of lipid extracts and determination of phospholipids by phosphorus analysis of spots separated by thin layer chromatography (TLC) were employed to determine the phospholipid class distributions of vertebrate (human, bovine, rat, mouse, frog) kidney, liver and spleen. The absence of significant changes arising from postmortem enzymatic degradation was demonstrated by analysis of lipids of organs after standing for different times postmortem. Intraspecies variability was evaluated by separate analysis of several rat organs. Accuracy of analytical results was insured by demonstrating the absence of spot overlap by two-dimensional TLC and low values for standard deviations. The values for kidney and liver demonstrate little or no species variability, whereas values for spleen indicate two groups which differ in cellular composition. The findings for kidney and liver are in keeping with data obtained from heart, skeletal muscle, lung and highly purified subcellular particulates which indicate that, among vertebrates, there is little or no species variability of phospholipid class distribution of organs and most subcellular particulates.

Precise quantitative determination of human blood lipids by thin-layer and triethylaminoethylcellulose column chromatography. II. Plasma lipids.

Abstract Two-dimensional thin-layer chromatography (TLC) alone or following ion-exchange (TEAE) cellulose column chromatography is shown to provide improved separation of human plasma polar lipids. With both procedures, a number of uncharacterized phospholipids occurring in small amounts are detectable in human plasma. Determination in quadruplicate of phosphorus in spots after two-dimensional TLC is shown to be a rapid, reproducible procedure for accurate analysis of plasma phospholipid composition, standard deviations for major components being in the 1–2% range.

DETERMINATION OF POLAR LIPIDS: QUANTITATIVE COLUMN AND THIN-LAYER CHROMATOGRAPHY.

The structures of the polar lipid classes of plants and animals are presented, their nomenclature discussed, and suggestions are presented for clarification of nomenclature. The three general types of quantitative chromatographic procedures (column chromatography, thin-layer chromatography, and combinations of column and thin-layer chromatography) available for polar lipids are reviewed and a new quantitative two-dimensional thin-layer chromatographic procedure is presented. Useful quantitative procedures employing columns of cellulose, silicic acid, silicic acid mixed with silicate, magnesium silicate, and ion exchange celluloses are presented. New findings with diethylaminoethyl cellulose columns are described. New quantitative procedures employing silicic acid, magnesium silicate, or diethylaminoethyl cellulose column chromatography with quantitative thin-layer chromatography are described.