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.

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

Phospholipid synthesis in a membrane fraction associated with mitochondria.

The Surface of Lipid Droplets Is a Phospholipid Monolayer with a Unique Fatty Acid Composition

The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes.

A universal reagent for phospholipid analysis.

Quantitative Analysis of Phospholipids by Thin-Layer Chromatographyand Phosphorus Analysis of Spots p ROCEDURES FOR ANALYSIS of phospholipid composition by thin-layer chromatography (TLC) and phospho~nts analysis have been reported from a number of laboratories. These procedures usually depend upon one-dimensional TLC and elution of spots before analysis. The method reported here has the advantage of improved separations by two-dimensionM TLC, direct aspiration of spots by suction, and phosphorus analysis without pr ior elution. Our procedure depends upon two-dimensional TLC with the solvent pairs 1) chloroform/ methanol/water 65/25/4 a~ld n-butanol/acetic acid/water 60/20/20; and 2) chloroform/ methanol/2S% aqueous ammonia 65/35/5 followed by chloroform/acetone/methanol/acetic acid/water 5/271/1/0.5. The adsorbent composed of silica gel plain/magnesium silicate 9/1 (1) after spreading with a conventional Desaga spreader (0.25 nnn layer) is heat activated for 20 rain at 120C, cooled for 30 rain, spotted, and ehromatograms developed in chambers lined with solvent-saturated paper (2). Spots are detected by spraying with a 0.6% solution of potassium dichromate in 55% (by wt) sulfuric acid followed by heating for 30 rain at 180C in a forced draf t oven or by exposure to iodine vapors. Af ter development, spots are circled and lettered for identification and several blank areas corresponding in size to the sample spots are marked off. A typical ehromatogram of each series is photographed (Polaroid camera) and the spots recovered by aspiration. Aspirat ion of the spots directly into 30 ml Xjeldahl digestion flasks is accomplished by fitting a rubber stopper with two plastic tubes removed from plastic wash bottles. One tube with a pointed end serves as the intake and the other tube for attachment to a water pump for suction. Adsorbent is prevented from passing out of the digestion flask during aspiration by adding 0.9 ml of 72% perehlorie acid (used subsequently for digestion) to the flask to act as a liquid t rap by moistening the lower bulb portion of the flask and by insertion of a 1 cm square of \"Kimwipe\" or similar light weight paper into the end of the suction tube to serve as a filter. After aspiration, the plastic tubes are tapped to remove any dry powder and the paper filter pushed with a wire plunger into the flask. Digestion of the flask contents is carried out on an electrically heated Kjeldahl rack with water-pump suction to remove any escaping fumes. The heaters are adjusted to give gentle refluxing so that digestion is complete in about 20 rain. After digestion, the sides of the flask are rinsed with 5 ml of distilled water, 1 ml of 2.5% ammonium molybdate solution is added, the flask swirled for mixing, 1 ml of 10% ascorbic acid solution is added, and finally 2 nfl of distilled water are added. The solution is transferred to a centrifuge tube~ heated in a boiling water bath for 5 rain, cooled, and suspended adsorbent removed by eentrifugation for 5-10 rain. Samples and blanks are transferred to euvettes and the optical density determined at 820 m/x af ter zero adjustment with water. Sensitivity can be increased by using a 10 nfl digestion flask and one half of the specified amounts of reagents. Glassware should be acid eleaned. Corrected optical densities are determined by subtraction of the reading obtained from a blank area corresponding in size to that of the sample. The values are then converted to tLg of phosphorus using a factor derived from a standard curve prepared using Na~HPO~. The factor in our laboratories is 11.0 for standard amounts and twice that for half amounts of reagents. Molar ratios of phospholipids are obtained by expression of results as percent of the total phosphorus in the sample. Deterruination of the total phosphorus is conveniently accomplished by spotting 50-100 t~g of total sample in a blank area (upper right corner) after development with both solvents. The total sample is then charred, etc., in the same manner as the samples. For expression of results as percent of the total lipid, phosphorus values for brain lipids are multiplied by the following' factors: phosphatidyl bmsitol, 31.4; phosphatidyl serine, 26.2; lecithin and phosphatidyl ethanolamine, 25.4 ; phosphatidic acid, 25.0; sphingomyelin, 24.8; and cardiolipin, 24.4 Aninml tissue lipid extracts are spotted at levels of 200-1000 ~g for determinations and at least four ehromatograms are developed with each of the two-dimensional systems. Average values for the major lipid classes (lecithin, sphingomyelin, phosphatidyl ethanolamine and phosphatidyl serine) are thus obtained from eight determinations. Usually spots from two eh roma tog ra ms are pooled for minor components. The values obtained from a normal adult human brain by the present procedure and the

Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots.

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.

A. N. Siakotos

Papers

Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots.

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

Quantitative thin-layer chromatography of lipids by photographic densitometry