Quantitative densitometric thin-layer chromatography of lipids using copper acetate reagent.
TL;DR: A copper acetate spray reagent has been used for the quantitative densitometric thin-layer chromatography of glycolipids and phospholipids from human CNS myelin and the precision ranges between zero and ± 4.5%.
About: This article is published in Journal of Chromatography A.The article was published on 1969-01-01. It has received 396 citations till now. The article focuses on the topics: Thin-layer chromatography & Reagent.
Citations
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TL;DR: An improved method for extracting the lipids from tissues consists of the use of hexane:isopropanol, followed by a wash of the extract with aqueous sodium sulfate to remove nonlipid contaminants.
2,400 citations
TL;DR: The mobile phases described permit separation of the six major phospholipids of amniotic fluid in one dimension with either conventional or high performance thin layer chromatography.
Abstract: The mobile phases described permit separation of the six major phospholipids of amniotic fluid in one dimension with either conventional or high performance thin layer chromatography. An example of this separation with an extract of amniotic fluid is given.
470 citations
TL;DR: Crassostrea gigas spat were fed diets of Pavlova lutheri, Dunaliella tertiolecta or Tetraselmis suecica as discussed by the authors.
Abstract: Crassostrea gigas spat were fed diets of Pavlova lutheri, Dunaliella tertiolecta or Tetraselmis suecica . The D. tertiolecta diet contained no fatty acid longer or more unsaturated than linolenic acid (18:3ω3) and the T. suecica diet was deficient in decosahexaenoic acid (22:6ω3). The algal diets were supplemented with micro-encapsulated triolein, oyster lipid extract or 22:6ω3. Spat were also starved or fed carbohydrate diets.
451 citations
TL;DR: The high-performance thin-layer chromatography method can be applied to lipids extracted from marine organisms including the complex polar lipids derived from phytoplankton and fish neural tissue.
397 citations
TL;DR: The membrane composition of late endosomes by suborganellar fractionation in the absence of detergent is analyzed and it is found that the internal membranes of this multivesicular organelle can be separated from the limiting membrane and that each membrane population exhibited a defined composition.
373 citations
References
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TL;DR: 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.
Abstract: 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
1,513 citations
1,329 citations
TL;DR: The extra-myelin portion of white matter had a lipid composition that was very similar to that of myelin, but quite different from that of gray matter.
718 citations
TL;DR: Detailed analyses of the individual lipids present in such subfractions are described by using a recently developed method for the determination of all known phospholipids (Dawson, Hemington & Davenport, 1962); it provides new data on the lipid composition of brain mitochondria, nuclei and microsomes, as well as the subcellular particles peculiar to brain: myelin fragments, synaptosomes and synaptic vesicles.
Abstract: It is becoming increasingly apparent that lipids act as essential structural elements in the multienzyme systems associated with the cell organelles. Many of the enzymes of the electron-transport chain in mitochondria can be isolated as discrete lipoproteins (Green, 1959), and the phospholipid contained in these is essential for enzymic activity (Reich & Wainio, 1961). Moreover, lipids play a fundamental role in the microstructure of cell meimbranes and may even participate metabolically in the transport of cations (Hokin & Hokin, 1964). Clearly, therefore, the distribution of individual lipids within the cell becomes of fundamental importance. The technique of differential centrifugation has enabled various subcellular fractions to be prepared and numerous lipid analyses of these fractions have been carried out. With liver tissue, consisting predominantly of hepatic cells, the relative cellular homogeneity allows these analyses to apply to defined morphological entities of the cell such as the nuclei or mitochondria (Spiro & McKibbin, 1956; Macfarlane, Gray & Wheeldon, 1960; Getz & Bartley, 1961). However, in tissues like the brain with its complicated cytological differentiation, the application of the same centrifugation technique produces fractions that are grossly heterogeneous. Lipid analyses of such fractions (Peterson & Schou, 1955; Biran & Bartley, 1961) therefore, although ofgreat value, must be regarded as preliminary. In recent years, the use of density-gradient centrifugation (Hebb & Whittaker, 1958; Whittaker, 1959, 1961; Whittaker, Michaelson & Kirkland, 1963, 1964), coupled with morphological characterization in the electron microscope (Gray & Whittaker, 1960, 1962; Whittaker, 1960; Whittaker etal. 1963, 1964), has permitted the isolation of certain morphologically defined structures in relatively pure form. These include pinched-off nerve endings or 'synaptosomes', and synaptic vesicles which are contained within the synaptosomes and can be released from them by suitable disruptive procedures. The present paper describes detailed analyses of the individual lipids present in such subfractions by using, among other techniques, a recently developed method for the determination of all known phospholipids (Dawson, Hemington & Davenport, 1962). It provides new data on the lipid composition of brain mitochondria, nuclei and microsomes, as well as the subcellular particles peculiar to brain: myelin fragments, synaptosomes and synaptic vesicles.
432 citations
TL;DR: An improved procedure for the quantitative assay of phospholipids separated by TLC is described, in which a specially washed Silica Gel H and a newly designed chromatography unit are employed.
365 citations