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Journal ArticleDOI

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

01 May 1970-Lipids (John Wiley & Sons, Ltd)-Vol. 5, Iss: 5, pp 494-496
TL;DR: 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.
Abstract: 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.
Citations
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Journal ArticleDOI
TL;DR: It is demonstrated that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.
Abstract: Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4(-/-) mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4(-/-) mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.

1,875 citations

Journal ArticleDOI
TL;DR: It is hypothesize that carvacrol destabilizes the cytoplasmic membrane and, in addition, acts as a proton exchanger, thereby reducing the pH gradient across the cytopsomal membrane.
Abstract: The natural antimicrobial compound carvacrol shows a high preference for hydrophobic phases. The partition coefficients of carvacrol in both octanol-water and liposome-buffer phases were determined (3.64 and 3.26, respectively). Addition of carvacrol to a liposomal suspension resulted in an expansion of the liposomal membrane. Maximum expansion was observed after the addition of 0.50 μmol of carvacrol/mg of l-α-phosphatidylethanolamine. Cymene, a biological precursor of carvacrol which lacks a hydroxyl group, was found to have a higher preference for liposomal membranes, thereby causing more expansion. The effect of cymene on the membrane potential was less pronounced than the effect of carvacrol. The pH gradient and ATP pools were not affected by cymene. Measurement of the antimicrobial activities of compounds similar to carvacrol (e.g., thymol, cymene, menthol, and carvacrol methyl ester) showed that the hydroxyl group of this compound and the presence of a system of delocalized electrons are important for the antimicrobial activity of carvacrol. Based on this study, we hypothesize that carvacrol destabilizes the cytoplasmic membrane and, in addition, acts as a proton exchanger, thereby reducing the pH gradient across the cytoplasmic membrane. The resulting collapse of the proton motive force and depletion of the ATP pool eventually lead to cell death.

1,461 citations

Journal ArticleDOI
TL;DR: Results of this study show that oleic acid is as effective as linoleic acid in lowering LDL-C levels in normo-triglyceridemic patients, and oleoic acid seemingly reduces HDL-C Levels less frequently than does linolesic acid.

1,310 citations

Journal ArticleDOI
TL;DR: The impairment of microbial activity by the cyclic hydrocarbons most likely results from hydrophobic interaction with the membrane, which affects the functioning of the membrane and membrane-embedded proteins.

1,155 citations

Journal ArticleDOI
TL;DR: It is speculated that the antimicrobial effect of (+)menthol, thymol, and linalyl acetate may result, at least partially, from a perturbation of the lipid fraction of microorganism plasma membrane, resulting in alterations of membrane permeability and in leakage of intracellular materials.
Abstract: In the present paper, we report the antimicrobial efficacy of three monoterpenes [linalyl acetate, (+)menthol, and thymol] against the gram-positive bacterium Staphylococcus aureus and the gram-negative bacterium Escherichia coli. For a better understanding of their mechanisms of action, the capability of these three monoterpenes to damage biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein from unilamellar vesicles with different lipidic compositions (phosphatidylcholine, phosphatidylcholine/phosphatidylserine [9:1], phosphatidylcholine/stearylamine [9:1], and phosphatidylglycerol/cardiolipin [9:1]). Furthermore, the interaction of the terpenes tested with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry. Finally, the results were related to the relative lipophilicity and water solubility of the compounds examined. Taken together, our findings lead us to speculate that the antimicrobial effect of (+)menthol, thymol, and linalyl acetate may result, at least partially, from a perturbation of the lipid fraction of microorganism plasma membrane, resulting in alterations of membrane permeability and in leakage of intracellular materials. Besides being related to physicochemical characteristics of the drugs (such as lipophilicity and water solubility), this effect seems to be dependent on lipid composition and net surface charge of microbial membranes. Furthermore, the drugs might cross the cell membranes, penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.

1,029 citations

References
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Journal ArticleDOI
01 Jan 1966-Lipids
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

Journal ArticleDOI
01 Mar 1966-Lipids
TL;DR: Proper procedures preventing introduction of contaminants are presented including descriptions of equipment and precautions to eliminate or minimize contamination.
Abstract: 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.

194 citations