Phosphorus Assay in Column Chromatography
TL;DR: If the highest accuracy was not required, the following manipulations simplified and speeded multiple total phosphorus determinations on the eluates from column chromatographic separations.
About: This article is published in Journal of Biological Chemistry.The article was published on 1959-03-01 and is currently open access. It has received 12381 citations till now. The article focuses on the topics: Column chromatography & Phosphorus.
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TL;DR: Using this method, the liped peroxide level in the liver of rats suffering from carbon tetrachloride intoxication was investigated and was in good agreement with previously reported data obtained by measuring diene content.
24,847 citations
TL;DR: The high content of plasmalogen phospholipids in these sediments suggests that the anaerobic prokaryotic Clostridia are found in the aerobic sedimentary horizon, which would require anaer aerobic microhabitats in the aerated zones.
Abstract: The measurement of lipid phosphate is proposed as an indicator of microbial biomass in marine and estuarine sediments. This relatively simple assay can be performed on fresh, frozen or frozen-lyophilized sediment samples with chloroform methanol extraction and subsequent phosphate determination. The sedimentary lipid phosphate recovery correlates with the extractible ATP and the rate of DNA synthesis. Pulse-chase experiments show active metabolism of the sedimentary phospholipids. The recovery of added 14C-labeled bacterial lipids from sediments is quantitative. Replicate analyses from a single sediment sample gave a standard deviation of 11%. The lipid extract can be fractionated by relatively simple procedures and the plasmalogen, diacyl phospholipid, phosphonolipid and non-hydrolyzable phospholipid content determined. The relative fatty acid composition can be readily determined by gas-liquid chromatography. The lipid composition can be used to define the microbial community structure. For example, the absence of polyenoic fatty acids indicates minimal contamination with benthic micro-eukaryotes. Therefore the high content of plasmalogen phospholipids in these sediments suggests that the anaerobic prokaryotic Clostridia are found in the aerobic sedimentary horizon. This would require anaerobic microhabitats in the aerated zones.
1,693 citations
TL;DR: Phospholipids may be measured colorimetrically (as dipalmitoyl lecithin) without conventional acid digestion and color development procedures by forming a complex with ammonium ferrothiocyanate.
1,673 citations
TL;DR: Fresh plasma freed of intact platelets can be shown to contain minute particulate material (platelet‐dust) which can be separated by ultracentrifugation and shows a corresponding deficiency in Platelet Factor 3 activity.
Abstract: SUMMARY
Fresh plasma freed of intact platelets can be shown to contain minute particulate material (platelet-dust) which can be separated by ultracentrifugation. Much of this material is rich in phospholipid and shows coagulant properties resembling those of Platelet Factor 3. The supernatant plasma, freed of these particles, shows a corresponding deficiency in Platelet Factor 3 activity.
Electron microscopic studies suggest that the lipid-rich particles may originate from the osmophilic granules of platelets and that they are extruded on storage of platelets, even in the absence of coagulation. It is suggested that the liberation of this material is the basis of the process of ‘activation’ of platelets known to occur with storage.
When clotting occurs, coagulant particulate material is released from platelets, considerably in excess of what is required for thrombin generation and it can be detected in serum. Its presence accounts for the platelet-like activity of serum.
The material can be distinguished from intact platelets, red cell stroma and chylomicra. It was observed that chylomicra, freed of platelet-dust, were inactive in a thrombin generation test, which thus appeared responsive only to platelet type lipid. Both chylomicra and platelet-dust were effective in a Stypven time test. Attention is drawn to possible errors of interpretation which may arise from the use of the latter test as an index of Platelet Factor 3 activity.
Preliminary immunological investigation of platelets revealed the presence of fibrinogen and at least three other constitutents which appear to be primarily platelet components.
1,342 citations
Cites background or methods from "Phosphorus Assay in Column Chromato..."
...An immunological demonstration of fibrinogen in human platelets has previously been reported by Seligman, Goudemand, Janin, Bernard and Gragar (1951) and by Salmon and Bounameaux (1958), and this was the only immunoprecipitable human platelet antigen they found. However, Salmon and Bounameaux (1958) reported in bovine platelets...
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...k l e f h o d s f o r Exarwinatioiz o f G.M. Si)ctioiis For Electron Microscopy the techniques and reagents listed and described by Kay (1965) were used....
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...The Hutanol extraction method described by Grette (1962) was used....
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...An immunological demonstration of fibrinogen in human platelets has previously been reported by Seligman, Goudemand, Janin, Bernard and Gragar (1951) and by Salmon and Bounameaux (1958), and this was the only immunoprecipitable human platelet antigen they found....
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...Grette’s work (1962) makes it probable that Platelet Factor 3, along with small molecular weight platelet products ( 5 hydroxy-tryptamine and adenine nucleotides), originate from the platelets by an active process of expulsion; the expulsion being affected by a contractile actomyosin-like protein present in platelets (Grette, 1962)....
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TL;DR: The modified method involved application of crude egg phosphatides to a column of alumina in the proportion of 1 g phosphatide/25 g alumina, and elution of the lecithin fraction with the 2-component solvent system chloroform:methanol, 9:1 by vol.
Abstract: Chromatographically homogeneous egg lecithin, as determined by TLC on Silica Gel G, has been isolated from crude egg phosphatides by column chromatography on alumina through modification of existing, lengthy methods. The modified method involved application of crude egg phosphatides to a column of alumina in the proportion of 1 g phosphatide/25 g alumina, and elution of the lecithin fraction with the 2-component solvent system chloroform:methanol, 9:1 by vol. This method of purification separated lecithin from other choline and non-choline components of crude phosphatides, avoided overloading of the alumina column, and made unnecessary the need for a second chromatographic fractionation of partially purified lecithin on silicic acid, which is needed in existing methods of purification of lecithin. The use of fresh yolks permitted easier removal of pigment from the final product than was possible with commercially dried yolks. Phosphatides extracted from dried yolks were much more highly colored than were the phosphatides extracted from fresh yolks and the color presisted through chromatography on alumina. The fatty acid/phosphorus molar ratio of the purified lecithin was 2.00, which is the theoretical FA/P molar ratio of phosphatidylcholine; other materials with this ratio were not present.
1,259 citations
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TL;DR: This communication describes how several of the metabolic intermediates may be isolated in good yield by column chromatography on ion exchange resins and summarizes the values which were found for their normal equilibrium levels.
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