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Journal ArticleDOI

A simple method for the isolation and purification of total lipides from animal tissues.

01 May 1957-Journal of Biological Chemistry (American Society for Biochemistry and Molecular Biology)-Vol. 226, Iss: 1, pp 497-509

AbstractWork from this laboratory resulted in the development of a method for the preparation and purification of brain lipides (1) which involved two successive operations. In the first step, the lipides were extracted by homogenizing the tissue with 2: 1 chloroform-methanol (v/v), and filtering the homogenate. In the second step, the filtrate, which contained the tissue lipides accompanied by non-lipide substances, was freed from these substances by being placed in contact with at least 5-fold its volume of water. This water washing entailed the loss of about 1 per cent of the brain lipides. This paper describes a simplified version of the method and reports the results of a study of its application to different tissues, including the efficiency of the washing procedure in terms of the removal from tissue lipides of some non-lipide substances of special biochemical interest. It also reports some pertinent ancillary findings. The modifications introduced into the method pertain only to the washing procedure. A chloroformmethanol extract of the tissue, prepared as described in the original version of the method, is mixed with 0.2 its volume of water to which, for certain purposes, different mineral salts may be added. A biphasic system without any interfacial fluff is obtained (2). The upper phase contains all of the non-lipide substances, most of the strandin, and only negligible amounts of the other lipides. The lower phase contains essentially all the tissue lipides other than strandin. In comparison with the original method, the present version has the advantage of being simpler, of being applicable to any scale desired, of substantially decreasing the losses of lipides incidental to the washing process, and, finally, of yielding a washed extract which can be taken to dryness without foaming and without splitting of the proteolipides (3).

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Citations
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Journal ArticleDOI
E. G. Bligh1, W. J. Dyer1
TL;DR: The lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials that has been applied to fish muscle and may easily be adapted to use with other tissues.
Abstract: Lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials. The entire procedure can...

43,580 citations

Journal ArticleDOI
TL;DR: The procedure developed is simple, rapid, and generally applicable t o lipids, and the results did not affect the validity of the method.
Abstract: Fatty acid methyl esters and dimethylacetals suitable for gas chromatographic analysis were prepared by treatment of lipids with boron fluoride–methanol (140 g BF3 per liter of methanol). This reagent is stable and easy to handle. Reaction conditions were investigated for triglycerides, diglycerides, monoglycerides, free fatty acids, sterol esters, phosphatidyl ethanolamines, phosphatidyl serines, phosphatidyl cholines, monophosphoinositides, monogalactosyl glycerides, phosphatidal cholines (choline plasmalogens), digalactosyl glycerides, and sphingomyelins. The methyl esters and dimethylacetals were readily purified by thin-layer chromatography, and yields were quantitative. There were few undesirable side reactions, and they did not affect the validity of the method. The procedure developed is simple, rapid, and generally applicable to lipids.

5,603 citations

Journal ArticleDOI
TL;DR: Kinetic analysis indicates that TPA can substitute for diacylglycerol and greatly increases the affinity of the enzyme for Ca2+ as well as for phospholipid, and various phorbol derivatives which have been shown to be active in tumor promotion are also capable of activating this protein kinase in in vitro systems.
Abstract: Tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) directly activate in vitro Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), which normally requires unsaturated diacylglycerol. Kinetic analysis indicates that TPA can substitute for diacylglycerol and greatly increases the affinity of the enzyme for Ca2+ as well as for phospholipid. Under physiological conditions, the activation of this enzyme appears to be linked to the receptor-mediated phosphatidylinositol breakdown which may be provoked by a wide variety of extracellular messengers, eventually leading to the activation of specific cellular functions or proliferation. Using human platelets as a model system, TPA is shown to enhance the protein kinase C-specific phosphorylation associated with the release reaction in the total absence of phosphatidylinositol breakdown. Various phorbol derivatives which have been shown to be active in tumor promotion are also capable of activating this protein kinase in in vitro systems.

4,525 citations

Journal ArticleDOI
TL;DR: The effects of the ionic strength and pH of the hemolyzing solution on the hemoglobin content of human erythrocyte ghosts were studied in phosphate buffers and suggest an electrophysical interaction of hemoglobin with membrane constituents.
Abstract: The effects of the ionic strength and pH of the hemolyzing solution on the hemoglobin content of human erythrocyte ghosts were studied in phosphate buffers and found to have a pronounced influence upon hemoglobin binding in the ghosts. Buffer concentrations between 10 and 20 ideal milliosmolar (imOsm), at pH values 5.8 – 8.0, resulted in maximum hemoglobin removal from ghosts. The pH optimum for hemoglobin binding to ghosts was between 5.8 and 5.9 in a 20 imOsm buffer. The influence of these variables suggest an electrophysical interaction of hemoglobin with membrane constituents. This study provides a basis for comparison of existing methods for ghost preparation, as well as a means for prediction of the conditions required for preparation of ghosts containing any desired amount of hemoglobin. Conditions were found that allowed the preparation of hemoglobin-free ghosts by single-stage hemolysis and washing. Hemoglobin-free ghosts were prepared in 20 imOsm phosphate buffer at pH 7.4. Essentially all the lipid was recovered in the ghosts, but non-hemoglobin nitrogen-containing substances were lost. The pyridine hemochromogen method for hemoglobin determination was adapted for the measurement of very small quantities of hemoglobin through use of the Soret band (418 mμ) for absorbancy measurements.

4,199 citations

Journal ArticleDOI
07 Apr 2011-Nature
TL;DR: Discovery of a relationship between gut-flora-dependent metabolism of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for the development of new diagnostic tests and therapeutic approaches for atherosclerotic heart disease.
Abstract: Metabolomics studies hold promise for the discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. Here we used a metabolomics approach to generate unbiased small-molecule metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine—choline, trimethylamine N-oxide (TMAO) and betaine—were identified and then shown to predict risk for CVD in an independent large clinical cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted upregulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a critical role for dietary choline and gut flora in TMAO production, augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary-choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases, an enzymatic source of TMAO, segregated with atherosclerosis in hyperlipidaemic mice. Discovery of a relationship between gut-flora-dependent metabolism of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for the development of new diagnostic tests and therapeutic approaches for atherosclerotic heart disease.

3,302 citations


References
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Journal ArticleDOI
Jordi Folch1, I. Ascoli1, Marjorie B. Lees1, J.A. Meath1, F. N. LeBaron1 
TL;DR: A simple method is described for the preparation of extracts of total pure lipides from brain tissue by homogenizing the tissue with a chloroform-methanol mixture and washing free of non-lipide contaminants.
Abstract: For the preparation of lipide extracts from tissues, the method of Bloor (I), either in its original form or with slight modifications, has been a standard procedure. This method consists in extracting the tissue with a mixture of ethyl alcohol and ether. Since the extract obtained is known to contain non-lipide contaminants, it is usually taken to dryness and the residue extracted with a solvent, such as chloroform or petroleum ether, which exhibits a highly specific solvent power for lipides. However, the secondary extracts obtained have been shown to contain substances other than lipides (2, 3). In the case of nervous tissue, it has been common experience that all of the lipides present in tissue are not extracted by Bloor’s procedure (4, 5). Thus, different workers have found it necessary to introduce a subsequent extraction of the tissue with another solvent of higher solvent power for lipides than Bloor’s mixture. This second solvent has usually been chloroform (4, 5). The methods thus developed are time-consuming, complicated, and, owing to the fact that they involve protracted treatment of the tissue with boiling solvents, they are open to the general objection that the procedure followed results in changing the chemical nature of some of the lipides. Furthermore, the extracts thus obtained are known to contain non-lipide contaminants (4, 5). This paper describes a simple method for the preparation of extracts of total pure lipides from brain tissue. The method consists of homogenizing the tissue with a chloroform-methanol mixture. The clear lipide extract thus obtained is then washed free of non-lipide contaminants by being placed in contact with a large amount of water. The whole procedure can be run at 0” and thus any danger of chemical changes in lipides is reasonably excluded.

1,173 citations

Journal ArticleDOI
TL;DR: The values for cerebrosides found in the current literature include gangliosides, and other investigators (JOHNSON, MCNABB, and ROSSITER, 1950; CUMINGS, 1953; BLACKWOOD and CUMings, 1954) have neglected the ganglariosides.
Abstract: THE extensive research work in the physiological and pathological processes in which lipids are involved has increased the necessity for accurate micromethods for their quantitative estimation. BRANTE (1949) reviewed modern micromethods for the determination of the lipids in nervous tissue, and made a thorough investigation of the various factors which may influence them. Like most other investigators, BRANTE determined the cerebroside content only by estimating the reducing substances in a lipid extract before and after hydrolysis. From the important work of KLENK and collaborators (KLENK, 1941, 1942; KLENK and LANGERBEINS, 1941 ; SCHUWIRTH. 1940) we know that cerebrosides are not the only lipids containing carbohydrates in the central nervous system. They have isolated gangliosides from the brain and determined their amount in different nervous tissues. Besides these two glycolipids, a third has been described by ARSOVE, FOLCH, and MEATH (1951a). They named the new lipid strandin, but my preparations like DAWN'S (1952), showed that strandin consisted of gangliosides in a different physico-chemical state contaminated with low-molecular substances and mucopolysaccharides. CHATAGNON and CHATAGNON (1953, 1954) have suggested that sphingomyelin is also a part of the complex. BRANTE (1949) discussed the interference of gangliosides on the cerebroside values, and we (BRANTE and SVENNERHOLM, 1949, 1951) determined both total glycolipids and gangliosides in some foetuses. Other investigators (JOHNSON, MCNABB, and ROSSITER, 1950; CUMINGS, 1953; BLACKWOOD and CUMINGS, 1954) have neglected the gangliosides. Therefore the values for cerebrosides found in the current literature include gangliosides. EDGAR (1955) who has thoroughly discussed the problem, used the term glyco-sphingosides instead of cerebrosides. He has also made some estimations of both gangliosides and cerebrosides. Consequently, the carbohydrates in a lipid extract are derived from at least two lipid sources. But lipid extracts are contaminated to some extent with low-molecular substances, partly of carbohydrate nature. It is impossible to remove them completely by the methods generally used, i.e., precipitation of the lipids before extraction or re-extraction of a primary lipid extract. Generally, the error introduced by the contaminants is negligible i n the determination of glycolipids in adult nervous tissue, but this is not so in foetal tissue, where the amount of glycolipids is low and that of the contaminants is both relatively and absolutely higher. FOLCH, ASCOLI, LEES. MEATH, and LEBARON (1951b), however, succeeded in removing these substanccs by partition dialysis. By this procedure the gangliosides too are separated from the other lipids. It therefore appeared that it might be possible to use this method to separate cerebrosides from other substances containing carbohydrates.

531 citations

Journal ArticleDOI
TL;DR: The isolation from brain tissue of a substance to which the name of strandin has been given for descriptive purposes is reported, which has the property of forming long strands that show perfect orientation under polarized light.
Abstract: This paper reports the isolation from brain tissue of a substance to which the name of strandin has been given for descriptive purposes. This term has been chosen because, when dried from aqueous solution, strandin has the property of forming long strands that show perfect orientation under polarized light. As isolated from brain by any one of the three procedures described below, strandin is an electrophoretically homogeneous compound. By ultracentrifuge studies, it is shown to have a main component with a minimal molecular weight of 250,000. It is soluble in water and chloroform and is extracted quantitatively from the tissue with chloroform-methanol mixtures (1). Chemically, it can be classified as a lipide, since among its constituents are found fatty acids and sphingosine or a sphingosine-like substance. However, many of its properties are quite different from those of a typical lipide. Strandin is found in gray matter in relatively large concentration; i.e., 6 to 7 mg. per gm. of wet tissue. It is found in white matter at one-tenth its concentration in gray matter and in brain tumors in concentrations larger than in white matter and smaller than in gray matter. In other tissues that have been studied, namely, heart, skeletal muscle, uterus, lung, liver and kidney, strandin is found in very small amounts; i.e.,

253 citations