Extraction gangliosides

In order to extract lipids from tissues, it is necessary to find solvents which will not only dissolve the lipids readily but will overcome the interactions between the lipids and the tissue matrix. Various solvents or solvent combinations have been suggested as extractants for lipids, and currently some interest is being shown in isopropanol-hexane (2 3 by volume), because its toxicity is relatively low [355,743], but it does not yet appear to have been tested with a sufficiently wide range of tissues. It does not extract gangliosides quantitatively. Most lipid anaiysts use chloroform-methanol (2 1 by volume), with the endogenous water in the tissue as a ternary component of the system, to extract iipids from animal, plant and bacterial tissues. Usually, the tissue is homogenized in the presence of both soivents, but better results may be obtained if the tissue is first extracted with methanol alone before the chloroform is added to the mixture. With difficult samples, more than one extraction may be needed, and with lyophilised tissues, it may be necessary to re-hydrate prior to carrying out the extraction. The... 

In Table V reference is presented for various sources, from which gangliosides have been extracted. Gangliosides are widely distributed in different organs and it is diflBcult to draw any conclusions from their localization as to a possible specific physiological significance of this group of glycosphingolipids. 

Partially purified brain gangliosides Chloroform-methanol-extracted >500... 

Included among drugs that alter the permeability of the membrane are phosphatidylserine, S-adenosylmethionine, and ganglioside extracts. Phosphatidylserine, produced from purified extracts of bovine brain cortex, alters the permeability and functionality of the neuronal membrane. A study... 

The muscles were freed by gross dissection of extraneous tissue which was mainly fat and peripheral nerves, and then stored at -40°C. For an experiment, approximately 1 kg tissue was macerated by a meat grinder and homogenized in ten volumes of tetrahydrofuran 0.01 M KC1 (4 1, v/v), stirred for 3 hours, and filtered through a Buchner funnel. The extraction was repeated twice and the filtrates then combined and concentrated in a rotary evaporator. One liter of chloroform-methanol (2 1, v/v) was added to the lipid extract which has the appearance and consistency of syrup. Gangliosides were partitioned into the upper layer by the addition of 200 ml of water (11) and the lower layer extracted two additional times with theoretical upper phase containing 0.027% KC1. The combined upper layers were then concentrated and dialyzed exhaustively at 4°C with five changes of distilled water. 

Buffered Tetrahydrofuran. In 1973, Tettamanti et al. [19) described an improved procedure for the extraction, separation and purification of brain gangliosides. In this method, the brain tissue was subjected to homogenization and extraction with buffered [potassium phosphate buffer, pH 6.8) tetrahydrofuran. Following centrifugation, diethyl ether was added and the mixture separated into organic and aqueous phase. The gangliosides, recovered exclusively in the aqueous phase, were then freed of residual phospholipids and other minor contaminants [i.e. peptides)by column chromatography on silica gel.This procedure, as shown by the authors,was superior to the commonly used chloroform/methanol... 

Extracts from —3 X 107 cells and a mixture of GM, and GMg were compared by TLC after staining with resorcinol. To improve sensitivity of technique, the spray was applied heavily, giving rise to some nonspecific staining. Only those bands that reproducibly stained the characteristic blue of gangliosides are bracketed. 

Glycolipid analysis. Gangliosides were extracted with chloroform-methanol (2 1), purified on Sephadex G-25 columns and separated into individual species by thin-layer chromatography as described previously (12). 

Since interferons appear to be species-specific, we investigated whether the ganglioside fraction from mouse brain was more potent in inhibiting antiviral activity of mouse fibroblast interferon than that obtained from heterologous brain extracts. As seen in Figure 1 bovine brain gangliosides were almost as potent... 

Preparation Of Gangliosides. The pooled tissues were homogenized in 7 volumes of cold acetone to remove neutral lipids and water. Gangliosides were extracted from the acetone-dried powder with 3 volumes of chloroform/... 

Germany). Dialyzed upper phase samples of brain and lymphoma cells with from 30,000 to 50,000 cpm were used for each TLC plate. All radiolabeled samples were chromatographed in parellel with mouse brain ganglioside standards extracted as described above. 

Gangliosides labeled by intracranial injection of l-t(C]ManNAc were extracted from 1 g of brain, applied with solvent 1 (vertically) and solvent 2 (horizontally). Assayed fractions indicated by numbers (and ganglioside abbreviations) correspond to the PFC assay results in Figure 4. Fraction 6 refers to the area surrounding all the assayed spots (15). 

Suzuki, K. (1965) The pattern of mammalian brain gangliosides—II. Evaluation of the extraction procedures, post-mortem changes and the effect of formalin preservation, J. Neurochem. 12, 629-638. 

The GM2 ganglioside (14) is an example of a carbohydrate antigen, specific for melanoma, sarcoma and kidney carcinoma. It could suit the purpose of an immuno therapy with monoclonal antibodies. In previous examinations the used GM2 was extracted from human and animal tissues. In this, one ran the risk of biological contamination of the GM2, which could influence the results of an examination. For this reason R. R. Schmidt et al. developed a total synthesis of the GM2 ganglioside to sustain sufficient substance for further immunological examinations without faking artifacts. ... 

Extraction parameters such as solvent type, mixture ratios, metal ion concentration, pH of the aqueous phase, extraction time, and temperature influence the recovery of extracted lipids and must be validated to ensure reliable results. For example, the recovery of the acidic lipids PA and phosphatidylglycerol (PG) can be less than 30% in classic Folch and Bligh Dyer extraction, where these lipids can become bound to proteins tightly (17). Lipids bound to proteins covalently are only released under appropriate conditions, which depend on the type of lipid-protein linkage. For example, ceramides bound to protein of the comi-fled envelop in the human skin (18) can be extracted after mild alkaline hydrolysis of the ester linkage between hpid and protein. Special conditions are required for extraction of more polar lipids such as gangliosides, lysophospholipids and lysosphin-golipids, or phosphatidylinositol-phosphates. 

Gangliosides extracted from bovine brain tissue (Cronassial, Sygen) have been widely used in Western Europe and South America for several neurological disorders. 

In some, if not all of these sialoproteins, the nature of the nonulosaminic acid appears to vary with the species. The mucin extracted from sub-maxillary glands is a common source for isolation of the acids. Human saliva affords IV-acetylneuraminic acid, which is also present in human milk, human-brain gangliosides, and human-serum proteins. 

Burg, J., Banerjee, A., and Sandhoff, K., Activating proteins for ganglioside GM2 degradation by P-hexosaminidase isozymes in tissue extracts from different species. Biol. Chem. Hoppe-Seyler. 366, 887-891 (1985). 

In his early studies, Klenk138 used the solvent-extraction method for the isolation of gangliosides this was later combined with chromatography.158... 

The unusual solubility behavior of, for example, the Tay-Sachs brain tissues is due to qualitative and quantitative differences in the composition of the appropriate lipid.211 The isolation and purification of these anomalous gangliosides were carried out similarly to those for the gangliosides of normal tissues, for example, by the method of Folch and coworkers.182 In this method, the brain tissues are extracted with chloroform-methanol-water, the extract is washed with potassium chloride solution, and the extracted material is separated on a column of silicic acid by elution with an increasing concentration of methanol in chloroform. 

Wang W.Q., Gustafson A., Ganglioside extraction from erythrocytes a comparison study, Acta Chemica Scandinavica 49 (1995) 929-936. 

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