On the loss of gangliosides by dialysis

The Gangliosides represent a family of sialic acidcontaining sphingoglycolipids which have ceramide (N‐acyl‐sphingosine) as the basic hydrophobic portion (Svennerholm, 1972). A heteropolysaccharide is glycosidically linked through the primary hydroxyl group of ceramide and the aldehyde of glucose. There are usually three or four different carbohydrates and up to a total of seven in the chain. The isolated gangliosides are soluble both in organic and aqueous solvents presumably because of their content of both hydrophobic and hydrophilic groups. The most commonly employed extraction procedure is based upon their solubility in chloroform‐methanol (2: 1, v/v) and subsequent partitioning into an aqueous methanol phase (FOLCH, LEES and SLOANE‐STANLEY, 1957). This methanolic solution is then dialysed against water to remove small molecular‐weight contaminants. This paper reports the loss of gangliosides upon dialysis when they are present at low concentrations. Gangliosides were prepared from fresh calf brain as previously described (Kanfer, 1969) and contained 25‐25% GMl, as judged by quantitative thin‐layer chromatography (Suzuki, 1964). GM2 was prepared from this material by treatment with neuraminidase as previously described (Kolodny, Brady, Quirk and Kanfer, 1970) and purified by the procedure of Winterbourne (1971). GM2 was isolated from brain tissue of a Tay‐Sachs child provided by Dr. B. Volk. Thin‐layer chromatography was carried out on Analtech Silica Gel G plates (Analtech Co., Wilmington, Del.) with chloroformmethanol‐2.5 N NH4OH (60 : 30 : 8, by vol.) as developing solvent. Total sialic acid was quantified by the resorcinol procedure according to Suzuki (1964). Duplicate known quantities of gangliosides were dissolved in 2 ml of methanol 41 M KCI(1 : 1, v/v) and dialysed overnight in untreated Visking tubing (0.22‐in. dia.; Fisher Scientific Co., Pittsburgh, Pa.; Cat. No. 1000) against a 500‐fold vol. of distilled water at 4°C. The contents of the sacs were removed and the total volume was measured; a 10‐1 5 per cent increase in volume was usually observed. Portions were analysed in duplicate for total sialic acid content, and the remainder was lyophilized for examination by TLC.