Purification of gangliosides by liquid-liquid partition chromatography

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Purification of gangliosides by liquid-liquid partition chromatography ( purification-gangliosides-by-liquid-liquid-partition-chromat )

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Purification of gangliosides by liquid-liquid partition chromatography Tomohisa Kato*,† and Kenichi Hatanaka1,§ Japan Chemical Innovation Institute,* 1-3-5 Jimbocho, Chiyoda-ku, Tokyo 101-0051, Japan; Kaneka Corporation,† 3-2-4, Nakanoshima, Kita-ku, Osaka 530-8288, Japan; and Institute of Industrial Science,§ The University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153-8505, Japan Abstract A liquid-liquid partition chromatographic tech- nique was applied to separate amphiphilic glycolipids. A two-phase solvent system composed of n-butanol-t-butyl methyl ether-acetonitrile-water at a volume ratio of 3:1:1:5 was found to be suitable for separating the gangliosides from total lipids extracted from rat brain by liquid-liquid partition chromatographic systems, namely centrifugal parti- tion chromatography (CPC) and high-speed counter-current chromatography. GM1 could be separated rapidly by using the upper phase as stationary phase for both systems. Moreover, various kinds of gangliosides (GM1, GD1a, GD1b, GT1b) could be separated individually by using the lower phase as stationary phase by CPC. The sample can be recovered without loss by these systems.—Kato, T. and K. Hatanaka. Purification of gangliosides by liquid-liquid parti- tion chromatography. J. Lipid Res. 2008. 49: 2474–2478. Supplementary key words ganglioside • separation • centrifugal parti- tion chromatograph • counter-current chromatography Gangliosides are acidic glycolipids widely distributed in most vertebrate tissues and body fluids, and are particu- larly abundant in the nervous system. Ganglioside is a sphingoglycolipid that contains one or more sialic acid res- idues (N-acetylneuraminic acid, N-glycolylneuraminic acid) linked to an oligosaccharide chain. In recent years, their various roles in biological functions, such as cell-cell inter- action, cell proliferation and differentiation, cell-substrate interaction, carcinogenesis, bacteria or virus infection, im- mune response, and inflammation, have been reported (1). Isolation and purification of gangliosides from complex lipid mixtures are generally accomplished by preparative TLC (2), HPLC using anion-exchange columns such as DEAE-Sephadex (3, 4), Q-Sepharose (5) and MonoQ (6), or by using a strong anion exchanger cartridge (InertSep SAX) (7). This work was supported by a grant for Development of Novel Diagnostic and Medical Applications through Elucidation of Sugar Chain Functions from the New Energy and Industrial Technology Development Organization. Manuscript received 10 June 2008 and in revised form 21 July 2008. Published, JLR Papers in Press, July 21, 2008. DOI 10.1194/jlr.D800033-JLR200 In this research, we employed centrifugal partition chro- matography (CPC) to separate and purify pseudo-glycolipids that were obtained by the saccharide primer method (8). CPC and high-speed counter-current chromatography (HSCCC) are both liquid-liquid partition chromatographic techniques that have been widely used for separation and purification of natural products (9–13). The difference between CPC and HSCCC is the component (rotor and tubing, respectively), in which liquid-liquid partition is per- formed (11). It is thought that because mixing conditions for these systems are not identical, amphiphilic com- pounds such as glycolipids may be separated differently. These liquid-liquid partition chromatographic techniques have advantages over chromatographic techniques such as normal column chromatography and HPLC using solid support. Because irreversible adsorption does not occur, simple rinsing of the instrument allows full recovery of noneluted compounds. Moreover, pretreatment of samples is unnecessary and purification can be accomplished in a short time. Significantly, liquid-liquid partition chromato- graphic techniques do not require expensive solid support. In this paper, we describe the successful separation and purification of gangliosides (from complex lipid mixtures extracted from rat brain) by two liquid-liquid partition chromatographic systems, CPC and HSCCC. MATERIALS AND METHODS Materials All organic solvents were of analytical grade and were pur- chased from Nacalai Tesque, Kyoto, Japan. Rat brain tissue (Rock- land) was purchased from Funakoshi, Tokyo, Japan. The strong anion exchanger cartridge (InertSep SAX) was purchased from GL Science, Tokyo, Japan. Apparatus CPC was performed using the CPC240 (Sensyu Scientific, To- kyo, Japan). Total rotor volume of this model is 240 ml, and num- 1 To whom correspondence should be addressed. e-mail: hatanaka@iis.u-tokyo.ac.jp Copyright © 2008 by the American Society for Biochemistry and Molecular Biology, Inc. methods 2474 Journal of Lipid Research Volume 49, 2008 This article is available online at http://www.jlr.org Downloaded from www.jlr.org by guest, on June 7, 2018

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