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Journal of Lipid Research, Vol. 45, 574-581, March 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Methods

Microwave-mediated analysis for sugar, fatty acid, and sphingoid compositions of glycosphingolipids

Saki Itonori^1,*, Masato Takahashi^*, Tomonori Kitamura^*, Kazuhiro Aoki, John T. Dulaney and Mutsumi Sugita^*

^* Department of Chemistry, Faculty of Liberal Arts and Education, Shiga University, Hiratsu, Otsu, Shiga 520-0862, Japan
Department of Applied Molecular Biology, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
Department of Medicine, Division of Nephrology, The University of Tennessee, Court Avenue, Memphis TN 38163

¹ To whom correspondence should be addressed. e-mail: itonori{at}sue.shiga-u.ac.jp

For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of by-products, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH₃OH for 2 min followed by 1 M HCl/CH₃OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of by-products such as the O-methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N-acetylated and remethanolyzed for 30 s by microwave exposure.

Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.

Abbreviations: CMS, ceramide monosaccharide; CTS, ceramide trisaccharide; ganglioside GM1, Galß1-3GalNAcß1-4(NeuAc2-3) Galß1-4Glcß1-1Cer; GC, gas-liquid chromatography; GC-MS, gas-liquid chromatography-mass spectrometry; Glc, glucose; GlcNAc, N-acetylglucosamine; HexNAc, N-acetylhexosamine; lyso-CMS, Galß1-1sphingosine; lyso-CTS, GlcNAcß1-3Manß1-4Glcß1-1sphingosine; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Man, mannose; TMS, trimethylsilyl

Supplementary key words acid hydrolysis • alkaline hydrolysis • lysoglycosphingolipid

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