SUGAR COMPOSITION  OF
GLYCOLIPIDS

Several procedures were reported for the analysis of the hexoses found in lipid compounds. We give below one of the simplest method used frequently for preliminary studies.

The glycolipid sample is first hydrolyzed with aqueous acid, thus cleaving the glycosidic bonds, and the released hexoses are converted into the corresponding alditols which are acetylated before GLC analysis (Kannan R et al.  J Chromatogr 1974, 92, 95). This procedure is also efficient for the analysis of glycosphingolipids.

Procedure:

The glycolipid sample is treated for one night at 100°C with 1 ml of 1M HCl in water.
The reaction mixture is washed 2 times with 3 ml hexane to remove fatty acids.
The aqueous solution is kept warm and dried under a stream of nitrogen.
A solution of sodium borohydride (30 mg/ml in water) is added to the dry residue and kept for 2 h at room temperature. The reaction is stopped by addition of 2 drops of acetic acid and the solution is evaporated or lyophilized.
Acetic anhydride (0.3 ml) is added and the solution is kept at 100°C for 2 h. After evaporation of the reagents (addition of toluene or ethanol helps the drying), 1 ml of chloroform is added  and washed with 2 ml water.
The alditol acetates are analyzed by GLC with an apolar column and the amount of each hexose is estimated using arabinose as an internal standard.  

Comments :

Another practical approach to the technical problem of the hydrolysis of glycosphingolipids has been described using a one-spot heating in a household microwave oven with 0.1 M NaOH in methanol for 2 min followed by 1M HCl in methanol for 45 s (Itonori S et al., J Lipid Res 2004, 45, 574).

Oligosaccharides released from glycolipids following ceramide glycanase digestion may be analyzed by HPLC after labeling with a fluorescent tag, anthranilic acid (Neville D et al., Anal Biochem 2004, 331, 275).