QUANTITATIVE DETERMINATION OF
The estimation of the total amount of glycolipids in a tissue extract must be estimated after a specific chromatographic separation isolating glycolipids present in the extract. Thus, a prior isolation of the whole glycolipid fraction must be carried out on a silicic acid column. The general procedure involving an acetone elution, as previously described, is recommended. The isolated fraction contains not only glycoglycerolipids but also steryl glycosides and glycosphingolipids.
As only glycoglycerolipids may be hydrolyzed in alkaline conditions, their amount is estimated from the amounts of sugar or fatty acids measured before and after a mild saponification.
These determinations may be also done on purified chromatographic fractions obtained by TLC or column chromatography.
Determination of the total sugar residues
1 – Method for sugar amounts higher than 10 mg per sample
Dry lipids are heated at 80°C for 20 min with 2 ml of a solution of orcinol (5-methylresorcinol) (2 mg/ml of 70% sulfuric acid, v/v). On cooling, the absorbance of the solution is measured at 505 nm. A blank sample is analyzed simultaneously. The amount of sugar is read from a calibration curve prepared by performing the reaction on known amounts of glucose (up to 40 mg from an aqueous solution containing 5 mg/ml of sugar).
The same procedure may be used with glycolipid fractions separated by TLC. Add 3 ml of orcinol solution to the scraped silica gel and vortex thoroughly before and after warming. Centrifuge the tubes and measure the absorbance of the supernatant.
2 – Method for sugar amounts lower than 10 mg per sample
We suggest the microdetection procedure using 5-hydroxy-1-tetralone which forms a fluorescent product (Momose T et al., Talanta 1959, 3, 151).
To aliquots containing 1 to 5mg of glucose as standards, to dried glycolipid samples (containing 1 to 10 mg of glycolipids) or to scraped silica gel spots from TLC plates, 0.1 ml water is added. It must be noted that we could only use Whatman TLC plates, other brands being unreliable. Similar restrictions were reported when running the reaction on TLC plates followed by fluorometric detection (Watanabe K et al., J Lipid Res 1995, 36, 1848).
Under ice cooling, 0.4 ml of reagent solution (25 mg of hydroxytetralone in 100 ml of conc. sulfuric acid, stored at 4°C) is added to each tube. The mixture is heated either at 120°C for 10 min or at 100°C for 40 min.
The reaction mixture is then diluted with 1.5 ml of water and the fluorescence intensity is measured (exc/em: 490 nm/530 nm) after cooling at room temperature in the dark (about 15 min). The tubes containing silica gel are strongly vortexed for 1 min after addition of water and after centrifugation the supernatant is collected for fluorescence measurement.
As difference of intensity is seen with various glycolipids, the use of purified standards closely related to the samples studied is recommended.
Determination of glycoglycerolipids
An aliquot of the total glycolipid fraction is mildly saponified as previously described for the preparation of ceramide-containing lipids. The water soluble sugar moieties from the glycoglycerolipids are thus eliminated in the supernatant and only the steryl glycosides and glycosphingolipids remain in the lower chloroform solution.
The amount of glycoglycerolipids is calculated as the difference between the sugar content of the intact extract and the saponified extract.
The direct determination of the sugar moieties of glycoglycerolipids can also be determined on the aqueous supernatant after drying by lyophilisation.
As the molar proportions of the sugar residues in a given glycolipid or in an extract may not be known, the procedure is not definitive. The measurement of the fatty acid content is thus required to provide an accurate estimation of the amount of glycoglycerolipid present in a crude sample or in a chromatographic fraction.
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.
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.
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).