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.
FATTY ACID DETERMINATION
The fatty acid moieties of the total glycoglycerolipid fraction may be determined after a mild saponification of the glycolipid fraction isolated from a lipid sample
The total glycolipid fraction is mildly saponified as described above for the analysis of the sugar moiety and fatty acids are extracted from the mixture by 2 hexane washes after acidification with 4M HCl in the presence of phenolphthalein.
After addition of a known amount of C17:0 as an internal standard, the fatty acid extract is methylated as previously described for free fatty acids.
This procedure is also useful to determine the fatty acid profile of a crude glycoglycerolipid fraction or isolated fractions.