The quantitative analysis of mixtures of glycosphingolipids by acetylation with radioactive acetic anhydride and separation by thin-layer chromatography is described. The method was primarily developed for screening of glycosphingolipid composition of tissues, cultured cells, and virus preparations. The sensitivity and linearity of the method is demonstrated. Although theoretically a much higher sensitivity is achievable, in practice about 10m9 g of glycosphingolipid can be detected and tentatively identified. As an example, the acetylation pattern of lipids from a preparation of herpes simplex virus is demonstrated.
Glycosphingolipid
analysis is frequently based upon quantitation of the constituent monosaccharides, either by calorimetry (l-5), gas chromatography (6,7), or enzymatic procedures (8,9). They can also be quantitated by analysis of the sphingosine (10) or fatty acid (11) moieties.
Determination of hydroxyl groups by acetylation is a well-established method (12)(13)(14)(15)(16)(17). By the use of radioactive acetyl groups (18-21) it has been made more sensitive. No application of this procedure for glycosphingolipids and oligosaccharides seems to have been made, although other radioisotopic methods have been applied (22)(23)(24)(25).
The purpose of this paper is to demonstrate the usefulness of the sensitive radioderivatization technique in the glycosphingolipid field. The method was based on the procedure of Saito and Hakomori (26) for the isolation of glycosphingolipids as their acetylated derivatives. After acetylation, glycosphingolipids were purified from phospholipids by chromatography on Florisil columns. Alternatively, they could be directly chromatographed on thin-layer plates coated with silica gel-Florisil. Radioacetylation is a nondestructive alternative to densitometric quantitation. In contrast to most thin-layer densitometric procedures, the peak