Gas–liquid chromatographic assay of lipid-bound sialic acids: measurement of gangliosides in brain of several species

Abstract

A method is described for analysis of gangliosides by GLC assay of the sialic acid component. Mild acid treatment in methanol converted the latter to methyl ketoside methyl ester, which was then chromatographed as the TMS derivative. The major methanolysis product was shown to be the beta-anomer, and its chromatographic peak was used for quantification. NANA and NGNA could be analyzed simultaneously, while an O-acetylated derivative of NGNA was detected qualitatively. Standard curves were obtained for the three following representative samples: (a) a mixture of beef brain gangliosides, (b) Tay-Sachs ganglioside, and (c) hematoside-NANA. These had different slopes which reflected the variation in yield of beta-NANA obtained from methanolysis. The smallest sample analyzed in the present study contained 0.3 micro g of NANA. The advantage of GLC in solving the problem of false chromogens is illustrated in a comparative study with two colorimetric procedures. Two columns are described whose combined use is highly effective in establishing identity and in eliminating false peaks when they arise. The GLC method has been applied to analysis of the total brain ganglioside content of several species, and a general trend was observed toward decreasing levels in the lower vertebrates. In addition, NGNA was detected and quantified in several of these samples.