A high performance liquid chromatography method for the analysis of glycosphingolipids using galactose oxidase/NaB 3H 4 labeling of intact cells and synaptosomes

Abstract

The major objective of this study was to combine an HPLC method with a galactose oxidase/NaB 3H 4 labeling method to allow both a chemical quantitation of individual glycolipids and an analysis of their 3H labeling. Neutral glycolipids in whole cells were oxidized with galactose oxidase, and the resultant aldehydes were radiolabeled by reduction with tritiated sodium borohydride. Gangliosides, oxidized with galactose oxidase, either were reduced while in the native state in the whole cell or were first extracted and then reduced. Tritiated glycolipids were perbenzoylated and separated by HPLC. Ultraviolet detection of the derivatives was at 230 nm. Incorporated radioactivity was determined either by collecting fractions from the HPLC separation and counting on a liquid scintillation spectrometer or with a flow-through counter. The order of the derivatization and reduction is critical. Reduction of glycolipids prior to derivatization yielded sharp uv and radioactive peaks. Perbenzoylation of the oxidized glycolipids prior to reduction yielded multiple uv peaks, a noisy baseline, and broad radioactive peaks which did not always have a corresponding uv peak. The labeled neutral glycolipids were stable at −40°C for at least 14 days, and gangliosides were stable at −15°C for at least 14 days. When samples were stored at 20°C there was a time-dependent decrease in the glycolipid/internal standard uv peak area ratio for GbOse 4 and GbOse 3 apparent by 28 days after perbenzoylation. The distribution of radiolabel among peaks showed no change with time or temperature. We adapted the technique to allow 3H labeling of glycolipids from monolayers of cultured glioma cells and from mouse brain synaptosomes. This method permits simultaneous quantification of individual glycolipids and analysis of their surface exposure.