HPLC analysis of neutral glycolipids: an aid in the diagnosis of lysosomal storage disease

Abstract

Concentrations of GL-la (glucocerebroside) (8.36 nmol/ml), GL-2a (lactosyl-ceramide) (4.03 nmol/ml), GL-3a (globotriosylceramide) (2.25 nmol/ml) and GL-4a (globotetraosylceramide) (2.87 nmol/ml) have been determined in normal plasma and compared to concentrations in the plasma from patients with Gaucher, Krabbe, Fabry, Sandhoff and Tay-Sachs diseases as well as with hypercholesterolemia. HPLC analysis of perbenzoylated glycolipid derivatives (isolated and purified by modification of an existing procedure) was performed on samples equivalent to 50 to 100 μl of plasma. The sensitivity could be readily increased ten-fold. We have employed a novel internal standard-monogalactosyl diglyceride, a plant glycolipid, commercially available in pure form. Analysis was performed on a 5 micron ultrasphere silica column, using a gradient of isopropanol in hexane rather than the more usual dioxane in hexane. Our gradient exhibited an essentially flat baseline precluding the necessity of a reference cell. Recoveries of glycolipids added to plasma (95%), experimental yields (60%) and standard curves are presented and discussed. A method is also presented for the separation of GL-la and monogalactosyl diglyceride derivatives for rapid (8 minute) isocratic analysis of multiple samples from Gaucher patients. The benefits of such a simple, reproducible HPLC technique are discussed.