Determination of Neutral Glycolipids in Plasma and Erythrocytes of Fabry's Disease Family by High Performance Liquid Chromatography

Abstract

Method of determination of neutral glycosylceramides in human plasma and erythrocytes by high performance liquid chromatography was studied.
Neutral glycosylceramides extracted by chloroform; methanol (2;1) were separated from lipids such as glycerides and phospholipids by 50% methanol inactivated Unisil column and derivatized by 4-nitrobenzoyl chloride in pyridine at 37°C overnight. After excess reagents were removed by solvent extraction, pernitrobenzoylated neutral glycosylceramides were injected onto TSK Silica 60 column (4×250mm) and eluted with linear gradient of n-hexane; methylene chloride; acetonitrile=10; 4; 0.5 to 10; 4; 4 for 20min at a flow rate of 2.0 ml/min at room temperature. Eluent was monitored at 254 nm.
N-Palmitoyl-D, L-dihydro-glucosyl- and lactosyl-cerebroside were obtained as single peaks of their pernitrobenzoylated derivatives. A good calibration curve was obtained by injecting 0.1 to 2.0 μg of the samples. Recoveries of Glc- and Lac-cermides from plasma were 89% and 78 % and those from erythrocytes were 88% and 66%, respectively.
Every glycosyl ceramide in plasma and erythrocytes was separated as multiple peaks. In the family of Fabry's disease, ceramide trihexoside concentrations in plasma and erythrocytes of hemizygote were 3 to 5 times higher than those of normal and heterozygotes diagnosed by α-D-galactosidase activities in leukocytes.
This method will be useful for the biochemical diagnosis of other neutral glycosylceramide and ganglioside storage diseases.