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.
