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Published on: November 8, 2016
Determination of gangliosides in human cerebrospinal fluid by high-performance thin-layer chromatography and direct
1Institute of Clinical Laboratory Diagnostics, Zagreb University School of Medicine, Yugoslavia.
Abstract:
A method for the separation and quantification of a complex ganglioside mixture from a clinically available amount (5 ml) of human cerebrospinal fluid (CSF) is described. After reduction of the CSF volume by ultrafiltration, gangliosides are extracted with methanol/chloroform, then separated and quantified by high performance thin layer chromatography (HPTLC) and direct densitometry. For purification of crude ganglioside extract, the method of choice was microdialysis against water. Recovery for the present method including all methodological steps was 78%. No delective loss of gangliosides was demonstrated. The CSF ganglioside pattern from 'normal' CSF samples resembles that of brain gangliosides, particularly cerebellum gangliosides. Based on chromatographic comparison with standards, the percentages of lipid-bound NeuAc-positive fractions were: GM1 = II3NeuAc-GgOse4Cer (3%), GD3 = II3NeuAc2-Lac-Cer (3%), GD1a = IV3NeuAc,II3NeuAc-GgOse4Cer (15%), X1 (3%), GD1b = II3(NeuAc)2-GgOse4Cer (16%), X2 (3%), GT1b = IV3NeuAc,II3NeuAc2-GgOse4-Cer (41%), and GQ1b = IV3NeuAc2-,II3NeuAc2-GgOse4-Cer (16%). The total ganglioside content varied between 616-944 micrograms/l. Within-run and between-run assay precision (relative standard deviation) for 'normal' pooled CSF ranged from 0.04 to 0.12 for the predominant CSF ganglioside fractions (GD1a, GD1b, GT1b, GQ1b), and from 0.13 to 0.23 for the less pronounced fractions (GM1, GD3).
