A sensitive assay of lysogangliosides using high-performance liquid chromatography.
Lysogangliosides, LGM1, LGM2 and LGM3, each carrying a single sphingoid base (i.e., C18:1, C18:0, C20:1, C20:0), were prepared and a sensitive assay method of these lipids using HPLC was developed. The method involves fluorescence derivatization of the free amino group of the molecule with o-phthalaldehyde, separation of the molecular species of each lysoganglioside using reversed-phase HPLC and assay on the basis of a known amount of one of the lysogangliosides, as the internal standard. Using this method, lysoganglioside can be accurately assayed in the range of 5-1000 pmol. For assay of the lipid in the tissue, crude isolation procedures including extraction of lipids, Folch's partition and DEAE-Sepharose and AG 1-X2 column chromatographies were required before the fluorescence derivatization. In the normal human and the bovine cerebral cortex, 0.4-2.0 pmol/mg protein of LGM1 containing C18:1 and C20:1 sphingosine residues were detected. In the frontal cortex from a patient with Sandhoff disease, an abnormal accumulation (55-78 pmol/mg protein) of LGM2 was noted. Among various molecular species, LGM2 containing C18:1 was the most abundant.
Kobayashi
T
T
Department of Neurology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Goto
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I
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005732:Gangliosides / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry; D012497:Sandhoff Disease / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005732:Gangliosides