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Simultaneous quantitation of ceramides and 1,2‐diacylglycerol in tissues by latroscan thin‐layer chromatography‐flame‐ionization detection
Author(s) -
Okumura Kenji,
Hayashi Kazunori,
Morishima Itsuro,
Murase Kichiro,
Matsui Hideo,
Toki Yukio,
Ito Takayuki
Publication year - 1998
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-998-0237-3
Subject(s) - chromatography , glyceride , chemistry , diacylglycerol kinase , thin layer chromatography , ceramide , flame ionization detector , acetone , silicic acid , lipidology , elution , sphingomyelin , solvent , chloroform , gas chromatography , membrane , biochemistry , fatty acid , organic chemistry , apoptosis , protein kinase c , enzyme
Ceramides and 1,2‐diacylglycerol have been demonstrated in intracellular signaling pathways. A method of simultaneous mass determination of ceramides and 1,2‐diacylglycerol in tissues was developed using the latroscan which combines thin layer chromatography and flame ionization detection (TLC/FID) techniques. Because of relatively low amounts of these components in tissues, the fraction of nonpolar lipids, which included ceramides and glycerides, was eluted with chloroform/acetone mixture (3∶1, vol/vol) through a silicic acid column to eliminate the polar phospholipids. Development of Chromarods was carried out using three solvent systems in a four‐step development technique. The relationship of the peak area ratio to weight ratio compared with cholesteryl acetate added as an internal standard was linear. The amount of ceramides increased with incubation of rat heart homogenate and human erythrocyte membranes in the presence of sphingomyelinase (E.C. 3.1.4.12). The latroscan TLC/FID system provided a quick and reliable assessment of ceramides and 1,2‐diacylglycerol.