SIMULTANEOUS QUANTITATION OF CERAMIDES AND 1,2-DIACYLGLYCEROL IN TISSUES BY LATROSCAN THIN-LAYER CHROMATOGRAPHY FLAME-IONIZATION DETECTION

Ceramides and 1,2-diacylglycerol have been demonstrated in intracellul ar signaling pathways. A method of simultaneous mass determination of ceramides and 1,2-diacylglycerol in tissues was developed using the la troscan which combines thin layer chromatography and flame ionization detection (TLC/FID) techniques. Because of relatively low amounts of t hese components in tissues, the fraction of nonpolar lipids, which inc luded ceramides and glycerides, was eluted with chloroform/acetone mix ture (3:1, vol/vol) through a silicic acid column to eliminate the pol ar phospholipids. Development of Chromarods was carried out using thre e solvent systems in a four-step development technique. The relationsh ip of the peak area ratio to weight ratio compared with cholesteryl ac etate added as an internal standard was linear. The amount of ceramide s increased with incubation of rat heart homogenate and human erythroc yte 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.