Bound lipids liberated by alkaline hydrolysis after exhaustive extraction of pulverized clavus

In the present study, covalently bound lipids were found in clavus material and their lipid classes were determined by high-performance thin-layer chromatography (HPTLC). Clavus material, pulverized completely in a Mikro-Dismembrator II, was exhaustively extracted three times with chloroform/methanol (2:1, 1:1 and 1:2 v/v) at 80 degrees C for 1 h each time and this sequence of extractions was repeated to obtain the unbound lipid-free residue which was saponified and then extracted with chloroform. The extract proved to comprise several bands of lipids covalently bound through an ester-like linkage. These were identified as free fatty acids, cholesterol, ceramides and glucocerebrosides by HPTLC. However, omega-hydroxy fatty acids were not detected in the lipids. To analyse the fatty acids amide-linked to the bound ceramides, the latter were isolated by preparative HPTLC and subjected to mild acid hydrolysis. Since the bound ceramides constituted neither omega-hydroxy fatty acids nor alpha-hydroxy fatty acids, they were not identified as hydroxyl-acylsphingosines.