Determination of glucocerebroside, sphingomyelin, free fatty acid and total lipid by thin-layer chromatography and charring-scintillation quenching

Abstract

A previously described method has been extended to various specific lipids of liver and brain. The basic method involves thin-layer chromatography followed by charring to reveal the bands. The intensity of each band is determined by suspending the silica gel in a radioactive scintillation gel and measuring the optically quenched activities. The lipids are extracted with hexane/isopropanol and, in the case of total lipid determinations, the extract is simply applied to a silica gel plate and charred without use of a development step. For brain cerebroside, the extract is applied to the plate and developed in the usual way. For liver cerebroside, the dried lipid extract is fractionated with a silica gel column to purify the glycolipid, which is then purified further by development with a plate. For sphingomyelin the ester type lipids in the extract are cleaved by alkali for 1 min and the resultant lipids are applied directly to the thin-layer plate. Free fatty acids are chromatographed and measured after a preliminary solvent partitioning to remove most lipids. The method is useful for samples of 5-40 [mu]g. Methods for quantitative application of samples to plates are described. A modification of the Camag sample streaker is described which yields precise 1-cm streaks.