This chapter discusses assay of enzymes of lipid metabolism with colored and fluorescent derivatives of natural lipids. Fatty acid containing the colored or fluorescent probe was condensed with cholesterol, or glycerol, or deacylated sphingolipid, or glycerophospholipid. The colored or fluorescent lipid product was then isolated from the reaction mixture and purified by column or by thin-layer chromatography. The yellow substrates are dispersed and incubated with lipase. The reaction is terminated with alkaline ethylene glycol, and the unreacted neutral substrate is extracted into benzene. Acid is then added, the fatty acid produced by enzymatic hydrolysis is extracted into benzene, and its absorbance is determined spectrophotometrically. The activity of the lysosomal sphingomyelinase was determined at pH 5.0 using a solubilized preparation of enzyme from a lysosome-enriched preparation of rat brain, as well as extracts of skin fibroblasts of normal humans and Niemann-Pick patients and of amniotic cells. Fluorescent sphingomyelin and nonfluorescent sphingomyelin are mixed in the molar ratio used in the assay procedure, and the concentration is estimated by determining the phosphorus content.
ASJC Scopus subject areas
- Molecular Biology