LIPASE-BASED QUANTITATION OF TRIACYLGLYCEROLS IN CELLULAR LIPID EXTRACTS - REQUIREMENT FOR PRESENCE OF DETERGENT AND PRIOR SEPARATION BY THIN-LAYER CHROMATOGRAPHY

A protocol, based on the use of Pseudomonas lipase, is presented to me asure quantitatively the amount of triacylglycerols in extracts from c ultured cells or tissues. Since the lipase also acts on di-and monoacy lglycerols, separation of the extracts by thin-layer chromatography is recommended. In order to allow the lipase-catalyzed hydrolysis to pro ceed efficiently, lipid extracts or eluates from silica scrapings were mixed with the detergent Thesit [dodecylpoly(ethylene glycol ether)], prior to drying. After dissolution of the dried residues in water, th e amount of triacylglycerols was quantified using Pseudomonas sp. lipa se, glycerol kinase, glycerol-phosphate oxidase, and peroxidase. The a ctivity of the latter enzyme was followed either colorimetrically in t he presence of 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic a cid or fluorimetrically in the presence of homovanillic acid.