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 measure quantitatively the amount of triacylglycerols in extracts from cultured cells of tissues. Since the lipase also acts on di- and monoacylglycerols, separation of the extracts by thin-layer chromatography is recommended. In order to allow the lipase-catalyzed hydrolysis to proceed 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, the amount of triacylglycerols was quantified using Pseudomonas sp. lipase, glycerol kinase, glycerol-phosphate oxidase, and peroxidase. The activity of the latter enzyme was followed either colorimetrically in the presence of 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic acid or fluorimetrically in the presence of homovanillic acid.
Van Veldhoven
P P
PP
Katholieke Universiteit Leuven, Belgium. Paul.VanVeldhoven@med.kuleuven.ac.be
Swinnen
J V
JV
Esquenet
M
M
Verhoeven
G
G
D002855:Chromatography, Thin Layer; D003124:Colorimetry; D003902:Detergents; D005991:Glycerol Kinase / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism; D006801:Humans; D008049:Lipase / Q000378:metabolism*; D009195:Peroxidase / Q000378:metabolism; D000077423:Polidocanol; D011092:Polyethylene Glycols; D011549:Pseudomonas / Q000201:enzymology; D012233:Rhizopus / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D014280:Triglycerides / Q000032:analysis*; D014407:Tumor Cells, Cultured
D003902:Detergents; D014280:Triglycerides; D000077423:Polidocanol; D011092:Polyethylene Glycols; D005993:Glycerolphosphate Dehydrogenase; C036140:L-alpha-glycerol-phosphate oxidase; D009195:Peroxidase; D005991:Glycerol Kinase; D008049:Lipase