Lipase-based Quantitation of Triacylglycerols in Cellular Lipid Extracts: Requirement for Presence of Detergent and Prior Separation by Thin-Layer Chromatography

Lipids. 1997 Dec;32(12):1297-300. doi: 10.1007/s11745-006-0166-1.

Abstract

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.

MeSH terms

  • Chromatography, Thin Layer
  • Colorimetry
  • Detergents
  • Glycerol Kinase / metabolism
  • Glycerolphosphate Dehydrogenase / metabolism
  • Humans
  • Lipase / metabolism*
  • Peroxidase / metabolism
  • Polidocanol
  • Polyethylene Glycols
  • Pseudomonas / enzymology
  • Rhizopus / enzymology
  • Spectrometry, Fluorescence
  • Triglycerides / analysis*
  • Tumor Cells, Cultured

Substances

  • Detergents
  • Triglycerides
  • Polidocanol
  • Polyethylene Glycols
  • Glycerolphosphate Dehydrogenase
  • L-alpha-glycerol-phosphate oxidase
  • Peroxidase
  • Glycerol Kinase
  • Lipase