Skip to main content
SpringerLink
Log in

Lipase-based quantitation of triacylglycerols in cellular lipid extracts: Requirement for presence of detergent and prior separation by thin-layer chromatography

  • Method
  • Published:
Lipids

Abstract

A protocol, based on the use of Pseudomonas lipase, is presented to measure quantitatively the amount of triacylglycerols in extracts from cultured cells or 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 scraping 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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AAP:

4-aminoantipyrine

LNCaP:

lymph node carcinoma of the prostate

TAG:

triacylglycerol

TBHB:

2,4,6-tribromo-3-hydroxybenzoic acid

TLC:

thin-layer chromatography

References

  1. Swinnen, J.V., Van Veldhoven, P.P., Esquenet, M., Heyns, W., and Verhoeven, G. (1996) Androgens Markedly Stimulate the Accumulation of Neutral Lipids in the Human Prostatic Adenocarcinoma Cell Line LNCaP, Endocrinology 137, 4468–4474.

    Article  PubMed  CAS  Google Scholar 

  2. Fossatu, P., and Prencipe, L. (1982) Serum Triglycerides Determined Colorimetrically with an Enzyme That Produces Hydrogen Peroxide, Clin. Chem. 28, 2077–2080.

    Google Scholar 

  3. McGowan, M.W., Artiss, J.D., Strandbergh, D.R., and Zak, B. (1983) A Peroxidase-Coupled Method for the Colorimetric Determination of Serum Triglycerides, Clin. Chem. 29, 538–542.

    PubMed  CAS  Google Scholar 

  4. Nägele, U., Hägele, E.O., Sauer, G., Wiedermann, E., Lehmann, P., Wahlefeld, A.W., and Gruber, W. (1984) Reagent for the Enzymatic Determination of Serum Total Triglycerides with Improved Lipolytic Efficiency, J. Clin. Chem. Clin. Biochem. 22, 165–174.

    PubMed  Google Scholar 

  5. Van Veldhoven, P.P., and Bell, R.M. (1988) Effect of Harvesting Methods, Growth Conditions and Growth Phase on Diacylglycerol Levels in Cultured Human Adherent Cells, Biochim. Biophys. Acta 959, 185–196.

    PubMed  Google Scholar 

  6. Van Veldhoven, P.P., Croes, K., Casteels, M., and Mannaerts, G.P. (1997) 2-Methylacyl-CoA Racemase: A Coupled Assay Based on the Use of Pristanoyl-CoA Oxidase/Peroxidase and Reinvestigation of Its Subcellular Distribution in Rat and Human Liver, Biochim. Biophys. Acta 1347, 62–68.

    PubMed  Google Scholar 

  7. Van Veldhoven, P.P. (1995) Activity Measurements of Acyl-CoA Oxidases in Human Liver, J. Inher. Metab. Dis. 18 (Suppl. 1), 125–134.

    Article  PubMed  Google Scholar 

  8. Nägele, U., Wahlefeld, A.-W., and Ziegenhorn, J. (1983) Triglycerides—Colorimetric Methods, Methods in Enzymatic Analysis (3rd Edition) VIII, 1–12 VCN Weinheim, Germany.

    Google Scholar 

  9. Trinder, P., and Webster, D. (1984) Determination of HDL-Cholesterol Using 2,4,6-Tribromo-3-Hydroxybenzoic Acid with a Commercial CHOD-PAP Reagent, Ann. Clin. Biochem. 21, 430–433.

    PubMed  CAS  Google Scholar 

  10. Guilbault, G., Kramer, D.N., and Hackley, E. (1967) New Substrate for Fluorimetric Determination of Oxidative Enzymes, Anal. Chem. 39, 271.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000, Leuven, Belgium

    Johannes V. Swinnen, Murielle Esquenet & Guido Verhoeven

  2. Afd. Farmakologie, K.U. Leuven-Campus Gasthuisberg, Herestraat, B-3000, Leuven, Belgium

    Paul P. Van Veldhoven

Authors
  1. Paul P. Van Veldhoven
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Johannes V. Swinnen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Murielle Esquenet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guido Verhoeven
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paul P. Van Veldhoven.

About this article

Cite this article

Van Veldhoven, P.P., Swinnen, J.V., Esquenet, M. et al. Lipase-based quantitation of triacylglycerols in cellular lipid extracts: Requirement for presence of detergent and prior separation by thin-layer chromatography. Lipids 32, 1297–1300 (1997). https://doi.org/10.1007/s11745-006-0166-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-006-0166-1

Keywords

Search

Navigation