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. 1993 Sep 15;294(Pt 3):885–891. doi: 10.1042/bj2940885

Use of pyrenemethyl laurate for fluorescence-based determination of lipase activity in intact living lymphoblastoid cells and for the diagnosis of acid lipase deficiency.

A Nègre-Salvayre 1, A Dagan 1, S Gatt 1, R Salvayre 1
PMCID: PMC1134545  PMID: 8397511

Abstract

Pyrenemethyl laurate (PMLes), a fluorogenic substrate for determining in vitro lipase activity [Nègre, Salvayre, Dagan and Gatt (1989) Biochim. Biophys. Acta 1006, 84-88], has been administered to cultured lymphoblastoid cells from normal subjects and from a patient affected with Wolman disease, which is characterized by a deficiency of lysosomal acid lipase. The intracellular degradation of PMLes was dependent on the mode of administration of the substrate into the cells, and occurred by two separate pathways involving lysosomal and extra-lysosomal hydrolases. PMLes incorporated into LDL was taken up by normal lymphoblastoid cells through the apolipoprotein-B/E-receptor-mediated pathway and degraded in the lysosomal compartment, as suggested by the degradation block in Wolman cells. In contrast, when PMLes dissolved in 2% dimethyl sulphoxide was added directly to the culture medium, its hydrolysis was similar in lymphoblastoid cells from controls and from patients affected with Wolman disease, neutral lipid storage disease or familial hypercholesterolaemia. This suggested that the administered PMLes was degraded by a non-lysosomal enzyme which is not deficient in Wolman cells. This enzyme also differs from the neutral lipase system which is deficient in lymphoblastoid cells from patients with neutral lipid storage disease. When pyrenemethanol was administered directly to the cell culture, it was only poorly acylated and was rapidly released into the culture medium. These results and the fluorescence properties of PMLes ('monomeric' emission in a hydrophobic environment and 'excimeric' emission in a hydrophilic environment) and pyrenemethanol ('monomeric' emission in a hydrophilic environment) allowed us to design a 'direct reading' procedure by monitoring (without any lipid extraction) the fluorescence of intact living cells and that of the culture medium during pulse-chase experiments. This method allowed the direct evaluation of the time course of in situ degradation of PMLes. In pulse-chase experiments with LDL-PMLes, the fluorescence of normal cells decreased relatively rapidly with time whereas the fluorescence of the culture medium increased concomitantly. With Wolman cells, the cellular fluorescence decreased only very slightly, whereas that of the culture medium remained at the basal level; this demonstrates the catabolic block in intact living cells from patients with Wolman disease. In vitro degradation of PMLes indicated the existence of two PMLes-degrading enzymes in lymphoblastoid cell homogenates: one is the acid lipase which is involved in PMLes degradation in the lysosomal compartment (and is deficient in Wolman cells), while the second is a cytoplasmic enzyme (not deficient in Wolman cells).

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Selected References

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  1. Di Donato S., Garavaglia B., Strisciuglio P., Borrone C., Andria G. Multisystem triglyceride storage disease is due to a specific defect in the degradation of endocellularly synthesized triglycerides. Neurology. 1988 Jul;38(7):1107–1110. doi: 10.1212/wnl.38.7.1107. [DOI] [PubMed] [Google Scholar]
  2. Esterbauer H., Dieber-Rotheneder M., Waeg G., Striegl G., Jürgens G. Biochemical, structural, and functional properties of oxidized low-density lipoprotein. Chem Res Toxicol. 1990 Mar-Apr;3(2):77–92. doi: 10.1021/tx00014a001. [DOI] [PubMed] [Google Scholar]
  3. Goldstein J. L., Brown M. S. Binding and degradation of low density lipoproteins by cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. J Biol Chem. 1974 Aug 25;249(16):5153–5162. [PubMed] [Google Scholar]
  4. Goldstein J. L., Dana S. E., Faust J. R., Beaudet A. L., Brown M. S. Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease. J Biol Chem. 1975 Nov 10;250(21):8487–8495. [PubMed] [Google Scholar]
  5. HAVEL R. J., EDER H. A., BRAGDON J. H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955 Sep;34(9):1345–1353. doi: 10.1172/JCI103182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ho Y. K., Brown M. S., Kayden H. J., Goldstein J. L. Binding, internalization, and hydrolysis of low density lipoprotein in long-term lymphoid cell lines from a normal subject and a patient with homozygous familial hypercholesterolemia. J Exp Med. 1976 Aug 1;144(2):444–455. doi: 10.1084/jem.144.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Morand O., Fibach E., Dagan A., Gatt S. Transport of fluorescent derivatives of fatty acids into cultured human leukemic myeloid cells and their subsequent metabolic utilization. Biochim Biophys Acta. 1982 Jun 11;711(3):539–550. doi: 10.1016/0005-2760(82)90070-4. [DOI] [PubMed] [Google Scholar]
  9. Negre-Salvayre A., Abouakil N., Lombardo D., Salvayre R. Hydrolysis of fluorescent pyrene-acyl esters by human pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase. Lipids. 1990 Aug;25(8):428–434. doi: 10.1007/BF02538084. [DOI] [PubMed] [Google Scholar]
  10. Negre-Salvayre A., Lopez M., Levade T., Pieraggi M. T., Dousset N., Douste-Blazy L., Salvayre R. Ultraviolet-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cells. II. Uptake and cytotoxicity of ultraviolet-treated LDL on lymphoid cell lines. Biochim Biophys Acta. 1990 Aug 6;1045(3):224–232. doi: 10.1016/0005-2760(90)90124-g. [DOI] [PubMed] [Google Scholar]
  11. Negre A. E., Salvayre R. S., Dagan A., Gatt S. New fluorometric assay of lysosomal acid lipase and its application to the diagnosis of Wolman and cholesteryl ester storage diseases. Clin Chim Acta. 1985 Jun 30;149(1):81–88. doi: 10.1016/0009-8981(85)90276-1. [DOI] [PubMed] [Google Scholar]
  12. Negre A., Salvayre R., Durand P., Lenoir G., Douste-Blazy L. Enzyme studies on Epstein-Barr virus-transformed lymphoid cell lines from Wolman's disease. Lipases, cholesterol esterase and 4-methylumbelliferyl acyl ester hydrolases. Biochim Biophys Acta. 1984 Jun 6;794(1):89–95. doi: 10.1016/0005-2760(84)90301-1. [DOI] [PubMed] [Google Scholar]
  13. Negre A., Salvayre R., Vuillaume M., Durand P., Douste-Blazy L. Acid lipase and carboxylesterases in EBV-transformed lymphoid cell line from Wolman's disease: influence of fatty acid structure of substrate. Enzyme. 1984;31(4):241–246. doi: 10.1159/000469533. [DOI] [PubMed] [Google Scholar]
  14. Nègre-Salvayre A., Salvayre R. Protection by Ca2+ channel blockers (nifedipine, diltiazem and verapamil) against the toxicity of oxidized low density lipoprotein to cultured lymphoid cells. Br J Pharmacol. 1992 Nov;107(3):738–744. doi: 10.1111/j.1476-5381.1992.tb14516.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nègre A., Dagan A., Gatt S. Pyrene-methyl lauryl ester, a new fluorescent substrate for lipases: use for diagnosis of acid lipase deficiency in Wolman's and cholesteryl ester storage diseases. Enzyme. 1989;42(2):110–117. doi: 10.1159/000469017. [DOI] [PubMed] [Google Scholar]
  16. Nègre A., Salvayre R., Dagan A., Borrone C., Gatt S. New spectrophotometric assays of acid lipase and their use in the diagnosis of Wolman and cholesteryl ester storage diseases. Anal Biochem. 1985 Mar;145(2):398–405. doi: 10.1016/0003-2697(85)90380-x. [DOI] [PubMed] [Google Scholar]
  17. Nègre A., Salvayre R., Dagan A., Gatt S. Pyrenemethyl laurate, a new fluorescent substrate for continuous kinetic determination of lipase activity. Biochim Biophys Acta. 1989 Nov 6;1006(1):84–88. doi: 10.1016/0005-2760(89)90326-3. [DOI] [PubMed] [Google Scholar]
  18. Nègre A., Salvayre R., Dousset N., Rogalle P., Dang Q. Q., Douste-Blazy L. Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice. Biochim Biophys Acta. 1988 Nov 25;963(2):340–348. doi: 10.1016/0005-2760(88)90300-1. [DOI] [PubMed] [Google Scholar]
  19. Nègre A., Salvayre R., Douste-Blazy L. Lipases et cholestérol estérases acides: maladie de Wolman et "cholesteryl ester storage disease" (polycorie cholestérolique de L'adulte). Pathol Biol (Paris) 1988 Feb;36(2):167–181. [PubMed] [Google Scholar]
  20. Nègre A., Salvayre R., Rogalle P., Dang Q. Q., Douste-Blazy L. Acyl-chain specificity and properties of cholesterol esterases from normal and Wolman lymphoid cell lines. Biochim Biophys Acta. 1987 Mar 13;918(1):76–82. doi: 10.1016/0005-2760(87)90011-7. [DOI] [PubMed] [Google Scholar]
  21. Patrick A. D., Lake B. D. Deficiency of an acid lipase in Wolman's disease. Nature. 1969 Jun 14;222(5198):1067–1068. doi: 10.1038/2221067a0. [DOI] [PubMed] [Google Scholar]
  22. Price P., McMillan T. J. Use of the tetrazolium assay in measuring the response of human tumor cells to ionizing radiation. Cancer Res. 1990 Mar 1;50(5):1392–1396. [PubMed] [Google Scholar]
  23. Radom J., Salvayre R., Levade T., Douste-Blazy L. Influence of chain length of pyrene fatty acids on their uptake and metabolism by Epstein-Barr-virus-transformed lymphoid cell lines from a patient with multisystemic lipid storage myopathy and from control subjects. Biochem J. 1990 Jul 1;269(1):107–113. doi: 10.1042/bj2690107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Radom J., Salvayre R., Maret A., Nègre A., Douste-Blazy L. Metabolism of 1-pyrenedecanoic acid and accumulation of neutral fluorescent lipids in cultured fibroblasts of multisystemic lipid storage myopathy. Biochim Biophys Acta. 1987 Jul 31;920(2):131–139. doi: 10.1016/0005-2760(87)90252-9. [DOI] [PubMed] [Google Scholar]
  25. Radom J., Salvayre R., Negre A., Douste-Blazy L. Metabolism of pyrenedecanoic acid in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and from a patient with multisystemic lipid storage myopathy. Biochim Biophys Acta. 1989 Sep 25;1005(2):130–136. doi: 10.1016/0005-2760(89)90178-1. [DOI] [PubMed] [Google Scholar]
  26. Radom J., Salvayre R., Negre A., Maret A., Douste-Blazy L. Metabolism of neutral lipids in cultured fibroblasts from multisystemic (or type 3) lipid storage myopathy. Eur J Biochem. 1987 May 4;164(3):703–708. doi: 10.1111/j.1432-1033.1987.tb11183.x. [DOI] [PubMed] [Google Scholar]
  27. Roberts D. C., Miller N. E., Price S. G., Crook D., Cortese C., La Ville A., Masana L., Lewis B. An alternative procedure for incorporating radiolabelled cholesteryl ester into human plasma lipoproteins in vitro. Biochem J. 1985 Feb 15;226(1):319–322. doi: 10.1042/bj2260319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Salvayre R., Negre A., Maret A., Lenoir G., Douste-Blazy L. Separation and properties of molecular forms of alpha-galactosidase and alpha-N-acetylgalactosaminidase from blood lymphocytes and lymphoid cell lines transformed by Epstein-Barr virus. Biochim Biophys Acta. 1981 Jun 15;659(2):445–456. doi: 10.1016/0005-2744(81)90070-x. [DOI] [PubMed] [Google Scholar]
  29. Salvayre R., Negre A., Maret A., Radom J., Douste-Blazy L. Extracellular origin of the lipid lysosomal storage in cultured fibroblasts from Wolman's disease. Eur J Biochem. 1987 Dec 30;170(1-2):453–458. doi: 10.1111/j.1432-1033.1987.tb13721.x. [DOI] [PubMed] [Google Scholar]
  30. Salvayre R., Nègre A., Radom J., Douste-Blazy L. Independence of triacylglycerol-containing compartments in cultured fibroblasts from Wolman disease and multisystemic lipid storage myopathy. FEBS Lett. 1989 Jun 19;250(1):35–39. doi: 10.1016/0014-5793(89)80679-9. [DOI] [PubMed] [Google Scholar]
  31. WOLMAN M., STERK V. V., GATT S., FRENKEL M. Primary familial xanthomatosis with involvement and calcification of the adrenals. Report of two more cases in siblings of a previously described infant. Pediatrics. 1961 Nov;28:742–757. [PubMed] [Google Scholar]

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