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. 1987 Jul;128(1):141–150.

Immunocytochemical localization of peroxisomal enzymes in human liver biopsies.

J A Litwin, A Völkl, J Müller-Höcker, T Hashimoto, H D Fahimi
PMCID: PMC1899788  PMID: 2886050

Abstract

The immunocytochemical localization of catalase and three enzymes of the peroxisomal lipid beta-oxidation system--acyl-CoA oxidase, the bifunctional protein enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase--in human liver biopsies was investigated by means of light and electron microscopy. The antisera raised against all four enzymes from rat liver cross-reacted with the corresponding proteins in homogenates of human liver as revealed by immunoblotting. For light-microscopic localization in glutaraldehyde-fixed Epon-embedded material, the removal of resin and controlled digestion with trypsin was necessary. At the ultrastructural level specific labeling for all four antigens was found by the protein A-gold technique in peroxisomes of liver parenchymal cells fixed with formaldehyde-low glutaraldehyde concentrations and embedded in Lowicryl K4M. In biopsies fixed with glutaraldehyde and embedded in Epon, treatment with metaperiodate or etching with sodium ethoxide improved the immunolabeling. After such treatment catalase showed the most intense labeling and acyl-CoA oxidase the weakest, the two other proteins exhibiting an intermediate immunoreaction. In material postfixed with osmium only catalase could be visualized in peroxisomes. The immunocytochemical investigation of peroxisomal proteins in human liver biopsies provides a simple and highly promising approach for further elucidation of the pathophysiology of peroxisomal disorders.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altman L. G., Schneider B. G., Papermaster D. S. Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy. J Histochem Cytochem. 1984 Nov;32(11):1217–1223. doi: 10.1177/32.11.6436366. [DOI] [PubMed] [Google Scholar]
  2. Angermüller S., Leupold C., Völkl A., Fahimi H. D. Electron microscopic cytochemical localization of alpha-hydroxyacid oxidase in rat liver. Association with the crystalline core and matrix of peroxisomes. Histochemistry. 1986;85(5):403–409. doi: 10.1007/BF00982670. [DOI] [PubMed] [Google Scholar]
  3. Bendayan M., Reddy J. K. Immunocytochemical localization of catalase and heat-labile enoyl-CoA hydratase in the livers of normal and peroxisome proliferator-treated rats. Lab Invest. 1982 Oct;47(4):364–369. [PubMed] [Google Scholar]
  4. Bendayan M., Reddy M. K., Hashimoto T., Reddy J. K. Immunocytochemical localization of fatty acid metabolizing heat-stable and heat-labile enoyl-coenzyme A (CoA) hydratases in liver and renal cortex. J Histochem Cytochem. 1983 Apr;31(4):509–516. doi: 10.1177/31.4.6338108. [DOI] [PubMed] [Google Scholar]
  5. Bendayan M., Zollinger M. Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J Histochem Cytochem. 1983 Jan;31(1):101–109. doi: 10.1177/31.1.6187796. [DOI] [PubMed] [Google Scholar]
  6. Bronfman M., Inestrosa N. C., Nervi F. O., Leighton F. Acyl-CoA synthetase and the peroxisomal enzymes of beta-oxidation in human liver. Quantitative analysis of their subcellular localization. Biochem J. 1984 Dec 15;224(3):709–720. doi: 10.1042/bj2240709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dell'Orto P., Viale G., Colombi R., Braidotti P., Coggi G. Immunohistochemical localization of human immunoglobulins and lysozyme in epoxy-embedded lymph nodes: effect of different fixatives and of proteolytic digestion. J Histochem Cytochem. 1982 Jul;30(7):630–636. doi: 10.1177/30.7.6179982. [DOI] [PubMed] [Google Scholar]
  8. Fahimi H. D. Cytochemical localization of peroxidatic activity of catalase in rat hepatic microbodies (peroxisomes). J Cell Biol. 1969 Nov;43(2):275–288. doi: 10.1083/jcb.43.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goldfischer S., Collins J., Rapin I., Coltoff-Schiller B., Chang C. H., Nigro M., Black V. H., Javitt N. B., Moser H. W., Lazarow P. B. Peroxisomal defects in neonatal-onset and X-linked adrenoleukodystrophies. Science. 1985 Jan 4;227(4682):67–70. doi: 10.1126/science.3964959. [DOI] [PubMed] [Google Scholar]
  10. Goldfischer S., Moore C. L., Johnson A. B., Spiro A. J., Valsamis M. P., Wisniewski H. K., Ritch R. H., Norton W. T., Rapin I., Gartner L. M. Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome. Science. 1973 Oct 5;182(4107):62–64. doi: 10.1126/science.182.4107.62. [DOI] [PubMed] [Google Scholar]
  11. Goldfischer S., Reddy J. K. Peroxisomes (microbodies) in cell pathology. Int Rev Exp Pathol. 1984;26:45–84. [PubMed] [Google Scholar]
  12. Hajra A. K., Bishop J. E. Glycerolipid biosynthesis in peroxisomes via the acyl dihydroxyacetone phosphate pathway. Ann N Y Acad Sci. 1982;386:170–182. doi: 10.1111/j.1749-6632.1982.tb21415.x. [DOI] [PubMed] [Google Scholar]
  13. Hand A. R. Cytochemical detection of peroxisomal oxidases. J Histochem Cytochem. 1979 Oct;27(10):1367–1370. doi: 10.1177/27.10.92494. [DOI] [PubMed] [Google Scholar]
  14. Hashimoto T. Individual peroxisomal beta-oxidation enzymes. Ann N Y Acad Sci. 1982;386:5–12. doi: 10.1111/j.1749-6632.1982.tb21403.x. [DOI] [PubMed] [Google Scholar]
  15. Jacobsen M., Clausen P. P., Smidth S. The effect of fixation and trypsinization on the immunohistochemical demonstration of intracellular immunoglobulin in paraffin embedded material. Acta Pathol Microbiol Scand A. 1980 Nov;88(6):369–376. doi: 10.1111/j.1699-0463.1980.tb02508.x. [DOI] [PubMed] [Google Scholar]
  16. Kase F., Björkhem I., Pedersen J. I. Formation of cholic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid by rat liver peroxisomes. J Lipid Res. 1983 Dec;24(12):1560–1567. [PubMed] [Google Scholar]
  17. Lazarow P. B., Black V., Shio H., Fujiki Y., Hajra A. K., Datta N. S., Bangaru B. S., Dancis J. Zellweger syndrome: biochemical and morphological studies on two patients treated with clofibrate. Pediatr Res. 1985 Dec;19(12):1356–1364. doi: 10.1203/00006450-198512000-00030. [DOI] [PubMed] [Google Scholar]
  18. Lazarow P. B., De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2043–2046. doi: 10.1073/pnas.73.6.2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LeHir M., Herzog V., Fahimi H. D. Cytochemical detection of catalase with 3,3'-diaminobenzidine. A quantitative reinvestigation of the optimal conditions. Histochemistry. 1979 Nov;64(1):51–66. doi: 10.1007/BF00493354. [DOI] [PubMed] [Google Scholar]
  20. Litwin J. A. Light microscopic histochemistry on plastic sections. Prog Histochem Cytochem. 1985;16(2):1–84. doi: 10.1016/s0079-6336(85)80001-2. [DOI] [PubMed] [Google Scholar]
  21. Litwin J. A., Yokota S., Hashimoto T., Fahimi H. D. Light microscopic immunocytochemical demonstration of peroxisomal enzymes in epon sections. Histochemistry. 1984;81(1):15–22. doi: 10.1007/BF00495395. [DOI] [PubMed] [Google Scholar]
  22. Miyazawa S., Osumi T., Hashimoto T. The presence of a new 3-oxoacyl-CoA thiolase in rat liver peroxisomes. Eur J Biochem. 1980 Feb;103(3):589–596. doi: 10.1111/j.1432-1033.1980.tb05984.x. [DOI] [PubMed] [Google Scholar]
  23. Novikoff A. B., Goldfischer S. Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J Histochem Cytochem. 1969 Oct;17(10):675–680. doi: 10.1177/17.10.675. [DOI] [PubMed] [Google Scholar]
  24. Osumi T., Hashimoto T. Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver. Arch Biochem Biophys. 1980 Aug;203(1):372–383. doi: 10.1016/0003-9861(80)90189-7. [DOI] [PubMed] [Google Scholar]
  25. Poulos A., Sharp P., Fellenberg A. J., Danks D. M. Cerebro-hepato-renal (Zellweger) syndrome, adrenoleukodystrophy, and Refsum's disease: plasma changes and skin fibroblast phytanic acid oxidase. Hum Genet. 1985;70(2):172–177. doi: 10.1007/BF00273077. [DOI] [PubMed] [Google Scholar]
  26. Roels F., Goldfischer S. Cytochemistry of human catalase. The demonstration of hepatic and renal peroxisomes by a high temperature procedure. J Histochem Cytochem. 1979 Nov;27(11):1471–1477. doi: 10.1177/27.11.92501. [DOI] [PubMed] [Google Scholar]
  27. Roth J., Bendayan M., Carlemalm E., Villiger W., Garavito M. Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J Histochem Cytochem. 1981 May;29(5):663–671. doi: 10.1177/29.5.6166664. [DOI] [PubMed] [Google Scholar]
  28. Roth J., Bendayan M., Orci L. Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem. 1978 Dec;26(12):1074–1081. doi: 10.1177/26.12.366014. [DOI] [PubMed] [Google Scholar]
  29. Schutgens R. B., Heymans H. S., Wanders R. J., van den Bosch H., Tager J. M. Peroxisomal disorders: a newly recognised group of genetic diseases. Eur J Pediatr. 1986 Feb;144(5):430–440. doi: 10.1007/BF00441734. [DOI] [PubMed] [Google Scholar]
  30. Slot J. W., Geuze H. J. Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J Cell Biol. 1981 Aug;90(2):533–536. doi: 10.1083/jcb.90.2.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Suzuki Y., Orii T., Mori M., Tatibana M., Hashimoto T. Deficient activities and proteins of peroxisomal beta-oxidation enzymes in infants with Zellweger syndrome. Clin Chim Acta. 1986 Apr 30;156(2):191–196. doi: 10.1016/0009-8981(86)90152-x. [DOI] [PubMed] [Google Scholar]
  32. Tager J. M., Van der Beek W. A., Wanders R. J., Hashimoto T., Heymans H. S., Van den Bosch H., Schutgens R. B., Schram A. W. Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome. Biochem Biophys Res Commun. 1985 Feb 15;126(3):1269–1275. doi: 10.1016/0006-291x(85)90322-5. [DOI] [PubMed] [Google Scholar]
  33. Völkl A., Fahimi H. D. Isolation and characterization of peroxisomes from the liver of normal untreated rats. Eur J Biochem. 1985 Jun 3;149(2):257–265. doi: 10.1111/j.1432-1033.1985.tb08920.x. [DOI] [PubMed] [Google Scholar]
  34. Yokota S., Deimann W., Hashimoto T., Fahimi H. D. Immunocytochemical localization of two peroxisomal enzymes of lipid beta-oxidation in specific granules of rat eosinophils. Histochemistry. 1983;78(4):425–433. doi: 10.1007/BF00496194. [DOI] [PubMed] [Google Scholar]
  35. Yokota S., Fahimi H. D. Immunocytochemical localization of catalase in rat liver. J Histochem Cytochem. 1981 Jul;29(7):805–812. doi: 10.1177/29.7.6790603. [DOI] [PubMed] [Google Scholar]

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