The Histochemical Journal

, Volume 27, Issue 11, pp 897–905 | Cite as

Ultrastructural localization of xanthine oxidase activity in the digestive tract of the rat

  • Rosier J. M. Van Den Munckhof
  • Helena Vreeling-Sindelárová
  • Jacques P. M. Schellens
  • Cornelis J. F. Van Noorden
  • Wilma M. Frederiks


Precise localization of xanthine oxidase activity might elucidate physiological functions of the enzyme, which have not been established so far. Because xanthine oxidase is sensitive to chemical (aldehyde) fixation, we have localized its activity in unfixed cryostat sections of rat duodenum, oesophagus and tongue mounted on a semipermeable membrane. Previous studies had shown that this procedure enables the exact localization of activities of peroxisomal oxidases with maintenance of acceptable ultrastructure. Moreover, leakage and/or diffusion of enzyme molecules was prevented with this method. The incubation medium to detect xanthine oxidase activity contained hypoxanthine as substrate and cerium ions as capturing agent for hydrogen peroxide. After incubation, reaction product in the sections was either visualized for light microscopy or sections were fixed immediately and processed for electron microscopy. At the ultrastructural level, crystalline reaction product specifically formed by xanthine oxidase activity was found to be present in the cytoplasmic matrix of enterocytes and goblet cells and in mucus of duodenum. Moderate activity was found in the cytoplasm of apical cell layers of epithelia of oesophagus and tongue, with highest activity in the cornified layer. Moreover, large amounts of reaction product were found to surround bacteria present between cell remnants of the cornified layer of the oesophagus. Many bacteria surrounded by the enzyme showed signs of destruction and/or cell death. The intracellular localization of xanthine oxidase activity in the cytoplasm of epithelial cells as well as the extracellular localization suggest that the enzyme plays a role in the lumen of the digestive tract, for instance in the defence against microorganisms.


Cerium Digestive Tract Xanthine Oxidase Goblet Cell Hypoxanthine 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Amaya,Y.,Yamazaki,K-I.,Sato,M.,Noda,K.,Nishino,T. &Nishino,T. (1990) Proteolytic conversion of xanthine dehydrogenase from the NAD-dependent type to the O2-dependent type. Amino acid sequence of rat liver xanthine dehydrogenase and identification of the cleavage sites of the enzyme protein during irreversible conversion by trypsin.J. Biol. Chem. 265, 14170–5.PubMedGoogle Scholar
  2. Angermüller,S. (1989) Peroxisomal oxidases: cytochemical localization and biological relevance.Progr. Histochem. Cytochem. 20, 1–63.Google Scholar
  3. Angermüller,S. &Fahimi,H. D. (1988) Light microscopic visualization of the reaction product of cerium used for localization of peroxisomal oxidases.J. Histochem. Cytochem. 36, 23–8.PubMedGoogle Scholar
  4. Angermüller,S.,Bruder,G.,Völkl,A.,Wesch,H. &Fahimi,H. D. (1987) Localization of xanthine oxidase in crystalline cores of peroxisomes. A cytochemical and biochemical study.Eur. J. Cell Biol. 45, 137–44.PubMedGoogle Scholar
  5. Arborgh,B.,Ericsson,J. L. E. &Helminen,H. (1971) Inhibition of renal acid phosphatase and aryl sulfatase activity by glutaraldehyde fixation.J. Histochem. Cytochem. 19, 449–51.PubMedGoogle Scholar
  6. Becker,B. F. (1993) Towards a physiological function of uric acid.Free Rad. Biol. Med. 14, 615–31.CrossRefPubMedGoogle Scholar
  7. Becker,B. F.,Reinholz,N.,Özçelik,T.,Leipert,B. &Gerlach,E. (1989) Uric acid as radical scavenger and antioxidant in the heart.Pflügers Arch. 415, 127–35.CrossRefPubMedGoogle Scholar
  8. Björk,L. &Cleasson,O. (1979) Xanthine oxidase as a source of hydrogen peroxide for the lactoperoxidase system in milk.J. Diary Sci. 62, 1211–5.Google Scholar
  9. Briggs,R. T.,Drath,D. B.,Karnovsky,M. L. &Karnovsky,M. J. (1975) Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method.J. Cell Biol. 67, 566–86.CrossRefPubMedGoogle Scholar
  10. Chalmers,G. R. &Edgerton,V. R. (1989) Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons.J. Histochem. Cytochem. 37, 899–901.PubMedGoogle Scholar
  11. Dikov,A.,Alexandrov,I.,Russinova,A. &Boya-Djieva-Michailova,A. (1988) Ultracytochemical detection of enzymes by reduction of potassium ferricyanide. I. A method for detection of xanthine oxidase.Acta Histochem. 83, 107–15.PubMedGoogle Scholar
  12. Engerson,T. D.,Mckelvey,T. G.,Rhyne,D. B.,Boggio,E. B.,Snyder,S. J. &Jones,H. P. (1987) Conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues.J. Clin. Invest. 79, 1564–70.PubMedGoogle Scholar
  13. Frederiks,W. M. &Marx,F. (1993) A histochemical procedure for light microscopic demonstration of xanthine oxidase activity in unfixed cryostat sections using cerium ions and a semipermeable membrane technique.J. Histochem. Cytochem. 41, 667–70.PubMedGoogle Scholar
  14. Frederiks,W. M.,Kooij,A. &Marx,F. (1993) The effect of ischemia on xanthine oxidase activity in rat intestine and liver.Int. J. Exp. Pathol. 74, 21–6.PubMedGoogle Scholar
  15. Frederiks,W. M.,Bosch,K. S.,van DenMunckhof,R. J. M. &vanNoorden,C. J. F. (1994) A quantitative histochemical study on xanthine oxidase activity in rat liver using the cerium capture method in the presence of polyvinyl alcohol.J. Histochem. Cytochem. 42, 1091–7.PubMedGoogle Scholar
  16. Gossrau,R.,vanNoorden,C. J. F. &Frederiks,W. M. (1989) Enhanced light microscopic visualization of oxidase activity with the cerium capture method.Histochemistry 92, 349–53.CrossRefPubMedGoogle Scholar
  17. Gossrau,R.,Frederiks,W. M. &vanNoorden,C. J. F. (1990) Histochemistry of reactive oxygenspecies (ROS)-generating oxidases in cutaneous and mucous epithelia of laboratory rodents with special reference to xanthine oxidase.Histochemistry 94, 539–44.PubMedGoogle Scholar
  18. Granger,D. N.,Höllwarth,M. E. &Parks,D. A. (1986) Ischemia-reperfusion injury: role of oxygen-derived free radicals.Acta Physiol. Scand. 126 (Suppl. 548), 47–63.Google Scholar
  19. Granger,D. N.,Rutili,G. &Mccord,J. M. (1981) Superoxide radicals in feline intestinal ischemia.Gastroenterology 81, 22–9.PubMedGoogle Scholar
  20. Hopwood,D. (1991) Fixation of tissue for histochemistry. InHistochemical and Immunocytochemical Techniques. Applications to Pharmacology and Toxicology (edited byBach,P. H. &Baker,J. R. J.), pp. 147–65. London: Chapman & Hall.Google Scholar
  21. Ichikawa,M.,Nishino,T.,Nishino,T. &Ichikawa,A. (1992) Subcellular localization of xanthine oxidase in rat hepatocytes: high resolution immunoelectron microscopic study combined with biochemical analysis.J. Histochem. Cytochem. 40, 1097–1103.PubMedGoogle Scholar
  22. Jarasch,E.-D.,Grund,C.,Bruder,G.,Heid,H. W.,Keenan,T. W. &Franke,W. W. (1981) Localization of xanthine oxidase in mammary-gland epithelium and capillary endothelium.Cell 25, 67–82.PubMedGoogle Scholar
  23. kooij,A. (1994) A re-evaluation of the tissue distribution and physiology of xanthine oxidoreductase.Histochem. J. 26, 889–915.CrossRefPubMedGoogle Scholar
  24. Kooij,A.,Schiller,H. J.,Schijns,M.,vanNoorden,C. J. F. &Frederiks,W. M. (1994) Conversion of xanthine dehydrogenase into xanthine oxidase in rat liver and plasma at the onset of reper-fusion after ischemia.Hepatology 19, 1488–95.CrossRefPubMedGoogle Scholar
  25. Kooij,A.,Bosch,K. S.,Frederiks,W. M. &vanNoorden,C. J. F. (1992) High levels of xanthine oxidoreductase in rat endothelial, epithelial and connective tissue cells.Virchows Archiv. B. Cell. Pathol. 62, 143–50.Google Scholar
  26. Kooij,A.,Frederiks,W. M.,Gossrau,R. &vanNoorden,C. J. F. (1991) Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor tetranitro BT.J. Histochem. Cytochem. 39, 87–93.PubMedGoogle Scholar
  27. Massey,V.,Komai,H.,Palmer,G. &Elion,G. B. (1970) On the mechanism of inactivation of xanthine oxidase by allopurinol and other pyrazolo [3,4-d] pyrimidines.J. Biol Chem. 245, 2837–44.PubMedGoogle Scholar
  28. Mcmillan,P. J. (1967) Differential demonstration of muscle and heart type lactic dehydrogenase of rat muscle and kidney.J. Histochem. Cytochem. 15, 21–31.Google Scholar
  29. Meijer,A. E. F. H. (1972) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections.Histochemie 30, 31–9.CrossRefPubMedGoogle Scholar
  30. Meijer,A. E. F. H. (1980) Semipermeable membrane techniques in quantitative enzyme histochemistry. InTrends in Enzyme Histochemistry and Cytochemistry. Ciba Foundation Symposium 73, pp. 103–20. Amsterdam: Excerpta Medica.Google Scholar
  31. Patel,H. R. H.,Frederiks,W. M.,Marx,F.,Best,A. J. &vanNoorden,C. J. F. (1991) A quantitative histochemical study ofd-amino acid oxidase activity in rat liver in relationship with feeding conditions.J. Histochem. Cytochem. 39, 81–6.PubMedGoogle Scholar
  32. Peden,D. B.,Hohman,R.,Brown,M. E.,Mason,R. T.,Berkebile,C.,Fales,H. M. &Kaliner,M. A. (1990) Uric acid is a major antioxidant in human nasal airway secretions.Proc. Natl Acad. Sci. USA 87, 7638–42.PubMedGoogle Scholar
  33. Richardson,K. C.,Jarrett,L. &Finke,E. H. (1960) Embedding in epoxy resins for ultrathin sectioning in electron microscopy.Stain Technol. 35, 313–23.PubMedGoogle Scholar
  34. Sackler,M. L. (1966) Xanthine oxidase from liver and duodenum of the rat: histochemical localization and electrophoretic heterogeneity.J. Histochem. Cytochem. 14, 326–33.PubMedGoogle Scholar
  35. Schellens,J. P. M.,Frederiks,W. M.,vanNoorden,C. J. F.,Vreeling Sindelárová,H.,Marx,F. &Mcmillan,P. J. (1992) The use of unfixed cryostat sections for electron microscopic study ofd-amino acid oxidase activity in rat liver.J. Histochem. Cytochem. 40, 1975–9.PubMedGoogle Scholar
  36. Terao,J. &Nagao,A. (1991) Antioxidative effect of human saliva on lipid peroxidation.Agric. Biol. Chem. 55, 869–72.Google Scholar
  37. Tubaro,E.,Lotti,B.,Cavallo,G.,Croce,C. &Borelli,G. (1980a) Liver xanthine oxidase increase in mice in three pathological models. A possible defence mechanism.Biochem. Pharmacol. 29, 1939–43.PubMedGoogle Scholar
  38. Tubaro,E.,Lotti,B.,Santiangeli,C. &Cavallo,G. (1980b) Xanthine oxidase increase in polymorphonuclear leukocytes and macrophages in mice in three pathological situations.Biochem. Pharmacol. 29, 1945–8.PubMedGoogle Scholar
  39. van DenMunckhof,R. J. M.,Vreeling-sindelárová,H.,Schellens,J. P. M. &Frederiks,W. M. (1994) Localization of uric acid oxidase activity in core and matrix of peroxisomes as detected in unfixed cryostat sections of rat liver.J. Histochem. Cytochem. 42, 177–83.PubMedGoogle Scholar
  40. vanNoorden,C. J. F. &Frederiks,W. M. (1992)Enzyme Histochemistry: a Laboratory Manual of Current Methods. Oxford: Oxford University Press.Google Scholar
  41. vanNoorden,C. J. F. &Frederiks,W. M. (1993) Cerium methods for light and electron microscopical histochemistry.J. Microsc. 171, 3–16.PubMedGoogle Scholar

Copyright information

© Chapman & Hall 1995

Authors and Affiliations

  • Rosier J. M. Van Den Munckhof
    • 1
  • Helena Vreeling-Sindelárová
    • 1
  • Jacques P. M. Schellens
    • 1
  • Cornelis J. F. Van Noorden
    • 1
  • Wilma M. Frederiks
    • 1
  1. 1.Department of Cell Biology and HistologyAcademic Medical Centre, University of AmsterdamAmsterdamThe Netherlands

Personalised recommendations