The Histochemical Journal

, Volume 12, Issue 6, pp 669–685 | Cite as

Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films

  • C. J. F. Van Noorden
  • J. Tas


The cytochemical determination of the activity of glucose-6-phosphate dehydrogenase (G6PDH) with tetranitro blue tetrazolium (TNBT) was studied with model films of polyacrylamide gel incorporating purified enzyme. This model system enabled a quantitative study to be made of different parameters involved with the cytochemical assay as it is applied to sections or smears. The enzyme activity of G6PDH incorporated in the model films was also assayed biochemically. Optimal conditions for retaining the maximum amount of enzymic activity are described. The behaviour of G6PDH towards enzyme inhibitors was found to be similar in model films and in solution. With TNBT, absorbance measurements at a single wavelength (535 nm) were used to estimate the enzyme activity quantitatively. When carried out under standardized conditions, both the cytochemical and biochemical assay showed a linear relation with the time of incubation and obeyed the Beer-Lambert law. The correlation between biochemical and cytochemical data was very high, which enabled cytochemical data to be converted into absolute units of enzyme activity. The data obtained in this way closely resembled the data of enzyme activity calculated from the absorbance of formazan produced inside polyacrylamide model films and afterwards extracted into a suitable solvent.


Tetrazolium Formazan Absorbance Measurement Biochemical Assay Quantitative Aspect 
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. Altman, F. P. (1974) Studies on the reduction of tetrazolium salts. III. The products of chemical and enzymic reduction.Histochemistry 38, 155–71.Google Scholar
  2. Altman, F. P. (1975) Quantitation in histochemistry: a review of some commercially available microdensitometers.Histochem. J. 7, 375–95.Google Scholar
  3. Altman, F. P. (1976a) The quantification of formazans in tissue sections by microdensitometry. I. The use of neotetrazolium chloride.Histochem. J. 8, 373–81.Google Scholar
  4. Altman, F. P. (1976b) Tetrazolium salts and formazans.Prog. Histochem. Cytochem. 9 (3).Google Scholar
  5. Altman, F. P. (1976c) Tetrazolium salts: a consumer's guide.Histochem. J. 8, 471–85.Google Scholar
  6. Altman, F. P. (1976d) The determination of section thickness. InHistochemistry and Cytochemistry 1976, Proceedings of the 5th International Congress of Histochemistry and Cytochemistry, Bucharest, Romaina (edited byDiculescu, I., Onicescu, D. andEskenasy, A.), p. 19.Bucharest.Google Scholar
  7. Altman, F. P. &Butcher, R. G. (1973) Studies on the reduction of tetrazolium salts. I. The isolation and characterisation of a half-formazan intermediate produced during the reduction of neotetrazolium chloride.Histochemie 37, 333–50.Google Scholar
  8. Altman, F. P., Høyer, P. E. &Andersen, H. (1979) Dehydrogenase histochemistry of lipid-rich tissues: a tetrazolium-metal chelation technique to improve localisation.Histochem. J. 11, 485–8.Google Scholar
  9. Andersen, H. &Høyer, P. E. (1974) Simplified control experiments in the histochemical study of coenzyme-linked dehydrogenases.Histochemistry 38, 71–83.Google Scholar
  10. Bergmeyer, H. U., Gawehn, K. &Grassl, M. (1970) Die biochemischen Reagentien. I. Enzyme. InMethoden der Enzymatischen Analyse, Vol. 1, 2nd edn (edited byBergmeyer, H. U.), pp. 388–483. Weinheim: Verlag Chemie.Google Scholar
  11. Brederoo, P., Daems, W. Th., Van Duijn, P. &Van Der Ploeg, M. (1968) Cytochemica investigations of the lead method for acid phosphatase by use of a model system.Proc. R. microsc. Soc. 3, 153.Google Scholar
  12. Butcher, R. G. (1971) The chemical determination of section thickness.Histochemie 28, 131–6.Google Scholar
  13. Butcher, R. G. (1978) The measurement in tissue sections of the two formazans derived from nitroblue tetrazolium in dehydrogenase reactions.Histochem. J. 10, 739–44.Google Scholar
  14. Butcher, R. G., &Altman, F. P. (1973) Studies on the reduction of tetrazolium salts. II. The measurement of the half reduced and fully reduced formazans of neotetrazolium chloride in tissue sections.Histochemie 37, 351–63.Google Scholar
  15. Cavins, J. F. &Friedman, M. (1968) Specific modification of protein sulfhydryl groups with α, β-unsaturated compounds.J. biol. Chem. 243, 3357–60.Google Scholar
  16. De Jong, A. S. H., Van Duijn, P. &Daems, W. Th. (1976) Cytochemical model system for microsomal rat liver glucose-6-phosphatase.J. Histochem. Cytochem. 24, 643–51.Google Scholar
  17. Harrison, R. A. P. (1974) The detection of hexokinase, glucose-phosphate isomerase and phosphoglucomutase activities in polyacrylamide gels after electrophoresis: a novel method using immobilized glucose-6-phosphate dehydrogenase.Analyt. Biochem. 61, 500–7.Google Scholar
  18. Hosemann, W., Teutsch, H. F. &Sasse, D. (1979) Identification of G6PDH-active sinusoidal cells as Kupffer cells in the rat liver.Cell Tissue Res. 196, 237–47.Google Scholar
  19. Löhr, G. W. &Waller, H. D. (1970) Glucose-6-phosphat-Dehydrogenase. InMethoden der Enzymatischen Analyse, Vol. 1, 2nd edn (editedBergmeyer, H. U.), pp. 599–606. Weinheim: Verlag Chemie.Google Scholar
  20. Lojda, Z., Gossrau, R. &Schiebler, T. H. (1976)Enzymhistochemische Methoden. pp. 239–259. Heidelberg: Springer-Verlag.Google Scholar
  21. Lojda, Z., Van Der Ploeg, M. &Van Duijn, P. (1967) Phosphates of the naphthol AS series in the quantitative determination of alkaline and acid phosphatase activitiesin situ studied in polyacrylamide membrane model systems and by cytospectrophotometry.Histochemie 11, 13–32.Google Scholar
  22. Marzotko, D., Warchol, J. B. &Wachowiak, R. (1973) Spektrophotometrische Untersuchungen über die Lichtempfindlichkeit von PMS in wässriger Lösung.Acta histochem. 46, 53–9.Google Scholar
  23. Møller, M. &Høyer, P. E. (1979) Histochemical demonstration of a circadian rhythm of succinate dehydrogenase in rat pineal gland. Influence of coenzyme Q10 addition.Histochemistry 59, 259–69.Google Scholar
  24. Mosbach, K. &Mattiasson, B. (1976) Multistep enzyme systems. InMethods in Enzymology, Vol. XLIV,Immobilized Enzymes, (edited byMosbach, K.), pp. 453–478. New York: Academic Press.Google Scholar
  25. Papadimitriou, J. M. &Van Duijn, P. (1970) Effects of fixation and substrate protection on the isoenzymes of aspartate aminotrasferase studied in a quantitative cytochemical model system.J. Cell Biol. 47, 71–83.Google Scholar
  26. Pette, D. &Wimmer, M. (1979) Kinetic microphotometric activity determination in enzyme containing gels and model studies with tissue sections.Histochemistry 64, 11–22.Google Scholar
  27. Pool, C. W., Diegenbach, P. C. &Scholten, G. (1979) Quantitative succinate dehydrogenase histochemistry. I. A methodological study on mammalian and fish muscle.Histochemistry 64, 251–62.Google Scholar
  28. Rosenquist, T. H. (1976) On the use of quantitation in histochemistry: a review of the literature.Histochem. J. 8, 205–8.Google Scholar
  29. Satterfield, C. N. &Sherwood, T. K. (1963)The Role of Diffusion in Catalysis. pp. 56–63. Reading, Massachusetts: Addison-Wesley Publishing Co.Google Scholar
  30. Tas, J. (1975) Histochemical conditions influencing metachromatic staining. A comparative study by means of a model system of polyacrylamide films.Histochem. J. 7, 1–19.Google Scholar
  31. Tas, J., De Vries, A. C. J. &Berndsen, R. G. (1979) A method for the quantitative determination of protein incorporated in solubilizable polyacrylamide gels.Analyt. Biochem. 100, 264–70.Google Scholar
  32. Tas, J. &Roozemond, R. C. (1973) Direct recording of metachromatic spectra in a model system of polyacrylamide films.Histochem. J. 5, 425–36.Google Scholar
  33. Van Der Ploeg, M. (1975) Quantitative model film studies: a link between enzyme cytochemistry and biochemistry.Acta histochem. Suppl.XIV, 69–77.Google Scholar
  34. Van Der Ploeg, M. &Van Duijn, P. (1968) Cytophotometric determination of alkaline phosphatase activity of individual neutrophilic leukocytes with a biochemically calibrated model system.J. Histochem. Cytochem. 16, 693–706.Google Scholar
  35. Van Duijn, P. (1974) Fundamental aspects of enzyme cytochemistry. InElectron Microscopy and Cytochemistry, Proceedings of the 2nd International Symposium, Drienerlo, The Netherlands, 1973 (edited byWisse, E., Daems, W. Th., Molenaar, I. andVan Duijn, P.), pp. 3–23. Amsterdam: North-Holland.Google Scholar
  36. Van Duijn, P. (1976) Prospects for microscopical cytochemistry.Histochem. J. 8, 653–76.Google Scholar
  37. Van Duijn, P., Pascoe, E. &Van Der Ploeg, M. (1967) Theoretical and experimental aspects of enzyme determination in a cytochemical model system of polyacrylamide films containing alkaline phosphatase.J. Histochem. Cytochem. 15, 631–45.Google Scholar
  38. Van Duijn, P. &Van Der Ploeg, M. (1970) Potentialities of cellulose and polyacrylamide films as vehicles in quantitative cytochemical investigations on model substances. InIntroduction to Quantitative Cytochemistry, Vol. 2 (edited byWied, G. L. andBahr, G. F.), pp. 223–262. New York: Academic Press.Google Scholar
  39. Van Noorden, C. J. F., Tas, J. & Sanders, J. A. H. (1980) Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetrazolium salts studied in a model system of polyacrylamide films.Acta histochem. Suppl.XXIV (in press).Google Scholar
  40. Weber, G. (1977) Enzymology of cancer cells.New Engl. J. Med. 296, 541–51.Google Scholar
  41. Weiss, G., Vergani, G. &Pentz, S. (1979) Histochemical demonstration of G6PDH- and SDH-activity on cultured fibroblastoid cells from rat pancreas using a membrane technique.Histochemistry 64, 111–4.Google Scholar

Copyright information

© Chapman and Hall Ltd. 1980

Authors and Affiliations

  • C. J. F. Van Noorden
    • 1
  • J. Tas
    • 1
  1. 1.Laboratory of Histology and Cell BiologyUniversity of Amsterdam, Jan Swammerdam InstituteAmsterdamThe Netherlands

Personalised recommendations