Summary
In the histochemical detection of the disaccharidases and glucosidases the reliability of methods with coupled oxidation of glucose (with various buffers, tetrazolium salts and concentrations of substrates, tetrazolium salts and PMS) and azo-dye methods with the postincubation as well as simultaneous azo-coupling in cryostat sections (unfixed, fixed with Baker's formol and acetone) and frozen sections after fixation in cold Bakers's formol and glutaraldehyde was tested. Various rat organs and human enterobiopsies were used. The methods were modified.
Despite the fact that glutaraldehyde and formol fixation does not completely destroy enzyme activities splitting maltose, sucrose, trehalose and lactose (as it could be shown by a simple Glukophan test) the use of the fixed sections is not recommended. Activity of these enzymes is not completely structurally bound and a part of them escapes from the unfixed cryostat sections into the solutions used for rinsing or for incubation. Activities of these enzymes were demonstrated in the content of the rat jejunum as well. The results of the detection of disaccharidases with a coupled oxidation of glucose are dependent on buffer (type and pH), on the tetrazolium salt (type and concentration), on the concentration of phenazine methosulfate and of disaccharides, on the conditions during the incubation (temperature, anaerobic or aerobic conditions, aqueous or gel media) and on the type of sections. With all the substrates used (maltose, sucrose, trehalose and lactose) a positive reaction in the enterocytes (both of rat and human) and in the cells of convoluted tubules in rat kidney was obtained. With lactose the reaction was weak and irregular and could be obtained under anaerobic conditions only. A proximodistal gradient in the rat intestine was revealed. In the detection of lactose the use of galactose oxidase in combination with glucose oxidase decreased the intensity of the staining. In evaluating the validity of the localization the artifacts caused by the diffusion of disaccharidases and by the method with coupled oxidation of glucose were considered, the latter being their main source. By no means such artifacts could be avoided. The positive staining is revealed in the sites of the bound tetrazolium salt where it is contacted by the reduced PMS. No reaction can be obtained in sites lacking affinity for the tetrazolium salts even if they contained an active enzyme. The technique allows at the most the localization on the cellular but not intracellular level. The “disaccharidase granules” of Dahlquist and Brun are artifacts.
When the sections are incubated individually with the described gel media or in the incubation chambers the amount of produced formazan may serve as a measure of the activity of the respective disaccharidase. Such technique proved to be of value in investigating the changes of activities of disaccharidases in the jejunum of patients with primary malabsorption syndrome. These activities were reduced in comparison with the normal jejunum.
The limitations in localization of the postincubation azo-coupling methods for the deection of glucosidases and galactosidases are much the same as those of the methods with coupled oxidation of glucose. In addition to it the relative substrate specificity of the intestinal disaccharidases has to be considered, because identical enzymes may not be detected with synthetic and natural substrates. Using our new method with hexazo-p-rosaniline in the simultaneous azocoupling an improved localization of 6-Br-2-naphthyl-α-D-glucosidase was achieved. In the enterocytes the enzyme was localized in the microvillous zone and apical part of the cytoplasma.
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References
Auricchio, S., A. Rubino, A. Prader, J. Rey, J. Jos, J. Frézal, and M. Davidson: Intestinal glycosidase activities in congenital malabsorption of disaccharides. J. Pediat. 66, 555–564 (1965).
Barka, T., and P. J. Anderson: Histochemistry. Theory, practice, and bibliography. New York-Evanston-London: Harper & Row, Inc. 1963.
Bartoníček, V., and Z. Lojda: Incubation chambers made of plastic. Acta histochem. (Jena) 15, 389–391 (1963).
Bentley R., Glucose aerodehydrogenase (Glucose oxidase). In: Methods in enzymology (S. P. Colowick and N. O. Kaplan, eds.), p. 340. New York: Academic Press 1955.
Borgström, D., A. Dahlquist, G. Lundh, and J. Sjövall: Studies in intestinal digestion and absorption in the human. J. clin. Invest. 36, 1521–1536 (1957).
Cohen, R. B., S. H. Rutenburg, K-C. Tsou, M. A. Woodburg, and A. M. Seligman: The colorimetric estimation of β-D-glucosidase. J. biol. Chem. 195, 607–614 (1952a).
—, K-C. Tsou, S. H. Rutenburg, and A. M. Seligman: The colorimetric estimation and histochemical demonstration of β-D-galactosidase. J. biol. Chem. 195, 239–249 (1952b).
Dahlquist, A.: The intestinal disaccharidases and disaccharide intolerance. Gastroenterology 43, 694–696 (1962).
—, and B. Borgström: Digestion and absorption of disaccharides in man. Biochem. J. 81, 411–418 (1961).
—, and A. Brun: A method for the histochemical demonstration of disaccharidase activities: application to invertase and trehalase in some animal tissues. J. Histochem. Cytochem. 10, 294–302 (1962).
—, B. Bull, and B. E. Gustafson: Rat intestinal 6-bromo-2-naphthyl glycosidase and disaccharidase activities. I. Enzymic properties and distribution in the digestive tract of conventional and germ-free animals. Arch. Biochem. 101, 150–158 (1965a).
— and D. L. Thomson: Rat intestinal 6-bromo-2-naphthyl glycosidase and disaccharidase activities. II. Solubilization and separation of the small-intestinal enzymes. Arch. Biochem. 109, 159–167 (1965b).
—, J. B. Hammond, R. K. Crane, J. V. Dunphy, and A. Littman: Intestinal lactase deficieny and intestinal lactose intolerance in adults. Preliminary report. Gastroenterology 45, 488–491 (1963).
Dixon, M., and E. C. Webb: Enzymes, 2nd ed. London: Longmans, Green & Co. Ltd. 1964.
Doell, R. G., and K. Kretchmer: Studies of small intestine during development. 1. Distribution and activity of β-galactosidase. Biochim. biophys. Acta (Amst.) 62, 353–360 (1962).
Duve, Ch. de: The lysosome concept. In: Lysosomes (Ciba Foundation Symposium, A. V. S. de Reuck, and M. P. Cameron, eds.), pp. 1–31, Boston: Little, Brown & Co. 1963.
Frič, P., and Z. Lojda: Enzymes of the human jejunum mucosa. Acta gastro-ent. belg. 27, 528–530 (1964).
Gottschalk., A.: α-D-glucosidases. In: The enzymes (J. B. Sumner, and K. Myrbäck, eds.), vol. I, part I, pp. 551–582. New York: Academic Press 1950.
Haemmerli, U. P., H. Kistler, R. Ammann, T. Marthaler, G. Semenza, S. Auricchio, and A. Prader: Acquired milk intolerance in the adult caused by lactose malabsorption due to a selective deficiency of intestinal lactase activity. Amer. J. Med. 38, 7–30 (1965).
Heilskov, N. S. C.: Studies in animal lactase. Diss. Copenhagen 1956.
Klotz, A. P.: Intestinal lactase deficiency and diarrhea in adults. Amer. J. dig. Dis. 9, 345–354 (1964).
Lifschitz, F., and G. H. Holman: Disaccharidase deficiencies with steatorrhea. J. Pediat. 64, 34–44 (1964).
Littman, A., and J. B. Hammond: Diarrhea in adults caused by deficiency in intestinal disaccharidases. Gastroenterology 48, 237–249 (1965).
Lojda, Z.: Some remarks on the histochemical demonstration of dehydrogenases. Čs. Morfol. 9, 179–189 (1961).
—: Remarks on histochemical demonstration of dehydrogenases. II. Intracellular localization. Folia morph. (Prague) 13, 84–96 (1965).
—, and P. Frič: Histoenzymatic study of jejunal biopsies in malabsorption syndromes. In: Second Internat. Congr. of Histo- and Cytochemistry (T. H. Schiebler, A. G. E. Pearse, and H. H. Wolff, eds.), p. 204. Berlin-Göttingen-Heidelberg: Springer 1964.
—, B. Večerek, and H. Pelichová: Some remarks concenrning the histochemical detection of acid phosphatase by azocoupling reactions. Histochemie 3, 428–454 (1964).
Miller, D., and R. K. Crane: The digestive function of the epithelium of the small intestine. II. Localization of disaccharide hydrolysis in the isolated brush border portion of intestinal epthelial cells. Biochim. biophys. Acta (Amst.) 52, 293–298 (1961).
Monis, B., K-C. Tsou, and A. M. Seligman: Development of a histochemical method for α-D-galactosidase and its distribution in the rat. J. Histochem. Cytochem. 11, 653–661 (1963).
Pearse, A. G. E.: Histochemistry, theoretical and applied. London: J. & A. Churchilll, Ltd. 1960.
Pearson, B., P. L. Wolf, and M. Andrews: The occurence of β-glucosidase in mammalian tissues (abstr). Fed. Proc. 21, 249 (1962).
— and J. Vasquez: A comparative study of a series of new indolyl compounds to localize β-galactosidase in tissues. Lab. Invest. 12, 1249–1259 (1963).
Plotkin, G. R., and K. J. Isselbacher: Studies of disaccharidase splittting enzymes of intestinal mucosa in nontropical sprue and other malabsorption states. Amer. J. dig. Dis. 9, 807–808 (1964).
Prader, A., G. Semenza, and S. Auricchio: Intestinale Absorption und Malabsorption der Disaccharide. Schweiz, med. Wschr. 93, 1272–1274 (1963).
Rosen, G., and N. Kretchmer: Cellular localization of invertase and lactase with fluorescence antibodies (1964 in press). Quoted according to P. Sunshine and N. Kretchmer 1964.
Rutenburg, A. M., J. A. Goldbarg, S. H. Rutenburg, and T. R. Lang: The histochemical demonstration of α-D-glucosidase in mammalian tissues. J. Histochem. Cytochem. 8, 268–272 (1960).
—, S. H. Rutenburg, B. Monis, R. Teague, and A. M. Seligman: Histochemical demonstration of β-D-galactosidase in the rat. J. Histochem. Cytochem. 6, 122–129 (1958).
Scarpelli, D. C., R. Hess, and A. G. E. Pearse: The cytochemical localization of oxidative enzymes. I. Diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase. J. biophys. biochem. Cytol. 4, 747–751 (1958).
Sonntag, W. M., M. L. Brill, W. G. Troyer, J. D. Welsh, G. Semenza, and A. Prader: Sucrose-isomaltose malabsorption in an adult woman. Gastroenterology 47, 18–25 (1964).
Sunshine, P., and N. Kretchmer: Studies of small intestine during development. III. Infantile diarrhea associated with intolerance to disaccharides. J. Pediat. 34, 38–40 (1964).
Ugolev, A. M., N. N. Jesuitova, and P. D. Lafy: Localization of invertase activity in small intestinal cells. Nature (Lond.) 203, 879–880 (1964).
Veibel S., β-glucosidase. In: The enzymes (J. B. Sumner, and K. Myrbäck, eds.), vol. I, part I, pp. 551–582. New York: Acad. Press 1950a.
—: Hydrolysis of galactosides, mannosides and thioglycosides. In: The enzymes (J. B. SumNer and K. Myrbäck, eds.), vol. I, part I, pp. 621–634. New York: Acad. Press 1950b.
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Lojda, Z. Some remarks concerning the histochemical detection of disaccharidases and glucosidases. Histochemie 5, 339–360 (1965). https://doi.org/10.1007/BF00285800
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DOI: https://doi.org/10.1007/BF00285800