INTESTINAL {3-GALACTOSIDASE ACTIVITIES IN MALABSORPTION SYN DRoMEs

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1 GASTROENTEROLOGY Copyright 1966 by The Williams & Wilkins Co. Vol. 50. No.4 P,inted in U.S.A. INTESTINAL {3-GALACTOSIDASE ACTIVITIES IN MALABSORPTION SYN DRoMEs GIUSEPPE ZOPPI, M.D., BEAT HADORN, M.D., RICHARD GITZELMANN, M.D., HANSJORG KISTLER, M.D., AND ANDREA PRADER, M.D. University Pediatric Department, Kinderspital, and University Department of Internal Medicine, Zurich, Switzerland In the past few years, evidence has ac -cumulated indicating multiplicity of intestinal,b-galactosidases in different species including man. 1-7 In man, two,b-galactosidases have been separated by gel filtration with Sephadex.3. 4, 4a Activity of intestinal {3-galactosidases has usually been assayed with the use of lactose (the "natural" substrate) and of synthetic substrates (o-nitrophenyl-{3-galactoside, o-npg; 6-bromo-2-naphthyl-{3- D-galactopyranoside, 6-BNG). In some intestinal preparations from the calfl and rat5, 6 there were indications for the existence of enzymes hydrolyzing synthetic substrates, but not disaccharides of the corresponding configuration. On the other hand, Wallenfels and Fischers purified from calf intestine an enzyme exhibiting high hydrolytic activity with lactose but less activity with o-npg and other synthetic substrates. Thus the problem of substrate specificity must be considered when,b-galactosidase activity is studied in human intestinal mucosa. In rabbit and rat?' 9 activities with lactose and the synthetic substrates have been found to dissociate in different tissue fractions: In most fractionation experiments a Received October 11, Accepted December 3,1965. Address requests for reprints to: Dr. Andrea Prader, Kinderspital ZUrich, Steinwiesstrasse 75, 8032 ZUrich, Switzerland. This investigation was supported in part by Grants 3105 and 3586 of Schweizerischer Nationalfonds zur Forderung der wissenschaftlichen Forschung, and by the Association for the Aid of Crippled Children, New York. The authors are indebted to Dr. D. H. Shmerling for performing the biopsies and to Mrs. G. Schircks for skillful technical assistance. 557 major proportion of {3-galactosidase activity with lactose sedimented with the particulate fraction, whereas most of the activity with synthetic substrates was found in the supernatant fluid. In man several forms of intestinal lactase deficiency are known (reviewed in references 10 and 11). The hereditary congenital lactose malabsorption and the acquired lactose malabsorption are characterized by an isolated deficiency of intestinal lactase activity. In gluten-sensitive enteropathy (celiac disease or sprue) and in other malabsorption syndromes with partial or subtotal atrophy of the intestinal mucosa, there is a general deficiency of disaccharidase activity. In all these conditions the intestinal,b-galactosidase activity has been studied only with lactose as substrate. In the rare cases of infantile lactose intolerance with severe lactosuria (Durand syndrome) the question of whether intestinal lactase activity is present or absent remains open. The demonstration of 6- BNG-ase activity in the mucosa of one patientl 2 still does not imply the presence of lactase activity in this syndrome. In order to elucidate the intestinal enzyme defects more precisely,,b-galactosidase activity of human intestinal mucosa was assayed with both lactose and 6-BNG as substrates. Peroral intestinal biopsies were taken from normal adults and children, from patients with acquired lactose malabsorption, and from patients with malabsorption syndromes characterized by various degrees of mucosal atrophy. Materials and Methods Substrates Maltose (4- (a-d-glucopyranosyi) -D-glucopyranoside, The British Drug Houses Ltd., Poole,

2 TABLE 1. Clinical data and fj-galactosidase and a-glucosidase activities in homogenates of jejunal mucosa of adults fl-galactosidases a-glucosidases Blood glucose Group, patients Clinical diagnosis Homogenate increase after oral lactose 6-BNG- asea i L actase Maltase I Sucrase ilsomaltase load (50 g) mg protein/ml I U / g protein 1, Controls L. G. Healthy B.G. Suspected lactose malabsorp mg/100 ml tion M. P. Protein-losing enteropathy S. E. Ulcerative colitis mg/100 ml 2, Isolated lactase deficiency M.F. Lactose malabsorption < mg/ioo ml C. R. Pernicious anemia , Generalized disaccharidase deficiency B. A. Sprue < < mg/100 ml L. P. Chronic diarrhea (abuse of < laxatives) R.M. Chronic diarrhea (hyperthy mg/ioo ml roidism) a 6-BNG-ase 6-bromo-2-naphthyl-fj-galactosidase. Histology Subtotal atrophy '"" 00 t>:l '"'3 t- - 0/>.

3 April 1966 INTESTINAL {3-GALACTOSIDASE'ACTIVITIES 559 TABLE 2. Clinical data and (3-galactosidase and a-glucosidase activities in homogenates of duodenal mucosa of children Patient Age Clinical diagnosis Homogenate p-galactosidases 6-BNG-1 asea Lactase a-glucosidases Maltase I Sucrase I m1se Histology yr mgprotein/ ml G. B. 2 Healthy R.E. 7 Exudative en teropathy V. V. 7 Celiac disease S. M. 5 Celiac disease N. R. 5 Celiac disease IU/g protein < Subtotal atrophy < Subtotal atrophy <0.05 <0.05 <0.05 <0.05 Subtotal atrophy a Abbreviation as in table 1. England), sucrose (a-d-glucopyranosyl-,8-dfructofuranoside, The British Drug Houses Ltd., Poole, England), lactose (4-(,8-D-galactopyranosyl) -D-glucopyranoside, Nutritional Biochemicals Co., Cleveland, Ohio), and 6- bromo-2-naphthyl-,8-d-galactopyranoside (Sigma Chemical Co., St. Louis, Mo.) were obtained commercially. Isomaltose (6- (a-d-glucopyranosyl) - D-glucopyranoside) was a generous gift from Dr. Allene Jeanes, Agricultural Research Service, United States Department of Agriculture, and from Prof. R. Weidenhagen, N euoffstein/ Pfalz, Germany. Tissue Fractionation Procedure Peroral jejunal biopsies were performed in adults with a Crosby capsule" and duodenal biopsies in children with a Brandborg-Rubin Quinton tube;14 pieces of mucosa weighing 25 to 50 mg were frozen immediately and kept at -20 C until homogenization in cold M NaCI with a hand-operated all glass homogenizer. Centrifugation was performed in the cold at 113,000 X g for 75 min. Protein was estimated by the method of Lowry et a1. 15 Enzyme Assays Enzyme assays were performed on the initial homogenate and on the supernatant and the pellet; the pellet was suspended in 0.5 to 0.8 ml of cold M NaC1. The procedure described by Auricchio et a1. '6 was used for the disaccharidase assays. 6-Bromo-2-naphthyl-,8- galactosidase assays were performed according to Cohen et a1.17 The coupling reaction was carried out with naphthanil diazo blue B (Echtblausalz B, Merck, Darmstadt, Germany) using commercial 6-bromo-2-naphthol (6- bromo-2-naphthol, Aldrich Chemical Co., Milwaukee, Wis.) as the standard. TABLE 3. Distribution of 6-bromo-2-naphthyl-{3- galactosidase and lactase activities from jejunal mucosa of adults in the supernatant fluid and pellet after centrifugation at 113,000 X g for 75 m1n Group, patient 6-BNG-asea Lactase super-i natant Pellet super-i natant Pellet fluid fluid IU X 100 IU X 100 1, Controls L.G < B.G < M.P S. E , Isolated lactase deficiency M.F < C.R < , Generalized disaccharidase deficiency B.A < a Abbreviation as in table 1. Results Tables 1 and 2 clearly demonstrate that in the pathological conditions studied, 6- BNG-ase activity remained unchanged although the activities of lactase or of all disaccharidases were lowered. Table 3 shows the distribution of lactase and 6-BNG-ase activities in the supernatants and the pellets. Roughly 60% of

4 560 ZOPFI ET AL. Vol. 50, No.4- the total 6-BNG-ase activity was found in the supernatant and about 20% in the pellet. This distribution was found in both the controls and the patients. The lactase activity was, on the other hand, demonstrated mainly in the pellet. Discussion The occurrence of significant 6-BNG-ase activity in intestinal mucosa of patients with isolated lactase deficiency and of patients with generalized disaccharidase deficiency confirms that at least two enzymes capable of hydrolyzing,8-galactosidic links are present. It would also indicate that the 6-BNG-ase activity is not bound to the brush border region of the epithelial cells since in malabsorption syndromes. this part of the cell undergoes serious alt'erations18 (histology reports in tables 1 and 2). Although the homogenates were not subjected to any special solubilization procedure, 6-BNG-ase activity was found in the supernatant fluid indicating that it had originally been in a soluble form or associated with a particulate (e.g. lysosomal) fraction destroyed by the freezing and fractionation applied to the biopsy specimens. The distribution of 6-BNG-ase activity between supernatant fluid and pellet in the controls was the same as in the patients with lactase deficiency. Thus, the presence of 6-BNG-ase activity in the pellets of three cases (M. F., C. R., B. A., table 3) with only insignificant lactase activity may have been due to incomplete tissue disruption, to contamination of the pellets with supernatant fluid, or to redistribution of the enzyme (s) during, the fractionation procedure.19 In conclusion, the results of this study provide further evidence for the multiplicity of intestinal,8-galactosidases in man. The presence of considerable 6-BNG-ase activity together with a deficiency of lactase activity in patients with clear-cut lactose malabsorption (M. F., B. A., table 1) suggests further that only the particulate enzyme, hydrolyzing mainly lactose, plays a physiological role in the intestinal absorption of lactose while the soluble,8- galactosidase may be the lysosomal,8- galactosidase which has been found m many other tissues.2o It should be noted that lactase deficiency of human small intestine can only be diagnosed when lactose is used as the substrate. Summary In homogenates of intestinal mucosa from normal adults and children, and from patients with acquired lactose malabsorption and with malabsorption syndromes characterized by various degrees of mucosal atrophy,,8-galactosidase activity was assayed using lactose and 6-bromo-2- naphthyl-,8-galactoside (6-BNG) as the substrates. Significant 6-BNG-ase activity was found in the mucosa of patients with an isolated lactase defect and in patients with generalized disaccharidase deficiency. After separation of supernatant fluids and pellets by centrifugation, 6-BNG-ase activity was found mainly in the supernatant fluid and lactase activity mainly in the pellet. This was the same for all subjects examined. These results are interpreted as additional evidence for the existence of more than one,8-galactosidase in human intestinal mucosa. It is thought that only one of these,8-galactosidases, the particulate enzyme hydrolyzing mainly lactose ("true lactase") plays a physiological role in the intestinal absorption of lactose, while the other, the soluble,8-galactosidase, might be compared to the lysosomal,8-galactosidase found in many other tissues. REFERENCES 1. Heilskov, N. S, C Studier over animalsk lactase. Ejnar Munksgaard, Copenhagen. 2. Doell, R. G., and N. Kretchmer Studies of small intestine during development. I. Distribution and activity of,a-galactosidase. Biochim. Biophys. Acta 62: Semenza, G" and S. Auricchio Chromatographic separation of human intestinal disaccharidases. Biochim. Biophys. Acta 65: Semenza, G., S. Auricchio, and A. Rubino Multiplicity of human intestinal disac-

5 April 1966 INTESTINAL fj-galactosidase ACTIVITIES 561 charidases. I. Chromatographic separation of maltases and of two lactases. Biochim. Biophys. Acta 96: a. Hsia, D. Y. Y., M. Makler, G. Semenza, and A. Prader fj-galactosidase activity in human intestinal lact.ases. Biochim. Biophys. Acta 113: Dahlqvist, A., B. Bull, and B. E. Gustafsson Rat intestinal 6-bromo-2-naphthylglycosidase and disaccharidase activities. I. Enzyme properties and distribution in the digestive tract of conventional and germfree animals. Arch. Biochem. Biophys. 109: Dahlqvist, A., B. Bull, and D. L. Thomson Rat intestinal 6-bromo-2-naphthylglycosidase and disaccharidase activities. II. Solubilization and separation of the small intestinal enzymes. Arch. Biochem. Biophys. 109: Koldovsky, 0., R Noak, G. Schenk, V. Jirsova, A. Heringova, H. Brana, F. Chytil, and M. Fridrich Activity of fjgalactosidase in homogenates and isolated microvilli fraction of jejunal mucosa from suckling rats. Biochem. J. 96: Wallenfels, K, and J. Fischer Untersuchungen uber milchzuckerspaltende Enzyme. X. Die Lactase des Kalberdarms. Z. PhysioI. Chern. 321: Doell, R G., and N. Kretchmer Studies of small intestine during development. II. The intracellular location of intestinal fjgalactosidase. Biochim. Biophys. Acta 67: Haemmerli, U. P., H. J. Kistler, R Ammann, S. Auricchio, and A. Prader Lactasemangel der Dunndarmmucosa als Ursache gewisser Formen erworbener Milchintoleranz beim Erwachsenen. Helv. Med. Acta 30: Prader, A., and S. Auricchio Defects of intestinal disaccharide absorption. Ann. Rev. Med.16: Arakawa, T., J. Akabane, and M. Akabane Lactose intolerance with normal activity of lactase. Tohoku J. Exp. Med. 83: Crosby, W. H., and H. W. Kugler Intraluminal biopsy of the small intestine; the intestinal biopsy capsule. ArneI'. J. Dig. Dis. 2: Brandborg, L. L., C. E. Rubin, and W. E. Quinton A multipurpose instrument for suction biopsy of the esophagus, stomach, small bowel, and colon. Gastroenterology 37 : Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R J. Randall Protein measurements with the Folin phenol reagent. J. BioI. Chern. 193: Auricchio, S., A. Rubino, R Tosi, G. Semenza, M. Landolt, H. Kistler, and A. Prader Disaccharidase activities in human intestinal mucosa. Enzym. BioI. Clin. (Basel) 3: Cohen, R B., K C. Tsou, S. H. Rutenburg, and A. M. Seligman The colorimetric estimation and histochemical demonstration of fj-d-galactosidase. J. BioI. Chern. 195: Rubin, C. E., L. L. Brandborg, A. L. Flick, W. C. MacDonald, R A. Parkins, C. M. Parmentier, P. Phelps, S. Sribhibhadh, and J. S. Trier, Intestinal biopsy. Ciba Foundation study group no. 14, p J. & A. Churchill Ltd., London. 19. Gitzelmann, R, E. A. Davidson, and J. Osinchak Disaccharidase of rabbit small intestine: intracellular distribution, solubilization, purification and specificity. Biochim. Biophys. Acta 85: Sellinger, O. Z., H. Beaufay, P. Jacques, A. Doyen, and C. de Duve Tissue fractionation studies 15: Intracellular distribution and properties of fj-n-acetylglucosaminidase and fj-galactosidase III rat liver. Biochem. J. 74: