ACTIVITIES OF BRUSH BORDER LACTASE, ACID fj-galactosidase, AND HETERO-fj-GALACTOSIDASE IN THE JEJUNUM OF THE ZAMBIAN AFRICAN

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1 GASTROENTEROLOGY 64: , 1973 Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No. 3 Printed in U.S.A. ACTIVITIES OF BRUSH BORDER LACTASE, ACID fj-galactosidase, AND HETERO-fj-GALACTOSIDASE IN THE JEJUNUM OF THE ZAMBIAN AFRICAN G. C. COOK, M.D., F.R.C.P., N.-G. Asp, M.D., AND A. DAHLQVIST, M.D. Department of Medicine, The University of Zambia, Lusaka, Zambia; and Department of Medicine, University Hospital, and the Department of Nutrition, Chemical Center, the University of Lund, Lund, Sweden The activities of brush border lactase, lysosomal acid fj-galactosidase, and cytoplasmic hetero-fj-galactosidase have been measured in jejunal biopsies from 26 Zambian African subjects. All of them had adult hypolactasia. A low activity of brush border lactase was demonstrated, and that amounted to 5 to 90% (mean 62) of the total lactase activity at ph 6.0. Acid fj-galactosidase and hetero-fj-galactosidase activities were not decreased. Oral lactose tolerance tests were abnormal in 25 of the subjects, and 21 had gastrointestinal symptoms. There was a significant correlation between brush border lactase activity and the maximum blood glucose rise after oral lactose (P < 0.05), indicating that the low concentration of that enzyme in Zambian subjects is adequate for the hydrolysis of a significant fraction of oral lactose. A very high incidence of adult hypolactasia has been shown in the Bantu tribes of Uganda 1-3 and South Africa. 4 A similar prevalence would be expected in the Zambian. African who is probably of ethnologitally similar stock. Recent investigations have shown that the human intestine contains three different fj-galactosidases: a brush border lactase, a lysosomal acid fj-galactosidase, and a hetero-fj-galactosidase which is Received August 29, Accepted November 1, Address requests for reprints to: Dr. N.-G. Asp, Department of Nutrition, Chemical Center, P.O. Box 740, s , Lund 7, Sweden. The authors acknowledge the financial support from The University of Zambia Research Fund, The Swedish Medical Research Council (Project nos. B69-13P-2262, and B69-13X-157), The Swedish Nutrition Foundation, A. PAhlsson's Foundation, and the Medical Faculty of the University of Lund. The authors are also grateful to Mr. James Tembo for his help with the patients, and for explaining the procedures to them, and to Mrs. G. Andersson, Mrs. B. NorEm, and Miss U. Iwarson for the chemical analyses. probably cytoplasmic in origin. 5-7 The activity of each of these enzymes has been studied in a group of Zambian African subjects, with recently developed methods. S Subjects and Methods Table 1 gives details of the 26 subjects investigated. Twenty were male and six female. The mean age was 36 (17 to 59) years. Most of the main tribal groups of Zambia were represented. None had clinical evidence of malnutrition or of gastrointestinal disease. They were all inpatients at the University Teaching Hospital, Lusaka. They were taken at random from one medical ward of the hospital. Lactose (n = 26) and glucose plus galactose (n = 5) tolerance tests were done and jejunal biopsy specimens (n = 26) were obtained as previously reported. I Blood glucose was estimated by a glucose-oxidase method. The biopsy specimens were all divided into two as soon as they were obtained. The main part was immediately frozen to -20 C and transported by air to Sweden in Dry Ice; all of the specimens were wrapped in parafilm as soon as they were obtained and dispatched in plastic tubes, also sealed with parafilm, in three separate batches (nos. 1 to 8, 9 to 18, and 19 to 26). The other 405

2 406 COOK ET AL. Vol. 4, No.3 TABLE 1. Clinical details and results of oral carbohydrate tolerance tests Maximum Patient Sex Age Tribe Diagnosis glucose Maximum rise after Estimated Symptoms glucose glucose milk after rise after (25 g) intake lactose a lactose plus (50 g) galactose (25 g) yr pints/day mg/loo ml 1 F 27 Tonga No abnormality '/2 C 8 2 F 20 Bemba Iron-deficiency anemia 1;' C 9 3 F 48 Lungu Unexplained abdominal pain? C 3 4 M 20 Kasai Acute malaria (recovered) '/ M 31 Tumbuka Post-traumatic epilepsy <,;' C 14 6 F 26 Kunda Right lower lobar pneumonia 1 D F 19 Inamwanga Schistosoma mansoni infection < '12 D 7 56 (mild) 8 M 58 Zulu Chronic malaria '/2 D M 23 Tonga No abnormality 0 D M 51 Kaluchazi Pulmonary tuberculosis M 59 Ngoni No abnormality 1 D M 52 Zulu Tuberculous pleural effusion 1 C 8 13 M 44 Bisa Pulmonary tuberculosis 1 C 0 14 M 28 Kunda Acute malaria (recovered) 1 C M 40 Bemba Alcoholic coma (recovered) 1,1~ M 43 Lozi Contact dermatitis '12 C M 37 Chewa No abnormality 1 C " 18 M 22 Lunda No abnormality 1;' D M 17 Nsenga Iron-deficiency anemia '12 D M 52 Soli Mild hypertension '12 D F 22 Nsenga Chronic calcific pancreatitis 1/2 D 4 22 M 24 Lozi Right lower lobar pneumonia '/2 D M 50 Kaonde-Ila Mild hypertension M 30 Chewa Polyarthropathy of unknown '/2 D+ 9 etiology 25 M 59 Ngoni Left lower lobar pneumonia '12 D M 35 Nsenga Right lower lobar pneumonia 1 13 a C, cohc; D, diarrhea; D+, severe diarrhea. " Mild diabetic curve. part of the biopsy specimen was immediately placed in 10% (w/v) formol-saline for histological examination. Dissecting microscope and histological appearances will be reported elsewhere (G. C. Cook, N. O. Berg, unpublished observations). All of the biopsy specimens were similar to those taken from Ugandan patients,9 and very few, if any, would have been considered normal for a British or Swedish population. In the intact biopsy specimen the enzyme activities studied in this investigation are stable for many months at -20 C. After homogenization some of the enzyme activities decrease on storage, and all were estimated within 30 hr of that procedure. Disaccharidase activities were measured by methods previously reported ' When total lactase was assayed at ph 6 with the glucose oxidase-peroxidase system 10 internal standards were used, i.e., standards containing the same amount of mucosal homogenate as in the assay procedure, since the high concentrations of homogenate necessary for the assay of very low lactase activity interferes with the glucose oxidase-peroxidase system. 13 In most of samples total lactase was also estimated by measuring liberated galactose with a galactose dehydrogenase-nad+ system. 12 The two methods gave concordant results. The different /3-galactosidases were assayed specifically as described by Asp and Dahlqvist. 8 Brush border lactase was estimated in two different ways. (1) Lactase activity at ph 6 was assayed in the presence of 0.2 mm p-chloromercuribenzoate which in-

3 March 1973 JEJUNAL fj-galactosidases IN ZAMBIANS 407 TABLE 2. Results of brush border lactase, acid fjgalactosidase, and hetero- fj-galactosidase assaysa Patient HeteroiJ-galac- tosidase Brush border lactase Acid ij-galactosidase 2-NG' at As lactase ph 4.5 at ph Mean a All results are expressed as units per gram of protein. The technique used for the separate assay of the three different fj-gaiactosidases is given in detail in reference 8. 2-NG, 2-naphthyl fj-galactosidase. hibits the contribution of the acid p-galactosidase. (2) The lactase activity at ph 6 exerted by the acid p-galactosidase was calculated from the specific assay of that enzyme and subtracted from the total lactase activity, the difference representing the activity of the brush border lactase. The two methods gave concordant results and the mean value is given in table 2. Enzyme activities have been calculated as units (micromoles of substrate hydrolyzed per minute at 37 C) per gram of mucosal protein. Results Tolerance tests. Table 1 gives the results for the maximum rise in blood glucose after oral lactose, and glucose plus galactose loads. Twenty-five of the 26 subjects had a maximum blood glucose rise of less than 20 mg per 100 ml after 50 g of lactose. In 1 subject (no. 18) the maximum rise was 27 mg per 100 ml. Twenty-one of the subjects had symptoms after lactose. There were no symptoms after glucose plus galactose. Disaccharidases. Table 3 gives the results of the disaccharidase assays. Total lactase activity was low in all of the biopsy specimens, and within the range reported in subjects with adult hypolactasia in other populations. There was a significant correlation between lactase activity and the maximum rise in blood glucose after oral lactose in the different individuals (P < 0.02) (fig. 1). The maltase, isomaltase, sucrase, and trehalase concentrations were all within the normal TABLE 3. Results of jejunal disaccharidase assays at ph 6.0" Patient Total lactase Maltase Isomaltase Sucrase Trehalase Mean a All results are expressed as Units per gram of protein at ph 6.0.

4 408 COOK ET AL. Vol. 4, No ~ E UJ <.fl C( o. ~~~~~~~-~~~~- ~ ~~--~ CO ~~-~ ~~,~-/ III ~/O~O \ ~ o. ~ olo--4,..._-' '--'--..j..--l-.l..---' o ~ W LACTASE ACTIVITY (UNITS Ig PROTEIN) FIG. 1. Correlation between lactase activity in the biopsy specimens and the maximum rise in blood glucose after oral lactose. The correlation is significant for both total lactase (e) (r = ; P < 0.02) and brush border lactase (0) (r = ; P < 0.05). The calculated regression lines for total lactase (- - -) [y = (x)] and brush border lactase (-) [y = (x)] are shown. range, although the means were slightly higher than in European and American subjects. Brush border lactase. Table 2 gives the results of the brush border lactase assays. A low residual activity was demonstrated in 24 of the biopsy specimens. The very low concentrations in subjects 7 and 10 (0.1 U per g of protein) are not significantly higher than zero using the present methods. There was a significant correlation between brush border lactase activity and the maximum rise in blood glucose after oral lactose in the different individuals (P < 0.05) (fig. 1). Acid (3-galactosidase. Table 2 gives the results of the acid (3-galactosidase assays. The mean is within the normal range for other population groups. 16 Hetero-(3-galactosidase. Table 2 gives the results of the hetero-(3-galactosidase assays. The mean is within the normal range for other population groups. 16 The very low concentration in subject 9 is not significantly higher than zero by the method used. There was not a significant positive correlation between either acid (3-galactosidase (r = ) or hetero-(3-galactosidase (r = ) activities and the maximum blood glucose rise after oral lactose in the different individuals (fig. 2). Discussion In the 26 adult Zambian African subjects studied there is a 100% incidence of adult hypolactasia. This is similar to that in the Bantu people of East 1 and South Africa. 4 Recent work in Israel confirms that adult hypolactasia has agenetic basis. 14 The activities of the other disaccharidases are slightly higher than those in most Caucasians; the reason for this is not clear, and is surprising in view of the fact that only a few of the biopsy specimens in this study were completely normal when compared with those from subjects studied in Sweden (G. C. Cook, N. O. Berg, unpublished observation). All of the disaccharidase activities are similar to those in Ugandan African Bantu subjects. 2 Recent work has demonstrated three separate (3-galactosidases in the jejunum of subjects in North America and Sweden, where adult hypolactasia is unusual. 5-7 The (3-galactosidase activity is predominantly due to the brush border enzyme (brush border lactase), which is presumably the only enzyme involved in the hydrolysis of dietary lactose. That enzyme l UJ 20 <.fl o U ;j...j (!) c c c 9 10 FIG. 2. Correlation between activities of acid fj-galactosidase (0) and hetero-fj-galactosidase (_), and the maximum blood glucose rise after oral lactose. The correlations are not significant. For acid fj-galactosidase, r = ; for hetero-f3-galactosidase, r = The calculated regression lines for acid f3-galactosidase ( ) [y = (x)] and hetero-fj-galactosidase (- - -) [y = (x)] are shown.

5 March 1973 JEJUNAL {j-galactosidases IN ZAMBIA'VS 409 can also hydrolyze some synthetic heterofj-galactoside substrates, although lactose is most rapidly hydrolyzed. Lysosomal fjgalactosidase has an acid ph optimum and is known as acid fj-galactosidase. It also hydrolyzes lactose and synthetic substrates and is relatively nonspecific. It is confined to the lysosomes,15 and probably does not contribute to the digestion of dietary lactose in vivo. The third enzyme is present in a soluble form in the cytoplasm. It can hydrolyze a number of synthetic hetero-fj-galactoside subiitrates, but not lactose; it is known as hetero-fjgalactosidase. When the three fj-galactosidase activities in the Zambian African subjects are compared with Scandinavian subjects with high lactase 16 it is clear that the mean acid fj-galactosidase and hetero-fjgalactosidase activities are similar in both groups. In some biopsy specimens the hetero-fj-galactosidase was very low, but that occurred as often in the Zambian African as in Scandinavian subjects. An association between low activities of hetero-fj-galactosidase and low brush border lactase has not been shown, and the hypothesis put forward by Gray et ai.,17 that hetero-fj-galactosidase is either a precursor or a fragment of brush border lactase, is not substantiated. The correlations between brush border lactase and acid fj-galactosidase (r = ) and hetero-fj-galactosidase (r = ) in this study are not significant. Although the total lactase activity was very low, brush border lactase could be demonstrated in 24 of the 26 subjects studied. In the remaining 2 subjects the enzyme was probably present although the value (0.1 U per g of protein) was not significantly different from zero with the methods used. The mean concentration of brush border lactase was 1.8 U per g of protein, which is approximately 5% of the mean in Caucasian adults with high lactase activity. In subjects with adult hypolactasia from Finland 16 the mean brush border lactase was 4.1 U per g of protein and that is significantly higher than in our Zambian African subjects. The mean age of the Finnish subjects was 46 years (range 26 to 59), i.e., 10 years older than the Zambians now studied. Thus, age differences between the groups are not likely to account for the different levels of residual lactase. These are perhaps due to a complex multifactorial regulation of the level of brush border lactase. In methods generally used for the assay of jejunal lactase, lactose is used as the substrate, and the assay is carried out at ph The brush border lactase is then active at an optimum ph, but the acid fj-galactosidase also contributes to the resultant activity, usually with about 1 U per g of protein, and constitutes a considerable fraction of the total lactase activity in biopsies from subjects with adult hypolactasia. In this investigation 9 to 95% (mean 38) of the total lactase activity at ph 6.0 was due to the acid fj-galactosidase. There was a significant positive correlation between lactase activity (brush border lactase and total lactase) and the rise in blood glucose after oral lactose. That indicates some in vivo hydrolysis of lactose by the residual brush border lactase. There was no positive correlation between the activities of the other two fj-galactosidases and the rise in blood glucose after lactose. These enzymes are presumably not involved in the hydrolysis of dietary lactose. REFERENCES 1. Cook GC, Kajubi SK: Tribal incidence of lactase deficiency in Uganda. Lancet 1: , Cook GC, Dahlqvist A: Jejunal hetero {j-galactosidase activities in Ugandans with lactase deficiency. Gastroenterology 55: , Cook GC: Lactase deficiency: a probable ethnological marker in East Africa. Man N S 4: , Jersky J, Kinsley RH: Lactase deficiency in the South African Bantu. S Afr Med J 41: , Asp NG, Dahlqvist A, Koldovsky 0: Human small intestinal {j-galactosidases. Separation and characterization of one lactase and one hetero {j-galactosidase. Biochem J 114: , 1969

6 410 COOK ET AL. Vol. 4, No.3 6. Asp NG: Human small intestinal It-galactosidases. Separation and characterization of three forms of an acid It-galactosidase. Biochem J 121: , Gray GM, Santiago NA: Intestinal It-galactosi 'dases. I. Separation and characterization of three enzymes in normal human intestine. J Clin Invest 48: , Asp NG, Dahlqvist A: Human small intestine It-galactosidases. Specific assay of three different enzymes. Anal Biochem 47: , Cook GC, Kajubi SK, Lee FD: Jejunal morphology of the African in Uganda. J Pathol 98: , Dahlqvist A: Assay of intestinal disaccharidases. Anal Biochem 22:99-107, Dahlqvist A: Assay of intestinal disaccharidases. Enzymol Bioi Clin (Basel) 11:52-66, Dahlqvist A, Asp NG: Accurate assay of low intestinal lactase activity with a fluorometric method. Anal Biochem 44: , Asp NG, Koldovsky 0, Hoskova J: Use of the glucose oxidase method for assay of disaccharidase activities in the small intestine-a limitation. Physiol Bohemoslov 16: , Gilat T, Kuhn R, Gelman E, et al: Lactase deficiency in Jewish communities in Israel. Am J Dig Dis 15: , Alpers DH: Separation and isolation of rat and human intestinal It-galactosidases. J Bioi Chern 244: , Asp NG, Berg NO, Dahlqvist A, et al: The activity of three different small-intestinal It-galactosidases in adults with and without lactase deficiency. Scand J Gastroenterol 6: , Gray GM, Santiago NA, Colver EH, et al: Intestinal It-galactosidases. II. Biochemical alteration in human lactase deficiency. J Clin Invest 48: , 1969