LACTOSE TOLERANCE TESTS IN ADULTS WITH NORMAL LACTASE ACTIVITY

GASTROENTEROLOGY Copyright 1966 by The Williams & Wilkins Co. Vol. 50, No.3 Printed in U.S.A. LACTOSE TOLERANCE TESTS IN ADULTS WITH NORMAL LACTASE ACTIVITY ALBERT D. NEWCOMER, M.D., AND DOUGLAS B. MCGILL, M.D. Gastrointestinal Research Unit, Mayo Clinic and Mayo Foundation, Section of Medicine, and the Mayo Graduate School of Medicine, University of Minnesota, Rochester, Minnesota Lactose intolerance has been well documented by several groups in recent years. 1-6 Pediatricians have long suspected this condition, and current interest has been stimulated by several technical and conceptual advances. 7-9 Dahlqvist 9 has developed a simple method for the quantitative study of these enzymes in homogenates of the mucosa of the small bowel. He and others have been able to separate several enzymes which split disaccharides with varying specificities for different substrates. 10-12 Prior to these studies, lactose tolerance was defined in terms of response to an oral lactose load. Failure of the blood glucose to rise significantly over fasting levels was and is taken as evidence of lactose intolerance and presumably deficient concentrations of lactase in the small bowel. Studies of the normal subject, however, are incomplete with respect to the critical dose of lactose, patterns of symptomatic response, appearance time, height of rise of blood sugar, and correlation between such a rise and the concentration of mucosal enzyme. Our purpose is to report the findings in 18 normal subjects who were given an oral lactose load of 100 g and to correlate the peak rise in blood sugar with the concentrations of lactase in the jejunum at the ligament of Treitz. Lactose tolerance tests were repeated in the same persons after a 50-g dose, and the patterns of response Received October 16, 1965. Accepted November 22, 1965. Address requests for reprints to: Section of Publications, Mayo Clinic, Rochester, Minnesota 55902. This investigation was supported in part by Research Grants AM-6908 and AM-5259 from the National Institutes of Health, United States Public Health Service. 340 were compared. Limitations in the diagnostic usefulness of the test are apparent. Methods From a group of 69 volunteers 18 subjects were chosen on the basis of normal lactase activity in the jejunum. The 69 subjects were healthy, ambulatory, free of gastrointestinal disease, and without a history of milk intolerance. The five women and 13 men, all Caucasian, ranged in age from 21 to 48 years, with a mean age of 31 years. All had been on normal diets. The 18 subjects chosen were given 36 oral lactose tolerance tests. Two different doses of lactose were used: 50 g in 500 ml of water and 100 g in 200 ml of water. The 10% solution is nearly isotonic and the 50'70 solution is moderately hypertonic. All tests were done after an overnight fast and at least 1 week after the previous tolerance test. Ingestion of the lactose solution took less than 5 min. Venous blood sugar was measured with the subject fasting (0), and 15, 30, 60, 90, and 120 min after ingesting the lactose. Two subjects underwent the following two additional tests on separate occasions: (a) 25 g of glucose and 25 g of galactose in 500 ml of water given orally, and (b) 50 g of lactose in 500 ml of water instilled into the duodenum through a Dreiling tube over 5 min. In the former test, samples of blood were obtained at 0, 15, 30, 60, 90, and 120 min; in the latter, samples were obtained at 0, 5, 15, 3 0, 60, 90, and 120 min. An image intensifier was used for accurate placement of the tube. All biopsies were obtained at the ligament of Treitz. A double port capsule on a Rubin biopsy tube produced two mucosal specimens which were placed on filter paper. One specimen was placed in 10% formalin for histological examination (hematoxylin and eosin, periodic acid-schiff, and methanine silver stains). The other specimen was quickly weighed on a Sartorius balance (model 2603), frozen to -10 to -15 F within several minutes, and later used for the disaccharidase assay. Enzyme assays were done in less than 1 week. Values were shown to

March 1966 LACTOSE TOLERANCE TESTS 341 be unchanged after tissue had been frozen 8 weeks. Lactase activity was determined by the quantitative method of Dahlqvist 9 with one minor modification: the maleate buffer which contained lactose was adjusted to a ph of 5.8 rather than 5.6. Optimal enzyme activity was present at both values. Lactase activity is expressed as micromoles of lactose substrate hydrolyzed per gram of wet weight of mucosa per minute at 37 C. Values are a lso given in terms of lactose split per gram of protein per minute at 37 C. Blood sugar was determined in an autoanalyzer b y utilizing the potassium ferricyanide-ferrocyanide oxidation reduction reaction. Total protein was determined by the method of Lowry and associates." p-hydrion paper was used to establish the ph of the stool. Results Jejunal biopsies. The jejunal specimens for biopsy averaged 13.4 mg in wet weight (6.8 to 19.3 mg), and the mean mucosal protein content averaged 111 mg per g of wet weight (79 to 154 mg). Jejunal lactase activity (table 1). The range of lactase activity in the 18 subjects was 2.0 to 5.4 units per g of wet weight with a mean of 3.5 ± 1 SD of 1.1 (18 to 51 units per g of protein and a mean of 32 ± 1 SD of 10.6). The range of lactase activity in the normal population of 69 was 0 to 7.5 units per g wet weight with a mean of 3.1 ± 1 SD of 1.7 (0 to 65 units per g protein and a mean of 27). The enzyme activity in the group of 18 fell within the 21st and 78th percentile of the normal population of 69. Histological findings. All unfixed specimens showed a normal villous pattern under low power microscopy. All 17 specimens that were available for fixed sections appeared normal histologically. Sugar tolerance tests. All the blood sugar values are listed in table 1. The maximal rise in blood sugar above the fasting value after administration of 50 and 100 g of lactose is shown in figure 1. The mean rise for both doses of lactose was 28 mg per 100 ml (table 2). After the 50-g dose the increase in blood sugar ranged from 0 to 50 mg per 100 ml and after 100 g the range was 5 to 55 mg per 100 m!. TABLE 1. Results oj jejunal lactase activity and lactose tolerance tests J ejunal lactase Venous blood sugar Oral dose of Oral dose of Subject Age, sex lactose (50 g lactose 1100 Wet Protein in 500 ml gin 200 ml weight water), water)~ maximal maximal rise rise - - - --- - - - - Imits/g unitsj g mgj/ OO ml 1 29, M 2.0 19 50 46 2 35, M 3. 0 27 18 5 3 29, M 2.1 22 21 41 1 29, M 5.1 48 31 13 5 32, M 3.3 22 40 55 6 24, M 2.2 20-7 18 7 48, M 2.9 28 36 20 8 48, M 2.8 25 34 47 9 21, M 4.7 43 34 25 10 33, M 4.9 38 37 28 11 48, M 4. 0 44 33 49 12 30, M 5.4 44 29 32 13 24, M 3.5 40 0 29 14 29, F 3.8 31 37 15 15 24, F 2.3 18 42 20 16 27, F 3.0 28 16 23 17 21, F 3.5 30 29 18 18 33, F 4.0 51 20 11 A flat curve is defined in our study as an increase in blood sugar of less than 20 mg per 100 m!. Four subjects had flat curves after 50 g of lactose and six after 100 g (P > 0.7). Two subjects (2 and 6) had flat curves after both 50 and 100 g of lactose. The oral administration of 25 g of glucose and 25 g of galactose in 500 ml of water also resulted in flat curves with increases in blood sugar of 15 and 16 mg per 100 m!. Duodenal infusion of 50 g of lactose caused a prompt and substantial increase in blood sugar of 38 and 58 mg per 100 ml in the two subjects. An increase was detected at 5 min but a maximum was not reached until 15 min. Symptoms did not develop in either subject after instillation of lactose into the duodenum. The tolerance curves in case 6 are compared in figure 2. In the lactose tolerance tests using 50 g the maximal increase in blood sugar occurred at 15 min in 12 subjects and at 30 min in four. Two had no increase above the fasting level. With 100 g of lactose, the peak elevation in blood sugar occurred at

342 NEWCOMER AND MaGILL Vol. 50, No.3 60 50 40 30 20 10 o -10 '------' Lactose 50gm/500ml 100gm /200ml FIG. 1. Maximal rise in blood sugar after ingestion of 50 and 100 g of lactose in 18 subjects. I TABLE 2. Results of lactose tolerance tests Lactose Tests 50 g 100 g ---------------- Mean maximal rise in blood sugar 28 28 above fasting level (mg/loo ml) Range of maximal rise in blood sug- 0-50 5-55 ar (mg/loo ml) Number of tests with maximal rise 4 6 less than 20 mg/loo ml Time (minutes) of maximal rise: ~ ~ 6 W 4 7 60 *a 4 90 1 Two subjects having no rise above the fasting level. a * 15 min in six persons, at 30 min in seven, at 60 min in four, and at 90 min in one. There is a significant difference between the number of subjects in each group that have a peak rise at 15 min (P < 0.01). There is no correlation between lactase activity in the jejunum and maximal increase in blood sugar after either the 50 or 100 g of lactose. Symptoms. Except for initial fullness after drinking the lactose solution, symptoms did not develop in any of the 18 subjects during or after the test. Stool ph was checked for 2 days after all 50-g lactose tolerance tests. In two subjects the ph of the stool dropped to 5.5 but with no change in the normal formed character of the stool. In the other tests the stool ph was 6.0 or more. Comment Early reports describe flat curves after lactose tolerance tests in normal subjects but the incidence was exaggerated because samples were not obtained before 1 hr.14,15 More recently, Girardet and Richterich,16 using an 80-g dose in 200 ml of water, found peak rises of less than 25 mg per 100 ml in four of 16 normal adults. Lactase activity was not measured. Isselbacher and Senior 17 refer to five "flat" curves among 15 tolerance tests in normal subjects. The concept of an "absorptive ratio" was introduced by Cuatrecasus and associates. 5 This was defined as the ratio of the area under the lactose curve during the first 40 min to the area under the glucose-pius-galactose curve for the same period. Peak rises were not recorded. The "absorbers" had a ratio of more than 70% and the "nonabsorbers," less than 34%. Some of the subjects had assays for mucosal lactase, and the authors concluded that the tolerance tests accurately predicted lactase activity. The relation between lactase activity and peak rise has been reported by four groups whose findings are summarized in table 3. Littman and Hammond 18 reported that 22 of 23 subjects with normal lactase activity had an increase of more than 25 mg

March 1966 LACTOSE TOLERANCE TESTS 343... ~ C) " 1:: 150 130 I ~ 110 In 500 ml water x....,...-...,. I --.!t "- x,duodenallactose,50gm I / ' / ' / '\. t;' / ' \. \:oral glucose, 25gm and 90 '" -... galactose, 25 gm ~ ~ "- :;, 0 0 Ci5, "."'"... x-----x 70, """"" """",.----------------_. x... 50 ' -\Oral lactose,50gm 30 15 30 45 60 75 90 105 120 Time, minutes FlO. 2. Oral lactose, oral glucose-galactose, and duodenal lactose tolerance tests in subject 6. TABLE 3. Lactose tolerance tests in adults with normal lactase reported in literature Lacto s~ dosage Time of blood sugar delermina tions Norm.llactase activity in small bowel No. subjects Blood sugar rise g/ml H,O Littman and Hammond, 100/250 1965 18 Peternel, 1965 19 100/ 400 McMichael et ai., 1965 6 50/? Haemmerli et ai., 1965 4 50/400 Total min <20 mg/loo 1111 0, 30, 60, 90, 120 23 0, 30, 45, 60, 75, 90, S 120 0, 30, 60, 90, 120 21 1 0, 10, 20, 30, 40, 60, 12 1 90, 120 64 2 per 100 ml. One had a peak of 22 mg per 100 ml. PeterneP9 studied eight nondiabetic subjects and found that an increase of more than 20 mg per 100 ml distinguished these persons from a lactase-deficient group. McMichael and associates 6 found that only one of 21 patients with a variety of disorders had a maximal increase in blood sugar of less than 25 mg per 100 ml (15 mg per 100 ml). One of 12 subjects with normal lactase studied by Haemmerli et a1. 4 had a peak rise of less than 20 mg per 100 m!. In these 12, plus five additional subjects with "low normal" lactase values, peak rises occurred between 40 and 90 min with only one showing an increase at less than 40 min. There is, therefore, some uncertainty regarding the frequency and significance of the so-called flat curve for the

344 NEWCOMER AND McGILL Vol. 50, No.3 lactose intolerance test. The definition of a peak rise is arbitrary, but most authors have come to regard rises of 20 to 25 mg per 100 ml as tending to separate the normal from the lactase-deficient person. In the last four studies cited, normal values for jejunal lactase were most often associated with peak rises in blood sugar of this magnitude (table 3). Our study, however, indicates that increases of less than 20 mg per 100 ml may be anticipated in a significant number of normal subjects who do not have symptoms of intolerance to milk and who have normal values for jejunal lactase at the ligament of Treitz. Such persons will not have symptoms from the lactose load. A peak increase in excess of 25 mg per 100 ml in the nondiabetic, however, is good evidence against a lactase deficiency since we, like others, found flat curves in most subjects who had symptoms of milk intolerance and low levels of jejunal lactase at the ligament of Treitz (less than 0.5 units per g wet weight). In such subjects symptoms develop from an oral lactose load of 50 g. The reason for the discrepancy is not clear. Since many factors other than lactase activity in the small bowel influence the time and amount of increase in blood sugar, one would expect considerable variation with some flat curves in normal subjects; such has been shown to be the case. In 10 of 36 tests (28%), the maximal rise in blood sugar was less than 20 mg per 100 ml and in three it was less than 10 mg per 100 ml. The dosage and concentration of the lactose solution could not be incriminated as factors since no significant difference was apparent in the number of flat curves when the two methods of administration were compared. The mean and range of peak rises were the same with each dose. Some persons had a "normal" peak with the larger dose and flat curves with the smaller dose, while in others the converse was true. A flat lactose-tolerance curve may be a manifestation of an absorptive defect in the small bowel. 20 21 Such defects have been ruled out adequately in our subjects. The histology of the jejunal mucosa was normal in all 17 specimens examined. Six subjects who had a flat curve on one occasion had a peak rise of more than 25 mg on another. We did not consider it necessary to perform a glucose-galactose absorption test on these persons. The two subjects who had flat curves after both doses of lactose also had a low peak rise in blood sugar after the administration of glucose and galactose, but duodenal instillation of lactose resulted in a normal rise in blood sugar. In the studies reported to date, blood glucose has been sampled at different time intervals. In our opinion, it is important to begin with a 15-min value since 50% of our peaks occurred then. A flat lactose tolerance curve could result from exceedingly rapid gastric emptying. If such were the case, the peak rise in blood sugar would precede the 15-min value. In the two subjects who received lactose intraduodenally, the blood sugar was rising within 5 min and reached a peak at 15 min. At least in these two instances, rapid gastric emptying could not be implicated as a cause for the flat oral tolerance tests and it seems unlikely to explain the other flat curves. Delayed gastric emptying would appear to be a more likely mechanism. Failure to obtain the 15-min samples would have resulted in an additional five flat curves. Other workers have found that peak rises appeared later in the first hour. We were somewhat surprised to find 12 of 16 peak rises at 15 min when using the 50-g lactose dose in 500 ml of water. Six of 18 peaks in subjects using 100 g of lactose in 200 ml of water occurred at 15 min. This significant difference presumably reflects varying rates of gastric emptying. The more isotonic 50-g solution with its greater volume should promote more rapid gastric emptying. Hunt 22 has shown that the rate of gastric emptying is proportional to the log of the volume ingested and that osmoreceptors in the small bowel are sensitive to carbohydrate concentration. A hypertonic solution of sugar causes inhibition of gastric emptying through this osmoreceptor mechanism. All of the 18 subjects in this study had

March 1966 LACTOSE TOLERANCE TESTS 345 normal lactase activity in the jejunum. The values for this enzyme are lower than in some reported series. A major problem in the comparison of data from different laboratories and, in fact, between different patients is that small bowel values for lactase activity vary in the same subject, being very low in the proximal portion of the duodenum and rising near the ligament of Treitz. We have found that beyond this point there is considerable increase resulting in maximal levels further along in the small bowel. Thus, fluoroscopic control of the biopsy site is essential, but even then one is uncertain as to what point in the small bowel he is sampling. We have chosen to study the mucosa at the ligament of Treitz since this appears to be the most relatively fixed point. The relationship between values for lactase at this level and those at sites further along in the jejunum need to be studied further. It may be that some of our subjects have less than optimal levels of lactase in the more distal portion of the jejunum thus accounting for the flat curve. However, they denied symptoms of milk intolerance and symptoms did not develop after the lactose load. Data from other patients we have studied indicate that individuals with low levels of lactase throughout the jejunum will have symptoms of intolerance to milk and will develop symptoms after 50 g of lactose. The occurrence of 28% flat curves In normal persons after oral ingestion of lactose limits the usefulness of this test in the diagnosis of lactase deficiency. The subjective response to a lactose load taken orally is much more helpful than the peak rise in blood sugar in the recognition of lactase deficiency when a mucosal enzyme assay is not available. Although somewhat impractical, perhaps the peak rise in blood sugar after duodenal instillation of lactose would more clearly separate subjects with normal from those with deficient lactase activity. Summary Thirty-six oral lactose tolerance tests were done in 18 healthy persons who had normal jejunal lactase activity at the ligament of Treitz. Each subject received both a 50- and a 100-g dose of lactose in solutions of different tonicity. An absorptive defect was ruled out in each subject. A rise in blood sugar of less than 20 mg per 100 ml (flat curve) above the fasting level was observed in 28% of the tests. Failure to obtain a blood sugar at 15 min would have resulted in flat curves in an additional 14%. Flat curves were obtained with the oral administration of both doses of lactose. Two subjects had flat curves after the oral administration of both doses and also after ingesting the component monosaccharides. Duodenal instillation of lactose in these two subjects produced a normal curve. This suggested that delayed gastric emptying contributed to a low rise in blood sugar after oral intake of lactose. The limitations in the use of the oral lactose tolerance test to diagnose lactase deficiency are apparent. REFERENCES 1. Kern, F., Jr., J. E. Struthers, and W. L. Attwood. 1963. Lactose intolerance as a cause of steatorrhea in an adult. Gastroenterology 45: 477-487. 2. Dahlqvist, A., J. B. Hammond, R. K. Crane, J. V. Dunphy, and A. Littman. 1963. Intestinal lactase deficiency and lactose intolerance in adults: Preliminary report. Gastroenterology 45: 488-491. 3. Klotz, A. P. 1964. Intestinal lactase deficiency and diarrhea in adults. Am. J. Dig. Dis. 9: 345-354. 4. Haemmerli, U. P., H. Kistler, R. Ammann, T. Marthaler, G. Semenza, S. Auricchio, and A. Prader. 1965. 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. 5. Cuatrecaslls, P., D. H. Lockwood, and J. R. Caldwell. 1965. Lactase deficiency in the adult: A common occurrence. Lancet 1: 14-18. 6. McMichael, H. E., J. Webb, and A. M. Dawson. 1965. Lactase deficiency in adults: A cause of "functional" diarrhoea. Lancet 1: 717-720. 7. Miller, D., and R. K. Crane, 1961. The digestive function of the epithelium of the small intestine. 1. An intracellular locus of disac-

346 NEWCOMER AND McGILL Vol. 50, No.3 charide and sugar phosphate ester hydrolysis. Biochim. Biophys. Acta 62: 281-293. 8. Miller, D., and R. K. Crane. 1961. The digestive function of the epithelium of the small intestine. II. Localization of disaccharide hydrolysis in the isolated brush border portion of intestinal epithelial cells. Biochim. Biophys. Acta 62: 293-298. 9. Dahlqvist, A. 1964. Method for assay of intestinal disaccharidases. Anal. Biochem. 7: 18-25. 10. Dahlqvist, A. 1962. The intestinal disaccharidases and disaccharide intolerance (Editorial). Gastroenterology 43: 694-696. 11. Dahlqvist, A. 1962. Specificity of the human intestinal disaccharidases and implications for hereditary disaccharide intolerance. J. Clin. Invest. 41: 463-470. 12. Semenza, G., and S. Auricchio. 1962. Chromatographic separation of human intestinal disaccharidases. Biochim. Biophys. Acta 66: 172-175. 13. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193: 265-275. 14. Field, C. W. 1919. Blood sugar curves with glucose, lactose, maltose, mannite, and cane sugar. Proc. Soc. Exp. BioI. Med. 17: 29-30. 15. Koehler, A. E., 1. Rapp, and E. Hill. 1935. The nutritive value of lactose in man. J. Nutr. 9: 715-723. 16. Girardet, P., and R. Richterich. 1963. L'aspect statistique des courbes de charge orale en lactose chez Ie sujet normal. Schweiz. Med. Wschr. 93: 1808-1811. 17. Plotkin, G. R. 1964. Quoted by Isselbacher, K. J., and J. R. Senior. The intestinal absorption of carbohydrate and fat. Gastroenterology 46: 287-298. 18. Littman, A., and J. B. Hammond. 1965. Diarrhea in adults caused by deficiency in intestinal disaccharidases. Gastroenterology 48: 237-249. 19. Peternel, W. W. 1965. Lactose tolerance in relation to intestinal lactase activity. Gastroenterology 48: 299-306. 20. Jeejeebhoy, K. N., H. G. Desai, and R. V. Verghese. 1964. Milk intolerance in tropical malabsorption syndrome: Role of lactose malabsorption. Lancet 2: 666--668. 2l. Lifshitz, F., A. P. Klotz, and G. H. Holman. 1965. Intestinal disaccharidase deficiencies in gluten-sensitive enteropathy. Amer. J. Dig. Dis. 10: 47-57. 22. Hunt, J. N. 1963. The duodenal regulation of gastric emptying. Gastroenterology 46: 149-156.