Disaccharide Absorption in Normal and Diseased Human Intestine

Vol. 51, No.4

GASTROENTEROLOGY

Printed in U.S.A.

Copyright© 1966 by The Williams & Wilkins Co.

DISACCHARIDE ABSORPTION IN NORMAL AND DISEASED HUMAN INTESTINE GARY

M.

GRAY,

M.D.,

AND NILDA

A.

SANTIAGO, B.S.

United Stales Army T ropical Research Medical Labomto1'y, San Juan, Puerto Rico, and Evans MernoTial Department of Clinical Research, University Ho spital, University Medical Center, Boston, Massachusetts

In recent years, interest in intestinal absorption of carbohydrates has been concentrated on the study of monosaccharide absorption, especially glucose absorption, 1 - 3 despite the fact that man's carbohydrate intake is almost completely in the form of disaccharides and oligosaccharides. Since starch, the principal dietary oligosaccharide, is hydrolyzed intralumina lly by pancreatic amylase mainly to disaccharides, the intestinal absorptive cells are faced with the task of hydrolyzing disaccharides and transporting the released monosaccharide products. Whereas there has been intense interest in the clinical syndromes associated -.,vith disaccharide intolerance 4 - 8 and intestinal disaccharidase deficiency, 9 - 13 our present knowledge of disaccharide hydrolysis and absorption in man is based mainly on in vitro assays of intestinal biopsy homogenates and on changes in blood sugar concentrations after ingestion of elisaccharides. Except for recent information from rat experiments, 14 - 16 little is known about the hydrolysis process in vivo of the common dietary disaccharides other than sucrose, 17 - 19 and comparative data concerning the interrel ation of hydrolysis and

absorption in normal and abnormal intestine is lacking. In the present experiments, normal subjects and patients with tropical sprue or intestinal lactase deficiency were studied by an intestinal perfusion technique in order to evaluate more directly the disaccharide absorption process.

Methods Normal controls . Ten normal subjects (7 from Boston, 3 from Puerto Rico) were studied. Those from Puerto Rico had histologically normal jejunal biopsies by light microscopy, no evidence of malabsorption as measured by both 6-day quantitative stool fat excretion and the 25-g xylose excretion test, normal intestinal disaccharidases;• and no anemia. Despite recent reports of racial variation of intestinal disaccharidases,"1 ' 22 we found no difference in enzyme activities for 28 Puerto Rican and 14 North American subjects (table 1), and the incidence of lactase deficiency in the two groups was similar. In the intestinal perfusion experiments described below, t he mean hydrolysis and absorption rates were the same for both groups (3 Puerto Ricans and 7 North Americans), and hence they were combined to serve as the control group. Patients with intestinal disease. In the 11 patients with tropical sprue, stool fat excretion was greater than 15 g per day, urinary xylose Received April 4, 1966. Accepted May 16, 1966. Address requests for reprints to: Dr. Gary M. excretion after ingestion of 25 g was less than 4 Gray, Department of Medicine, Stanford Uni- g in 5 hr, and the jejunal biopsy showed (1) \·ersity School of Medicine, Palo Alto, California shortening of villi and loss of the normal scalloping, (2) semicuboidal epithelial cells rather 94304. This work was supported in part by United than the normal columnar type, and (3) inStates Public Health Service Research Grant crease in the number of lymphocytic cells in the AM 03560 and by Training Grant T1 AM 5025 lamina propria. All patients had mild to moderfrom the National Institute of Arthritis and ate megaloblastic anemias. None of the patients had received any vitamin or antibiotic Metabolic Diseases. The authors are grateful to Dr. F. J. Ingel- therapy. finger for support and guidance and for his helpIn addition, 2 patients were studied who had ful criticism of the manuscript. intestinal lactase deficiency as determined by 489

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GRAY AND SANTIAGO T ABLE

1. Comparison of disacchm·idases• Lactase-deficient

N ormal values

Group

Total no.

North American Puerto Ri can

·-

14 28

Lactase

Sucrase

Maltase

No .

%

50 (15-82) 42 (18-69)

87 (40-165) 93 (40-154)

241 (128-428) 262 (110-461)

2 6

21

14

• Assayed as described by Dahlqvist2° and expressed as micromoles split per minute per gram of protein. Mean va lues are given with range in p arentheses .

quantitative assay20 of jejunal mucosa. Both patients had histologically normal intestinal biopsies and no biochemical evidence of generalized malabsorption. They developed symptoms of cramping abdominal pain and diarrhea after ingesting milk or lactose. Perfusion experiments. The subject swallowed a double lumen t ube the evening before the study, and the · position of the tube was adjusted under fluoroscopy the following morning so that the proximal orifice was just beyond the ligament of Treitz.18 The carbohydrate solution, made isotonic with sodium chloride, was infused at 15 ml per min through the proximal orifice and samples collected by siphonage t hrough the distal tube whose orifice was 30 em from the point of infusion. Polyethylene glycol 4000 (PEG) was the nonabsorbable, watersoluble marker and was analyzed by Hyden's method.23 A steady state was reached within 30 min so that, thereafter, concentrations of sugar and PEG in successive 10- to 15-min samples did not vary more than ±10%. Solutions containing 80 mmoles per liter of disaccharide (lactose, sucrose, or maltose) or the equivalent monosaccharide mixture [glucose + galactose (80 mM each), glucose + fructose (80 mM each), or glucose (160 ruM)] were given successively in random order as previously outlined so that absorption of the various sugars could be compared.19 Assay of carbohydrates. Sugars were reagent grade and were chromatographically pure except galactose, which contained a trace of glucose and of an oligosaccharide.19 All were determined on Ba (OH) 2-ZnSO, filtrates" and recovery of any sugar added to intestinal juice was 100 ± 5%. Sucrose, glucose, and fructose were assayed enzymatically as previously described.18 Galactose was analyzed by the usual galactose oxidase (GaO) method19 " 25 except that the volume of sample was 0.1 ml, of reagent 0.25 ml, and absorbance was determined in microcuvettes (0.7-ml. capacity) with a 1-cm light

path. A linear relation prevailed when 5 to 13 f.tg of galactose was in t he reaction mixture. Since GaO also has a slight specificity for lactose,25 a correction was made when indicated by subtracting the absorbance found by GaO assay of that amount of lactose known to be in the sample. Lactose and maltose were analyzed by a 2step reaction involving (1) hydrolysis of disaccharide by the appropriate enzyme and (2) analysis by tris (hydroxymethyl) amino methane (tris)-buffered glucose oxidase18 of the glucose released from this hydrolysis. In the hydrolysis step, 1 ml of sample containing up to 1 mg of disaccharide was incubated with 1 ml of buffered disaccharidase solution for 1 hr at 37 C. These lactase and maltase (Nutritional Biochemicals, Cleveland, Ohio) solutions were made fresh daily by adding 250 mg of the enzyme to 25 ml of 0.1 M sodium phosphate buffer at pH 7.0, shaking and filtering . The difference between the glucose in the sample before and after hydrolysis represented glucose released from the disaccharide. Free monosaccharides in collected sample did not interfere with the analysis, and assays were within 5% of the expected glucose value. Disaccharidase assay . Sucrase, lactase, and maltase activities of intestinal samples were determined by a modification18 of Dahlqvist's method.17 Expression of results. When perfusion techniques are utilized, it is usually assumed that the test substance that disappears from the intestinal lumen has been absorbed. However, description of the disaccha ride absorption process is more complex. Since, as determined from urinary excretion studies, only 0.1 to 1.0 mmole of disaccharide is absorbed intact after in
Octobe1· 1966

491

DISACCHARIDE ABSORPTION I N MAN

drolysis. This assumption is supported by the finding of less than 0.5 mmole of intact disaccha ride excreted in the urine during our studies in which 72 mmoles of disaccharide were infused into the intestine. (Since maltose is metabolized after absorption,'"· ,. urinary excretion is not an accurate measure of the extent of its absorption. However, intestinal maltase activity is considerably greater than t hat of other disaccha ridases, and it is unlikely that intact maltose crosses the mucosal barrier even to the same extent as sucrose or lactose.) To determine absorption, the monosaccharide products that accumulate within the intestinal lumen must be subtracted from the disaccharide that disappears.'" The disaccharide absorption process can therefore be considered in terms of (1) disaccharide hydrolysis and (2) monosaccharide absorption . Further, since t he monosaccharide products can be measured separately, absorption may be expressed in terms of the individual monosaccha rides.'"

Results

H ydrolysis and absorption rates of maltose, sucrose, and lactose in normal snbjects . Figures 1 and 2 show the results of 30-cm segment perfusion studies in 10 norm a l subj ects. Hydrolysis rates for lactose were appreciably slower than those found for sucrose or maltose (P < 0.01 ) (fig. 1) . When considered in terms of the monosaccharide components, absorpt ion rat es for lactose were only half as rapid as those for su crose or maltose (P < 0.01) (fig. 2). Hydrolysis and absorption r ates of sucrose and maltose did not differ signifi cantly from each other (P > 0.1), and indeed, when individual studies were compared t here was a significant correlation of the hydrolysis rates for t hese two disaccharides (r = 0.78; P < 0.05). Absorption of disaccharides versus monosaccharide mixtures. Absorption from sucrose has been shown to occur at t he same rates as from the equivalent monosaccharide mixture. 19 Figure 3 demonstrates that glucose absorption was also no different whether maltose (80 mM) or the equivalent glucose (160 mM) solution was infused. However , as shown in the left portion of figure 3, absorption of glucose (30 mmoles per hr) and galactose (21 mmoles

ci :z:

"~ w

r+

60

so

0

40


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,--

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JO

0::0

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o-

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M

Fm. 1. Comparison of hydrolysis rates of lactose (L), maltose (M), and sucrose (S) in 10 normal subj ects. A 30-cm infusion-to-collection distance was used in jejunum, and brackets indicate ::!: 2 SE in this and subsequent figures.

per hr) from lactose was appreciably slower than from the monosaccharide mixture (glucose 48 mmoles per hr and galactose 30 mmoles per hr) (P < 0.05). Also notable is the fact that glucose absorption was more rapid than galactose absorption in both the lactose and glucosegalactose infusion studies (paired analysis, t-test P < 0.01). Hydrolysis and absorption in tropical sprue. Figure 4 compares hydrolysis for 11 patients with intestinal malabsorption due to tropical sprue to t hat for the 10 normal subj ects. Hydrolysis of all three disaccharides was markedly decreased from normal (P < 0.001 ). Lactose hydrolysis was impaired appreciably more ( -75%) than that for t he other two disaccharides, (paired analysis, t-test P < 0.02) and hydrolysis was lowered more for sucrose (-54%) than for maltose ( -40%) (P < 0.05). T able 2 shows that absorption in terms of t he individual monosaccharides was decreased similarly for both disaccharides and monosaccharide mixtures, except t hat glucose product absorption from lactose was signifi cant ly more impaired than was its absorption from the glucosegalactose mixture. Comparative kinetics of lactase, maltase, and sucrase . In our perfusion experiments, only one concentration of sugar (80 mM) was used. It is conceivable that the discrepancies in hydrolysis rates for t he three disaccharides might be due to differences in the kinetics of the specific

492

Vol. 51, No.4

GRAY AND SANTIAGO 100 90

80

70

60 50

z

Q

40

10..

a: 0

30

V)

al

<

20

a:

10

:I:

'_. V)

w

0 ::E :1

30 40

LACTOSE

SUCROSE

MALTOSE

FIG. 2. The fate of monosaccharides released from the disaccharide hydrolysis shown in figure 1 : G, glucose; Ga, galactose; F, fructose.

enzymes that split these sugars. How- these patients was depressed as much as ever, as shown in figure 5, assay of that for the sprue patients, whereas their human intestinal mucosa revealed that ability to hydrolyze sucrose and maltose the relation of substrate concentration to was essentially normal. I ntmluminal disaccharidase activity. splitting activity is nearly identical for lactase, sucrase, and maltase; also, it is Assay of intestinal contents from the pernotable that saturation is approached at fusion studies revealed enzymatic activity the 80 mM concentration. Thus the use of that could account for only a small portion this single concentration in the perfusion of the hydrolysis found in vivo except for studies appears to be adequate for com- maltase activity in the sprue patients which was 12% of the in vivo hydrolysis rate parison of the three disaccharides. Hydrolysis and absorption of lactose in (table 3). This small amount of activity patients with intestinal lactase deficiency. is consistent with that found in previous The 2 patients with depressed intestinal studies.17 - 19 lactase activity as measured by in vitro Discussion quantitative assay of their intestinal In contrast to the rapid hydrolysis and ab biopsies were able to hydrolyze lactose in vivo at rates that were only 25% of normal sorption rates for sucrose and maltose in nor(fig. 6). Indeed, lactose hydrolysis in mal subjects, lactose hydrolysis occurred at

DISACCHARIDE ABSORPTION I N MAN

October 1966

493

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0

f=

Q_

0::

0

(/)

co

<(

INY :

LACTOSE

INF: GLUCOSE &. GALACTOSE

INF : NAL TOSE

INF:GUJCOSE

FIG. 3. Comparison of disaccharides and equivalent monosaccharide mixtures in 10 normal subjects. Absorption of both glucose and galactose was significantly slower from lactose than from the glucose-galactose mixture (P < 0.05) . Note difference in scale of ordinates. NORNAL

TROPICAL SPRUE

20 10 ... oo;c. (10)

(11)

FIG. 4. Hydrolysis of disaccharides in tropical sprue. P ercentage values indicate degree of lowering from normal. Symbols are the same as in figure 1.

rates that were only about half of those for the other disaccharides so that absorption rates for the glucose and galactose products were appreciably below those found when the equivalent monosaccharide mixture was infused. This suggests that the hydrolysis step is rate-limiting in the over-all process of lactose hydrolysis-monosaccharide absorption, whereas hydrolysis is apparently not the rate-limiting step for sucrose 19 or maltose absorption. Dahlqvist and Borgstrom's studies m

man 17 suggesting that lactose is better handled than sucrose by jejunum are in marked contrast to our findings. However, using their technique, we previously found sucrose to be absorbed very rapidly in jejunum,18 and our present studies are compatible with in vitro assays of human intestinal homogenates which show lactase to be only 50% of sucrase activity. 20 • 29 Recently Forster and colleagues30 and Mehnert and colleagues, 31 using an intestinal segment isolated by balloons, 32

494

GRAY AND SANTIAGO

TABLE

2. I mpaiTment of caTbohydrate abs01·ption in tTopical spTuea Monosaccharide mixture

Disaccharides

%

%

decrease from normal

decrease from ttormal

Maltose Glucose

-53

Glucose Glucose

-63

-------Lactose Glucose Galactose

-74 -67

> 0.2 ---

Glucosegalactose Glucose Galactose

-58 -63

< 0.05 >0.7

---

----Sucrose Glucose Fructose

p valueb

-60 -64

a Data from 10 normal subjects (table 1) a nd 11 patients with tropica.! spru e. b Paired ana lysis, t-test comparing absorption from disaccharides and monosaccharide mixtures.

found that lactose disappeared more slowly than either glucose or galactose in man. However, their methods did not permit measurement of hydrolysis or component monosaccharide absorption, and they did not compare lactose to the equivalent monosaccharide mixture. Experiments by Dahlqvist and Thomson 16 in rats have demonstrated a relatively slow rate of hydrolysis and absorption for lactose, analogous to our findings in man. Considering the relatively slow lactose absorption in our normal subj ects, the rise in blood sugar concentration after lactose ingestion cannot be expected to be comparable to that found after ingestion of other sugars. Indeed, our findings may explain the fact that about 30% of individuals with normal intestinal lactase activity show little increase in blood sugar after ingestion of lactose. 33 It is somewhat surprising that sucrose and maltose hydrolysis rates were equal in our studies since intestinal maltase activity is about 3 times the sucrase activity .2 o, 29 There are at least two possible explanations for this discrepancy, however: (1) the rate of contact between mucosal surface and sugar molecules may not have

Vol. 51, No.4

been rapid enough to saturate all of the enzyme sites and may thus have been below the absolute capacity of either sucrase or maltase. The equal hydrolysis rates for the two disaccharides would thus be related to physical factors within the test segment; (2) it is known that maltase activity of intestinal homogenates in vitro is probably provided by five different enzymes two of which are also responsible for all sucrase activity. 29 Perhaps only the two enzymes having both sucrase and maltase activity are active in intact normal intestine in vivo thereby resulting in identical hydrolysis rates for the two disaccharides. However, as estimated from sugar tolerance tests, maltose appears to be better absorbed than sucrose in patients with sucrase deficiency 34 • 35 indicating that the other three maltases are probably capable of in vivo activity. Whether they express this capability in the intact normal intestine is unknown. That glucose was absorbed more rapidly than galactose whether lactose or the glucose-galactose mixture was infused is of interest, since these monosaccharides share the same mechanism for active transport. Although glucose and galactose are mutually inhibitory, the glucose effect on galactose absorption predominates. 36 • 3; Our results are consistent with this observation. The damaged intestine in tropical sprue may be expected to have a reduced capacity to digest and absorb carbohydrates; although both hydrolysis and absorption were equally depressed in our studies, lactose hydrolysis was most affected by disease and maltose splitting was least affected. This relative difference in the degree of impairment cannot be accounted for by reduction in the number of absorptive cells a lone, but rather appears to indicate that some functions of the intestinal absorptive cell are more sensitive to injury than others. Of course, it is impossible to determine whether the patients with sprue had an intestinal lactase deficiency which antedated their generalized intestinal disease; however, the incidence of lactase deficiency in apparently normal Puerto Ricans appears relatively low (table 1). The more severe impairment

495

DISACCHARIDE ABSORPTION IN MAN

October 1966 100

80

60 LACTASE ..--.-• SUCRASE 0------<:> MALTASE t>----tJ.

20

10

O•L---~~------------L--------------------------------------0 10 20 40 60 80 100 120 140 160 180 200 220 240 SUBSTRATE CONCENTRATION ( MMOLES/L)

FIG. 5. Relation of substrate concentration to the relative rate of hydrolysis in homagenates of human intestine. Incubation for 1 hr at 37 c:• The ordinate is expressed as percentage of calculated V max39 and each point represents the mean of four assays.

DNORMAL

D ¥

LACTASE DEFICIENCY

70

\ LLI

(/)a:0 iii~ ~u

ou

o::< o!!! >-O

:::t:Ul ~ 0 ::f:

...!

J.J.

R.O.

J.J.

R.O.

FIG. 6. Disaccharide hydrolysis in two lactase deficient patients. Symbols are the same as in figure 1.

496

GRAY AND SANTIAGO

Vol. 51, No.4

3. DisacchaTidase activity of intestinal con tents in vitm•

TA BLE

Sucrase activi ty

Maltase activity

Lactase activity

No. In vitro

o/o of in vivo

In vitro

0. 6 ± 0.1 1.1 ± 0 .3

1.5 4.2

0.4 ± 0.2 0.2 ± 0.1

o/o of in vivo

o/o

of

In vitro

in vivo

1.2 ± 0.2 4.1 ± 1.3

2.1 12

--Cont rols . . .. Tropical sprue . •

•

•

••

•

•

••

0

0

•

•

•

10 11

1.2 2.2

a Expressed as millimoles per hour per volume of fluid in t he m vivo perfusion experiment. Mean± sE.

of lactose splitting is unfortun ate because lactase activity is normally lower than other disaccharidases. Our findings in vivo are consistent with in vitro assays of intestinal homogenates indicating that lactase is relatively more depressed in disease than other disaccharidases.ll· 38 The 2 lactase-defi cient patients ·were able to split lactose no faster than patients wit h severely · damaged intestines due to tropical sprue, demonstrating a severe functional abnormality due to a specific enzymatic defect in the absence of any structural abn ormality by li ght microscopy. Summary

1. Hydrolysis and absorption of the common dietary disaccharides and absorption of the equivalent monosaccharide mixtures were determined in normal subjects, patients with tropical sprue, and patients with intestin al lactase deficiency. 2. I n normals, hydrolysis of lactose occurred at about half the rate for sucrose or maltose. Further , monosacch aride absorption from lactose was appreciably slower than from the equivalent glucosegalactose mixture suggesting t hat t he hydrolysis step is rate-limiting in the overall process of lactose absorption. 3. D espite the fact that maltase activity of intestinal homogenates is known to be 3 times that of sucrase activity, in vivo hydrolysis rates were the same for maltose and sucrose. 4. Patients with tropical sprue h ad impairment of all hydrolysis and absorptive processes, but ability to hydrolyze lactose was proportionally more affected t han other processes suggesting that simple re-

duction in the number of absorptive cells cannot by itself account for the function al abnormalities in sprue. 5. Although their intestinal biopsies were histologically normal, patients with lactase deficiency ha d as much impairment of in vivo lactose hydrolysis as the patients with severe intestinal dam age due to t ropica l sprue. REFERENCES 1. Fordtran, J . S., P. H. Clodi, K. H. Soergel, and F. J. Ingelfinger. 1962. Sugar absorption tests with special reference to 3-0 -methyld-glucose and d-xylose. Ann. Intern . M ed. 57: 883-891. 2. Goldenberg, J ., and A. J . Cummings. 1963. The effect of pH on the abso rption rate of glucose in the small intestine of humans. Gastroenterology 45: 189-195. 3. Holdsworth, C. D., and A. M . Dawson. 1964. The absorption of monosaccharides in man . Clin . Sci. 27 : 371-379. 4. Holzel, A., T. M ereu, and M . L . Thomson. 1962. Severe lactose intolera nce in infancy. Lancet 2 : 1346-1348. 5. Weij ers, H . A., J. H . van de Kamer, W. K., Dicke, and J. Ij sseling. 1961. Diarrhoea caused by deficiency of sugar splitting enzymes. J . Acta Paediat. (Uppsala) 50: 55-71. 6. Durand, P., and G . M. Lamedica. 1962. Disaccharid e intolerance . H elv. Paediat. Acta 17: 395-410. 7. Auricchio, S., A D ahlqvist, G. Mi.irset, and A. Prader. 1963 . I somaltose intolerance causing decreased ability to utilize starch. J. P ediat. 62: 165-176. 8. Sunshine, P ., and N. Kretchmer. 1964. Studies of small intestine during development. III. Infantile diarrhea associated with intolerance to disaccharides. Pediatrics 34: 38-50. 9. Anderson, C. M., M . Messer, R. R. W. Townley, M. Freeman, and M. J. Robinson . 1962.

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DISACCHARIDE ABSORPTION IN MAN

Intestinal isomaltase deficiency in patients with hereditary sucrose and starch intolerance. Lancet 2: 556- 557. 10 . Auricchio, S., A. Rubino, M. Landolt, G. Semenza, and A. Prader. 1963. I solated intestinal lactase deficiency in the adult. Lancet 2 : 324-326. 11. Plotkin, G. R., and K. J . I sselbacher. 1964. Secondary disaccharidase deficiency in adult celiac disease (nontropical sprue) and other malabsorption states. New Eng. J. Med. 271: 1033-1037. 12. Dunphy, J. V., A. Littman, J. B. Hammond,

G. Forstner, A. Dahlqvist, and R. K. Crane. 1965. Intestinal lactase deficit in adults. Gastroenterology 49 : 12-21. 13. vVeser, E., and M. H. Sleisenger. 1965. Lactosuria and lactase deficiency in adult celiac disease. Gastroenterology 48: 571-578. 14. Dahlqvist, A., and D. L. Thomson. 1963. The digestion and absorption of sucrose by the intact rat. J . Physiol. (London) 167: 193209. 15. Dahlqvist, A., and D. L. Thomson. 1963. The

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19.

digestion and absorption of maltose and trehalose by the intact rat. Acta Physiol. Scand. 59: 111-125. Dahlqvist, A., and D. L. Thomson. 1964. The digestion and absorption of lactose by the intact rat. Acta Physiol. Scand. 61: 20-33. Dahlqvist, A., and B. Borgstrom. 196 1. Digest ion and absorption of disaccharides in man. Biochem. J . 81 : 411-4 18. Gray, G. M., and F. J. Ingelfinger. 1965. Intestinal absorption of sucrose in man: The site of hydrolysis and absorption. J. Clin. Invest. 44: 390-398. Gray, G. M ., and F. J . Ingelfinger. 1966. Intestinal absorption of sucrose in man: Interrelation of hydrolysis and monosaccharide product absorption. J . Clin. Invest. 45: 388--398.

20. Dahlqvist, A. 1964. Method for assay of intestinal disaccharidases. Anal. Biochem. 7 : 18-25.

21. Cook, G. C., and S. K. K ajubi. 1966. Tribal incidence of lactase deficiency in Uganda. Lancet 1: 725- 729. 22. Rosensweig, N. S., and T . M. Bayless. 1966. Racial difference in the incidence of lactase deficiency (Abstr.). J. Clin. Invest. 45: 1064. 23. Hyden, S. 1955. A t urbidimetric method for the determination of higher polyethylene glycols in biologic materials. Ann. Roy. Agr. Coli. Sweden 22: 139-145. 24. Somogyi, M. 1945. D etermination of blood sugar. J. Bioi. Chern. 160 : 69-73.

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25. Avigad, G., D. Amaral, C. Asensio, and B. L. Horecker. 1962. The D-galactose oxidase of Polypon~os circinatus. J. Bioi. Chern. 237: 2736-2743 . 26. Bickel, H . 196 1. Mellituria, a paper chromatographic study. J. Pediat. 59: 641-656. 27. Gryboski, J . D ., W. R. Thayer, Jr., W. A.

Gryboski, I. W. Gabrielson, and H. M. Sprio. 1963. A defect in disaccharide metabolism after gastrojejunostomy. New Eng. J. Med. 268: 78-80. 28. Weser, E., and M. H . Sleisenger. 1966. Metabolism of circulating disaccharides in man and t he rat (Abstr.) . J. Clin . Invest. 45 : 1084. 29. Auricchio, S., A. Rubino, R. Tosi, G. Semenza, M. Landolt, H. Kistler, and A. Prader. 1963. Disaccharidase activities in human intestinal mucosa. Enzym. Bioi. Clin. 3 : 193-208. 30. Forster, H., H . Mehnert, K. Stuhlfauth, B. Mehnert, H . T ammen, and P. Bailer. 1963.

Vergleichende Untersuchungen zur Resorption von Glucose, Galaktose und Lactose am Menschen und an der Ratte, III. Lactose-Resorption, Schlussfolgerungen. Klin. Wschr. 41: 549-554. 31. Mehnert, H., H. Forster, K. Stuhlfauth, B. Mehnert, K . Geser, und M. Langer. 1963. Vergleichende Untersuchungen zur Resorption von Glucose, Galaktose und Lactose am Menschen und an der Ratte, II. Glucose-unci Galaktose-Resorption. Klin. Wschr. 41: 544-549. 32. Mehnert, H ., H. Forster, B. Mehnert, B. v. Kutzschenbach, I. Thoring, und K . Arbogast. 1963. Vergleichende Untersuchungen zur Reso rption von Glucose, Galaktose und Lactose am Menschen und an der Ratte, I. Untersuchungsgut und Methodik, Vonmtersuchungen, Kritik. Klin. Wschr. 41: 540-543. 33. Newcomer, A. D., and D. B. McGill. 1966. Lactose tolerance tests in adults with normal lactase activity. Gastroenterology 50: 340-346. 34. Sonntag, W. M., M . L. Brill, W. C. Troyer,

Jr., J . D . Welsh, G. Semenza, and A. Prader. 1964. Sucrose-isomaltose malabsorption in an adult woman. Gastroenterology 47: 18-25. 35. Burgess, E . A., B. Levin, D. Mahalanabis, and R. E. T onge. 1964. H ereditary sucrose

intolerance: Levels of sucrose activity in jejunal mucosa. Arch. Dis. Child. 39: 431443. 36. Fisher, R. B., and D. S. P arsons. 1953. Galac-

tose absorption from the surviving small

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GRAY AND SANTIAGO intestine of the rat. J. Physiol. (London) 119: 224-232.

37. Riklis, E ., B. Haber, and J. H . Quastel. 1958.

Absorption of mixtures of sugars by isolated surviving guinea pig intestine. Canad. J. Biochem. 36: 373-380.

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38. Bayless, T. M., W. Walter, and R. Barber. 1964. Disaccharidase deficiencies in tropical sprue (Abstr.). Clin. R es. 12 : 445. 39. Lineweaver, H., and D. Burk. 1934. The deter-

mination of enzyme dissociation constants. J. Amer. Chern. Soc. 56: 658-666.