Influence of Biliary Stasis on the Activity and Distribution of Maltase, Sucrase, Alkaline Phosphatase, and Leucylnaphthylamidase in the Small Intestine of the Mouse

Vol. 64, No.3 Printed in U.S.A.

GASTROENTEROLOGY 64:411-420, 1973 Copyright © 1973 by The Williams & Wilkins Co.

INFLUENCE OF BILIARY STASIS ON THE ACTIVITY AND DISTRIBUTION OF MALTASE, SUCRASE, ALKALINE PHOSPHATASE, AND LEUCYLNAPHTHYLAMIDASE IN THE SMALL INTESTINE OF THE MOUSE ROSEANNE M. COOK, PH.D., PATRICIA M. POWELL, AND FLORENCE MOOG, PH.D.

Department of Biology, Washington University, St. Louis, Missouri

Ligation of the common bile duct results in loss of the type of alkaline phosphatase peculiar to duodenum in the mouse, but activity increases in jejunum and ileum. Leucylnaphthylamidase activity decreases slightly in duodenum and jejunum. Sucrase and maltase increase substantially everywhere except in the most proximal part of the duodenum. Despite the fact that biliary stasis leads to loss of intestinal weight, total sucrase and maltase activities are higher than in the heavier intestines of sham-operated control mlce. Along the length of the 'small intestine, the surface differs in extent, enzymic constitution, and functional capacity. The mucosal surface of the rat intestine, for example, is greatest, per unit gut length, in the proximal region, 1 where the villi are tallest,2 and becomes steadily smaller toward the distal end. Among the enzymes that are part of the surface membrane, alkaline phosphatase is maximal in the duodenum in rat and mouse,3, • but sucrase, 5 maltase,6 and leucylnaphthylamidase 5 , 7 reach their peak within the jejunum. Absorption of specific substances varies regionally, phosphorus for example being absorbed most rapidly in the duodenum 8 and glucose in the proximal jejunum, 9 whereas vitamin B12 is absorbed in the ileum. 10 Such conditions, as they obtain in the normally functioning adult intestine, represent the net effect of the interplay at Received August 7, 1972. Accepted October 20, 1972. Address requests for reprints to: Dr. Florence Moog, Department of Biology, Washington Uni· versity, St. Louis, Missouri 63130. This work was supported by Research Grant HD· AM·03490 from the National Institutes of Health. The authors are indebted to Mr. Gary Olson for preparation of electron micrographs of some of the material reported in this paper. 411

a wide variety of regulatory agencies, including hormones 11 and dietary constituents, 12, 13 as well as unidentified factors that flow into the intestine or are produced by it.1HS In most cases the influence of these agencies has been examined in relation to a single enzyme, and usually to the intestine as a whole or one limited region of it. At critical stages in development, the entire intestinal surface may, in response to specific stimuli, advance to a new level of differentiation. Adrenocorticoids, for example, cut off the capacity of the surface to permit passage of large molecules, 17, 18 at the same time bringing about the elongation of the microvilli 19 and eliciting increase of alkaline phosphatase in the duodenum 18,20 and of disaccharidases 21 · 23 and leucylnaphthylamidase 7in the jejunum. Even drugs like actinomycin D and cycloheximide, which have been known to elicit paradoxical increases of jejunal phosphatase activity in the young mouse 24 . 26 actually evoke regionally appropriate responses in the activity of several enzymes along the entire length of the intestine. 7, 23 The extent to which factors known to affect the individual entities in local regions of the intestine may exert more di-

412

COOK ET AL.

verse and widespread effects is a largely open question. This paper is concerned with the influence of biliary stasis, which in the mouse results in severe loss of alkaline phosphatase activity only in the duodenum, where it is normally high. 15 We now find that blocking the influx of bile results in a substantial increase of sucrase and maltase in the midregion of the intestine, where these activities are normally maximal. Both specific and total activities are elevated.

Methods The mice used were males 4 to 5 months old and weighing 23 to 26 g. They were derived from an SWR/J substrain that has been maintained in this laboratory for more than eight generations. They were fed Purina laboratory chow and water ad libitum. Surgical procedures. Biliary stasis was effected by the same technique as previously used. I. The mice were anaesthetized with Nembutal, laparotomized, and the common bile duct tied off midway between the gaLlbladder and the duodenum. Since the pancreatic duct enters posterior to the bile duct in the mouse, it was not disturbed. Control mice were laparotomized and subjected to internal manipulation mimicking that required in the experimental mice. After surgery the animals were given free access to food and water. By the 2nd day after operation they appeared to be feeding normally, and were lively and alert. Tissue preparation. The mice were killed by cervical dislocation, 72 hr after surgery. The intestine was excised, laid out on a glass plate without stretching, and cut into segments (see "Results"). Any content was pressed out gently, and the piece was then weighed, placed in iced distilled water, and stored at - 24 C for periods up to 10 days; we have found that intestinal segments stored under these conditions for as long as 2 months lose no alkaline phosphatase, maltase, sucrase, or leucylnaphthylamidase activity. A small piece of the left lateral lobe of the liver was also taken from each mouse. The tissues were homogenized in a Ten Broeck grinder just before being assayed. In a few cases, fragments were removed from the intestine in situ, immediately after killing, and fixed in glutaraldehyde for subsequent examination with the electron microscope. The mice were killed in midafternoon, when intestinal contents are minimal. The ileal seg-

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ments generally contained 50 to 200 mg of undigested material, with no consistent difference in quantity between experimental and control mice. In these experiments the material stripped from the intestinal lumen was not assayed for enzyme activity; in intact animals, however, the total gut contents at about 3 PM contain from 1 to 3% of the phosphatase and maltase activities (If the whole intestine, with the percentage of sucrase sometimes being very slightly higher. Assay procedures. Maltase and sucrase activities were determined by the technique of Dahlqvist. 27 The reaction mixture consisted of 0.1 ml of 3.5% (0.1 M) maltose or 7% (0.2 M) sucrose in 0.1 M maleate buffer at pH 6.0, plus 0.1 ml of homogenate containing 100 Jl.g of tissue for maltase determination, or 500 Jl.g for sucrase determination. After 60-min incubation at 37 C, the reaction was stopped by addition of 2 ml of freshly mixed Tris-glucose oxidase prepared from glucostat reagent (Worthington Biochemical Co., Freehold, N. J .). After 60 min at 37 C, color was read in a Coleman Jr. IIA spectrophotometer at 420 mJl.. Each test was run in duplicate, with a blank to which substrate was added at the end of the incubation period. Standards containing 100, 150, and 200 Jl.g of glucose per ml were determined in parallel with each test. Leucylnaphthylamidase activity was determined by the method of Goldbarg and Rutenberg,28 as previously adapted to the mouse intestine. 7 The reaction mixture consisted of 0.5 ml of 0.2 M phosphate buffer, pH 7.4, containing 0.25 mg of l-Ieucine-{j naphthylamide, plus 0.5 ml of homogenate containing 1 mg of tissue. After lO-min incubation at 37 C, the reaction was stopped by the addition of 0.5 ml of 40% trichloroacetic acid, after which 1.5 ml of 0.1 % sodium nitrite was added to each tube. After 3 min, 1.5 ml of 0.5% ammonium sulfamate solution was added, followed by 3 ml of 0.5 mg per ml N(1-naphthyl)ethylene diamine in 95% ethyl alcohol. After 10 min, during which the tubes were shaken twice, color was read in a Klett-Summerson spectrophotometer with a no. 56 filter. Each homogenate was tested in duplicate, with a blank to which tissue was added after trichloroacetic acid. Standards containing 6.6, 13.3, or 20 Jl.g of {j-naphthylamine per ml were run with each series of tests. Alkaline phosphatase activity was determined with both phenylphosphate (PHP) and {j-glycerophosphate ({jGP) as substrates, according to methods in routine use in this laboratory."29 When PHP was used, the as-

March 1973

413

INTESTINAL ENZYMES AFTER BILIARY STASIS

say mixture consisted of O.I-ml substrate solution (300 mM for duodenum, 180 mM for more distal regions) and 1.5 ml of 0.2 M carbonatebicarbonate buffer, pH 9.8, containing 16 mM MgCI 2 ; after this mixture was preheated at 37 C, 0.5 ml of homogenate containing 62.5 f.Lg of duodenum or 500 f.Lg of jejunum or ileum was added, and incubation was continued for 10 min. Released phenol was determined by the King-Armstrong 30 method, color being read in the Klett-Summerson spectrophotometer with a no. 54 filter. Activity on fjGP was determined similarly, at pH 9.4, with initial concentration of 120 mM substrate in the assay medium for all regions. Released phosphorus was measured by the Fiske-Subbarow31 technique, with color being read with the no. 66 filter. Protein was determined by the method of Lowry et al. 32

Results Optimal conditions for disaccharidase determination. Although the conditions of pH and incubation time originally established by Dahlqvist 33 proved suitable for mouse intestine, the recommended final substrate concentration of 28 mM proved much too low. For sucrase, it was necessary to use a final concentration of 3.5% (102 mM) to obtain near-maximal velocity, and, contrary to Dahlqvist's experience with rat intestine, even 4.5% was not inhibitory (fig. 1). For maltose, a final concentration of 50 mM proved optimal. Sucrase and leucylnaphthylamidase in duodenum and jejunum. The first series of operations was performed to determine whether biliary stasis would exert an effect on enzymes other than alkaline phosphatase. 15 Bile ducts were obstructed in 14 mice, and 14 were sham-operated. The parts of the intestine used for enzyme determination were the 1st and 3rd cm (duodenum), and 4 cm from around the midpoint (jejunum). Leucylnaphthylamidase activity was slightly below that of control mice at all levels, but the difference was significant only in the jejunum (table 1). Sucrase was unaffected by the operation in the most proximal region, but elsewhere underwent large increases (table 1). Although no intact control mice were used in this series, the leucylnaphthylami-

.6

3

5

'lIO

Sucrose

7

9

FIG. 1. Influence of su,crose concentration on sucrose activity. Reaction mixture consisted of 0.1 ml of sucrose in 0.1 M maleate buffer, pH 6.0, plus 0.1 ml of adult jejunal homogenate containing 500 j.lg of tissue; abcissa shows initial concentrations before addition of homogenate. CU ~ units on Coleman Jr. Spectrophotometer.

dase values in sham-operated animals closely approximate those previously reported for mice of the same strain. 7 The sucrase activities also did not differ from those we have obtained on normal males. Hence the stress of the operation itself did not affect our results. Maltase, sucrase, and phosphatase throughout the intestine. Although biliary stasis elicits a substantial increase in specific activity of sucrase, the possibility exists that the result is more apparent than real. Since animals with obstructed bile ducts lose weight, the effect might be an epiphenomenon resulting from persistence of sucrase after other proteins had been degraded. A new series of operations was therefore performed to answer the following questions: (1) Are other disaccharidase activities affected in the same way as sucrase? (2) Is the effect on disaccharidases uniform throughout the intestine? (3) Is the total activity elevated above that of the control animals? In this series, alkaline phosphatase activity was also measured to determine the nature of its responses to biliary stasis throughout the intestine. The bile duct was ligated in 8 animals in this series, and there were 6 sham-operated control animals. The experimental

414

COOK ET AL. TABLE

l. Effect of obstruction of the common bile duct on enzyme activities in the mouse intestine

Enzyme activity Sucrase a . . . . . . . .. . Control" . .. , . .. .. Experimental . .. . ,. , % LNA' ... Control Experimental . ..... . . % Alkaline phosphatase' Control .. .. ......... . ... Experimental .. .. .... .. .. a Sucrase "Control type.

c

Vol. 64, No.3

~ ~

Duodenum 1st em

3rd em

630 ± 54.5 c 721 ± 94.5 -14.4 (Ns)a

1032 ± 97.5 1796 ± 207.0 +73.6 (P < 0.005)

111 80

± ±

-2~.3

12.7 9.4 (NS)

181 ± 11.5 160 c± 12.8 -11.5 (NS)

13,040 3,300

Jejunum

1331 ± 84.3 2236 ± 150.5 +68.0 (P < 0.001) 317 ± 24.2 231 ± 15.3 -27.0 (P < 0.01)

3762 1134

385 281

micrograms of sucrose hydrolyzed per milligram of protein per 60 min. sham-operated 72 hr before killing; experimental ~ bile duct-ligated. There were 14 of each

SEM.

NS, not significant. 'LNA (leucylnaphthylamidase) ~ micrograms of ~-naphthylamine produced per milligram of protein per 60 min. f Phosphatase ~ micrograms of phosphate from phenyl phosphate per milligram of protein per 30 min. Phosphatase activities are from reference 15, and are repeated here for comparison. d

animals lost 18.5% of their starting weight in the 72-hr period after the operation (table 2). Their small intestines weighed 18.7% less than those of the control animals at death, and the total intestinal protein was 22.4% less. The relation of intestinal weight to body weight, and intestinal protein to fresh weight, remained constant (table 2). The small intestine was divided into five pieces of equal length, with the most proximal piece then being cut into two equal parts; the six segments are designated A 1, A 2 , B, C, D, and E. A1 underwent only a small increase of both sucrase and maltase activity, but in all other segments the increase was substantial (fig. 2). Although the general form of the distribution profile-highest in the midregion and falling at both ends-was preserved in both cases, certain differences are evident. Sucrase was elevated close to its maximal level within the second tenth (A 2 ); with maltase a large increase took place between A 2 and B, the difference between these two regions being significant (P < 0.025). Again, elevated sucrase levels were quite high throughout jejunum and ileum, but

TABLE

2. Effect of ligation of the common bile duct

on body weif?ht and protein content of small intestine Duct-ligated (8)

Sham-operated (6)

Body weight before and after operation (g) o hr 24.8 ± 0.36 a 24.5 ± 0.53 72 hr 20.2 ± 0.64 23.9 ± 0.73" Change -18.5% -2.5% Intestinal weight and protein content (mg) after operation Wet weight 1043 ± 61 1283 ± 72" Intestinelbody 51.5 mg/g 53.5 mg/g Total protein Protein/intestine

166 ± 8.6 157 Il-g/mg

214 ± 9.6 167 Il-g/mg

·SEM.

For difference in final body weight between ductligated and sham-operated animals, P < 0.005; for difference in intestinal weight, P < 0.025. b

maltase fell to a greater extent in the ileum (fig. 2). For both activities, differences between experimental and control animals were maximal, and highly significant (P < 0.005 for both), in the terminal ileal

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415

INTESTINAL ENZYMES AFTER BILIARY STASIS

segment; percentagewise the difference was greater for sucrase ( + 181 %) than for maltase (+ 117%). The ratio of maltase to sucrase activity was 5.5 for the control group and 4.6 for the experimental animals in the first tenth (A I) of the intestine (fig. 2). In the A2 through D segments, ratios ranged from 3.3 to 4.4, with no significant differences between experimental and control animals. As might be expected from the near tripling of sucrase activity in the ileal segment, however, the maltase-sucrase ratio in this region was depressed from 4.40 for control animals to 3.38 for experimental animals; applying the Wilcoxon test for unpaired data to these E segment ratios yields a P value of 0.012, on the basis of a two-tailed test. Alkaline phosphatase activity suffered the anticipated decrease in the most proximal segment (table 3); the difference between bile-obstructed and sham-operated animals is less than we have previously reported 1 5 because in the former case we determined separately the 1st cm of the duodenum, in which phosphatase activity is extremely high (see table 1). As before, activity with PHP in the most proximal segment was lowered more than that with {jGP, and the PHP: {jGP ratio in this segment was . therefore significantly less in the experimental than in the control animals (table 3). In the more distal segments the phosphatase activities with both substrates were consistently higher in the animals with obstructed bile ducts, and in the terminal segment the difference is significant (P < 0.01); PHP: {jGP ratios were slightly but not significantly elevated (table 3). Using the light microscope, we observed previously that biliary stasis did not lead to any visible disturbance of the phosphatase-rich border of the intestine. 15 In this series of experiments we have also examined the ultrastructure of the epithelial surface in intestinal fragments fixed in glutaraldehyde and postfixed with osmium tetroxide. Inspection of electron micrographs at X 40,000 magnification revealed no loss of the integrity of the membranous coat of the microvilli, in which

I

c

D

E

FIG. 2. Influence of biliary stasis on maltase and

sucrase activity along the length of the mouse intestine from proximal duodenum (A ,) to terminal ileum (E) . 0 - - - 0, 8 experimental mice with bile duct ligated 72 hr before killing; •.. . .•, 6 sham-operated control mice. Vertical lines are ± 1 SEM. Numbers at top of graph represent ratio of maltose to sucrose (m/s) hydrolyzed by each segment in experimental (E) and control (C) animals.

phosphatase, disaccharidases, and leucylnaphthylamidase 3 4-36 are invested. There was no change in the length of the microvilli, or in the appearance of their fibrous cores or of the terminal web. As a check on the completeness of biliary stasis, the alkaline phosphatase activity of the liver was determined. 15. 37 Activity with PHP averaged 14.2 iJ.g of phenol per mg of protein per 30 min for control animals, and 84.0 iJ.g of phenol for animals with obstructed bile ducts. Total enzyme activities. Total activity for each enzyme in the entire small intestine was calculated from the weights of the individual segments and their activities per milligram of fresh weight. Total malt-

416

COOK ET AL. TABLE

Vol . 64, No.3

3. Effect of lif?ation of the common bile duct on alkaline phosphatase activity throughout the

small intestine PHP'

!3GP'

PHP:!3GP'

Segment Phosphated

P

%

Phosphate

Control ' ...... Experimental .

5522 3376

±

Control . Experimental .

1168 1289

±

Control. Experimental .

276 350

±

Control. Experimental .

205 252

Control .. Experimental .

193 285

±

Control . Experimental .

120 240

±

A,

±

±

P

Ilg

!1!?

AI

%

921 414

- 38 .8

118 207

0.025

1809 1739

+ 10.3

NS

1091 1141

±

14 46

+25.9

NS

400 493

±

12 30

+23 .0

NS

298 352

11 33

+48.2

< 0.05

282 371

< 0 .01

183 308

.

± ±

±

157 174

-3 .E

NS f

206 134

+ 4 .E

NS

1.15 1.06

24 70

+ 23.2

NS

0.70 0.73

12 39

+ 18.1

NS

0.69 0.72

17 33

+31.6

< 0.05

0.69 0 .73

12 33

+ 68.0

< 0.01

0.69 0.77

2.92 1.90

± ±

0 .072}jD 0 001 0.049 < .

B ±

±

C ± ±

± ±

D ±

± ±

E ±

9 32

+ 100

t

±

PHP, phenyl phosphate. iSGP, is-glycerophosphate. C Average of the individual ratios. d Micrograms of phosphate per milligram of protein per 30 min ± SEM. eThere were 6 sham-operated (control) and 8 bile duct-ligated (experimental) mice. f ]\ S, not significant. a b

ase activity was elevated 33.8% above that of the control animals, total sucrase 37.9%, the differences between experimental and control animals being significant at the 1% level (table 4). Expressed per gram of small intestine, or per gram of body weight, activities were respectively more than 70% and more than 60% above those of control animals. Total phosphatase activity with PHP was decreased by 27.5% in the experimental animals, but the difference was not significant; with /3GP the difference was only 8.9% (table 4) . Activities per unit of intestine or per unit of body weight differed only slightly, but as might be expected from the greater effect of biliary stasis on activity with PHP, relative activities with this substrate were a little higher in control animals, but with /3GP were a little higher in experimental animals (table 4).

Discussion The pattern of phosphatase distribu-

tion along the length of the small intestine agrees with previous observations in both the mouse' and the rat. 3, 5, 38 The pattern of sucrase distribution is also like that reported for both animals. 5,39 Maltase activity, however, although highest in proximal jejunum in our mice, has been found most abundant in the distal ileum in CF1 mice 39 (see "Addendum"). Regardless of this discrepancy, it is evident that in the SWR/J mice used in our study, phosphatase and disaccharidase activities are inversely related in the proximal half of the intestine, the former falling as the latter rise. The view that the intestinal surface is an integral organization in which a variety of regionally appropriate responses may be evoked by common stimuli 23 is supported by the results of bile duct obstruction. In the most proximal segment, disaccharidases are slightly increased, while alkaline phosphatase activity falls sharply, apparently by loss of isozymes of high

March 1973

PHP: fjGP ratio. 29 In the jejunum, phosphatase is very slightly decreased, but disaccharidase activities surge up. These results imply that the enzymic constitution of the normal intestine is at least in part regulated throughout the intestine by secretions from the liver. We have previously pointed out that general debility is probably not the cause of loss of high ratio phosphatase. 15 The fact that total sucrase and maltase increase in mice with biliary stasis further militates against such an explanation, especially since starvation in the rat brings about a significant decrease in total maltase and sucrase within 24 hr.40 The stress of the operation, being the same for experimental and control animals, is not a likely explanation either. The fact that the epithelial surface retains its integrity after bile duct obstruction is also in harmony with the fact that the result may be gain, as well as loss, of enzyme activity. How the effect of biliary stasis is mediated is, however, a matter of speculation. Since the turnover time of the intestinal TABLE

417

INTESTINAL ENZYMES AFTER BILIARY STASIS

epithelium of the mouse is considerably less than 72 hr, 41-43 complete replacement would be expected in our experiments, provided that lack of bile does not seriously inhibit epithelial cell proliferation or transit. It has been reported that in the rat, ligation of the bile duct results in diminished proliferation without change in rate of cell loss, although enigmatically this situation is not attended by loss of mucosal weight or protein or deoxyribonucleic acid content. 44 Changes in cell dynamics may be contributory, yet it is unlikely that our findings can be explained primarily in such terms, since large differences in enzymic constitution are normally maintained in the face of small differences in cell renewal time in different regions of the intestine. 45 The decrease of duodenal phosphatase might result from reduction of size of duodenal villi, since in the rat obstruction of the pancreaticobiliary duct results in shortening of the duodenal villi, which are the longest in the intestine. 2. 45 Altmann's16 recent findings indicate however that the shortening of duodenal villi

4. Effect of ligation of the common bile duct on total enzyme activity, and on enzyme activity relative

to intestinal weight and to body weight

Maltase Control" . . . . . . . . . . Experimental. . , .. . - , . % .......... .. .. .. . . . Sucrase Control . . . . . . . . . . . . .. . . Experiment .. ... . . % .. Phosphatase with PHp d Control ..... . .......... Experimental ....... % .. . ... Phosphatase with /3Gpt Control .. .. .. ..... Experimental .. - , .... ,% .. ... . .... .. . ...

Total activity"

Activity /g intestine

Activity/g body weight

554 ± 42.7C 740 ± 38.0 +33.8 (P < 0.01)

406 ± 30.5 715 ± 44.4 +76.0 (P < 0.001)

23.5 ± 0.98 38.6 ± 1.42 +64.3 (P < 0.001)

145 ± 13.5 200 ± 12.2 +37.9(P< 0.01)

114±7.4 195 ± 15.6 +71.5 (P< 0.005)

6.17 ± 0.44 10.1 ± 0.66 +63.0 (P < 0.001)

171 ± 18.7 124 ± 15.0 -27.6 (Ns)e

139 ± 16.4 119 ± 13.3 -14.4 (NS)

7.23 ± 0.84 6.30 ± 0.85 -13.4 (NS)

89 ± 6.6 98 ± 7.2 +10.1 (NS)

4.75 ± 0.34 5.28 ± 0.34 +11.2 (NS)

113 ± 7.7 103 ± 11.1 -8.9 (NS)

a For maltase and sucrase, total activity ~ milligrams of substrate utilized per intestine per 60 min; for phosphatase, total activity ~ milligrams of phosphate liberated per intestine per 30 min; all activities given in milligrams. "There were 6 sham-operated (control) and 8 bile duct-ligated (experimental) mice. C

SEM.

PHP, phenylphosphate. eNS, not significant. r /3GP, /3-glycerophosphate. d

418

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COOK ET AL.

is due primarily to loss of pancreatic secretion, which is not cut off in our experiments; and in any case, the loss of a villusenlarging factor would not account for the increase of disaccharidase activities in jejunum and ileum. The duodenal phosphatase activity of germ-free mice is 12 times that of conventional mice. 46 Since bile salts inhibit the growth of anaerobic organisms,47 the decrease of duodenal phosphatase that we have found might be accounted for by an increase in the anaerobic flora. These organisms become luxuriant in intestinal blind loops, and the transport of arbutin and fructose is correspondingly impaired. 48 In the distal ileum in vitro, however, sodium taurocholate depresses uptake of 3-o-methyl glucose, 49 a finding that is in agreement with the demonstration that after the establishment of a bile fistula, transport of this sugar is enhanced in perfused jejunal segments. 50 Because increase of transport is prevented by continuous infusion of bile into the segment in vivo, but not by acute infusion in vitro, Roy et al. 50 suggest that bile does not exert a simple detergent effect, but may have a "specific effect on the monosaccharide active transport system localized in the plasma membrane of the epithelial cell microvilli." Taking into account the probability that sucrase and maltase have glucose-binding sites,51 the view just quoted is clearly in harmony with our results. Although the influx of bile appears to promote the expression of phosphatase of high PHP: ~GP ratio in the duodenum and restrict that of disaccharidases in the jejunum, additional factors must be involved in the responses to cessation of bile flow. Thus leucylnaphthylamidase, which has a distribution in the mouse intestine roughly paralleling that of maltase and sucrase,7 is slightly depressed in the area in which sucrase is strongly elevated (table 1). This difference may bespeak regulatory systems that respond independently, or with differential sensitivity, to changes in bile supply. In the distal ileum, where bile salt absorption is nor-

mally maximal,52 both disaccharidases and also low ratio phosphatase rise to twice their control level, or higher, after obstruction of the bile duct. These increases might be due to changes in intracellular metabolism, 53 but they might also reflect an alteration in the character of the surface membrane, 49 possibly resulting in diminished release of surface-associated enzymes into the lumen. 54 Since our work was done with whole homogenates, there is the additional possibility that the increased quantities of disaccharidases that we detect are not on the surface at all. The transfer of disaccharidases from sites of synthesis to the brush border seems to require an actinomycin D-sensitive step, 55 and this step might also be inhibited in biliary stasis. Addendum Because of the discrepancy between our findings and those of Madge 39 on distribution of maltase, we have examined the activity of this enzyme along the length of the small intestines of adult male CF1 mice obtained from Carworth, New City, New York, and also of C3H/He, C57B1/6J, and C57L!J mice, all from the Jackson Laboratories, Bar Harbor, Maine. All four strains yielded distribution profiles similar to that shown for SWR/J control animals in figure 2. The very high maltase activity in the distal ileum of Madge's39 CF1 mice thus represents a striking strain difference. REFERENCES 1. Fisher RB, Parsons DS: The gradient of muco-

2.

3.

4. 5.

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INTESTINAL ENZYMES AFTER BILIARY STASIS

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