Adaptation of rabbit small intestinal brush-border membrane enzymes after extensive bowel resection

Adaptation By Fresca Swaniker,

of Rabbit Small Intestinal Brush-Border Membrane Enzymes After Extensive Bowel Resection Weihong

Guo, Eric W. Fonkalsrud, Tammy Brown, Laura Newman, Los Angeles, California and Baltimore, Maryland

l Short lengths of small intestine may be resected without significantly affecting the digestive and absorptive capacity; however, extensive resection produces varying degrees of malnutrition. This study was undertaken to define the adaptive changes in the remaining small intestine of two of the jejunal and ileal mucosal brush-border membrane enzymes after extensive small bowel resection in rabbits. Thirty adult New Zealand White rabbits underwent a 50% to 60% jejunoileal-enterectomy with end-to-end anastomosis. Maltase activity (UE/g protein) increased from 152 (preoperative) to 392 at 3 weeks in the proximal segment and from 85 to 259 in the distal segment; these levels decreased to 222 and 155 in the respective segments at 6 weeks. AOP activity (UE/g protein) increased from 154 (preoperative) to 171 in the proximal segment and 171 to 256 in the distal segment at 3 weeks, and was 131 and 288 in the respective segments at 6 weeks. This marked increase in the mucosal brush-border enzymatic activities at 3 weeks postoperatively was associated with a 28% increase in bowel length. By 6 weeks the enzyme levels had decreased slightly; however, there was a persistent 41% increase in bowel length over that immediately postoperation. The mucosal surface area increased from 295 mm2 immediately postoperation to 5,337 mm2 at 3 weeks and 7,635 mm2 at 6 weeks, a 250% increase. The authors conclude that there is an immediate compensatory increase in villus length as well as brush-border enzymatic expression in the remaining intestine that gradually declines as the small intestinal surface area continues to increase and the bowel lengthens with time. Copyright o 1995 by W.B. Saunders Company INDEX WORDS: Adaptation, small syndrome; brush-border membrane

intestine; enzymes.

short

bowel

MALL BOWEL resection is performed for a variety of reasons: Small bowel ischemia, trauma, necrotizing enterocolitis, inflammatory bowel disease, and others. Although the small intestine has a large functional reserve, extensive resection may S

From the Dwision ofpediatric Surgery, UCLA School of Medicine, Los Angeles, CA and the Department of Surgery, St Agnes Hospital, Baltimore, MD. Presented at the 1994 Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, Dallas, Texas, October 21-23, 1994. Supported in part by the Hughes Employees Give Once Club, Los Angeles, CA. Address reprint requests to Eric W. Fonkalsrud, MD, Professor and Chiej D&ion of Pediatric Surgery, UCLA School of Medicine, 10833 Le Conte Ave, Los Angeles, CA 90024. Copyright o 1995 by W.B. Saunders Company 0022-346%/95/3007-0019$03.00/O

1000

and Marvin

Ament

cause nutritional depletion, and when severe, may lead to short bowel syndrome requiring months or years of total parenteral nutrition with its great costs and complications. Morphological changes in the remaining intestine after extensive small bowel resection include increased villus length and width, increase in crypt size, and production rate of crypt cells. There is increased cell migration to the villus tip and an increased mucosal surface area per unit of serosal length.1-5 In human beings, there is evidence for modest villus hyperplasia but not hypertrophy, and increased absorption.6,7 An early increase in blood flow to the intestinal remnant has been shown, which declines to basal levels after several weeks.8 Functionally, it has been shown that an increased proportion of the villus becomes enzymatically active, and there is an increased absorption of glucose per unit length of intestine in both animals and human beings.1,9 The distal small intestine has been shown to have a greater adaptive response than the proximal bowel.*JOJ1 The present study attempts to further define the functional adaptive changes occurring in both proximal and distal residual intestine after extensive small bowel resection in a rabbit model. MATERIALS

AND METHODS

Thirty adult male New Zealand White rabbits weighing 3 to 4 kg underwent a 50% to 60% midjejunoileal enterectomy with end-toend anastomosis of the remaining proximal and distal small intestine. At operation, mucosa was obtained from each rabbit’s intestine immediately proximal and distal to the site of resection to serve as control tissue. Four other rabbits underwent transection and reanastomosis without resection in the midileum to serve as surgical controls. Postoperatively, the diets were rapidly progressed to standard rabbit chow, with routine postoperative care. After initial weight loss, all rabbits gained to approximately 110% of preoperative weight by 3 weeks and 113% by 6 weeks. Rabbits were divided into two groups of 15 each. with one group euthanatized at 3 weeks and the other group at 6 weeks. Bowel length was measured, and specimens were taken for histological evaluation. Mucosal biopsy samples were again obtained from the intestine proximal and distal to the anastomosls (excluding a 5-cm segment on either side of the anastomosis). All mucosal biopsies were analyzed for maltase and aminooligopeptidase (AOP) activity, and for protein content.

Morphological Parameters Villus height, width, and number of villi per unit of serosal length were measured and used to obtain an estimate of the mucosal

JournalofPediatr/cSurgery,

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surface area using the method of Ecknauer et al (1982)12 using the following formula: Mucosal surface area (mm’) = (No. of villiimm width of serosa) x (No. of villiimm length of serosa) X (mean villus surface area) X (serosal surface area) Serosal surface area was calculated as the intestinal length (P-intestinal radius).

x

2

Assays Maltase activity was assayed using the method of Dahlqvist as modified by Tsuboi et a1.13AOP activity was assayed using the method of Wojnarowska and Gray. I4 Protein content was assayed using the method of Lowry et all5 using the Bio-Rad (Hercules, CA) reagent.

Statistical Analysis The data were analyzed using the Student’s t test, and results are expressed as mean f standard deviation of the mean. RESULTS

In the resected rabbits, after an initial postoperative weight loss, there was a gain of approximately 10% by 3 weeks and 13% at 6 weeks. There was a 28% increase in bowel length at 3 weeks after surgery, which increased to 41% at 6 weeks (Table 1). The estimated mucosal surface area increased 250% at 6 weeks in the resected rabbits compared with the immediate postoperative period. There was a 43% increase in mucosal surface area between the third and sixth weeks. There was an approximate 150% increase in villus height and 70% increase in villus Table

1. Changes

in Intestinal Extensive Small

maltase

(UE/g

Function Bowel

and Morphology Resection

Preoperatlvely Proximal protem) Distal

maltase

protein) Proximal protein) Distal

(UEIg

protein)

392

2 184*

222

k 49t

85 CL 58

259

k 89*

155 f 60t

154 t 60

171 * 96

131 r 37

171 -+ 93

256

I? 51*

288

f 89t

(g/g

Distal protein (g/g mucosa) Length (cm from pylorus to ileocecal valve) % increase in length height (mm)

Villus width (mm) Estimated mucosal area (mm*) NOTE.

152 +- 98

(lJE/g

Proximal protein mucosa)

Mean Villus

6 Weeks

(UE/g

AOP

AOP

3 Weeks

After

0.07

-t 0.04

0.06

-+ 0.01

0.07

t- 0.01

0.07

2 0.05

0.06

r 0.01

0.06

f 0.01

111 a9*

143 2 29”

0.44

f 0.05

28% 1.1 f 0.1*

0.06

2 0.01

0.1 t 0.02*

295

k 30

155 -+ 25t 41% 0.8 2 0.05t 0.1 f 0.02t

surface

Data are expressed

*P I .Ol 3 weeks compared tP I- .Ol 6 weeks compared *Immediately postoperative.

as mean with with

5337

F standard preoperatively. preoperatively.

f 600* deviation.

7635

2 800t

width at 3 weeks postoperation, which persisted until 6 weeks. Three weeks postoperatively there was a marked increase in maltase levels in both the proximal and distal intestine from 152 + 98 to 392 2 184 UE/g protein, and from 85 + 58 to 259 f 89 UE/g protein respectively (Table 1). These levels decreased at 6 weeks to 222 +- 49 and 155 + 60 WE/g protein respectively, although they remained significantly higher than preoperative levels. The enzymatic activity of AOP similarly increased 3 weeks after resection from 154 f 60 UE/g protein to 171 + 96, and from 171 + 93 to 256 + 51 UE/g protein in the proximal and distal intestine respectively; these levels remained relatively consistent at 6 weeks. The ileum distal to the anastomosis showed a threefold increase in maltase activity/g protein, and a 50% increase in AOP activity compared with a twofold increase in maltase activity and no increase in AOP activity shown by the proximal jejunum (Table 1). The four surgical control rabbits showed little change in their maltase, AOP, and protein levels at 3 weeks postoperation. DISCUSSION

Short bowel syndrome is an increasingly frequent condition in clinical practice, and efforts to ameliorate the condition, eg, increasing the function of the residual bowel, have been coupled with the search to develop diets that require less digestion and that can be absorbed by the residual intestine to provide adequate nutrition. Although small bowel transplantation has the potential of providing a complete cure for short bowel syndrome, clinical success with this procedure has been elusive despite several decades of investigative studies. It is hoped that a greater understanding of the intestinal adaptive process will lead to other therapies that approach the problem using alternate, and possibly more effective, techniques. In the small bowel response to extensive resection, there is a change in the morphological characteristics of the residual small boweF including a greater than twofold increase in villus height and width, which we observed in our present rabbit model. We also noted a compensatory increase in bowel length, varying degrees of which are seen in certain animal models as well as in human beings. 3~4~16-18 These changes in villus size together with the increase in bowel length appear to account for the great increase in surface area (250% increase by 6 weeks). Although the numerical value of the mucosal surface area is an estimate, it serves to illustrate the morphological extent of adaptation observed and is in agreement with other reports.3J9

1002

SWANIKER

The brush-border membrane enzymes maltase and aminooligopeptidase in the present rabbit model increased, consistent with other recent reports that show augmented function of the bowel remnant after extensive small intestinal resection. These studies are at variance with earlier reports that indicated a decrease in function with presumed enterocyte immaturity.20-23 The increased maltase and AOP enzyme activity levels were more marked in the distal ileum than in the proximal jejunum in the present study, which is consistent with previous studies showing increased adaptational changes seen in the distal small bowel.lJOJ1 After resection there is an increase in the concentration of nutrients reaching the distal remnant, which may stimulate an augmentation of enzymatic expression. On the other hand, there may be a limit to the absolute level of enzyme production per enterocyte, and therefore this portion of intestine

ET AL

may have a greater functional reserve by virtue of a lower initial level. The mechanisms involved in adaptation after extensive small bowel resection are multifactorial. The present study focuses on specific aspects of both morphological and functional adaptation. A major part of the initial and short-term adaptive response to extensive small bowel resection seems to be the up-regulation of enzymatic expression per enterocyte, and an increase in the functionally active portion of the villus.1J4 There also is an earlier expression of enzyme activity in the enterocyte life-cycle,4J4 with more rapid migration of functional enterocytes to the villus tip. The long-term response, however, is an increase in villus length/surface area of intestine; hyperplasia as opposed to hypertrophy,19 possibly allowing enzyme activity to decrease to levels that may be more energy conserving.

REFERENCES 1. Dowling RH, Booth CC: Structural and functional changes following small intestinal resection in the rat. Clin Sci 32:139-149. 1967 2. Menge H, Hopert R, Alexopoulos T, et al: Three-dimensional structure and cell kinetics at different sites of rat intestinal remnants during the early adaptive response to resection. Res Exp Med (Berl) 181:77-94,1982 3. Sigalet DL, Lees GM, Aherne F, et al: The physiology of adaptation to small bowel resection in the pig: An integrated study of morphological and functional changes. J Pediatr Surg 25:650657,199O 4. Menge H, Sepulveda FV, Smith MW: Cellular adaptation of amino acid transport following intestinal resection in the rat. J Physiol334:213-223.1983 5. Chaves M, Smith MW, Williamson RCN: Increased activity of digestive enzymes in ileal enterocytes adapting to proximal small bowel resection. Gut 28:981-987,1987 6. Porus RL: Epithelial hyperplasia following massive small bowel resection in man. Gastroenterology 48:753-757,1965 7. Weinstein LD. Shoemaker CP, Hersh T, et al: Enhanced intestinal absorption after small bowel resection in man. Arch Surg 99:560-562, 1969 8. Touloukian RJ. Spencer RP: Blood flow to the ileal remnant following massive intestinal resection. Surg Forum 22:370-371, 1971 9. Dowling RH, Booth CC: Functional compensation after small-bowel resection in man: Demonstration by direct measurement. Lancet 2:146-147,1966 10. Hanson WR, Osborne JW, Sharp JG: Compensation by the residual intestine after intestinal resection in the rat. 1. Influence of amount of tissue removed. Gastroenterology 72:692-700,1977 11. Williamson RCN, Buchholtz TW, Malt RA: Humoral stimulation of cell proliferation in small bowel after transection and resection in rats. Gastroenterology 75:249-254, 1978 12. Ecknauer R, Vadakel T, Wepler R: Intestinal morphology and cell production rate in aging rats. J Gerontol37:151-155, 1982 13. Tsuboi KK, Kwong LK, Burrill PH, et al: Sugar hydrolases and their arrangement on the rat intestinal microvillus membrane. J Membr Biol50:101-122, 1979

14. Wojnarowska F, Gray GM: Intestinal surface peptide hydrolases: Identification and characterization of three enzymes from rat brush-border. Biochim Biophys Acta 403:147-160,1975 15. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin Phenol reagent. J Biol Chem 193:265-275,1951 16. Shin CS, Chaudry AG, Khaddam MH, et al: Early morphological changes in intestine following massive resection of the small intestine and parenteral nutrition therapy. Surg Gynecol Obstet 151:246-250,198O 17. Wilmore DW, Dudrick SJ, Daly JM, et al: The role of nutrition in the adaptation of the small intestine after major resection. Surg Gynecol Obstet 132:673-680,197l 18. Postuma R, Moroz S, Friesen F: Extreme short-bowel syndrome in an infant. J Pediatr Surg 18:264-268, 1983 19. Thomson ABR: Resection of rabbit ileum: Effect on jejunal structure and carrier-mediated and passive uptake. Q J Exp Physiol 71:29-46,1986 20. Weser E, Hernandez MH: Studies of small bowel adaptation after intestinal resection in the rat. Gastroenterology 60:69-75, 1971 21. Bury KD: Carbohydrate digestion and absorption after massive resection of the small intestine. Surg Gynecol Obstet 135:177-187, 1972 22. Menge H, Robinson JWL: The relationship between the functional and structural alterations in the rat small intestine following proximal resection of varying extents. Res Exp Med (Berl) 173:41-53, 1978 23. McCarthy DM, Kim YS: Changes in sucrase, enterokinase and peptide hydrolase after intestinal resection. The association of cellular hyperplasia and adaptation. J Clin Invest 52942-951, 1973 24. Gutschmidt S, Kaul W, Menge H, et al: The adaptive response of disaccharidase activities at different sites along the villus epithelium after proximal intestinal resection in the rat. A microdensitometric study of enzyme kinetics. Res Exp Med 182:203213,1983 25. Nightingale JM, Lennard-Jones JE: The short bowel syndrome: What’s new and old? Dig Dis 11:12-31,1993 26. Schulzke JD, Fromm M, Bentzel CJ, et al: Ion transport in the experimental short bowel syndrome of the rat. Gastroenterology 102:497-504,1992

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Discussion MM. Ziegler (Cincinnati, OH): What you have done is confirmed some of the things that morphologically we have known about. The functional changes in both disaccharidase and peptidase enzyme activity is interesting. I have a question about methodology. Was your control a nonoperated animal or was it a division and then reanastomosis control? I really would question whether you would have to have that control in the comparative group. Classically I think most surgeons who manage a lot of short-bowel patients typically use lactose-free formulas, especially early after resection. What you have shown is that disaccharidase enzyme activity is enhanced early after a resection, which would make one speculate that you could use any kind of formula for feeding and that absorption and digestion should be preserved. The final question is what is it better to have, jejunum or ilium? Do you know if the distal segment that you have sampled here is ilium or is it really distal jejunum? F. Swaniker (response): We did have four control rabbits that underwent transsection only, and after 3 weeks the levels of enzyme in those rabbits were

about the same as in nonoperated controls. The controls we chose were actually nonoperated controls, but the resected animals did not have a significantly different value. Regarding the disaccharidase levels in adult rabbits, unfortunately the level of lactate activity in the bowel is so low that any decrease or increase in the activity of that enzyme may not have been easy to detect. It is true that clinically, deficiency in lactase is seen in human beings, and most people have to change to a lactose-free diet. But we did not test the activity of lactose. It may be that the maltase enzyme in human beings actually does increase, but we have not found any studies to that effect. We did notice a marked change in adaptation in the distal segments of the resected animals. The distal segment was actually distal ileum. The proximal segment was actually duodenum plus early jejunum. Those segments, as I said, did have more adaptation in terms of maltase enzyme activity and AOP activity. And if I had to choose between which segment of bowel to have left, I would choose to have the distal part left.