International Journal ofPancreatology, vol. 16, no. 1, 45-49, August 1994 �9 Copyright 1994 by Humana Press Inc. All rights of any nature whatsoever reserved. 0 t 69-4197/94/16:45-49/S5.00

Colonic Proteolysis Following Pancreatic Duct Ligation in the Rat

Daniel Bustos,*,l Osvaldo Tiscornia, 1 Maria L Caldarini/ Gustavo Negri, 2 Silvina Pons/ Kumiko Ogawa/ and Juan A. De Paula

llnstituto de Gastroenterologia "Dr. Jorge Perez Companc," Buenos Aires, Argentina, and 2Departamento de Bioquimica Clinica, Seccion Gastroenterologia y Enzimologia, Buenos Aires, Argentina

Summary

Luminal proteolytic activity (PA) of different colonic segments was ascertained in animals subjected to pancreatic duct ligation (PDL) and in control rats. The PDL rats revealed a significant PA reduction in the cecum, proximal colon (P < 0.01), and distal colon (P < 0.05). Proteolytic activity, trypsin, and chymotrypsin activity in control rats diminished progressively from the cecum to the distal colon. Conversely in PDL rats, we found maximal PA in distal colon. The conclusion is drawn that a significant proportion of colonic proteolytic activity can be attributed to pancreatic proteases with a maximal contribution at cecum level.

Key W o r d s : Proteolysis; pancreatic protease; trypsin; chymotrypsin; large bowel.

Introduction

It has been estimated that between 1 and 3 g each of trypsin and chymotrypsin and 0.5 g of elastase are produced by the human pancreas every day (1). These enzymes take part in the hydrolytic-digestive pro- cesses of the small bowel, before passing into the large intestine with food residues and other undi- gested substances (2). Pancreatic proteases probably have a multifunctional role in the large intestine, serving as substrates for bacterial proteases (3) and also acting as hydrolytic enzymes, which participate in protein breakdown. In this regard, comparative studies on protease activities in ileal effluent and feces have demonstrated that a significant proportion

Received January 3, 1994; Accepted March 8, 1994. *Author to whom all correspondence and reprint requests

should be addressed: Departamento de Bioqmmica Clinica, Facultad de Farmacia y Bioquimica, UBA, Yunin 956 (1113), Buenos Aires, Argentina.

of proteolytic activity in the human colon can be attributed to pancreatic proteases, and the studies sug- gest that considerable breakdown or inactivation of these enzymes occurs during gut transit (2,4-6). The extent of the contribution of pancreatic proteases to the proteolysis in the colon is unknown. In an attempt at assessing the contribution of host-produced pro- tease to proteolytic activity in the different colonic segment, this activity was compared in cecum, proxi- mal and distal colon of control rats, and in those that had undergone pancreatic duct ligation.

Materials and Methods

Twenty-four male Wistar rats, weighing 200-250 g each (Charles Rivers, Laboratory inc.), initially free from specific pathogens, were used. Half of these animals were anesthetized with ether, and following a midline abdominal incision were subjected to pan- creatic duct ligation (PDL) of the multiple pancreatic

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46 Bustos et al.

ducts discharging into the bile duct. The pancreatic ducts were transectioned between silk 6-0 sutures under a dissecting microscope. The other half were control animals that underwent sham operation. After surgery, all the animals were kept in individual cages and were given 50% glucose (1 part) and isotonic saline (5 parts) to drink for 3 d. The rats were then fed a standard chow. Since preliminary studies had shown that PDL rats ate more than controls, pair- feeding was introduced starting 3 d after surgery. In this way, each of the PDL animals was fed the same quantity of food as eaten by its pair in the control group on the previous day. Water intake and fecal weight were measured daily as from 3 d after surgery. The body weight was recorded initially at the moment of surgery and finally at the moment of autopsy.

All rats were exsanguinated through the aorta 10 d after surgery following the end of the dark period, which is a time of maximal food consump- tion, between 8 and 10 AM. Ligatures were placed at the ileocecal junction, between the cecum and the branches given off the middle colonic artery. The luminal contents of the cecum, proximal colon, and distal colon were collected separately. The pro- teolytic activity, trypsin, chymotrypsin activity, and soluble protein were measured in each luminal gut segment from control and PDL rats.

Measurement o f Trypsin and Chymotrypsin Activity Five hundred milligrams of intestinal contents

were homogenized in 5 mL of a 0.05 mol/L Tris buffer, pH 7.4. After centrifugation, the supernatant was assayed for trypsin and chymotrypsin activity. Chymotrypsin activity was measured using 0.1 mM of succinyl-Ala-Pro-Phe 4 nitroanilide (Sigma) as substrate (7). The assays were performed at 25~ in 0.1M Tris-HC1 and 0.01M CaC12, pH 7.8. For trypsin activity, a 0.01 solution of benzoyl-DL-arginine P- nitroanilide (Sigma) as substrate was used (8). The assays were performedat 25~ in 0.05MTris buffer, pH 8.2, containing 0.02M CaC12. The absorbance of the P-nitroanilide produced from the specific sub- strates was measured continuously on a Beckman model 35 spectrophotometer at 405 nm.

Measurement of Proteolytic Activity

One gram of fresh intestinal contents was homog- enized in 0.1 mol/L sodium phosphate buffer, pH

7.0, to produce 10% (w/v) slurries. The proteolytic activity assays were carried out immediately. The use of azocasein as a proteolytic substrate has been validated in a number of studies (9-11), and mea- surements of proteolytic activity were carried out using this substrate as follows: 1.0 mL of the sus- pension sample was dispensed in duplicate into 1.5-mL capacity Eppendorf tubes and mixed with 0.2 mL of 0.1 molYL sodium phosphate buffer, pH 7.0~ Reactions were initiated by the addi t ion of 0.3 mL azocasein (Sigma) stock solution, which gave an initial substrate concentration of 10 mg/mLo The capped tubes were then incubated for 1 h at 37~ The reactions were terminated by transferring the tube contents into centrifuge tubes containing 1 mL of 10% w/v TCA. Control tubes were incubated as above, but without azocasein, which was added after inactivation of the sample with TCA. Inacti- vated samples were allowed to stand at room tem- perature for 30 min and were then centrifuged at 27,000g for 8 min at 4~ The supematant fluid (1.0 mL) was then mixed with an equal volume of IN OHNa, and the absorbance of this solution was read at 450 nm.

Soluble Protein in Colonic Contents

Luminal contents were homogenized with 0.9% w/v NaC1 to give 10% (w/v) slurries. Samples were then centrifuged at 30,000g for 10 min to produce cell-free supernatants. Samples were precipitated with 10% (w/v) TCA and centrifuged at 30,000g for 10 rain. The pellets were solubilized and resuspended in 1M NaOH. Proteins were then determined by the Lowry method (12).

Results are presented as means + SD. Statistical evaluation was made by analysis of variance and, where appropriate, by the unpaired Student's t-test, a value of P < 0.05 being considered significant.

Results

In the PDL group the surgery was considered suc- cessful if the luminal trypsin and chymotrypsin activity were undetectabte. We included in this group 10 animals. Rats with PDL increased the fecal weight compared with control group (P < 0.01) (Table 1).

As Fig. 1 shows, the proteolytic activity in con- trol rats was maximal at cecum level, decreasing

International Journal of Pancreatology Volume 16, 1994

Colonic Proteolysis 47

Table 1 Water Intake, Fecal Weight, and Change in Body Weight in Controls and PDL Rats

Body wt,

Water intake,* Fecal weight,* g mL/d g/d Initial Final

Control group 51.7 + 9.2 10.9 + 1.4 236 + 14 24l +_ i2 (n = t2)

PDL 54.6 + 4.5 18.1 _+_ 5.3** 227 + 10 214 + 29 (N = 10)

*Mean + SD of daily measurements between 3 and 10 d after surgery. **p < 0o01 compared with control group.

11

"~1o

o09

7

O

4 g ,.- 3

i o" 2 O

CECUM PROXIMAL COLON DISTAL COLON

Fig. 1. Proteolytic activity of the colonic luminal con- tents from controls ~ a n d PDL [----]. Results are given as means + SD. *P < 0.0t compared with control group. **P < 0.05 compared with control group. ***P < 0.01 compared with distal colon from PDL rats. ****P < 0.05 compared with cecum from control group.

s teadi ly f rom c e c u m to distal colon. Converse ly in PD L rats, we found max ima l p ro teo ly t ic act ivi ty in distal colon. In the P D L series, when compared with the cont ro l group, the p ro teo ly t i c act ivi ty was s ignif icant ly reduced in all co lon ic segments (Fig. 1). As shown in Fig. 2, t rypsin and chymot ryps in act ivi ty in cont ro l rats d imin i shed progress ive ly f r o m the c e c u m to the distal co lon . The same de te rmina t ions in the P D L group gave undetect- able values.

Considerable amounts o f soluble protein were found in the colonic segments (Table 2). It was increased in cecum f rom PDL rats (P < 0.01) com- pared with the control group.

120 -

100 -

8 0 - . J

6 0 -

4 0 ~

2 0

0 CECUM PROXIMAL DISTAL

COLON COLON

Fig. 2. Chymotrypsin - - and Trypsin --- activity in the luminal content of different colonic segments from control rats. Results are given as mean + SD. *P < 0.01 compared with cecum.

Table 2 Soluble Protein in Colonic Contents from Controls

and PDL Rats (mg/g of Content)*

Control group, PDL Group n = 1 2 n = 1 0

Cecum 3.05 + 0.70 4.25 + 0.80* Proximal colon 3.55 _+ 0.60 3.95 _+ 0.20 Distal colon 4.55 + 0.80 4.75 + 0.30

*Results a r e g i v e n a s m e a n + SD. *p 0.01 compared with cecum from control rats.

Discussion

In our work, proteolytic activity, as well as trypsin and chymotrypsin, decreased in control rats f rom cecum to distal colon, indicat ing that substantial

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48 Bustos et al.

degradation or inactivation of pancreatic protease occurs during passage through the large gut. Earlier works have shown that colonic contents are strongly proteolytic, and it seems probable that this activity results from the combined actions of both bacterial and host-produced proteases (10,13). The results presented in this article show that the PA in the luminal contents from PDL rats was statistically less than in the control group in all of the segments stud- ied, thereby establishing that pancreatic proteases provide a substantial contribution to proteolytic ac- tivity in the large intestine. By subtracting the nonpancreatic proteolytic activity obtained in PDL group from the mean proteolytic activity measured in the control group, we calculated the participation of pancreatic proteases in the total proteolytic activ- ity in each colonic segment. By this method, the pan- creatic proteases showed a significant participation in the total, amounting to 84% in the cecum, 78% in the proximal colon, and 45% in the distal colon. The activity of pancreatic proteases in the large intestine may contribute to the intracolonic degradation of proteins. We found increased soluble protein in cecum from PDL rat indicating that a lower proteolytic activity and/or an overload of protein to the colon owing to absence of pancreatic secretion may occur in this group of rats. The products of proteolysis, amino acids, and small peptides may be available to a wide range of bacteria in the gut. Small peptides have been shown to stimulate the growth of many intestinal bacteria (14-16), and in vitro experiments on protein fermentation showed that free amino acids never accumulated in more than trace amounts, indicating that peptide hydrolysis was the limiting step in protein utilization by gut bacteria (11). On the other hand, the presence of considerable amounts of pancreatic protease in the colonic lumen may be important in pathological conditions. Animals sub- jected to hemorrhagic shock showed pronounced mucosal damage in the small bowel, but the damage to the mucosa was less pronounced after inhibition of the pancreatic proteases by means of trasylol (17), or by previous ligation of the pancreatic duct (18,19). This fact may also be important in the colon, since a substantial amount of pancreatic proteases are present.

In conclusion, we have shown that a large propor- tion of luminal colonic proteolysis is of pancreatic ori-

gin, with a maximal contribution at the level of the cecum and decreasing steadily to the distal colon. How- ever, because of differences in the digestive metabo- lism between humans and rats, the extrapolation of our findings to the human situation is quite difficult.

References

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2 Bohe M, Borgestrom A, Genell S, Ohlsson K. Determi- nation of inmunoreactive trypsin, pancreatic elastase and chymotrypsin in extracts of human feces and ileostomy drainage. Digestion 1983; 27: 8-15.

3 MacFarlane GT, MacFarlane S. Utilization of pancreatic trypsin and chymotrypsin by proteolytic and non-proteo- lyric Bacteroides fragilis~type bacteria. Curr Microbiol t991; 23: 143-148.

4 Genell S, Gustafsson B, Ohlsson K. Immunochem{cal quantitation of pancreatic endopeptidases in the intestinal contents of germfree and conventional rats. Scand J Gastroenterol 1977; 12: 811-820.

5 Genell S, Bengt E, Gustafsson B, Ohlsson K. Quantitation of active pancreatic endopeptidases in the intestinal contents of germfree and conventional rats. Scand J Gastroenterol 1976; 11: 757-762.

6 MacFarlane GT, Cummings JH, MacFarlane S, Gibson GR. Influence of retention time on degradation of pancreatic enzyme by human colonic bacteria grown in a 3-stage continuous culture system. J Appl Bacteriol 1989; 76(5): 520-527.

7 Del Mar EG, Largman C, Brodrick J, Geokas UM. A sensitive new substrate for chymotrypsin. Anal Biochem 1979; 99: 316-320.

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9 Brock FM, Forsberg CW, Buchanan-Smith JG. Proteolytic activity of m men microorganisms and effects of proteinase inhibitors. Appl Environ Microbiol 1982; 44: 561-569.

10 MacFarlane GT, Cummings JH, Allison C. Protein degra- dation by human intestinal bacteria. J Gen Microbio11986; 132: 1647-1656.

11 MacFarlane GT, Allison C. UtiLization of protein by human gut bacteria. Microbiol Ecology t986; 38: 19-24.

12 Lowry DH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1953; 193: 265-275.

13 MacFarlane GT, Allison C, Gibson SAW, Cummings JH. Contribution of the microflora to proteolysis in the human large intestine. JAppl Bacteriol 1988; 64: 37-46.

14 Harschild AHW. Incorporation of C 14 from amino-acids and peptides into protein by Clostridium perfringes type D. JBacteriot 1985; 29: t019-1028.

15 Leach FR, Snell EE. The absorption of glycine and alanine and their peptides by lactobacillus Casei. JBiol Chem 1980; 235: 3523-3531.

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Colonic ProteoIysis 49

16 Russell JB. Fermentation of peptides by Bacteroides mminicola B14. Appl Environ Microbiol 1983; 45:1566- 1574.

17 Bounous G, Menard D, De Medicis E. Role of pancreatic proteases in the pathogenesis of ischemic enteropathy. Gastroenterology 1977; 73: 102-108.

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Bounous G, Brown R, Nulder D, et al. Abolition of"tryptic enteritis" in the shocked dog. A rch Surg 1965; 91: 371-375. Montgomery A, Borgstrom A, Haglund VLF. Pancreatic proteases and intestinal mucosal injury after ischemia and reperfusion in the pig. Gastroenterology 1992; 102: 216-222.

International Journal of Pancreatofogy Volume 16, 1994