International Journal ofPancreatology, vol. 12, no. 2:121-125, October 1992 9 Copyright 1992 by The Humana Press Inc. All rights of any nature whatsoever re.served. 0169-4197/92112:21121-125/$2.00
Influence of Feeding Regimen and Postnatal Developmental Stages on Antibacterial Activity of Pancreatic Juice Stefan Pierzynowski/,3 Peeyush Sharma, *'2Jerzy Sobczyk/ Stanislaw Garwacki/ and Wieslaw Baref ZDepartment of Zoophysiology, University of Lund, Lund, Sweden; 2Department of Surgery, University Hospital, Lund, Sweden; and 3Department of Animal Physiology, Agricultural University, Warsaw, Poland
Summary Antibacterial activity of pancreatic juice in the pig (n = 8) was investigated during early postnatal development and in cattle (n = 6) receiving a different feeding regimen. For pancreatic juice collection, a catheter was surgically implanted in the pancreatic duct. Reintroduction of pancreatic juice was achieved through a T-shaped cannula in the duodenum. Pancreatic juice was collected for 30 min in all cases. In piglets, collections were carried out at 2, 5--6, and 7-10 wk of age, and in cattle, after a standard meal, 48 h starvation, and following 24 h intraduodenal glucose infusion~ Antibacterial activity was tested on Micrococcus Pyogenes strain ATTC 6538P by disc agar diffusion technique using nonactivated pancreatic juice, before and after heat treatment for 15 min at 65 and 100~ respectively. Piglets showed a significant rise in antibacterial activity of pancreatic juice after weaning. In cattle, 48 h of starvation resulted in a marked suppression of antibacterial activity. This activity was found to be normal after a standard meal and comparable to that after 24-h intraduodenal glucose infusion. Heating of pancreatic juice to 65~ caused a 35% increase in the antibacterial potency, whereas heating to 100~ completely abolished it. Additionally, dilution of pancreatic juice to 1:10 did not affect antibacterial potency. Key Words: Antibacterial activity; pancreatic juice; development stages; feeding regimen; piglets; cattle; starvation.
Introduction
gastrointestinal microbial homeostasis has been debated recently (4-6). Pancreatic juice possesses antimicrobial properties against many organisms (4). The beneficial effect of exocrine pancreatic extracts on the course and severity of gastrointestinal infections has also been investigated (7). Pancreatic juice from many mammalian species has been demonstrated to possess antimicrobial properties (8). It also has been shown that human pancreatic juice enhances the bactericidal actions of some antimicrobial drugs (9).
Presence of antimicrobial properties in certain body secretions has been demonstrated by earlier studies (1-3). The role of pancreatic juice in maintenance of Received January 5, 1987; Revised February 5, 1992; Accepted February 10, 1992 *Author to whom all correspondence and reprint requests should be addressed: Department of Surgery, University Hospital, Lund-221 85, Sweden
121
t22 Not m u c h is k n o w n about the i n f l u e n c e o f different feeding regimens and postnatal development on the antibacterial activity of pancreatic juice. We decided to investigate these two aspects in an experimental study.
Material and Methods Y o u n g , 2 to 10-wk old piglets o f either sex (n = 8) and adult cattle 4 to 5 yr of age (n = 6) were used ~br the experiments. The animals were bred at a standard breeding center. The piglets were weaned at 4 wk of age. Surgery was performed on unfasted piglets under general anesthesia using ketamine 40 mg/kg (Ketalar, Parke Davis, N J) and on cattle after an overnight fast under neuroleptic analgesia, using 2% x y l a z i n e 0.015 m L / k g ( R o m p u n , B a y e r , G e r m a n y ) . A silicon catheter (Dow C o m i n g , Midland) was implanted in the pancreatic duct and a T-shaped cannula in the duodenum, as described earlier (10,11), both of which were exteriorized percutaneously and interconnected for reintroduction of pancreatic juice under resting conditions. Pancreatic juice collections were started after a few days of postoperative recovery. Piglets were used to study the antibacterial activity of pancreatic juice during various postnatal developmental stages and cattle to study the effect of various feeding regimens on it. In piglets, pancreatic juice was collected for 30 min right after suckling or ingestion of a standard weaning food. This was done between 10:00 and 10:30 AM. The 30-min pancreatic juice collections in cattle were carried out under three different conditions: after a 48-h fast, after ingestion of a standard meal, and following 24-h intraduodenal glucose infusion (0.15 g/kg/h). This was done between 8:00 and 8:30 AM, after discarding the initial few milliliters of the juice. During the 48-h fast and 24-h intraduodenal glucose infusion, the animals had free access to water alone, and the pancreatic juice was reintroduced into the duodenum. The samples were kept in an ice bath during collection and then stored at -70~ after noting the pH. All animals were subjected to these procedures. Micrococcus pyogenes strain ATTC 6538P was used as a standard microorganism in all cases for determination of antibacterial activity of the pancreatic juice. None of the animals had previously International Journal of Pancreatology
Pierzynowski et al. been immunized against this microorganism. The antibacterial activity of the pancreatic juice was expressed in terms of neomycin, using a modified disc agar diffusion technique (12). The aim of the method was to compare the antibacterial potency of the pancreatic juice in each case with that of a standard minimum inhibitory concentration (MIC) of neomycin against the test microorganism. One unit of antibacterial activity o f pancreatic juice w a s equivalent to 10 gg/mL neomycin. The pancreatic juice showed no change in pH on thawing in any of the samples. Pure nonactivated juice was used for microbiological studies, since it has been demonstrated that antimicrobial properties are independent of activation and enzymatic action. Each sample was tested for antibacterial activity before and after heat treatment for 15 min at 65 and 100~ respectively. In addition, the pancreatic juice samples were diluted in a buffer solution to 1:2, 1:4, 1:5, 1:10, and 1:100, and antibacterial activity was tested.
Results The antibacterial potency o f pancreatic juice in the two groups of animals is presented in Table 1. Incubation of pancreatic juice at 65~ for 15 min caused an increase of the antibacterial potency by about 35% on average, whereas incubation at 100~ for the same duration c o m p l e t e l y abolished it. Antibacterial activity remained unaltered in samples diluted up to 1:10 but became successively weaker until it became undetectable at 1:100. Antibacterial activity of pancreatic juice increased with age in piglets, being weakest at 2 wk o f age and showing a significant rise after weaning at 4 wk. The differences were statistically significant. The rate of secretion of pancreatic juice in piglets increased from 0.7 + 0.4 mL/kg/h at 2 wk of age to 3.1 + 2.2 mL/kg/h at 7-10 wk. The weakest antibacterial activity in cattle pancreatic juice was observed after a 48-h fast. Cattle receiving a 24-h intraduodenal glucose infusion showed antibacterial activity similar to those on a standard diet. The rate o f secretion o f the pancreatic juice was significantly reduced after starvation (0.2 + 0.1 mL/kg/h) as compared to that of animals on standard diets and those receiving intraduodenal glucose infusions. Volume 12, 1992
Antibacterial Activity of Pancreatic Juice
123
Table 1 Volume Outflow, Antibacterial Activity Concentration, and Output in the Pancreatic Juice of Pig and Cattle Animals Pig (age in weeks), n = 8 2 wk 5--6 wk 7-10 wk Cattle (feeding regimen), n = 6 Standard meal 48-h starvation 24-h glucose infusion
Volume, mL/kg/h
Fresh pancreatic juice UtmL U/hNg
0.7 +_0.4 ab 0.4 + 0.2a 3.1 + 2.2c
3.2 + 1.1a 37 +_15b 38+30 b
2.2 _+ 1.0a 15 + 7.0b 118+89 c
5.4 _+2.3 a 45 + 17c 55+14 c
3.8 +_2.0a 18 +__6.0 t' 170_+73c
0.5 +_0.1/' 0.2 + 0.1a 0.6 + 0.14 b
28 + 19b 7 _+13a 44 + 18b
14 ___8.1 b 1.7 + 2a 27 + 10b
51 +_8c 20 _+7 b 54 + 19c
25 _+9 *'b 4.8 + 0.8 *'a 33 + 17b
Heated pancreatic jtrice, 65~ U/mL U/h/kg
One unit of antibacterial activity corresponds to I0 ~tg/mL of neomycin. Letters in superscript denote statistical significance calculated with ANOVA (p < 0.05). Statistical significance for data on antibacterial activity output in heated and unheated pancreatic juice (asterisk) compared with paired Student's t-test (p < 0.05).
Discussion Antibacterial activity of pancreatic juice in piglets increased with age, being strongest at 7-10 wk. This change was brought about soon after weaning, and the latter may have a role to play in stimulating the secretion of the antimicrobial factor. The rate of secretion o f the juice showed a tendency to decrease from 0.7 + 0.4 mL/kg/h at 2 wk to 0.4 + 0.2 mL/kg/h at 5-6 wk, i.e., shortly after weaning. But by contrast, antibacterial activity rose significantly from 3.2 + 1.1 U/mL to 37 + 15 U/mL during the same period and stayed at the latter level in piglets aged 7-10 wk, in whom the rate of secretion of pancreatic juice shot up to 3.1 + 2.2 mL/kg/ h. These observations indicate that the potency of antibacterial activity during early postnatal development in the pig is independent of flow rate of the pancreatic juice. An increase in the antibacterial activity of pancreatic juice around the time of weaning suggests that this might be a compensatory phenomenon in maintaining microbial homeostasis in the upper small bowel at a time when the small bowel is being presented with new food-borne microorganisms and still has not d e v e l o p e d an effective system of defense against them. Food intake is the major contaminant of the small bowel, and an effective system of microbial control is necessary, especially during early postnatal development. Some of the inherent International Journal of Pancreatology
factors involved in microbial homeostasis in suckling piglets are gastric acid (13), secretory immunoglobulins (14), and milk. Milk contains immunoglobulins (15), lysozyme (2), and nonlysozyme antimicrobial factors (16). All o f these may have a role to play in regulating bacterial growth in the upper small intestine, a role that is taken over by pancreatic juice after weaning. Our understanding of the factors that bring about such a significant rise in the antibacterial potency of pancreatic juice shortly after weaning remains incomplete. It is lucrative to implicate cholecystokinin (CCK) and secretin, but theoretically, one would expect the same C C K and secretin response after food ingestion as with milk, and therefore, this does not appear to be the likely mechanism of control. The answer probably lies in the breakdown products of ingested food, released as a result of bacterial action in the upper small bowel, that might be absorbed locally and cause a stimulation o f secretion of antibacterial factors by the exocrine pancreas. In cattle, starvation for 48 h caused a significant drop in the rate of secretion as well as the antibacterial potency of pancreatic juice, while being similar in animals on standard diet and those receiving intraduodenal glucose infusions. It is very likely that during starvation, the system o f exchange of information between the upper small bowel and pancreas fails to get activated and thus results in a weak Volume 12, 1992
124 antibacterial response in the pancreatic juice. This hypothetical system may be a crucial one that signals to the pancreas when to produce more or less of antibacterial factor. In contrast to the developing pig, bovine pancreatic juice antimicrobial activity appears to be flowdependent. Secretin and C C K do not potentiate antibacterial activity (8), and this further points to the existence of another system of control regulating the output of antibacterial factor by the pancreas. In this context, it would be interesting to study bacterial growth in the small bowel in fasting animals with a suppressed antibacterial activity. The effect of standard diet consumption and intraduodenal glucose infusion in terms o f rate of secretion of pancreatic juice, as well as the potency of antibacterial activity, was quite similar, and this indicates that the stimulation of secretion of the antibacterial factor is similar with these two regimens. This aspect should be investigated using other dietary fractions in the pure and mixed forms. We know that the rate of secretion of pancreatic juice on food ingestion is regulated partly by C C K and secretin and partly by vagus (17), but the r e g u l a t o r y m e c h a n i s m o f secretion of the antibacterial factor needs to be investigated. A very interesting feature of pancreatic juice, noted by our group as well as others, is that heat treatment up to 65~ does not affect antimicrobial potency (4,8), actually quite the contrary. On the other hand, heating the juice to 100~ completely a b o l i s h e s this p r o p e r t y , s u g g e s t i n g that the antimicrobial factor is heat-labile. Dilution does not seem to affect antibacterial activity of pancreatic juice up to around 1:10, and it also seems to be independent o f activation and enzymatic action. Rubinstein et alo (4), using column chromatography, demonstrated that the active substance had a presumptive mol wt of less than 4000 daltons. Interestingly, the antibacterial action of pancreatic juice appears to be very sensitive to pH, having an optimal activity at pH 8.5 with complete abolition of action at pH 7.0. It has been found that in patients with pancreatitis, there is a significant drop in the pH of pancreatic juice (18), which could pave the way Ior bacteri',d invasion since the inherent antimicrobial factor is set out of gear at a low pH. Our initial results International Journal of Pancreatology
Pierzynowski et al. stress the importance of regulating mechanisms governing the release of antimicrobial factor by the exocrine pancreas, both during early postnatal development as well as during starvation~ These mechanisms are yet to be identified, and further work in this field is necessary.
References 1 Galask RP, Snyder IS. Antimicrobial factors in amniotic fluid. Am J Obstet Gynecol 1970; 106: 59-65. 2 Reddy V, Bhaskaram C, Raghuramulu N, Jagadeesan V. Antimicrobial factors in human milk. Acta Paediatr Scand 1977; 66: 229-232. 3 Selsted ME, Martinez RJ. isolation and purification of bactericides from human tears. Exp Eye Res 1982; 34: 305-318. 4 Rubinstein E, Mark Z, Haspel I, Ben Ari G, Dreznik Z, Mirelman D, Tadmor A. Antibacterial activity of the pancreatic fluid. Gastroenterology 1985; 88: 927-932. 5 Boisen S, Agergaard N, Rotenberg S, Kragelund Z. Effects of gut flora on intestinal activities of trypsin, chymotrypsin, etastase and amylase in growing rats fed purified diets with cellulose pectin or sand. Z Tierphysiol Tiererniihrg u Futtermittelkde 1985; 53: 245-254. 6 Gyr K, Felsenfeld O, Zimmerli-Ning M. The effect of oral pancreatic enzymes on the intestinal flora of protein deficient vervet monkeys challenged with Vibrio Cholerae. Am J Clin Nutr 1979; 32: 1592-1596. 7 Gyr K, Felsenfeld O, Zimmerli-Ning M. Effect of oral pancreatic enzymes on the course of cholera in proteindeficient vervet monkeys~ Gastroenterology 1978; 74: 511-513. 8 Pierzynowski S, Sharma P, Sobczyk J, Garwacki S, Barej W, Westr6m B. A comparative study of antibacterial activity of pancreatic juice in mammals. (unpublished observations) 1992. 9 Mett H, Gyr K, Zak O, Vosbeck K. Duodeno-pancreatic secretions enhance bactericidal activity of antimicrobial drugs. Antimicrob Agents Chemother 1984; 26: 35-38. 10 Pierzynowski SG, Westr6m BR, Kartsson BW, Svendsen J, Nilsson B. Pancreatic cannutation of young pigs for long-term study of exocrine pancreatic function. Can J Anita Sci 1988; 68: 953-959. 11 Butler HC, Brinkman DC, Klavano PA. Cannulation of the bovine pancreatic duct. Am J Vet Res 1960; 21: 205-211. 12 Pilet Ch, Toma B. Recherche de residus de penicilline et de neomycine dans les tissus de poulets recevant une alimentation antibiosupplementee.Ann Nutr Alim 1969; 23: 277-281. 13 Knott FA. The gastric germicidal barrier. Guys Hosp Rep 1923; 73: 429-437. 14 Walker WA, Isslbacher KJ. Intestinal antibodies. NEng J Med 1977; 297: 767-773.
Volume 12, 1992
Antibacterial Activity of Pancreatic Juice
125
t5
Ahlstedt S, Carlsson B, Hanson L~, Goldblum RM. Antibody production by human colostral cells: immunoglobulin class, specificity and quantity. Scand J Immunol 1975; 4: 535-539. ;~6 Goldman AS, Smith CW. Host resistance factors in human milk. J Pediatr 1973; 82: 1082-1090. 17 Holst JJ, Schaffalitzky de Muckadell OB, Fahrenkrug J. Nervous control of pancreatic exocrine secretion in pigs~ Acta Physiol Scand 1979; 105: 33-51. 18 Dutta SK, Russel RN, Iber FL. Influence of exocrine pancreatic insufficiency on the intraluminal pH of the proximal small intestine. Am J Dig Dis 1979; 24: 529-534~
International Journal of Pancreatology
Volume 12, 1992
Influence of Feeding Regimen and Postnatal Developmental Stages on Antibacterial Activity of Pancreatic Juice Stefan Pierzynowski/,3 Peeyush Sharma, *'2Jerzy Sobczyk/ Stanislaw Garwacki/ and Wieslaw Baref ZDepartment of Zoophysiology, University of Lund, Lund, Sweden; 2Department of Surgery, University Hospital, Lund, Sweden; and 3Department of Animal Physiology, Agricultural University, Warsaw, Poland
Summary Antibacterial activity of pancreatic juice in the pig (n = 8) was investigated during early postnatal development and in cattle (n = 6) receiving a different feeding regimen. For pancreatic juice collection, a catheter was surgically implanted in the pancreatic duct. Reintroduction of pancreatic juice was achieved through a T-shaped cannula in the duodenum. Pancreatic juice was collected for 30 min in all cases. In piglets, collections were carried out at 2, 5--6, and 7-10 wk of age, and in cattle, after a standard meal, 48 h starvation, and following 24 h intraduodenal glucose infusion~ Antibacterial activity was tested on Micrococcus Pyogenes strain ATTC 6538P by disc agar diffusion technique using nonactivated pancreatic juice, before and after heat treatment for 15 min at 65 and 100~ respectively. Piglets showed a significant rise in antibacterial activity of pancreatic juice after weaning. In cattle, 48 h of starvation resulted in a marked suppression of antibacterial activity. This activity was found to be normal after a standard meal and comparable to that after 24-h intraduodenal glucose infusion. Heating of pancreatic juice to 65~ caused a 35% increase in the antibacterial potency, whereas heating to 100~ completely abolished it. Additionally, dilution of pancreatic juice to 1:10 did not affect antibacterial potency. Key Words: Antibacterial activity; pancreatic juice; development stages; feeding regimen; piglets; cattle; starvation.
Introduction
gastrointestinal microbial homeostasis has been debated recently (4-6). Pancreatic juice possesses antimicrobial properties against many organisms (4). The beneficial effect of exocrine pancreatic extracts on the course and severity of gastrointestinal infections has also been investigated (7). Pancreatic juice from many mammalian species has been demonstrated to possess antimicrobial properties (8). It also has been shown that human pancreatic juice enhances the bactericidal actions of some antimicrobial drugs (9).
Presence of antimicrobial properties in certain body secretions has been demonstrated by earlier studies (1-3). The role of pancreatic juice in maintenance of Received January 5, 1987; Revised February 5, 1992; Accepted February 10, 1992 *Author to whom all correspondence and reprint requests should be addressed: Department of Surgery, University Hospital, Lund-221 85, Sweden
121
t22 Not m u c h is k n o w n about the i n f l u e n c e o f different feeding regimens and postnatal development on the antibacterial activity of pancreatic juice. We decided to investigate these two aspects in an experimental study.
Material and Methods Y o u n g , 2 to 10-wk old piglets o f either sex (n = 8) and adult cattle 4 to 5 yr of age (n = 6) were used ~br the experiments. The animals were bred at a standard breeding center. The piglets were weaned at 4 wk of age. Surgery was performed on unfasted piglets under general anesthesia using ketamine 40 mg/kg (Ketalar, Parke Davis, N J) and on cattle after an overnight fast under neuroleptic analgesia, using 2% x y l a z i n e 0.015 m L / k g ( R o m p u n , B a y e r , G e r m a n y ) . A silicon catheter (Dow C o m i n g , Midland) was implanted in the pancreatic duct and a T-shaped cannula in the duodenum, as described earlier (10,11), both of which were exteriorized percutaneously and interconnected for reintroduction of pancreatic juice under resting conditions. Pancreatic juice collections were started after a few days of postoperative recovery. Piglets were used to study the antibacterial activity of pancreatic juice during various postnatal developmental stages and cattle to study the effect of various feeding regimens on it. In piglets, pancreatic juice was collected for 30 min right after suckling or ingestion of a standard weaning food. This was done between 10:00 and 10:30 AM. The 30-min pancreatic juice collections in cattle were carried out under three different conditions: after a 48-h fast, after ingestion of a standard meal, and following 24-h intraduodenal glucose infusion (0.15 g/kg/h). This was done between 8:00 and 8:30 AM, after discarding the initial few milliliters of the juice. During the 48-h fast and 24-h intraduodenal glucose infusion, the animals had free access to water alone, and the pancreatic juice was reintroduced into the duodenum. The samples were kept in an ice bath during collection and then stored at -70~ after noting the pH. All animals were subjected to these procedures. Micrococcus pyogenes strain ATTC 6538P was used as a standard microorganism in all cases for determination of antibacterial activity of the pancreatic juice. None of the animals had previously International Journal of Pancreatology
Pierzynowski et al. been immunized against this microorganism. The antibacterial activity of the pancreatic juice was expressed in terms of neomycin, using a modified disc agar diffusion technique (12). The aim of the method was to compare the antibacterial potency of the pancreatic juice in each case with that of a standard minimum inhibitory concentration (MIC) of neomycin against the test microorganism. One unit of antibacterial activity o f pancreatic juice w a s equivalent to 10 gg/mL neomycin. The pancreatic juice showed no change in pH on thawing in any of the samples. Pure nonactivated juice was used for microbiological studies, since it has been demonstrated that antimicrobial properties are independent of activation and enzymatic action. Each sample was tested for antibacterial activity before and after heat treatment for 15 min at 65 and 100~ respectively. In addition, the pancreatic juice samples were diluted in a buffer solution to 1:2, 1:4, 1:5, 1:10, and 1:100, and antibacterial activity was tested.
Results The antibacterial potency o f pancreatic juice in the two groups of animals is presented in Table 1. Incubation of pancreatic juice at 65~ for 15 min caused an increase of the antibacterial potency by about 35% on average, whereas incubation at 100~ for the same duration c o m p l e t e l y abolished it. Antibacterial activity remained unaltered in samples diluted up to 1:10 but became successively weaker until it became undetectable at 1:100. Antibacterial activity of pancreatic juice increased with age in piglets, being weakest at 2 wk o f age and showing a significant rise after weaning at 4 wk. The differences were statistically significant. The rate of secretion of pancreatic juice in piglets increased from 0.7 + 0.4 mL/kg/h at 2 wk of age to 3.1 + 2.2 mL/kg/h at 7-10 wk. The weakest antibacterial activity in cattle pancreatic juice was observed after a 48-h fast. Cattle receiving a 24-h intraduodenal glucose infusion showed antibacterial activity similar to those on a standard diet. The rate o f secretion o f the pancreatic juice was significantly reduced after starvation (0.2 + 0.1 mL/kg/h) as compared to that of animals on standard diets and those receiving intraduodenal glucose infusions. Volume 12, 1992
Antibacterial Activity of Pancreatic Juice
123
Table 1 Volume Outflow, Antibacterial Activity Concentration, and Output in the Pancreatic Juice of Pig and Cattle Animals Pig (age in weeks), n = 8 2 wk 5--6 wk 7-10 wk Cattle (feeding regimen), n = 6 Standard meal 48-h starvation 24-h glucose infusion
Volume, mL/kg/h
Fresh pancreatic juice UtmL U/hNg
0.7 +_0.4 ab 0.4 + 0.2a 3.1 + 2.2c
3.2 + 1.1a 37 +_15b 38+30 b
2.2 _+ 1.0a 15 + 7.0b 118+89 c
5.4 _+2.3 a 45 + 17c 55+14 c
3.8 +_2.0a 18 +__6.0 t' 170_+73c
0.5 +_0.1/' 0.2 + 0.1a 0.6 + 0.14 b
28 + 19b 7 _+13a 44 + 18b
14 ___8.1 b 1.7 + 2a 27 + 10b
51 +_8c 20 _+7 b 54 + 19c
25 _+9 *'b 4.8 + 0.8 *'a 33 + 17b
Heated pancreatic jtrice, 65~ U/mL U/h/kg
One unit of antibacterial activity corresponds to I0 ~tg/mL of neomycin. Letters in superscript denote statistical significance calculated with ANOVA (p < 0.05). Statistical significance for data on antibacterial activity output in heated and unheated pancreatic juice (asterisk) compared with paired Student's t-test (p < 0.05).
Discussion Antibacterial activity of pancreatic juice in piglets increased with age, being strongest at 7-10 wk. This change was brought about soon after weaning, and the latter may have a role to play in stimulating the secretion of the antimicrobial factor. The rate of secretion o f the juice showed a tendency to decrease from 0.7 + 0.4 mL/kg/h at 2 wk to 0.4 + 0.2 mL/kg/h at 5-6 wk, i.e., shortly after weaning. But by contrast, antibacterial activity rose significantly from 3.2 + 1.1 U/mL to 37 + 15 U/mL during the same period and stayed at the latter level in piglets aged 7-10 wk, in whom the rate of secretion of pancreatic juice shot up to 3.1 + 2.2 mL/kg/ h. These observations indicate that the potency of antibacterial activity during early postnatal development in the pig is independent of flow rate of the pancreatic juice. An increase in the antibacterial activity of pancreatic juice around the time of weaning suggests that this might be a compensatory phenomenon in maintaining microbial homeostasis in the upper small bowel at a time when the small bowel is being presented with new food-borne microorganisms and still has not d e v e l o p e d an effective system of defense against them. Food intake is the major contaminant of the small bowel, and an effective system of microbial control is necessary, especially during early postnatal development. Some of the inherent International Journal of Pancreatology
factors involved in microbial homeostasis in suckling piglets are gastric acid (13), secretory immunoglobulins (14), and milk. Milk contains immunoglobulins (15), lysozyme (2), and nonlysozyme antimicrobial factors (16). All o f these may have a role to play in regulating bacterial growth in the upper small intestine, a role that is taken over by pancreatic juice after weaning. Our understanding of the factors that bring about such a significant rise in the antibacterial potency of pancreatic juice shortly after weaning remains incomplete. It is lucrative to implicate cholecystokinin (CCK) and secretin, but theoretically, one would expect the same C C K and secretin response after food ingestion as with milk, and therefore, this does not appear to be the likely mechanism of control. The answer probably lies in the breakdown products of ingested food, released as a result of bacterial action in the upper small bowel, that might be absorbed locally and cause a stimulation o f secretion of antibacterial factors by the exocrine pancreas. In cattle, starvation for 48 h caused a significant drop in the rate of secretion as well as the antibacterial potency of pancreatic juice, while being similar in animals on standard diet and those receiving intraduodenal glucose infusions. It is very likely that during starvation, the system o f exchange of information between the upper small bowel and pancreas fails to get activated and thus results in a weak Volume 12, 1992
124 antibacterial response in the pancreatic juice. This hypothetical system may be a crucial one that signals to the pancreas when to produce more or less of antibacterial factor. In contrast to the developing pig, bovine pancreatic juice antimicrobial activity appears to be flowdependent. Secretin and C C K do not potentiate antibacterial activity (8), and this further points to the existence of another system of control regulating the output of antibacterial factor by the pancreas. In this context, it would be interesting to study bacterial growth in the small bowel in fasting animals with a suppressed antibacterial activity. The effect of standard diet consumption and intraduodenal glucose infusion in terms o f rate of secretion of pancreatic juice, as well as the potency of antibacterial activity, was quite similar, and this indicates that the stimulation of secretion of the antibacterial factor is similar with these two regimens. This aspect should be investigated using other dietary fractions in the pure and mixed forms. We know that the rate of secretion of pancreatic juice on food ingestion is regulated partly by C C K and secretin and partly by vagus (17), but the r e g u l a t o r y m e c h a n i s m o f secretion of the antibacterial factor needs to be investigated. A very interesting feature of pancreatic juice, noted by our group as well as others, is that heat treatment up to 65~ does not affect antimicrobial potency (4,8), actually quite the contrary. On the other hand, heating the juice to 100~ completely a b o l i s h e s this p r o p e r t y , s u g g e s t i n g that the antimicrobial factor is heat-labile. Dilution does not seem to affect antibacterial activity of pancreatic juice up to around 1:10, and it also seems to be independent o f activation and enzymatic action. Rubinstein et alo (4), using column chromatography, demonstrated that the active substance had a presumptive mol wt of less than 4000 daltons. Interestingly, the antibacterial action of pancreatic juice appears to be very sensitive to pH, having an optimal activity at pH 8.5 with complete abolition of action at pH 7.0. It has been found that in patients with pancreatitis, there is a significant drop in the pH of pancreatic juice (18), which could pave the way Ior bacteri',d invasion since the inherent antimicrobial factor is set out of gear at a low pH. Our initial results International Journal of Pancreatology
Pierzynowski et al. stress the importance of regulating mechanisms governing the release of antimicrobial factor by the exocrine pancreas, both during early postnatal development as well as during starvation~ These mechanisms are yet to be identified, and further work in this field is necessary.
References 1 Galask RP, Snyder IS. Antimicrobial factors in amniotic fluid. Am J Obstet Gynecol 1970; 106: 59-65. 2 Reddy V, Bhaskaram C, Raghuramulu N, Jagadeesan V. Antimicrobial factors in human milk. Acta Paediatr Scand 1977; 66: 229-232. 3 Selsted ME, Martinez RJ. isolation and purification of bactericides from human tears. Exp Eye Res 1982; 34: 305-318. 4 Rubinstein E, Mark Z, Haspel I, Ben Ari G, Dreznik Z, Mirelman D, Tadmor A. Antibacterial activity of the pancreatic fluid. Gastroenterology 1985; 88: 927-932. 5 Boisen S, Agergaard N, Rotenberg S, Kragelund Z. Effects of gut flora on intestinal activities of trypsin, chymotrypsin, etastase and amylase in growing rats fed purified diets with cellulose pectin or sand. Z Tierphysiol Tiererniihrg u Futtermittelkde 1985; 53: 245-254. 6 Gyr K, Felsenfeld O, Zimmerli-Ning M. The effect of oral pancreatic enzymes on the intestinal flora of protein deficient vervet monkeys challenged with Vibrio Cholerae. Am J Clin Nutr 1979; 32: 1592-1596. 7 Gyr K, Felsenfeld O, Zimmerli-Ning M. Effect of oral pancreatic enzymes on the course of cholera in proteindeficient vervet monkeys~ Gastroenterology 1978; 74: 511-513. 8 Pierzynowski S, Sharma P, Sobczyk J, Garwacki S, Barej W, Westr6m B. A comparative study of antibacterial activity of pancreatic juice in mammals. (unpublished observations) 1992. 9 Mett H, Gyr K, Zak O, Vosbeck K. Duodeno-pancreatic secretions enhance bactericidal activity of antimicrobial drugs. Antimicrob Agents Chemother 1984; 26: 35-38. 10 Pierzynowski SG, Westr6m BR, Kartsson BW, Svendsen J, Nilsson B. Pancreatic cannutation of young pigs for long-term study of exocrine pancreatic function. Can J Anita Sci 1988; 68: 953-959. 11 Butler HC, Brinkman DC, Klavano PA. Cannulation of the bovine pancreatic duct. Am J Vet Res 1960; 21: 205-211. 12 Pilet Ch, Toma B. Recherche de residus de penicilline et de neomycine dans les tissus de poulets recevant une alimentation antibiosupplementee.Ann Nutr Alim 1969; 23: 277-281. 13 Knott FA. The gastric germicidal barrier. Guys Hosp Rep 1923; 73: 429-437. 14 Walker WA, Isslbacher KJ. Intestinal antibodies. NEng J Med 1977; 297: 767-773.
Volume 12, 1992
Antibacterial Activity of Pancreatic Juice
125
t5
Ahlstedt S, Carlsson B, Hanson L~, Goldblum RM. Antibody production by human colostral cells: immunoglobulin class, specificity and quantity. Scand J Immunol 1975; 4: 535-539. ;~6 Goldman AS, Smith CW. Host resistance factors in human milk. J Pediatr 1973; 82: 1082-1090. 17 Holst JJ, Schaffalitzky de Muckadell OB, Fahrenkrug J. Nervous control of pancreatic exocrine secretion in pigs~ Acta Physiol Scand 1979; 105: 33-51. 18 Dutta SK, Russel RN, Iber FL. Influence of exocrine pancreatic insufficiency on the intraluminal pH of the proximal small intestine. Am J Dig Dis 1979; 24: 529-534~
International Journal of Pancreatology
Volume 12, 1992
