Pathogenesis of Bile-Induced Acute Pancreatitis in the Dog

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Experiments with Detergents

Peter T. Sum, MD, PhD, Sergio A. Bencosme, MD, PhD and Ivan T. Beck, MD, PhD, FRCP(C), FACP

A series of recent observations on different aspects of the chemistry and meta- bolic actions of bile have led us to investigate the possibility that bile may induce pancreatitis through the physicochemical detergent properties of its salts. To determine the relative importance of proteolytic enzymes on the one hand, and the detergent action of bile on the other, in the pathogenesis of bile-induced experimental pancreatitis in the dog, the early morphoiogic changes (1-30 min) produced by the intraductal injection of the following substances were investi- gated= (1) autogenous bile; (2) bile salt; (3) an anionic detergent, sodium lauryl sulfate (SLS); (4) trypsin; (5) a cationic detergent, cetrimide; (6) a neutralized mixture of SLS and cetrimide; and (7) saline solution. Autogenous bile, bile salts and SLS produce pancreatitis of similar morphologic appearance, characterized by punched-out areas of coagulation necrosis. Trypsin-induced pancreatitis was distinctly different from that produced by bile, bile salts and SLS. In trypsin pancreatitis, irregular areas of necrosis and hemorrhage were seen. Inactivation of the detergent activity of SLS by mixing it with the cationic detergent, cetri- mide, reduced the capacity of SLS to induce pancreatitis similar to that induced by bile and bile salts. These data seem to indicate that the early phases of bile- induced pancreatitis in our experiments are unlikely to result from the liberation of trypsin, but are related instead to the detergent activity of bile salts.

I t has b e e n p o s t u l a t e ( l t h a t the d i r e c t c y t o t o x i c a c t i o n of b i l e m a y p l a y a m a j o r role in i n i t i a t i n g a c u t e p a n c r e a t i t i s ( 1 - 3 ) . From the I)ivisiml of (;astroenterology and the Gastroenterology Clinical Investigation Unit, De- partment of Medicine, Hotel Dieu Hospital; the Department of Pathology, QneerFs University and the Kingston General I{ospital; and the Depart- ments of Medicine and Physiology, Queen's Uni- versit,v, Kingston, Ontario, Canada.

Supported hy Grant MT-3240 and MA-3507 of the Medical Research Council of Canada, and a grant from the Canadian Foundation for the Ad- vancenlent of Therapeutics.

Address for reprint requests: Dr. I. Beck, Hotel Dieu Hospital, Kingston, Ontario, Canada.

O t h e r i n v e s t i g a t o r s h a v e e m p h a s i z e d t h a t s u d d e n l i b e r a t i o n of a c t i v a t e d p r o t e o l y t i c e n z y m e s causes this c o n d i t i o n to d e v e l o p ( 4 - 6 ) . T i l e u n i t a r i a n c o n c e p t t h a t e n z y m a t - ic atttolysis causes a l l p a n c r e a t i t i s has re- c e n t l y b e e n q u e s t i o n e d o n b i o c h e m i c a l , p h a r m a c o l o g i c a n d m o r p h o l o g i c g r o u n d s ( 7 - 1 0 ) . B i l e - i n d u c e d p a n c r e a t i t i s , for in- stance, e x h i b i t s m o r p h o l o g i c f e a t u r e s dis- t i n c t l y d i f f e r e n t f r o m t h o s e e x h i b i t e d b y t r y p s i n - i n d u c e d p a n c r e a t i t i s (7, 1 I ) .

A series o f r e c e n t o b s e r v a t i o n s d e a l i n g w i t h d i f f e r e n t a s p e c t s of t h e c h e m i s t r y a n d

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SUM ET AL

metabolic actions of bile (12-15) have led

us to investigate the possibility that bile

may induce pancreatitis through the physi-

cochemical detergent properties of bile

salts.

T o

test

this hypothesis, we investigated

the

ability of a synthetic anionic detergent

bile salt (Na taurocholate) and a simple

anionic detergent sodiunl lauryl sulfate

(SLS)

to

produce the ntorphologic chan-

ges that develop when atttologous bile is

injected into the pancreatic duct of dogs.

T o elucidate the possible role of trypsin-

liberation in the pancreatitis produced by

these agents, the histologic changes in-

duced by them were compared to those

seen after the injection of trypsin.

In addition, we investigated whether

morphologic changes induced

by SLS

could be prevented if its detergent action

was inactivated by the admixtttre of a ca-

tionic detergent (16) .

MATERIALS AND METHODS

Since t h e p r i m a r y m o r p h o l o g i c c h a n g e s p r o d u c e d by different s u b s t a n c e s m a y be altered by s e c o n d a r y e v e n t s s u c h as h e m o r r h a g e a n d i n f l a m m a t i o n , in o r d e r to correlate closely t h e p r i m a r y events, it was decided to use a m e t h o d t h a t provides i m m e d i a t e a n d u n i f o r m histologic changes, s u c h as t h o s e

p r o d u c e d by i n j e c t i n g test s u b s t a n c e s u n d e r pres- s u r e into t h e p a n c r e a t i c d u c t (8).

Forty-two a d u l t m o n g r e l clogs of b o t h sexes, w e i g h i n g 9-15 kg, were used. All a n i m a l s were fed w i t h R o c k l a n d L a b o r a t o r y A n i m a l Diets C h o w for 2-3 d a y s a n d t h e n fasted for 24 h r before t h e e x p e r i m e n t s . U n d e r p e n t o b a r b i t a l a n e s t h e s i a (60 m g / k g b o d y w e i g h t ) , t h e p a n c r e a s was exposed. T h e m a j o r p a n c r e a t i c d u c t was isolated as it enters t h e d u o d e n u m a n d c a n n u l a t e d w i t h PE 50 polyethy- lene t u b i n g c o n n e c t e d to a 23-gauge needle. T h i s t u b e was a d v a n c e d 4-5 cm into t h e duct. T h e different solutions, described below, were w a r m e d

to b o d y t e m p e r a t u r e a n d injected into t h e p a n - creatic d u c t t h r o u g h t h e p o l y e t h } l e n e c a t h e t e r tinder a p p r o x i m a t e l y 250 m m H g pressure• T h e a m o u n t injected was 10 m l for each s o l u t i o n .

P a n c r e a t i c biopsies were t a k e n for histologic sections i m m e d i a t e l y before injection (to serve as

i n d i v i d u a l controls) a n d biopsies of the most involved p a r t were taken at l, 5, 10, 20 a n d 30 rain after p a n c r e a t i t i s was i n d u c e d . All s p e c i m e n s were tixed in formol a n d in Z e n k e r - f o r m o l a n d e m b e d d e d in paraffin. Sections were s t a i n e d w i t h t m m a t o x y t i n , p h l o x i n e a n d saffron (HP5) a n d w i t h Masson's t r i c h r o m e .

Dogs were d i s t r i b u t e d i n t o 7 g r o u p s according to

t h e s o l u t i o n each h a d received.

Group I: bile (6 dogs). A u t o g e n o u s bile col-

lected f r o m the g a l l b l a d d e r .

Group 2: bile salt (6 dogs). Synthetic s o d i u m t a u r o c h o l a t e * in 2 % a q u e o u s solution ( w / v ) .

Group 3: anionic detergent (6 dogs). S o d i u m lauryl sulfate t in a 0.5% a q u e o u s solution ( w / v ) .

Group 4: trypsin (6 dogs). Fifty m i l l i g r a m of

crystalline trypsin+ + in 10 m l of s o h ' e n t c o n t a i n i n g 5 ~ d e n a t u r e d gelatin a n d 0.09% p r o p y l p a r a b e n e in

• / O

distilled water. It was s h o w n previously t h a t in the absence of trypsin this solvent d o e s n o t i n j u r e the p a n c r e a s (7).

Group 5: cationic detergent (6 dogs). Cctri- m i d e , c e t y l t r i m e t h y l a m m o n i u m b r o m i d e t was used in a 0.5% a q u e o u s s o l u t i o n ( w / v ) .

Group 6: mixture of anionic and cationic deter- gent (6 dogs). A m i x t n r e of SLS a n d cetrimide, b o t h in a 1% a q u e o u s w / v solution was used. The}, were m i x e d in e q u a l a m o u n t s to give a final c o n c e n t r a t i o n of 0.5% for each. T h e m i x t u r e was p r e p a r e d d r o p by d r o p while it was agitated con- s t a n t l y u n t i l a w h i t i s h opalescent e m u l s i o n was f o r m e d . T b i s m a t e r i a l was always p r e p a r e d i m m e d i - ately before i n j e c t i o n .

Group 7: saline controls (6 dogs). 0.9% of a q u e o u s s o d i u m chloride.

Numerical evaluation of results. O n c e t h e his- tologie characteristics p r o d u c e d by t h e different s u b s t a n c e s were established, n e w sections were cut f r o m t h e original blocks w h i c h were p r e p a r e d from s p e c i m e n s o b t a i n e d i m i n after p a n c r e a t i t i s was i n d u c e d . T h e slides (six for e a c h group) were coded, m i x e d a n d i n t e r p r e t e d i n d e p e n d e n t l y by t w o

different observers.

RESULTS

G r o u p I : bile. A s

previously

d e s c r i b e d

(7), bile-induced pancreatitis is macroscop-

ically characterized by the immediate ap-

pearance of diffuse swelling in the involved

*K & K Laboratories, Plainview, NY. J-British D r u g Houses, T o r o n t o , C a n a d a . +Frank W. H o r n e r , M o n t r e a l , C a n a d a .

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part. At first it is streaky, b u t within a minute, diffuse blackish discoloration of the injected area occurs.

Within the first minute, the interstitial tissue demonstrates the presence of abun- dant diffuse acidophilic and homogenous material. At that time, there is no inflam- matory cell infiltration nor significant ab- normality of vessels and ducts. In the parenchyma, there are sharply defined loci of coagulative necrosis involving all tissue ele- ments (Fig 1A and 1B). T h e acini in the necrotic area can still be recognized, and the nuclei of these acinar cells are pyknotic or karyorrhectic. Z y m o g e n . granules are usually not seen in necrotic cells although they are occasionally present. Adjacent to these necrotic areas, a certain n u m b e r of acinar cells show different degrees of cellu-

lar degeneration indicated by nuclear

changes, such as reduction in size, irregu- larity of their borders, hyperchromatisnl and considerable reduction in the n u m b e r of zymogen granules. H e a l t h y cells adjacent to these cells, however, have their full c o m p l e m e n t of zymogen granules. Ne- crosis of the p a r e n c h y m a becomes more extensive and confluent as time progresses. Necrosis equally involves the acinar a n d islet tissue.

G r o u p 2: b i l e s a l t . T h e changes induced are similar to those induced by autogenous bile (Fig 2).

Group 3: anionic detergent. SLS- induced pancreatitis, grossly and microscopically, appears similar to that induced by bile and bile salt (Fig. 3A and 3 B ) .

Group 4: trypsin. Macroscopically, the

Fig 1A (left) and 1B (right). Bile-induced pancreatitis 1 rain after intraductal injection. In Fig 1A, note focal character of coagulation necrosis of parenchyma. (HPS, X 125) In Fig 1B, same necrotic area is shown at higher magnification. (HPS, X 312)

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Fig 2. Sodium taurocholate-induced pancreatitis 1 rain after intraductal injection. Portion of focus of coagulation necrosis similar to that produced by autogenous bile occu- pies right side of micrograph. On left, edge of rim of normal tissue is seen. (HPS, X 312)

involved part o[ the pancreas i m m e d i a t e l y appears p i n k and swollen but, in contrast to the changes induced after the injection o[ bile, 5-10 rain elapse before it becomes a d a r k brown-red. Grossly, after 30 min, the pancreas resembles that of G r o u p s 1, 2 a n d 3. Histologically, however, in contrast to G r o u p s 1, 2 a n d 3, at 1 rain, the vessels of the interstitial tissue a p p e a r to be dilated a n d show m i n i m a l to m a r k e d perivascular e d e m a a n d h e m o r r h a g e . W i t h i n 1 m i n the p a r e n c h y m a shows a wlriegated p a t t e r n due to the presence of extensive areas of necros~s with cells in different stages of disintegration, i n t e r m i n g l e d with relatively well pre- served acinar tissue (Fig 4A a n d 4B). I n some of the less involved areas, lighter stained necrotic acinar cells with nuclei in

SUM ET AL

karyolysis are seen. A l t h o u g h karyolysis is the p r e d o m i n a n t feature, occasional pyknotic nuclei are seen. I n some of the necrotic cells, the zymogen granules are considerably reduced, whereas the r e m a i n i n g cells are well granulated. As tile lesion progresses, complete disintegration ol: tile aciJ;ar tissue occurs so that large areas are occupied by cellular debris. T h e pan(reas becomes m o r e extensively involved but the pattern r e m a i n s basically u n c h a n g e d until the end of the experiment. Punched-out areas of coagulative necrosis with pyknotic nuclei, as seen in G r o u p s 1, 2 and 3 are not a feature of this pancreatitis. At 30 rain, p o l y m o r p h o n u c l e a r leukocytic infiltration is seen. Some of the cellular debris is re- m o v e d a n d the space between the necrotic acini a p p e a r s wider.

Group 5: cationic detergent. Macro- scopically, the lesion induced by cetrimide resembles that produced by trypsin, but it appears to develop m o r e slowly and is less

severe. Histologically, the cetrimide-

induced lesion is distinctly dit[erent from that induced by bile, bile salt or SI,S. A l t h o u g h cetrimide-induce(l pancreatitis nlay have some features in connnon with tile trypsin-induced disease, the two are easily distinguishal)le. At first there is mini- m a l h y p e r e m i a of p a r e n c h y m a l capillaries a n d at 30

nlin,

there are small foci of hemorrhage. T h e n u m b e r of necrotic and disintegrating acinar cells is smaller than in G r o u p 1-4. T h e less affected cells show diminished basophilia, m a r k e d reduction of their zymogen content, and hyperchro- m a t i c nuclei (Fig 5A a n d 5B).

Group 6: mixture o f anionic and cati- onic detergents. Macroscopically, there is only a m o d e r a t e degree of swelling in the i n t e r l o b u l a r space, I)egitming soon after injection of the solution containing the m i x t u r e of detergents. N o (liscoloration of the pancreas is seen t h r o u g h o u t the experi-

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tion. In Fig 3A, several foci of coagulative necrosis similar to those of Fig 1A are seen. (NPS, X 125) Fig 3B shows one of these areas at higher magnification. (HPS, X 312)

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SUM E1 AL

Fig 5A (left) and 5B (right). Cetrimide-induced pancreatitis 1 min after intraductal injection. In Fig 5A, acinar tissue in lower left third of photomicrograph appears normal. Rest of photomicrograph appears considerably paler due to marked reduction of zymogen granules. (HPS, X 125) Fig 5B shows center of Fig 5A at higher magnification. Most of cells in this area show degranulation. Some show hyperchmmatic nuclei and few exhibit darkening of cytoplasm. These latter cells are probably necrotic. (HPS, X 312)

ment. Microscopically, in 3 animals, n o

change can be seen at l min. In the o t h e r 3 dogs, even in the most severely affected, the interstitial tissue is normal. W i t h diligent search, one finds in the p a r e n c h y m a groups of a few acinar cells with irregularly shaped and h y p e r c h r o m a t i c nuclei (Fig 6), possibly u n d e r g o i n g necrosis. At no time d u r i n g these experiments were large focal areas of coagulative necrosis similar to those seen in bile, bile salt and SLS- induced pancreatitis observed.

Group 7: saline controls.

Macroscop- ically, the injected part of the pancreas shows slight swelling b u t no discoloration. No appreciable damage is seen microscopically when c o m p a r e d with the biopsy taken before injection (Fig 7).

N u m e r i c a l E v a l u a t i o n of R e s u l t s ( T a b l e 1)

Groups I , 2 and 3 (bile type

p a n c r e a -

titis.

When" the coded slides were re- viewed, it was not possible to distinguish between the lesions induced by bile, sodi- u m taurocholate and SLS. All 18 slides were, however, diagnosed by both observers as a bile type pancreatitis.

Group 4 (trypsin pancreatltis).

All six slides were recognized by both observers as trypsin pancreatitis.

Group 5 (cetrimide pancreatitis).

O [ the six, five slides were recognized as cetrimide pancreatitis by both observers, but one observer called one of the slides "mix- ttlre."

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Fig 6 (left). Mixture of SLS and cetrimide 1 rain after intraductal injection. Photomicrograph demon- strates most severely affected area in animal which showed greatest amount of damage. Upper two-thirds of photomicrograph shows normal pancreas with different degrees of degranulation. There is portion of islet in left upper corner. Lower third shows admixture of normal and several degenerated acinar ceils. On photomicrograph, nuclei of these appear pyknotic, but under microscope nuclear structures can still be recognized. (HPS, X 312) Fig 7 (fight). Saline control 1 rain after intraductal injection. Pancreas appears normal. (HPS, X 312)

Group 6 (mixture). T h r e e slides of tile mixture could not be differentiated from tile saline controls by either observer and were called "normal." T w o slides were incorrectly interpreted as " m i x t u r e " by both observers, but one was called "mixture" by one and "cetrimide" by the other.

Group 7 (saline controls). T h e s e slides all appeared normal. It was impos- sible to tell whether they came from G r o u p 7 or from the normal-appearing specimens of G r o u p 6.

D I S C U S S I O N

T h e early histologic changes produced in dog pancreas by intraductal injection of

autogenous bile, bile salt, trypsin, an anionic detergent (sodium lauryl sulfate), and a cationic detergent (cetrimide) were studied. T h e injection of bile, bile salt and

sodium lauryl sulfate (SLS) produced

cases of pancreatitis which were grossly and microscopically alike. On the other hand, the pancreatitis induced by trypsin was different from tile type produced by bile, bile salt and SLS. T h e differences f o u n d in this study between bile and trypsin pancreatitis were similar to those described previously (7). W h e n SLS and cetrimide were m i x e d in equal amounts, a whitish opalescent emulsion was obtained. W e do not know the exact physicochemical ha-

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T a b l e 1, N u m e r i c a l Evaluation of Results

SUM ET AL

Group No.

Diagnosis

Observer 1 Observer 2

Group 1 1

2

Bile 3

4 5 6

Bile type* Bile Type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type*

Group 2 1

2

Bile salt 3

4 5 6

Bile type* Bite type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type*

Group 3 1

2 Anionic detergent 3 (sodium lauryl sulfate) 4 5 6

Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type* Bile type*

Group 4 1 Trypsin Trypsin

2 Trypsin Trypsin

Trypsin 3 Trypsin Trypsin

4 Trypsin Trypsin

5 Trypsin Trypsin

6 Trypsin Trypsin

Group 5 1

2 Cationic detergent 3

(cetrimide) 4

5 6

Cetrimide Cetrimide Cetrimide Cetrimide Cetrimide Mixture Cetrimide Cetrimide Cetrimide Cetrimide Cetrimide Cetrimide

Group 6 1 Normal Normal

2 Normal Normal

Mixture of anionic 3 Mixture Mixture

and cationic detergent 4 Mixture Cetrimide

5 Normal Normal

6 Mixture Mixture

Group 7 1 Normal Normal

2 Normal Normal

Saline 3 Normal Normal

4 Normal Normal

5 Normal Normal

6 Normal Normal

* Since the lesions found in Groups ], 2, and 3 could other they were all diagnosed as bile type.

not be distinguished histologically from each

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ture o[ this m i x t u r e , b u t w h a t e x e r it m a y be, the experilnents d e m o n s t r a t e d t h a t the same a m o u n t of SLS w h i c h causes severe pancreatitis w h e n injected as a free a n i o n i c detergent no l o n g e r induces the same type of lesion once it has been n e u t r a l i z e d (16) by m i x i n g it with a cationic detergent.

P u h l i s h e d data on b i o c h e m i c a l studies on the activation o1 proteolytic enzymes in the course o[ bile-induced p a n c r e a t i t i s have yiehled controversial results (5-10, 17-19). O u r studies show that bile a n d SLS- i n d u c e d pancreatitis not only (lifter in their early stages f r o m the one b r o u g h t a b o u t by trypsin but that, at no time was the exten- sion of bile or a n i o n i c d e t e r g e n t - i n d u c e d (lisease a c c o m p a n i e d by lesions r e s e m b l i n g the enzyme-indtlced pancreatitis. T h e r e - fore, on the basis of this study, it seems unlikely t h a t i n i t i a t i o n a n d early p r o p a g a - tion of bile a n d a n i o n i c d e t e r g e n t - i n d u c e d pancreatitis are m e d i a t e d by a c t i v a t i o n of trypsin. It is m o r e likely t h a t bile, bile salts a n d SLS in(luce the lesion on the basis of direct cytotoxicity.

Bile is chemically a c o m p l e x n l i x t u r e which contains several

COlllpotnlds Stlch

as cholesterol, lecithin, bite p i g m e n t s a n d bile salts. Of these substances, bile salts possess

physicochemical

properties similar to those of a n i o n i c detergents (14, 15, 20). T h e toxic action of bile salts a n d SLS was recently d e m o n s t r a t e d by D a v e n p o r t (12) to be associated with increased flux of ions across the canine gastric mucosa. Lee a n d W h i t e - house (21) theorize t h a t bile salts, by vir- tue of their d e t e r g e n t action, i n h i b i t oxida- tive p h o s p h o r y l a t i o n by d i s r u p t i n g spatial o r g a n i z a t i o n of enzymes in m i t o c h o n d r i a .

T h e similarity in m o r p h o l o g i c appearance of the lesions i n d u c e d by bile, bile salt a n d SLS suggests that the m e c h a n i s m of action of these three substances is similar. ~Ve w o u l d like to suggest tbat the toxic action is related to their c o m m o n a n i o n i c

d e t e r g e n t p r o p e r t y . T h i s suggestion is [ur- ther s u p p o r t e d by the fact that w h e n SI.S was m i x e d with cetrimide, the c h a n g e s pro- d u c e d in the pancreas by this m i x t u r e were n o t o n l y m i n i m a l , b u t were u n l i k e the p a n c r e a t i t i s i n d u c e d by bile, bile salt a n d SLS alone.

R E F E R E N C E S

1. Beck IT, Pinter E J, Solymar J, et al: The role o[ pancreatic enzymes in the pathogenesis of acute pancreatitis. II. The fate of pancreatic proteolytic enzymes in the course of acute pancreatitis. Gastroenterol- ogy 43:60-70, 1962

2. Dragstedt 1.R, Haymond HD, Ellis JC: Pathogenesis of acute pancreatitis (acute pancreatic necrosis). Arch Surg 28:232-291, 1934

3. Flexner S: T h e constituent of tile bile causing pancreatitis and the effect of col- loids upon its action. J Exp Med 8:167- 177, 1906

4. Eisenhardt RH, Hartzell GW, Kubista T, et ah The etiology o[ acute haemorrhagic pancreatitis (AHI') in relation to the mechanism of activation ot pancreatic proteases. Fed Proc 20:252e, 1961

5. Keith LM Jr, Barnes JE, Denkewaher FR: Experimental study of interstitial injection of trypsin and trypsinogen into the pancreas. Arch Surg 77:416-420, 1958

6. Rich AR, Duff GL: Experimental and pathological studies on the pathogenesis of acute haemorrhagic pancreatitis. Bull J Hopkins Hosp 58:212-258, 1936

7. Beck 1T, Kahn DS, Solymar J, et ah T h e role of pancreatic enzymes in the pathogenesis of acute pancreatitis. IIi. Com- parison of she padmlogic and biochemical changes in the canine pancreas to intraductal injection with bile and with trypsin. Gastroenterology 46:531-542, 1964

8. Beck IT, McKenna RD, Zylberszac B, et ah The role of pancreatic enzymes in the pathogenesis of acute pancreatitis. IV. T h e effect of a trypsin inhibitor, Trasylol, on the cause of bile and trypsin-induced

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SUM ET AL

pan(leatitis in dogs. Gastroenterology 48: 478-483, 1965

9. Hansson K, L u n d h G, Stenram U: Morpho- logical and secretory studies in experi- mentally induced pancreatitis in rats. Acta Chir Scand 121:274-283, 1961

10. Jenson CB, ]mamoglu K, Root HD, et all T h e effect of hypothermia on experimental- ly induced haemorrhagic pancreatic necrosis. Gastroenterology 40:532 535, 1961 11. W a n k e M: Experimentelle Pankreatitis.

Proteolytische, lypolytische und bili'are Form. Stuttgart, Georg T h i e m e Verlag, 1968

12. Davenport H W : Destruction of the gastric mucosal barrier by detergents and urea. Gastroenterology 54:175-181, 1968

13. Dawson AM, Isselbacher KJ, Bell VM: Studies on lipid metabolism in the small intestine with observations on the role of bile salts. J Clin Invest 39:730-740, 1960 14. H o f m a n n AF: Clinical implications of

physicochemical studies on bile salts. Gas- troenterology 48:484-494, 1965

15. H o f m a n n AF, Small DM: Detergent properties of bile salts: correlation with physio-

logical function. Ann Rev Meal 18:333-376, 1967

16. Sykes G: Disinfection and Sterilization. Second edition. London, E. and F. N. Spon Ltd., 1965, p p 370-371

17. Geokas MC, McKenna RD, Beck I T : The role of elastase in experimental pancreatitis in dogs. (abstract) Gastroenterology 50:882-883, 1966

18. Reid LC, Paulette RE, Challis JW, et all T h e mechanism of tile pathogenesis of pancreatic necrosis and the therapeutic effect of propyhhiouracil. Surgery 43:538- 549, 1958

19. White T T , Magee DR: Perfusion of tile dog pancreas with bile without production of pancreatitis. Ann Surg 151:245-250, 1960 20. Moore EW, Dietschy JM: Na+ and K+ activity coefficients in bile and bile salts determined by glass electrodes. Am J Physiol 206: 1111-1117, 1964

21. Lee MJ, ~Vhitehouse MW: Inhibition of electron transport and coupled phosphory- lation in liver mitochondria by cholanic (bile) acids and their conjugates. Biochem Biophys Acta 100:317-328, 1965