실험적 취장염에 관한 연구

Abstract

[한글]

Studios on Experimental Pancreatitis

-The Role of Active Enzymes in Relation to Ductal Obstruction-

Sae Whan Kim

Department of Medical Science The Graduate School, Yonsei University

(Directed by Prof. Sa Suk Hong)

Extensive studies have been devoted to clarify the pathogenesis of acute

pancreatitis since Opie et al. (1901, 1909) proposed the common channel theory as

primary etiological factor. However, it is evident that the etiology of acute

pancreatitis is not simple and the mechanism remains unclear. Clinically, most of

pancreatitis explained by the fellowing two factors; 1) the reflux of the bile into

the pancreatic duct will activate the proteolytic enzymes which then cause the

autodigestion of the pancreatic tissue, 2) over-eating or the ingestion of alcohol

will cause the outflow of pancreatic juice leading to the increase of resistance

and pressure of the ductal system which will eventually cause the rupture of the

duct-acinar system and obstruction of the duct resulting in acute pancreatitis.

Many investigators have been interested in the common channel theory and

extensive studies have been made to prove this theory. However, the evidences were

controversial. Wangensteen et al. (1931) who preyed that an experimentally-made

common channel, which let the bile flow through the pancreatic duct into the

duodenum, could not elicit acute pancreatitis. Furthermore, clinical observations

(Mann and Giordano, 1923) showed that the common channel did not exist anatomically

in all patients and there were acute parcreatitis cases with no common channel in

whom the regurgitation of bile was not expected (Ivy and Gibbs, 1952: McCutcheon,

1964)Experimentally, it is true that the instillation of the pathogenic factors

into the pancreatic duct and the ligation of the duct will produce acute

pancreatitis. However, it is uncertain that the resulting pancreatic lesion is due

to autodigestion by the activation of proteolytic enzyme as suggested by Opie and

Meakins (1909) . Dragstedt et al. (1937) demonstrated that experimental exposure of

pancreatic tissue to active proteolytic enzyme in the intestine could not cause the

digestion of the cell membranes. Thereafter, White an? Magee (1950), Hansson(1971)

and Hong et al. (1962) have objected the concept of the activation of proteolytic

enzyme as the cause of acute pancreatitis.

In spite of these controversies, one cannot deny that some active enzyme will

take part in the pathogenesis of acute pancreatitis, and recently, Anderson et at.

(1969) reported that the lipolytic enzymes, especially, lipase and phospholipase A,

in addition to the proteolytic enzyme, produced more severe hemorrhagic and

necrotic pancreatitis.

On the other hand, many investigators (Rich and Duff, 1936; Thal et al., 1957)

thought the obstruction of the pancreatic duct is the basic etiologic factor of all

pancreatitis; however, the main pitfall of this concept is the fact that the simple

obstruction of the duct did not produce classical pancreatitis, but only mild

edematous pancreatitis.

The present investigation deals with the pathogenesis of acute pancreatitis from

a viewpoint with an inquiry into the effect of regurgitation of the active enzymes

into the pancreatic duct and pancreatic duct occlusion or patency by changes in the

serum amylase and lipase level and by the pathological changes of the pancreatic

tissue using the rabbit.

Materials and Methods

Experimental Animals

White rabbits of both sexes weighing 2 kg in average was used and divided as

follows. All the animals were anesthetized with secobarbital (30mg/kg, I. V.) and

the procedures were carried out with sterile technique as far as possible through

the midline incision on a prone position.

1) Experiments with ductal obstruction

a) Saline 2.0ml

b) Lipase 800units

c) α-Acyllysolecithin 10∼20mg

d) Trypsin 17,000 BAEE units

e) L-Asparaginase 1,000 I. U.

f) Chenodeoxycholic acid 20mg

Two milliliters of each experimental solution dissolved in saline was instilled

into the pancreatic duct slowly and the pancreatic duct was ligated. Blood

specimens were obtained from the ear vein or femoral artery before and at 24, 48 or

72 hours after the instillation.

2) Experiments without ductal obstruction

As a preliminary study, trepan blue 2% saline solution was instilled into the

pancreatic duct leaving the pancreatic duct patent and the fate of the injected dye

was observed. The animals in this group were arranged as fellow;

a) Saline 2.0ml

b) Trypsin 10,000 BAEE units

c) Phospholipase A 1.Omg (9 units)

d) Pig pancreatic juice (inlclude amylase 9334mg maltose, lipase 224㎖ N/20 NaOH

in 2㎖

e) Human saliva (include amylase 1760mg maltose in 2㎖)

Each experimental solution was instilled int? the pancreatic duct in the same

manner during the experiment 1 and the duct was not ligated but left open. Blood

specimens were obtained at 1,3,5 and 24 hours after injection of the experimental

solution. The Determination of the Serum Enzymes

The serum amylase activity was measured by the method of Nelson(17M) and the

serum lipase by the method of Cherry and Crandall(1932) using the Titrator TTT 2b.

Pathological Study

Histopathologic examinations were carried out on routine hematoxylin-eosin

stained section after 10% formalin fixation and paraffin embedding.

Results and Summary

To evaluate the roles of active enzymes and pancreatic duct obstruction or

patency on the pathogenesis and the progress of acute pancreatitis, several active

pancreatic enzymes were infused into the ligated or patent pancreatic duct of

rabbit ; then the changes of serum enzyme activities were determined and the

pathological lesions of the pancreatic tissue were examined.

1) There were marked increases in serum amylase and lipase levels 24 hours after

the instillation of trypsin, lipase, lysolecithin and cholate into the pancreatic

duct fellowing complete ligation in rabbits. Thereafter, both enzymes were

decreased and returned to the preoperative level after 72 hours. There were simlar

changes in the saline instilled group on a mild scale.

2) Following complete ligation of pancreatic duct, chenodeoxycholic acid

instillation produced severe parenchymal and fatty necrosis or hemorrhage, trypsin

caused hemorrhage and lysolecithin lead to marked parenchymal necrosis and

hemorrhage.

3) When the trypan blue was instilled into the pancreatic duct and the duct was

unobstructed, the dye, spread widely in the pancreatic tissue, rapidly evacuated

out through the patent pancreatic duct into the duodenum and had almost completely

disappeared grossly within 3 hours.

4) In the animals having unobstructed duct, instillation of trypsin,

phospholipase A and amylase caused early (1-5 hours) elevation of serum amylase and

lipase levels which gradually returned to the preoperative level within 24 hours.

However, the elevated serum amylase level persisted more than 24 hours in the group

where the phospholipase A was instilled.

5) In the animals having unobstructed duct, the instillation of trypsin and

phospholipase A produced typical acute pancreatitis; however, the animals, where

the saline, amylase and pig pancreatic juice had been instilled, showed mild

interstitial pancreatitis only without evidence of hemorrhage or parenchymal

necrosis.

In conclusion. it is clear that not only trypsin but also the lipases, especially

phospholipase A, have very important roles as pathogenic factors causing acute

pancreatitis and it is certain that the action of phospholipase A is due to the

conversion of lecithin to Iysolecithin. And finally, the obstruction or the patency

of the pancreatic duct effects mainly the progress of the disease.

[영문]

Extensive studies have been devoted to clarify the pathogenesis of acute pancreatitis since Opie et al. (1901, 1909) proposed the common channel theory as primary etiological factor. However, it is evident that the etiology of acute pancreatitis is not simple and the mechanism remains unclear. Clinically, most of pancreatitis explained by the fellowing two factors; 1) the reflux of the bile into the pancreatic duct will activate the proteolytic enzymes which then cause the autodigestion of the pancreatic tissue, 2) over-eating or the ingestion of alcohol will cause the outflow of pancreatic juice leading to the increase of resistance and pressure of the ductal system which will eventually cause the rupture of the duct-acinar system and obstruction of the duct resulting in acute pancreatitis.

Many investigators have been interested in the common channel theory and extensive studies have been made to prove this theory. However, the evidences were controversial. Wangensteen et al. (1931) who preyed that an experimentally-made common channel, which let the bile flow through the pancreatic duct into the

duodenum, could not elicit acute pancreatitis. Furthermore, clinical observations (Mann and Giordano, 1923) showed that the common channel did not exist anatomically in all patients and there were acute parcreatitis cases with no common channel in

whom the regurgitation of bile was not expected (Ivy and Gibbs, 1952: McCutcheon, 1964)Experimentally, it is true that the instillation of the pathogenic factors into the pancreatic duct and the ligation of the duct will produce acute pancreatitis. However, it is uncertain that the resulting pancreatic lesion is due to autodigestion by the activation of proteolytic enzyme as suggested by Opie and Meakins (1909) . Dragstedt et al. (1937) demonstrated that experimental exposure of pancreatic tissue to active proteolytic enzyme in the intestine could not cause the

digestion of the cell membranes. Thereafter, White an? Magee (1950), Hansson(1971) and Hong et al. (1962) have objected the concept of the activation of proteolytic enzyme as the cause of acute pancreatitis.

In spite of these controversies, one cannot deny that some active enzyme will take part in the pathogenesis of acute pancreatitis, and recently, Anderson et at. (1969) reported that the lipolytic enzymes, especially, lipase and phospholipase A,

in addition to the proteolytic enzyme, produced more severe hemorrhagic and necrotic pancreatitis.

On the other hand, many investigators (Rich and Duff, 1936; Thal et al., 1957) thought the obstruction of the pancreatic duct is the basic etiologic factor of all pancreatitis; however, the main pitfall of this concept is the fact that the simple obstruction of the duct did not produce classical pancreatitis, but only mild edematous pancreatitis.

The present investigation deals with the pathogenesis of acute pancreatitis from a viewpoint with an inquiry into the effect of regurgitation of the active enzymes into the pancreatic duct and pancreatic duct occlusion or patency by changes in the serum amylase and lipase level and by the pathological changes of the pancreatic tissue using the rabbit.

Materials and Methods

Experimental Animals

White rabbits of both sexes weighing 2 kg in average was used and divided as follows. All the animals were anesthetized with secobarbital (30mg/kg, I. V.) and the procedures were carried out with sterile technique as far as possible through the midline incision on a prone position.

1) Experiments with ductal obstruction

a) Saline 2.0ml

b) Lipase 800units

c) α-Acyllysolecithin 10∼20mg

d) Trypsin 17,000 BAEE units

e) L-Asparaginase 1,000 I. U.

f) Chenodeoxycholic acid 20mg

Two milliliters of each experimental solution dissolved in saline was instilled into the pancreatic duct slowly and the pancreatic duct was ligated. Blood specimens were obtained from the ear vein or femoral artery before and at 24, 48 or 72 hours after the instillation.

2) Experiments without ductal obstruction

As a preliminary study, trepan blue 2% saline solution was instilled into the pancreatic duct leaving the pancreatic duct patent and the fate of the injected dye was observed. The animals in this group were arranged as fellow;

a) Saline 2.0ml

b) Trypsin 10,000 BAEE units

c) Phospholipase A 1.Omg (9 units)

d) Pig pancreatic juice (inlclude amylase 9334mg maltose, lipase 224㎖ N/20 NaOH in 2㎖

e) Human saliva (include amylase 1760mg maltose in 2㎖)

Each experimental solution was instilled int? the pancreatic duct in the same manner during the experiment 1 and the duct was not ligated but left open. Blood specimens were obtained at 1,3,5 and 24 hours after injection of the experimental solution. The Determination of the Serum Enzymes

The serum amylase activity was measured by the method of Nelson(17M) and the serum lipase by the method of Cherry and Crandall(1932) using the Titrator TTT 2b.

Pathological Study

Histopathologic examinations were carried out on routine hematoxylin-eosin stained section after 10% formalin fixation and paraffin embedding.

Results and Summary

To evaluate the roles of active enzymes and pancreatic duct obstruction or patency on the pathogenesis and the progress of acute pancreatitis, several active pancreatic enzymes were infused into the ligated or patent pancreatic duct of rabbit ; then the changes of serum enzyme activities were determined and the pathological lesions of the pancreatic tissue were examined.

1) There were marked increases in serum amylase and lipase levels 24 hours after the instillation of trypsin, lipase, lysolecithin and cholate into the pancreatic duct fellowing complete ligation in rabbits. Thereafter, both enzymes were

decreased and returned to the preoperative level after 72 hours. There were simlar changes in the saline instilled group on a mild scale.

2) Following complete ligation of pancreatic duct, chenodeoxycholic acid instillation produced severe parenchymal and fatty necrosis or hemorrhage, trypsin caused hemorrhage and lysolecithin lead to marked parenchymal necrosis and hemorrhage.

3) When the trypan blue was instilled into the pancreatic duct and the duct was unobstructed, the dye, spread widely in the pancreatic tissue, rapidly evacuated out through the patent pancreatic duct into the duodenum and had almost completely disappeared grossly within 3 hours.

4) In the animals having unobstructed duct, instillation of trypsin, phospholipase A and amylase caused early (1-5 hours) elevation of serum amylase and lipase levels which gradually returned to the preoperative level within 24 hours.

However, the elevated serum amylase level persisted more than 24 hours in the group where the phospholipase A was instilled.

5) In the animals having unobstructed duct, the instillation of trypsin and phospholipase A produced typical acute pancreatitis; however, the animals, where the saline, amylase and pig pancreatic juice had been instilled, showed mild interstitial pancreatitis only without evidence of hemorrhage or parenchymal

necrosis.

In conclusion. it is clear that not only trypsin but also the lipases, especially phospholipase A, have very important roles as pathogenic factors causing acute pancreatitis and it is certain that the action of phospholipase A is due to the conversion of lecithin to Iysolecithin. And finally, the obstruction or the patency of the pancreatic duct effects mainly the progress of the disease.