Pancreatic Exocrine Enzymes and Intrapancreatic Protein Synthesis in Acute Oedematous Pancreatitis

Changes in serum and intrapancreatic enzyme content and protein synthesis in pancreas were studied in acute oedematous pancreatitis (AOP). Male Wistar rats (n = 111) were divided into 2 groups, controls with a sham operation and those with AOP. Serum amylase levels rose immediately after the procedure causing AOP and then fell gradually, while serum lipase and ribonuclease levels remained higher than control values over 48h. (p < 0.05, 0.01). Serum deoxyribonuclease (DNase) II levels were unchanged. Intrapancreatic enzyme levels were scarcely affected by AOP. 3H-leucine uptake into pancreatic tissue of rats with AOP was decreased throughout the study (p < 0.001), but some protein synthesis continued. Intrapancreatic enzyme contents are maintained despite diffusion into the blood because the pancreas retain its ability to synthesize enzymes.


INTRODUCTION
Acute oedematous pancreatitis (AOP) is classified as an attack with mild or moderate clinical features, which may develop into a severe form with parenchymal necrosis or haemorrhage 1. Several workers have studied the diffusion of exocrine enzymes from the pancreas to the blood [2][3][4] or changes in protein and enzyme synthesis in exocrine cells5-7, including some investigations on experimental pancreatitis8-1.
No elevation of exocrine enzymes in the blood may occur throughout the attack in patients with severe haemorrhagic or necrotizing pancreatitis -3, while in those with AOP enzyme levels rise in the absence of pancreatic necrosis 4. The relationship between serum and intrapancreatic enzyme levels and pancreatic protein synthesis in AOP was investigated in the persent study.

MATERIALS AND METHODS
Operations and samples One hundred and eleven male Wistar rats weighing 200-300 g were given a pellet diet and water ad libitum and were fasted for 24 h before the experiments. Sixtytwo rats (the AOP group) underwent laparotomy under ether anaesthesia with ligation of the lower bile duct using Block's method 15. Forty-two rats (controls) underwent a sham operation. Another 7 normal rats were simply fasted for 24 h.
Twelve rats in each group were used to examine only the 48 h mortality rates. Blood was taken quickly form the abdominal aorta of 7 rats in each group 12,24,36 and 48 h after the procedure. After saline lavage of the abdominal cavity the pancreas was removed. Likewise, blood samples and the pancreas were obtained from normal rats. Half the pancreas from 2 rats in each group was used to prepare histological sections stained with hematoxylin eosin.

Pancreatic enzyme assays
Five rats from each group (including normal rats) were used for pancreatic enzyme assay. Blood samples were centrifuged and serum obtained. Half the pancreas was homogenized at 0C in 10ml saline. The homogenate was centrifuged at 0C for 20 min at 10,000xg, and the supernatant was obtained. Pancreatic enzyme levels were measured in serum or supernatant as follows. Amylase was determined by an ultraviolet method and expressed in somogyi U per ml serum or mg pancreas protein. Lipase was determined by the Marupi Lipase Kit (Dainippon Pharmaceutical Co.) and was expressed in IU per serum or mg pancreas protein. Ribonuclease (RNase) was determined by Reddi's method16, using polycytidylic acid (P-L Biochemicals Inc.) a the substrate, and the absorbance was determined at 278 nm; levels were expressed as U per ml serum or mg pancreas protein. Deoxyribonuclease (DNase) II was determined as follows. To 0.5 ml sample was added 0.5ml DNA solution (4mg/ml; Sigma Chemical Co.) as substrate plus 2.5 ml 0.2 M pH 5.0 acetic acid buffer containing 20mM Mg 2 /. This mixture was allowed to react at 35C for 2 h before. The reaction was stopped by adding 0.5 ml 40% perchloric acid; the reaction mixture was centrifuged at 12,000 xg for 15 min. The supernatant was treated by Burton's method 17, and the absorbance was determined at 600 nm. DNase II level in the samples was expressed as U per 0.5 ml serum or mg pancreas protein. Protein estimations in pancreatic tissue were performed by Lowry's method 18.
Amino acid uptake Seven rats from each group (including normal rats) were used to determine the pancreatic uptake of labelled amino acid. Before sacrifice, 3.7 x 10 3 Bq/body wt 3H-leucine (DL-[4.5-H3] leucine, specific activity; 0.92-1.85 TBq/mmol) was injected into the tail vein. Half the pancreas was homogenized at 0C in 10ml 0.05 M Tris-HC1 buffer (pH 7.0). The homogenate was centrifuged at 0C for 20min at 10,000xg, and 4ml supernatant was centrifuged at 3,000 xg for 10 min after the addition of 1 ml 25% trichloroacetic acid (TCA).
TCA-soluble and TCA-insoluble fractions were assessed for radioactivity in a liquid scintillation counter after the addition of a scintillater and its level was expressed as dpm/mg pancreas protein. The percentage of radioactivity in the TCA-insoluble fraction of the homogenate was used to measure the incorporation rate of isotope.

Statistics
All data were presented as means _+ SD, and Student's t-test was used to compare data between groups.

Mortality rates and histologicalfindings
All controls undergoing the sham operation survived throughout the studyperiod. In the AOP group mortality rates were 1/12 at 12 h, 2/12 at 24 h, 3/12 at 36 h and 4/12 at 48 h. No histological changes were observed in control pancreas. At 12h in the AOP group the pancreas revealed oedema, inflammatory cell infiltration and slight interstitial haemorrhage. By 24 h, these changes had progressed and were accompanied by a slight decrease in zymogen granules in the exocrine cells. At 48h the pancreas exhibited intracellular vacuoles and cell necrosis. Serum enzyme levels ( Table 1) Controls showed no changes in serum amylase, but in rats with AOP amylese was elevated at 12h before  returning to control level. Control lipase levels rose slightly after operation, but in rats with AOP lipase was substantially higher throughout the experiment. Likewise, RNase levels remained constant in controls, but were greatly increases throughout in rats with AOP. Serum DNase II levels were unaltered in either group.
Intrapancreatic enzyme levels ( Table 2) Amylase levels were unchanged in controls on rats with AOP. The control procedure caused no enzyme changes. Amylase, RNase and DNase II levels were not altered in AOP, but lipase levels fell by 48 h.
Amino acid uptake into pancreatic tissue (  covered, values in AOP group persisted at a slightly lower level. The control operation reduced radioactivity in the TCA insoluble fraction but AOP produced a profound and persistent fall. The decrease in amino acid incorporation shows that there was reduced enzyme synthesis, though some activity continued during AOP. In summary, serum alterations in exocrine enzyme levels did not clearly reflect changes in pancreatic enzyme content, nor did it always indicate the pathological changes in the pancreas 11-14. Since enzyme synthesis is maintained within the pancreas, the results suggest the necessity for continuing anti-enzyme therapy in patients with AOP.

INVITED COMMENTARY
Although progress towards an effective therapy for acute pancreatitis has been limited by several factors, notably the absence of an ideal experimental model 1,2, considerable advances have been made in the detection and treatment of the complications of the disease. Unfortunately, these advances have not been paralleled by much improvement in understanding the basic pathophysiology of the disease.
In this study, Dr Kinami and his colleagues try to clarify the relationship between serum enzymes, intrapancreatic enzymes and pancreatic protein synthesis, a relationship that has been extensively studied over the past decade. Their results generally confirm previous publications on the subject, namely that serum enzyme levels do not accurately reflect the pathological changes in the gland and that enzyme synthesis persists, at least during the early phases of the disease process3.
The authors noted a slight decrease in acinar zymogen granule concentration at 24 h. This finding differs from most experimental models of acute pancreatitis, in which intracellular zymogen granule concentration is increased at an early stage 8 due to impaired acinar cell secretion4'5'6'7. Moreover, recent data suggest that digestive zymogen activation occurs within the acinar cell itself9'1. Thus, drugs directed at inhibiting acinar cell secretion, as a means of controlling pancreatitis, may actually worsen the disease by increasing intracellular zymogen concentration.
Most researchers now believe that intracellular enzyme activation proposed by Rao et al. 11 is a more crucial factor in the pathogenesis of acute pancreatitis than enzyme synthesis per se. Therefore, although anti-enzymatic therapy may be indicated in the treatment of acute pancreatitis, it should probably not be directed towards stopping enzyme synthesis, as suggested by the authors in their concluding remarks, but rather towards stopping intracellular enzyme activa-tion. Indeed, clinical trials on the systemic administration of the antiprotease aprotinin (Trasylol) have proved ineffective 2.
Newer studies on the pathogenesis of acute pancreatitis suggest that autoactivation of trypsinogen is the responsible factor for initiating intracellular zymogen activation8. This process requires a low pH of around 5 9 and probably takes place in newly formed intracellular vacuoles7'3'14, which appear as a result of an abnormal subcellular distribution of digestive zymogens and lysosomal hydrolases during the early phases of the disease3. The vacuoles noted on histological sections of the pancreas at 48 h in the present study (although reported to occur in experimental models involving diet and secretagogue-induced pancreatitis) were not usually noted in similar models of pancreatic duct obstruction3. Their appearance characterizes an early stage of the disease in many animal models and possibly in human disease15.
In conclusion we believe that to reach the authors' final suggestion about the "necessity of anti-enzymatic therapy to be carried out continuously on patients with acute oedematous pancreatitis", a number of important question remains to be answered: What enzyme, or combination of enzymes, should we aim at counteracting and what are the objective means of selecting our potential patients, i.e., the minority who may progress from acute oedematous pancreatitis to the necrotizing or haemorrhagic forms of the disease? It is hoped that cellular models of acute pancreatitis will provide some answers to similar questions in the near future.