Increased peritoneal permeability in acute experimental pancreatitis

International Journal of Pancreatology, 4 (1989) 091-098 $02.00 Copyright �9 1989 by The Humana Press Inc. All rights of any nature whatsoever reserved.

Increased Peritoneal Permeability in Acute Experimental Pancreatitis

Conny Svensson, ,.1 R u n e Sjb'dahl, ~ Christer Tagesson, 2 and Ingemar Ihse ~

ZDepartrnent of Surgery; and 2Department of Occupational Medicine, University Hospital, S-581 85 LinkiSping, Sweden

Received January 12, 1988; Accepted February 1, 1988

Summary

The peritoneal permeability to differently sized dextran molecules; Mw 4,000, 10,000, 20,000, and 40,000 was investigated in rats with bile-induced acute pancreatitis. Following intraperitoneal deposition, repeated blood samples for measurement of the different dextrans were collected. The results showed increased peritoneal permeability to molecules between 4,000-20,000 daltons in animals with pancreatitis, whereas there was no significant difference for molecules of 40,000 daltons compared to healthy control animals. Also, the passage of phospholipase Az (Mw 14,000) over the peritoneal membrane was increased during pancreatitis. It is suggested that the transperitoneal route may be of pathophysiological importance in the development of the systemic components of severe acute pancreatitis.

Key Words: Pancreatitis; permeability; dextran.

INTRODUCTION

Patients with severe acute pancreatitis generally have accumulation of large quantities of peritoneal fluid. In experimental studies this fluid after intravenous (1) or intraperitoneal (2) injection has been shown to cause hypotension, owing to its content o f vasoactive polypeptides of pancreatic origin like bradykinin or kallikrein. Fur thermore, since it is known to harbor other biologically active substances, the fluid has been suggested as a significant element in the pathophysiology of the systemic components of severe acute pancreatitis. Whether these toxic substances--originating from the pancreas - - reach the bloodstream via direct venous or lymphatic pathway of whether the transperitoneal route is involved is not yet settled (3,4). In the present study, the peritoneal permeability to differently sized molecules was investigated in rats with bile-induced acute pancreatitis.

*Author to whom all correspondence and reprint requests should be addressed.

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MATERIAL AND METHODS

Chemicals

Fluorescein-isothiocyanate-conjugated (FITC) dextrans with molecular weights of 4,000, 10,000, 20,000, and 40,000 daltons were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Porcine pancreatic phospholipase A2 (Mw 14,000) was obtained from Sigma Chemical Company, St. Louis, MO.

Induction of A cute Pancreatitis

Male Wistar rats (200-250 g body wt) fasted for 12 h but given free access to tap water were anesthetized by an intraperitoneal injection of Ketalar | (Parke- Davis & Co., Inc., USA, 80 mg/kg body wt) and Rompun | (Bayer AG, Leverkusen, FRG, 8 mg/kg body wt). The abdomen was opened via a mid- line incision and the duodenal loop as well as the bile-pancreatic duct were identified. Using a thin polyethyelene catheter (Portex, Hythe, Kent, UK), the bile-pancreatic duct was cannulated transduodenally. Acute experimental pancreatitis (AEP) was induced by intraductal injection during one minue of 0.2 mL 0.025 M glycyl-glycin-NaOH buffer, pH 10.3 containing 0.02 M CaC12, 4/zmol sodiumtaurodeoxycholate (NaTDC) and 0.2 nmol trypsin. To prevent leakage to the gut or flow into the liver during the infusion small artery clamps were put on the bile-pancreatic duct close to the duodenal wall and on the hepatic duct at the level of the hilum of the liver. After removal of the catheter, the abdominal wall incision was closed and the animals allowed to wake up. To prevent severe hypovolemia after the induction of AEP, the animals were given 10 mL 0.15 M saline subcutaneously. The control animals underwent sham operation (simple laparotomy).

Measurement of Peritoneal Permeability

After 24 h all animals were reanesthetized, a cannula inserted into a jugular vein, and the abdomen opened. The peritoneal fluid in animals with AEP was then removed and superseded by 1 mL 3.33 M FITC-dextran. Repeated blood samples for measurement of the different FITC-dextrans were taken from the jugular vein after 5, 10, 15, 20, 30, 45, and 60 min. All animals were sacrificed after the study period.

In experiment I four groups of rats, each one consisting of seven animals with AEP and seven control animals, were studied as follows: (1) FITC-dextran Mw 4,000, (2) FITC-dextran Mw 10,000, (3) FITC-dextran Mw 20,000, and (4) FITC-dextran Mw 40,000.

In experiment II one mL of a solution containing two different quantities of phospholipase A2, 100 and 1000 U (600 U/rag protein at pH 8.0 and 37 ~ respectively, was instilled intraperitoneally in two groups of healthy rats. Repeated blood samples were taken after 5, 10, 20, and 30 min.

In experiment III six rats with AEP had their peritoneal fluid superseded by one mL of a phospholipase Aa solution (1000 U/mL; 600 U/mg protein at pH 8.0 and 37 ~ The same amount of phospholipase A2 was instilled intra-

international Journal o f Pancreatology Volume 4, 1989

Peritoneal Permeability 93

peritoneally in six sham-operated controls. From the two groups blood samples were drawn at 5 and 20 min for determination of phospholipase A2.

Laboratory Assays

Amylase was measured by means of the Phadebas test (Pharmacia, Upp- sala, Sweden) and phospholipase A2 according to the method described by Tagesson and SjCSdahl (5). Measurement of the concentration of the different FITC-dextrans in serum was performed by emission spectrophotometry (6).

Statistics

Values were tested for statistical significance using the unpaired Student's t-test.

RESULTS

Six hours after the intraductal injection of NaTDC and trypsin the amylase level in serum was 915+385 /zkat/L (mean SD) as compared to 148_+20 txkat/L (mean + SD) in sham-operated animals suggesting the development of acute pancreatitis. The diagnosis was confirmed at the laparotomy done after 2 h.

Experiment I

The passage of FITC-dextran 4,000 and 10,000 across the peritoneal membrane into the bloodstream was significantly increased in animals with AEP, in comparison with sham-operated animals. As depicted in Figs. la and b, the difference was detectable already after 10 min. Also the passage of FITC- dextran 20,000 was increased, as shown in Fig. lc. The difference was, however, not statistically significant until after 30 rain. The largest molecules tested (FITC-dextran 40,000) could only be measured in minute amounts in peripheral blood and there was no difference between animals with AEP and those sham-operated (Fig. ld).

Experiment H

Following intraperitoneal deposition of phospholipase Az the activity of the enzyme in plasma increased rapidly. 1,000 U resulted in higher levels compared to 100 U (Fig. 2).

Experiment III

After instillation of phospholipase A2 activity into the peritoneal cavity the plasma levels of the enzyme were significantly higher in animals with AEP than in healthy, sham-operated controls (Fig. 3).

DISCUSSION

The results of the present study show that peritoneal permeability to molecules between 4,000-20,000 daltons is increased during bile-induced acute pancreatitis in rats compared to healthy controls. Whereas there was a

International Journal o f Pancreatology Volume 4, 1989

94 Svensson et al,

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96 Svensson et al.

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Fig. 3. Activity of phospholipase A2 in plasma 5 and 20 min after intraperitoneal deposition of 1000 U phospholipase A2. AEP=acute experimental pancreatitis; C =controls. Mean+_SEM, n=6, *** p<0.001.

significant difference between the two groups already from 10 min for molecules of 4,000 and 10,000 daltons, the difference did not appear until after 30 min, when molecules of 20,000 daltons were tested. Finally, the permeability of molecules of 40,000 daltons were not statistically different between pancreatitis and healthy rats, respectively. Therefore, it seems as if the observed increase in permeability during pancreatitis increased with decreasing molecular size. The findings further suggest that the passage of phospholipase A2 (Mw 14,000) over the peritoneal membrane is increased during AEP compared to normal conditions.

The ascitic fluid that accumulates during severe acute pancreatitis has been shown to contain several biologically active substances of pancreatic origin, such as phospholipase A2, lipase, proteases, vasoactive polypeptides, and a myocardial depressant factor. These substances have been held responsible for the multisystem failure frequently accompanying the severe form of the disease. It is, however, not known if they reach the bloodstream directly via pancreatic and peripancreatic veins and lymph vessels or if the transperitoneal route is the principle pathway. Waterman et at. (7) observed lower serum amylase levels if ascites were aspirated during the attack of induced pancrea- tiffs in dogs. If peritoneal fluid during acute experimental pancreatitis in guinea pigs was collected in a manner that prevented transperitoneal absorption serum amylase levels were similar to those in sham operated animals (8). Lankisch et al. (2) found that intraperitoneal injection of ascitic fluid to healthy rats caused hypotension. In a recent paper we reported an earlier

International Journal of Pancreatology Volume 4, 1989


decrease in amylase levels in serum and urine in patients with acute pancreatitis treated with peritoneal lavage as compared to nonlavaged controls (9). These data suggest that the transperitoneal route is essential, as do our findings of increased phospholipase A2 passage over the peritoneal membrane in AEP. On the other hand, Mayer et al. (3) recently reported results from an experimental study suggesting a prominent role of the direct lymphatic pathway.

Already, in 1958, Egdahl (t0) f rom a study on acute experimental pancreatitis in dogs, concluded that the initial rise in blood enzyme concentration may be owing to direct transfer into pancreatic or peripancreatic veins, and further suggested that in the later course transperitoneal absorption into peritoneal lymphatics was the main route. It has been suggested that relatively small molecules are responsible for the systemic effect in severe acute pancreatitis (11) and that pulmonary and hemodynamic effects of intravenous injection of the ascitic fluid are caused by substances of different molecular size, respectively (12). Therefore, our findings of increasing peritoneal permeability of molecules of decreasing molecular size during acute pancreatitis are of interest. Further, it may be speculated that the "permeability pat tern" of peritoneum in the individual patient can be determinant of the "pat tern of the systemic response" to the attack. Future studies on the cause or causes of the increased peritoneal permeability during acute pancreatitis are, therefore, important. In this context, the role of phospholipase A2 is of special interest as it has been demonstrated that the small intestinal permeability in- creases after mucosal application of lysolecithin that is formed by the action of phospholipase A2 on lecithin in the intestinal lumen (17)~

ACKNOWLEDGMENT

This work was supported by the Swedish Medical Research Council (Projekt No 17X-03012) and the County Council of Osterg~Stland.

This work was presented in part at the XVIII Meeting of the European Pancreatic Club, Nijmegen (The Netherlands), 18-20 September 1986.

REFERENCES

1 Amundsen E, Ofsted E, Hagen PD. Experimental acute pancreatitis in dogs. Scand. J. Gastroenterol. t968; 3: 659-664.

2 Lankisch PG, Koop H, Winckler K, Schmidt H. Continuous peritoneal dialysis as treatment of acute experimental pancreatitis in the rat. Dig. Dis. Sci. 1979; 24:117-122.

3 Mayer AD, Airey M, Hodgson J, McMahon MJ. Enzyme transfer from pancreas to plasma during acute pancreatitis. The contribution of ascitic fluid and lymphatic drainage of the pancreas. Gut 1985; 26: 876-881.

4 Lange JF, Beyaert P J, van Vugt H, Tytgat GNJ, van Gool J. Pathwys of enzyme transfer in sodium taurocholate-induced acute haemorrhagic pancreatitis. Digestion 1986; 35: 229-236.

5 Tagesson C, Sj6dahl R~ Rapid and sensitive assay of human plasma phospholipase A2 activity. Ann. Chir. Gyn. 1984; 73: 1-5.

6 Tagesson C, Sj6dahl R, Thor~n B. Passage of molecules through the wall of the gastrointestinal tract. A simple experimental model. Scand. J. Gastroenterol. 1978; 13: 519-524.

7 Waterman NG, Walsky RS. Transperitoneal absorption of amylase in acute experimental pancreatitis. Sug. Gynecol. Obstet. 1970; 131: 729-732.

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8 Waterman NG, Walsky R, Kasdan ML, Abrams BLo The treatment of acute haemorrhagic pancreatitis by sump drainage. Surg. Gynecol. Obstet. 1968; 126: 963-971.

9 !hse I, Evander A, Holmberg JT, Gustafson I. Influence of peritoneal lavage on objec- tive prognostic signs in acute pancreatitis. Ann. Surg. 1986; 204: 122-127.

10 Egdahl RH. Mechanism of blood enzyme changes following production of experimental pancreatitis. Ann. Surg. 1958; 148: 389-400.

11 Ellison EC, Pappas TN, Johnsson JA, Fabri P J, Carey LC. Demonstration and charac- terization of the hemoconcentrating affect of ascitic fluid that accumulate during haemorrhagic pancreatitis. J. Surg. Res. 1981; 30: 241-248.

12 Innes J. Fraser I, Carey LCo The vasoactive properties of ascitic fluid in acute pancreatitis in a porcine model. Arch. Surg. 1986; 121: 665-668.

13 Bolin T, Sj6dahl R, Sundqvist T, Tagesson C. Passage of molecules through the wall of the gastrointestinal tract. Increased passive permeability in rat ileum after exposure to lysolecithin. Scand. J. Gastroenterol. t98t; t6: 897-901.


Increased peritoneal permeability in acute experimental pancreatitis

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