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Mice deficient in either or both mouse alpha2-macroglobulin (MAM) and murinoglobulin-1 (MUG1) were generated and proved phenotypically normal under standard conditions. Acute pancreatitis was induced with a diet deficient in choline and methionine, supplemented with ethionine. The mortality was less than 25% in wild-type mice, as opposed to at least 56% in knockout mice, and was highest (70%) in MAM-/- mice, with earliest onset at 2 days. Plasma amylase and lipase levels were increased, but pancreatic tissue appeared histologically variable in individual mice. The clinical symptoms were most severe in MAM-/- mice and, surprisingly, were not aggravated in the double knockout mice, suggesting that the lack of proteinase inhibition capacity was not the major problem. Therefore, we analyzed the expression of 21 different cytokines and polypeptide factors in the pancreas of all experimental groups of mice. Interleukin-1-receptor antagonist mRNA was consistently induced by the diet in the pancreas of MAM-/- mice, and transforming growth factor-beta, tumor necrosis factor-alpha, tumor necrosis factor-beta, beta-lymphotoxin, and interferon-gamma mRNA levels were also increased. The data demonstrate the important role of alpha2-macroglobulin (A2M) in acute pancreatitis as both a proteinase inhibitor and a cytokine carrier. Mice deficient in MAM and/or MUG thus offer new experimental models for defining in vivo the role of the macroglobulins in pancreatitis and in other normal and pathological processes.

Background: Proteases and protease inhibitors are important in acute pancreatitis (AP), although little is known about the time course in cerulein-induced AP in the rat., Materials and Methods: AP was induced by supramaximal stimulation of cerulein, 10 microgram/kg/h, and during 72 h we measured lipase, amylase, albumin, prekallikrein, factor X, alpha(1)-protease inhibitor, alpha(1)-macroglobulin, alpha(2)-antiplasmin, antithrombin III (all in plasma) and macroscopic and histologic variables., Results: Within 12 h an edematous pancreatitis was evident with peak values of peritoneal exudate, pancreatic wet weight ratio, and plasma amylase and lipase activities. Histologically, edema and vacuolization were prominent already after 3 and 6 h, respectively, while inflammation, necrosis, and total histological score gradually increase to reach peak levels at 48 h. Proenzymes and most plasma protease inhibitors decreased to low levels after 6-12 h followed by a gradual increase. The sequential changes over time indicate that kallikrein - kinin activation, and plasminogen activation are probably early events in cerulein-induced AP in rats. alpha(1)-Macroglobulin and alpha(1)-protease inhibitor gradually decreased during the whole study period, probably being "second line" defense inhibitors. Levels above normal were seen for alpha(2)-antiplasmin and factor X at 48 h, normalizing at 72 h., Conclusions: These results suggest that protease activation and protease inhibitor consumption occur in cerulein-induced AP in the rat., (Copyright 1999 Academic Press.)

The views upon the value of general antiprotease capacity of the blood in evaluation of endotoxicosis in purulent-inflammatory processes of the abdominal cavity are presented. One hundred and thirty one patient with diffuse peritonitis, acute pancreatitis and obstructive jaundice was examined. A role of bacterial proteases in exhaustion of the inhibitors at the example of Pseudomonas aeruginosa strain has been established. Use of protease inhibitors (contrykal etc.) only for strict indications is substantiated.

The effect of new CCK receptor antagonist, lorglumid on taurocholate AEP in rats was studied. Lorglumid was applied intraperitoneally at a dose of 5.6 mg/kg BW immediately after taurocholate injection into choledochopancreatic duct. Activity of amylase, antithrombin III (AT III), alpha 1 protease inhibitor (alpha 1 PI), alpha 2 antiplasmin (alpha 2 AP) and alpha 2 M) in plasma, trypsin and chymotrypsin in pancreata were measured after 1, 3, 6 h of AEP. In AEP treated by lorglumid serum amylase activity and pancreatic wet weight was significantly reduced. The use of lorglumid prevented the increase of alpha 1 PI and alpha 2 AP compared to not treated animals. AT III and alpha 2 M in plasma and trypsin and chymotrypsin activity in pancreata did not change significantly in all groups. The mortality of the lorglumid treated rats was significantly lower in comparison with control group. It is concluded that lorglumid in taurocholate AEP moderates the changed plasma proteinase-antiproteinase balance. Our results indicate a protective effect of lorglumid in this model of acute experimental pancreatitis.

Heparin cofactor II is a proteinase inhibitor which inhibits both chymotrypsin and thrombin, and displays great similarities with antithrombin III, the main inhibitor of thrombin in human plasma. Since acute pancreatitis is known to be associated with modification of the proteinase-antiproteinase equilibrium, we studied heparin cofactor II and antithrombin III as well as other biochemical and haematological parameters in 10 patients experiencing attacks of acute pancreatitis. Heparin cofactor II activity decreased during the first week of illness, while its antigen concentration remained subnormal. This discrepancy between antigen concentration and activity which persisted during the first week of illness was due both to complex formation of heparin cofactor II with its target proteinases and to partial proteolysis of the inhibitor. Heparin cofactor II was shown to form a complex with chymotrypsin in the plasma of such patients. Antithrombin III levels remained unchanged throughout the study, with no discrepancy between its activity and antigen concentration. No modification of haemostasis was shown either, except for a rise in the fibrinogen level during the first days of illness. It is concluded that, unlike antithrombin III, heparin cofactor II is involved in the proteinase-inhibitor equilibrium in patients with acute pancreatitis, and that heparin cofactor II might react as an inhibitor of pancreatic proteinases rather than an inhibitor of thrombin.

We analyzed the role of polymorphonuclear granulocytes (PMN)-elastase in predicting the prognosis of patients with acute pancreatitis in comparison with C-reactive protein (CRP), lactate dehydrogenase (LDH), and the two antiproteases alpha 1-antitrypsin (alpha 1-AT) and alpha 2-macroglobulin (alpha 2-M). Fifty-two patients with acute pancreatitis were subdivided according to morphological criteria into 29 patients with edematous pancreatitis and 23 patients with necrotizing pancreatitis. Within 5 days after the onset of acute pancreatitis, the accuracy rates for detecting necrotizing pancreatitis were 86%, 84%, 82%, 72%, and 69%, using cutoff levels of 120 mg/L for CRP, 120 micrograms/L for PMN-elastase, 270 U/L for LDH, 1.5 g/L for alpha 2-M, and 3.5 g/L for alpha 1-AT, respectively. The median peak value of PMN-elastase was reached on day 1 of acute pancreatitis in contrast to the median peak of CRP, which was at its highest between days 3 and 4. PMN-elastase represents a reliable indicator, comparable with CRP, for the staging of acute pancreatitis. The advantage of PMN-elastase over CRP appears to be its earlier increase and the greater dynamism of its serum course. Finally, the results suggest that CT scanning for the evaluation of the extent of intra- and extrapancreatic necrosis could be restricted to those patients with increased values of PMN-elastase and CRP.

Biochemical signs of pancreatitis in plasma and pancreatic exudates were determined in 22 pigs subjected to pancreatic allograft transplantation after the graft had been in cold storage for 6 hr. Two perfusion and preservation media were used. We found signs of protease activation in the pancreatic exudate during the first hour after reperfusion. The local protease protection barrier was, however, not broken and no plasma changes indicating pancreatitis were seen during this period. On the first and second postoperative days, mild biochemical signs of pancreatitis were seen in the plasma, including a decrease in kininogen and C3 concentration as well as in plasma kallikrein inhibitory activity and the appearance of trypsin-protease inhibitor complexes. No correlation was seen between these biochemical signs of pancreatitis and graft appearance or function, indicating that the reperfusion pancreatitis seen after 6 hr of cold storage is of minor significance. No significant differences were seen between the two preservation media used (Perfadex and EuroCollins solution).

A radioimmunoassay for porcine, cationic, pancreatic elastase (irPE) is described. Normal porcine serum contains only small amounts of irPE (less than 3 micrograms/l). IrPE in serum and peritoneal exudate from 6 pigs with experimental pancreatitis was found mainly in a molecular form corresponding to free pro-enzyme. The presence of alpha 1-, alpha 2-macroglobulin-bound elastase-like enzymatic activity in the peritoneal exudates from pigs with pancreatitis, however, indicates that the proteinase is to some extent released as the active enzyme. In some pigs with pancreatitis, the elastase-like activity against Succ(Ala) in the peritoneal exudates increased during the experiment, arguing for a progressive activation of pro-elastase. Free proteolytic activity was not observed in any of the peritoneal exudates. This low degree of activation of elastase and the fact that the elastase inhibiting capacity is substantially larger than the trypsin inhibiting capacity in serum and biological fluids, leads us to the conclusion that active elastase is not a factor of principal importance in the pathogenesis of proteinase inhibitor consumption and tissue damage in our experimental pancreatitis model.

Changes of proteinase-inhibitory system values in blood plasma of dogs with experimental acute pancreatitis (EAP) were studied in dynamics. Their diagnostic and prognostic values was appraised. Essential shifts were revealed in the proteinase-inhibitory balance in blood plasma of dogs with EAP of various severity. The concentration of trypsin- and chymotrypsin-like proteinases exceeded the normal values 1.8-3.3 times in these animals. Coefficients expressing the relation of antitryptic and antichymotryptic activities to the activities of the corresponding proteinases in the blood plasma of animals with EAP were indicative of diminished blood plasma inhibitory potential as compared to that in intact animals. The authors are the first to show the diagnostic and prognostic value of the antichymotryptic activity/chymotrypsin in blood plasma of dogs with EAP.

Intramuscular administration of trypsin and kallikrein (andecaline) into rats contributed to more favourable development of acute traumatic pancreatitis. Trypsin and andecaline prevented the activation of proteinases in blood serum and pancreas and promoted an increase in content of trypsin inhibitor in blood serum. The proteinases efficiency appears to depend on elevation of general resistance of the organism to the trauma.

A significant decline (P = 0.047) in serum antiprotease concentration was detected in dogs with experimentally induced acute pancreatitis. Decreased serum antiprotease concentrations, similar in magnitude to those documented in the experimental dogs, were present in dogs with spontaneous, acute pancreatitis. These findings suggest that significant amounts of proteolytic enzymes escape into the systemic circulation during acute pancreatitis in dogs. These circulating enzymes may be involved in the extrapancreatic complications of acute pancreatitis.

Twenty-seven attacks of acute human pancreatitis of different severity were analysed concerning clinical outcome and activation of the coagulation and fibrinolytic systems. Consumptive coagulopathy was suggested by decreased platelet counts, decreased prothrombin values and consumption of fibrinogen during the first days in severe attacks. Factor X was slightly decreased the first 5 days in all attacks. Increased fibrinolysis was suggested by decreased plasminogen values in severe attacks. Fibrinogen degradation products were seen in 40% of the patients in blood and in 100% of the patients in the peritoneal fluid. The four main protease inhibitors of the two systems all showed protease-antiprotease complexation and lower functional than quantitative values. Plasma levels of antithrombin III and alpha 2-macroglobulin were low, while the levels of C1-inhibitor and alpha 2-antiplasmin were high. Functional levels of all the four protease inhibitors were almost zero in the peritoneal fluid in severe attacks. It is concluded that severe acute pancreatitis results in both consumptive coagulopathy and in increased fibrinolysis. A local antiprotease deficiency is seen in the peritoneal cavity and high levels of protease-antiprotease complexes are also seen in plasma. All these changes are closely correlated to the severity of the disease and may probably determine the clinical outcome of the acute attack.

A clinical and biochemical analysis of 27 attacks of acute pancreatitis was made throughout the course of the disease. In severe attacks alpha 2-macroglobulin (alpha 2-M) decreased during the first days, reaching values in blood below 40% of the normal value. In addition, this remaining alpha 2-M had a decreased trypsin-binding capacity, indicating circulating alpha 2-M protease complexes. The inter-alpha-trypsin inhibitor concentration was also decreased, whereas alpha 1-proteinase inhibitor, antichymotrypsin, and pancreatic secretory trypsin inhibitor increased. All changes were most pronounced in the peritoneal fluid and were also closely correlated to the severity of the disease, assessed by both Ranson's and McMahon's classification systems. All patients with clinical complications had profound biochemical changes. In accordance with earlier findings, activation of both the complement and kinin systems seems possible in both blood and peritoneal fluid at the low alpha 2-M concentrations found in severe attacks.

The correlation between clinical course, protease inhibitors, complement and kinin activation was studied in vivo in 27 attacks of acute pancreatitis and also in vitro. The value of peritoneal lavage was retrospectively analyzed in 73 pancreatitis attacks. The effect of aprotinin was studied in vitro. Complement and kinin activation occurred in vitro when alpha-macroglobulin (alpha 2-M) concentration was below 35%, in spite of 90% free and reactive alpha 1-protease inhibitor. These low alpha 2-M levels were found in the general circulation in severe attacks. Functional alpha 2-M levels were lower than electroimmunoassay values during the acute disease. Complement and kinin activation was present both in blood and in peritoneal fluid. The extent of activation was closely correlated to the severity of the pancreatitis attack. Classical as well as alternative complement activation occurred. Kinin activation was caused by plasma kallikrein as well as by other kininogenases. Functional kallikrein inhibition was lower than electroimmunoassay values for the C1 inactivator. High levels of activated trypsin was found in complex with alpha 1-protease inhibitor in severe attacks. These findings together with the low functional alpha 2-M and a possible functional deficiency also of the C1 inactivator make trypsin-induced activation of the complement as well as the kinin system possible in acute pancreatitis. Changes in proteases and protease inhibitors were most pronounced in the peritoneal fluid, functional alpha 2-M and C1 inactivator being zero, indicating that the primary event occurs locally in and around the pancreas. Peritoneal lavage thus ought to be beneficial. The good results achieved with peritoneal lavage in the retrospective analysis of the 73 clinically severe attacks seem to verify this conclusion. The biochemical changes seen in vivo indicate alpha 2-M and C1 inactivator to be most important against proteolytic activation of the complement and kinin systems. Treatment with purified protease inhibitors might be beneficial. This also seems to be true of aprotinin, according to the in vitro results, provided very high doses are used. All antiprotease therapy seems to be most important intraperitoneally.

alpha 2-macroglobulin is probably the most important of the antiproteases in plasma. In this study, the relationships of plasma alpha 2-macroglobulin to the clinical features of acute pancreatitis as well as to plasma levels of other antiproteases, immunoglobulins, and immunoreactive trypsin, were investigated in 55 patients with acute pancreatitis. The mean level of alpha 2-macroglobulin in 395 plasma samples from the patients was 2.12 g/liter compared with 2.41 g/liter in 29 healthy subjects and 2.93 g/liter in 17 patients with septicemia. Plasma levels were lower in 12 patients with severe pancreatitis than in 43 with mild attacks, and the lowest levels in three fatal attacks were less than half the mean of the normal range. Lowest levels were recorded at a mean time of 3 days after admission in the patients with mild attacks, at 5 days after admission in the patients with severe attacks, and 9 days after admission in those with fatal attacks. In contrast, plasma levels of the alpha 1-proteinase inhibitor antichymotrypsin and C-reactive protein increased to above normal levels during the attack, significantly more so in severe compared with mild attacks. Plasma levels of IgA, IgG, and IgM remained within the normal range or were increased. In patients with severe pancreatitis, plasma levels of immunoreactive trypsin remained elevated for longer than in those with mild attacks although there was little initial difference in the levels. These data suggest that decreasing levels of alpha 2-macroglobulin during the course of acute pancreatitis are due to a specific mechanism and unrelated, for the most part, to any generalized effect of pancreatitis on protein synthesis. The formation of rapidly cleared complexes between alpha 2-macroglobulin and active proteases is the most tenable explanation for the depletion of plasma levels, but the clinical significance of the changes remains unclear.

The plasma levels of certain digestive enzymes and protease inhibitors were determined in 40 patients with severe acute pancreatitis diagnosed as gallstone-induced (GP), alcoholic (AP), or idiopathic (IP). On admission, plasma levels of amylase and immunoreactive cationic trypsin(ogen) (IRCT) and elastase 2 (IRE 2) were found to be 50 +/- 10 ng/ml, 340 +/- 64 ng/ml, and 190 +/- 15 ng/ml, respectively, in all groups of patients. These enzymes levels remained high for the first 2 days following hospitalization and then decreased, although amylase and IRCT levels remained elevated above normal values throughout the study period (2 weeks). In general amylase and IRCT were lower in patients with concomitant ascites, pancreatic pseudocysts, or abscesses, and higher in patients who died, as compared to the pancreatitis group as a whole. In these patients, levels of immunoreactive alpha 1-protease inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-M) remained relatively constant at the lower end of the normal range throughout the study period. Inhibitor levels in plasma were unaffected by the etiology of pancreatitis or the occurrence of complications. A similar trend was seen with plasma levels of lysosomal hydrolases. The results indicate that in this group of patients, plasma levels of pancreatic digestive enzymes were reflective of the clinical features and severity of the disease, as well as the time following the acute attack that brought the patient to the hospital.

In a series of patients with acute pancreatitis we have studied complement factors, antiproteases (alpha 2-macroglobulin and alpha 1-antiprotease) and C-reactive protein to determine the value of their sequential measurement in the prediction of outcome relative to clinical assessment and current multiple factor scoring systems. Complement factors were unhelpful in predicting the severity of an attack. alpha 2-Macroglobulin levels were significantly lower in complicated attacks during days 3-8 and alpha 1-antiprotease levels were significantly higher during days 4-8. C-reactive protein concentrations showed the best discrimination between mild and complicated attacks, levels rising higher and persisting for longer in complicated attacks; these differences were highly significant from day 2 (the morning after admission) to day 8. The concentrations providing the best discrimination were found to be greater than or equal to 210 mg/l for the peak C-reactive protein (on the second, third or fourth day) and greater than or equal to 120 mg/l for the C-reactive protein at the end of the first week. Analysis demonstrated both the peak or seventh-day C-reactive protein concentration to be of similar accuracy to either the Ranson or Glasgow multiple factor scoring systems and slightly better for attacks associated with gallstones. The C-reactive protein assay is simple, quick to perform, provides useful clinical information and is more likely to be of value and to be adopted into routine clinical practice than multiple factor scoring systems.

This study has demonstrated an imbalance between the blood proteolytic and antiproteolytic system in acute pancreatitis. Serum elastase activity is markedly increased and elastase inhibitory capacity is decreased in this disease as compared with those in chronic pancreatitis and the control values. We have found that a return to normal values represents better evidence of resolution than amylase determinations. These results offer the clinician a biochemical guideline for the medical management of acute pancreatitis.

We employed a radioimmunoassay for human cationic trypsin to define the time course for alterations in the molecular forms of this enzyme in plasma from patients with pancreatitis. Six patients developed acute pancreatitis as a complication of a known disorder [three, Reye's syndrome; two, hemolytic uremic syndrome (HUS); one, choledochal cyst]. The immunoreactive forms of cationic trypsin were determined by gel filtration of each plasma sample followed by radioimmunoassay of the column fractions. Early in the course of the disease, predominantly free trypsinogen was released into the circulation in five patients. In the three patients with Reye's syndrome, subsequent plasma samples showed, in addition to free trypsinogen, increasing amounts of immunoreactive trypsin complexed to alpha 2-macroglobulin and alpha 1-protease inhibitor. In contrast, subsequent samples from the two patients with HUS contained little or no inhibitor-bound trypsin. The remaining patient had intermediate concentrations of cationic trypsin complexed to these two circulating protease inhibitors. Five patients died and postmortem studies showed a striking correlation between the histological severity of acute pancreatic inflammation and the amount of immunoreactive trypsin complexed to alpha 2-macroglobulin and alpha 1-protease inhibitor. This preliminary study suggests that measurement of alpha 2-macroglobulin or alpha 1-protease inhibitor-bound trypsin may be a useful method of monitoring the progression and severity of disease in patients with acute pancreatitis. Characterization of serial changes in the forms of circulating pancreatic proteases may enhance our understanding of time-dependent pathophysiologic events, possibly leading to improved forms of specific therapy.

Various factors in the kallikrein-kinin system were evaluated in acute and chronic pancreatitis. It was noted in particular that plasma trypsin and glandular kallikrein increased markedly in acute phase of pancreatitis and its correlation with amylase was observed. Plasma prekallikrein (PPK) decreased in acute pancreatitis, but increased in chronic pancreatitis. A negative correlation was noted between PPK and kallikrein like activity. Both HMW and LMW kininogen decreased in acute pancreatitis. It was presumed from these findings that the increase in kinin and its activation at the acute phase of pancreatitis might be due to kallikrein or trypsin originating from the pancreas.

The coagulation and fibrinolytic systems were analysed parallel to the clinical evaluation in 27 attacks of acute human pancreatitis of different severity. Consumptive coagulopathy was evident from decreased platelet counts, decreased prothrombin values and consumption of fibrinogen during the first days in severe attacks. Fibrinolysis was suggested by decreased plasminogen values and the presence of fibrinogen degradation products. All main protease inhibitors of the two systems showed protease-antiprotease complexation and lower functional than quantitative values. Functional levels of the protease inhibitors were almost zero in the peritoneal fluid in severe attacks. It is concluded that severe acute pancreatitis results in consumptive coagulopathy and fibrinolysis together with a local antiprotease deficiency. All the changes are closely correlated to the severity of the disease.

Serial measurements of circulating antiproteases were carried out on 42 consecutive patients admitted with acute pancreatitis. In the 7 days following admission the serum levels of alpha 1 antitrypsin (alpha 1 AT), alpha 1 antichymotrypsin (alpha 1 ACT) and the trypsin inhibitory capacity (TIC) increased by more than 300 per cent. The serum concentration of alpha 2 macroglobulin (alpha 2 M) alone showed a decline which was most profound in those patients with severe acute pancreatitis. Initial experience of fresh frozen plasma as a source of extrinsic antiproteases in severe acute pancreatitis is that it fails to prevent the decline in alpha 2 M or increase the already elevated TIC of the patients' serum.

Using an experimental model of pancreatitis in the rat, the role of trypsin and elastase in mediating lung vascular injury in this condition was examined. The induction of pancreatitis by injection of sodium cholate in the pancreas resulted in a significant decrease in serum trypsin inhibitory capacity, and in a complete saturation of serum elastase inhibitory capacity matched by the appearance of endothelial injury of pulmonary capillaries and edema formation. The complete lack of serum elastase inhibitory capacity was associated with the presence of elastase activity in serum and bronchoalveolar lavage (BAL) fluids. The pretreatment of animals with N-furoyl saccharin (a potent inhibitor of many serine proteinases) prevented lung capillary injury and the imbalance of serum proteinase-anti-proteinase activities as well as the appearance of any elastolytic activity in serum and BAL fluids. These findings which clearly demonstrate the protease dependence of the pulmonary vascular injury in our experimental model, strongly suggested a major role for elastase(s). The suppression, in the experimental model, of the serum elastase inhibitory capacity by using chloramine-T resulted in an earlier onset of lung vascular damage, a marked worsening of pulmonary lesions, and an increase of elastolytic levels in serum and BAL fluids. Furthermore the physical properties of the protein molecule with enzyme activity detected in BAL fluids were consistent with those of rat pancreatic elastase. The reported data strongly support the hypothesis that pancreatic elastase plays a major role in the development of pulmonary vascular injury after acute pancreatitis.

Extrapancreatic findings at computed tomography (CT), performed within 24 h in 42 consecutive episodes of acute pancreatitis, were classified according to a scoring system (EP score) and were correlated to Ranson's prognostic signs, to duration of hospital stay, biochemical changes in plasma and pancreatic ischaemia found at CT with contrast enhancement. Increasing EP score was found to be related to increasing number of positive Ranson's signs, longer hospital stay and pancreatic ischaemia. Plasma levels of immunoreactive cationic trypsin and amylase were not proportional to EP score. alpha 1-protease inhibitor, antichymotrypsin but not immunoreactive pancreatic secretory trypsin inhibitor increased proportionally to EP score. No changes related to EP score were seen in alpha 2-macroglobulin levels. Serum levels of trypsin-alpha 1-protease inhibitor complex were maximal after 3 days and most pronounced in cases with high EP scores. Plasma levels of factor X, alpha 2-antiplasmin and C1-esterase inhibitor were found to be inversely proportional to EP score.