Despite recent advances in the research on etiopathogenesis of acute pancreatitis (AP), not all factors that determine the onset and course of the disease have been explained. Classically, AP is defined as an acute inflammatory process developing within the pancreatic gland with lesser or greater involvement of adjacent tissues and/or other organs (1). In majority of cases it is a self-limiting process, yet 20% of patients develop severe form of necrosis with multiorgan complications. Severe form of AP accounts for up to 50% of mortality (1). In case of pancreatic necrosis, there are two periods of increased mortality: the one within the first 7 days from the onset due to multiorgan failure, and another, late period occurring commonly two weeks after the onset (late mortality) and usually due to infections with local complications (e.g. abscesses) or generalized infections (e.g. septicemia) (2).
Researchers concentrate on investigating the factors conditioning the development of severe AP. They consider genetic predisposition, the type of a possible responsible factor, and comorbidities. Experimental research pays much attention to the evaluation of ischemia effects on developing AP (3,4). The most essential task, however, remains to be the assessment of sensitivity and specificity of biochemical markers in prognosing the risk of severe AP development, and of their clinical availability (5).
The best known mechanisms of AP development are, e.g., intrapancreatic enzyme activation triggered by trypsinogen activation to trypsin followed by inflammatory mediators released from the infiltrated pancreatic connective stroma, i.e. cytokines, adhesive molecules, platelet activating factors (PAF), nitric oxide (NO), oxygen reactive forms (ORF) and lysosomal enzymes (6).
Molecular mechanisms of AP
The role of genetic factors
Recent research has suggested that trypsinogen activation is preceded by altered activation of myogen-activated protein kinase (MAP), p38 MAP kinase and c-jun amino-terminal (JUN) kinase. The level of p38MAP kinase increases most rapidly, with the peak of activity after three hours. JUN kinase activity is the highest after 12 hrs and after 24 hrs its activity becomes undetectable (7,8).
Nuclear transcription factor-κβ (NFκβ) is the link between subsequent stages of inflammatory response and immune reaction, and it is the primary known regulator of gene expression of many proinflammatory mediators. NF-κβ is activated by cytokines, oxidative stress or endotoxemia. NF-κβ is then translocated from the cytoplasm to the nucleus where NF-κβ binds specific DNA promoting items and induces transcription of proper genes. The number of thus triggered inflammatory mediators proves that it is highly involved in the initiation and expansion of inflammatory reaction from local to the generalized level (9, 10).
The role of proteolytic enzyme inhibitors
In a healthy body, the pancreas has many protective mechanisms against self-digestion by its own enzymes. Proteolytic enzymes, of which trypsinogen is the most important one, are produced by acinar cells as proenzymes and activated in the duodenum by intestinal endopeptidases. Brush border enterokinase hydrolyzes Lys23-Ile24 bond and releases the end-stage octapeptide called trypsinogen activation peptide (TAP). Trypsin itself can activate trypsinogen as well. Also, pancreatic secretory trypsin inhibitor (PSTI) protects the pancreas from trypsinogen activation in lobular cells. It binds to trypsin at the 1:1 ratio. The molar ratio of PSTI to trypsin is estimated to be 1:10. If more than 10% trypsinogen is activated, protective mechanisms are no longer effective (11). Apart from PSTI, trypsin itself may limit its activity by breaking down the Arg117 bond in a trypsinogen molecule. Additionally, investigations have confirmed the presence of human trypsinogen isoform, i.e. mesotrypsinogen resistant to trypsin inhibitors. The study of its biological function has produced two contradictory theories: that mesotrypsin may either prematurely activate or degrade pancreatic zymogenes, thus either triggering or protecting from the pathogenesis of AP. Mesotrypsin ability to bind inhibitors is due to a single mutation which gives mesotrypsin a unique new function. Researchers suggest that biological function of human mesotrypsin involves degradation of trypsin inhibitors in the alimentary tract. Improper mesotrypsinogen activation in the pancreas may contribute to the development of AP (12).
One of the rare causes of AP is genetic condition depending on cationic trypsinogen gene mutation. Genetic studies discovered mutation at 122 position (R122H), where histidine was replaced by arginine (13). The switch is responsible for spontaneous trypsin activation due to autolysis and it begins with Arg122-Lys123 binding. Once trypsinogen gets activated and its level exceeds PSTI concentration, other enzymes also get activated and AP develops. Mutations of the PSTI gene are thought to modify the course of AP via decreasing the threshold of AP and increasing the severity of the inflammatory process (14).
Biochemical markers of AP
Many biological markers are used to diagnose and predict the severity of AP. If convergent with clinical symptoms, they help in diagnosis and instituting prompt treatment. The most common markers include lipase and amylase. Both enzymes are secreted by pancreatic acinar cells (15,16). Their serum content depends on the time that has passed from the onset of first complaints of pain and other abdominal symptoms, triglyceride concentration and other chronic conditions, such as kidney failure (17). Amylase activity may remain normal in alcohol abusing patients despite evident clinical symptoms of AP. The peak of this enzyme activity occurs between the 2nd and 12th hr from the onset of symptoms and decreases as the symptoms subside.
Much more sensitive and specific is the activity of lipase, particularly in the alcohol abusing patients. The peak of its activity occurs between the 4th and 8th hr from the onset of symptoms. A common practice nowadays is to use lipase activity alone to diagnose AP since concomitant determination of amylase activity does not provide higher diagnostic accuracy (18). Another diagnostic marker is alanine aminotransferase (ALT) activity. Increased values correlating with amylase and lipase activity account for 95% billiary etiology of AP. The values higher than 150 mg/L are assumed to be cut-off values. Urine trypsinogen activated protein (TAP) is a useful marker to diagnose and assess the severity of AP. Increased values are observed several hours (6-12) after onset of symptoms. Unfortunately it is hardly clinically useful due to its limited availability (19).
Although medical history findings are most important in case of alcohol AP, in doubtful cases the diagnosis is easier when blood serum disialotransferrin is determined, a specific marker of high alcohol abuse. If determined after 24 hrs from admission, it increases the probability of diagnosing alcohol AP from 64% to 94% (20).
The role of C-reactive protein
Prognosing disease severity is important in managing patients with AP. The most common biological marker in that regard is C-reactive protein (CRP). CRP is an acute-phase protein abundantly produced by hepatocytes. The production is stimulated by cytokines, such as interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin 1beta (IL1-beta) (21). Enhanced CRP activity correlates closely with the severity of AP and the tendency of developing necrosis of the pancreas. The cut-off value is 150 mg/L. Unfortunately, CRP concentration increases significantly 24-48 hrs after the onset of disease (19, 22). Therefore it is useless within the first 24 hrs (23). CRP is 37-77% sensitive in confirming severe AP within 24 hrs and its sensitivity increases up to 83-100% 48 hrs after the occurrence of first symptoms (24). Gürleyik has obtained similar results. He found that the CRP determined 48 hrs after disease onset was 84% sensitive, 73% specific, and had 50.1% positive predictive value (PPV) (25).
Since CRP turned out to have little sensitivity within 24 hrs from the disease onset, the search for early markers turned to cytokines. It is a group of various peptides that exhibit signaling characteristics. The group is divided into monokines, lymphokines, chemokines, hematopoetins and interferons. These proteins often exhibit different biological properties though they have some common features: they are active in very small concentrations, they are not produced all the time but cells are stimulated to synthesize them, they produce biological effects via binding with highly specific receptors on the target cell surface, and they have pleiotrophic effects (2,6).
The role of cytokines
Most important for the prognosis of AP is IL-6, produced by monocytes, macrophages, lymphocytes T and B, endothelium, epithelium and fibroblasts, chondroblasts and osteocytes. Its concentration increases between 18 and 48 hrs from disease onset. It is a factor initiating the production of acute-phase proteins in the liver (CRP) and enhancing the synthesis of IL-2 and its receptor on T- lymphocyte surface. Contrary to other cytokines, it is detectable easily in the circulation, and it exhibits typical endocrine activity (2, 26). Jiang found that after 24 hrs IL-6 demonstrated higher sensitivity (100%), specificity (89.9%), and PPV (91%) than CRP and TNF-α, which emphasizes its usefulness in assessing disease severity within first 24 hrs (27).
IL-8 is another important cytokine. This chemokine is produced by numerous cells equipped with TNF- α and IL-1 receptors. Acinar cells of the pancreas are likely to be the primary source of IL-8 and other chemokines (28). Berney found that IL-8 concentration in serum correlated with disease severity, especially when organ inflammation became a generalized process (29). Its highest concentrations were observed between 12 and 24 hrs from disease onset (5). Similarly to other chemokines IL-8 converts partially inactive integrines into molecules of active configuration (26). Thus, IL-8 may be very useful to assess the severity of AP in its early stage.
IL-15 is a cytokine that has many biological properties of IL-2. Its concentrations have been examined with reference to AP severity, multiorgan complications and infections. Its level was high in patients with multiorgan dysfunction, infections and those who died during the course of AP. IL-15 is more useful than CRP, IL-6 or IL-8 to predict AP severity (30).
IL-18, a member of the monokine family, is also released at significantly higher concentrations in early stages of pancreatic necrosis in the course of AP as compared to mild forms (31). IL-18 can activate helper lymphocytes Th1 and B-lymphocytes and enhances defense against infections. On the other hand, it stimulates the release of TNF- α, chemokine, and INFγ. It has been suggested that IL-18 acts as a significant link in disordered immune response in a necrotic form of AP.
TNFα, like IL-18 from the monokine family, is a biological marker considered in the assessment of AP severity. In the course of AP it is released in the liver, lungs and spleen. TNFα and IL-1 are the main and most important inflammatory mediators in AP (26). TNFα is the key regulator of proinflammatory cytokines and leukocyte adhesion molecules. It might be useful in estimating the severity of disease and individual predisposition to develop organic complications and septic shock. TNFα, however, is washed out from serum rapidly so that its sensitivity and PPV depend closely on timing with the first symptoms (32). Since TNFα seems to be the key cytokine in developing AP, it has also become an important target of therapy. First research articles on etanercept, a TNF-α inhibitor applied in treatment of AP in mice, have been published but, despite promising results, further observation is necessary (33).
Other predicting factors
Procalcitonine (PCT) is also a useful marker to assess AP severity. Increased serum PCT concentrations occur between 24 and 36 hr, particularly in patients with infected necrosis (34). This observation has been confirmed in a multiple-center investigation which compared PCT levels with CRP. Both parameters were monitored for 21 consecutive days. Contrary to CRP, the level of PCT was significantly increased in case of pancreatic necrosis in patients with multiorgan dysfunction syndrome (MODS), in all patients who required subsequent surgery and in those who died of AP. Researchers also suggested the determination of PCT as a single parameter of the risk of developing complications (35,36).
Phospholipase A2 is a marker related not only to pancreatic necrosis but also to pulmonary complications in the course of AP (37). High blood serum values are determined 24 hrs after the onset of AP.
The presence of pulmonary complications is also related to matrix metalloproteinase-9 (MMP-9) occurring early in the blood serum. It is characterized by negative predictive value (NPV) of 96.2% and PPV reaching 100% (38,39).
Macrophage migration inhibitory factor (MIF) has also been investigated in this respect. It can intensify inflammatory reaction by cytokine involvement at the place of inflammation. Its high values were noted 24 hours after the onset of AP in patients who developed pancreatic necrosis; however, it did not correlate with multiple organ complications (40).
Increased values of soluble thrombomodulin (sTM) appear second to high MIF. After 48 hrs they were observed in the blood serum of patients at a risk to develop pancreatic necrosis (41).
Platelet-activating factor (PAF) is a proinflammatory phospholipid from the biologically active triglyceride family. These compounds take part in wound healing processes, angiogenesis and apoptosis, including the development of inflammation in the course of AP (42).
Another early prognostic marker of AP is polymorphonuclear elastase. Its sensitivity and specificity in the first 24 hrs of patients’ hospitalization in predicting severe form of AP are 92% and 91%, respectively. Polymorphonuclear elastase demonstrates 78% PPV and 96% NPV. All these characteristics make this parameter a valuable prognostic marker of AP. Besides, it is easily accessible in clinical practice (43).
Researchers concentrate on searching for the best possible prognostic factor of AP. In clinical practice, though, diagnosis is based on biological markers, clinical assessment and other lab results included in Ranson scale or APACHE II and imaging investigations used to define computer tomography severity index (CTSI). Accurate prognosis of AP course cannot be made until all those parameters have been determined so that the best possible treatment can be undertaken.