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Biomarkers of acute and chronic pancreatitis
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16 Biomarkers of acute and chronic pancreatitis Bhupendra S. Kaphalia
INTRODUCTION
etiological agents, the sequence of pancreatic injury follows a similar pattern for most of the etiologies. Some patients with pancreatitis can also develop pancreatic cancer. Overall, chronic pancreatitis is a devastating disease with great social, economic, and psychological impact. The prognosis of pancreatitis and pancreatic cancer is very poor and many patients die before reaching the clinical stage of the disease. Therefore, identification of biomarkers for both pre- and post-pancreatitis stages could be important for clinical interventions.
Early detection and prevention of pancreatic injury, progressing to inflammation and fibrosis and possibly pancreatic cancer, should be critical for the success of both clinical and surgical interventional therapies. Although a significant literature available on biomarkers of pancreatic diseases is focused on various forms of pancreatic cancer, identification of biomarkers for early pancreatic injury and precancerous stages should be the key to early prevention and therapy. Therefore, summarizing reliable and probable biomarkers identified for acute and chronic pancreatic toxicities/pancreatitis rather than pancreatic cancer is the focus of this chapter. Both biliary duct disease and chronic alcohol abuse constitute 70 75% of the etiologies of acute and chronic pancreatitis; the remaining third may be idiopathic, including those caused by chemical exposure, pesticide and metal poisoning, use of drugs, infections, and autoimmune and anatomical conditions. Congenital abnormalities, generally rare and asymptomatic, are related to two important events during embryological development of the gland: rotation and fusion. Anatomical disorders, deposition of cholesterol and related substances, and obstruction of the pancreatic duct due to tumor and cyst formation could be rare (Lankisch and Banks, 1998; Kaphalia, 2011). Pancreatic toxicity is generally characterized by dysregulation of lipid metabolism and edema in early reversible stages, followed by massive necrosis resulting in inflammation, with or without fibrosis (scarring of the tissue) at the advanced stages. Irrespective of
R. Gupta (Ed): Biomarkers in Toxicology. ISBN: 978-0-12-404630-6
ANATOMICAL, PHYSIOLOGICAL, AND METABOLIC CONSIDERATIONS FOR PANCREATIC INJURY The pancreas is an important digestive and glandular organ in vertebrates, consisting of endocrine and exocrine components. About 85% of the gland consists of the exocrine pancreas populated by acinar cells and a few duct cells. While the endocrine pancreas secretes insulin, a hormone needed to control the body’s glucose, the exocrine pancreas synthesizes and stores zymogens (inactive forms of digestive enzymes such as trypsinogen) required for the digestion of food. The digestive enzymes are secreted in the upper part of the intestine and activated by a gut hormone (enterokinase) for the digestion of food, as detailed in several textbooks and reviews. An activation of zymogens within exocrine acinar cells in the gland triggers massive necrosis followed by inflammation with or without
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© 2014 Elsevier Inc. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-404630-6.00016-6
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fibrosis, commonly termed pancreatitis, and classified as acute pancreatitis and chronic pancreatitis.
Acute pancreatitis Clinically, acute pancreatitis is characterized by an acute abdominal pain accompanied by elevated pancreatic enzymes in the blood or urine, or both. Acute pancreatitis could be a single episode or it may reoccur; it is induced by the release of activated proteases from the pancreatic acinar cells and is often hemorrhagic. It occurs suddenly and lasts for a short period of time and usually resolves by itself in most of the cases. The severity of acute pancreatitis depends upon histologic evaluation and may vary from mild (fat necrosis with enlarged gland (edema)) to severe (large confluent foci fat necrosis around the pancreas, focal hemorrhage and acinar cell necrosis).
Chronic pancreatitis Chronic or calcifying pancreatitis is a continuing inflammatory response characterized by severe morphological changes (such as irregular sclerosis and permanent loss of exocrine parenchyma), which may be focal, segmental, or diffused. Clinically, chronic pancreatitis is characterized by recurrent or persisting abdominal pain, although chronic pancreatitis may also present without pain. Chronic pancreatitis does not resolve by itself and could progress to a slow destruction of the pancreatic gland. Irrespective of etiology, the clinical pattern of chronic pancreatitis is characterized by the recurrent episodes of acute pancreatitis in the early stages followed by pancreatic insufficiency, steatorrhea, pancreatic calcification and, maybe, diabetes mellitus at the chronic stage. However, an intra-acinar activation of zymogens in the gland itself is the primary cause of pancreatic injury and pancreatitis (Lankisch and Banks, 1998).
Biomolecular basis of pancreatitis A large number of digestive enzymes are synthesized and stored by the exocrine pancreas to be released into the upper part of the intestine for digestion. The metabolic composition of the pancreas, particularly the exocrine pancreas, has been previously detailed (Kaphalia, 2011). Activation of zymogens within the acinar cells is potentially damaging to the gland and causes autodigestion of exocrine pancreas and surrounding tissue. This process involves several extra-acinar cellular events, both in the pancreas and elsewhere in the body, due to the generation of inflammatory mediators such as
cytokines, chemokines, and growth factors. It is also generally known that lipid degradation by-products produced due to oxidative stress are involved in various target organ pathogeneses. Clinically, the disease can be evident by noninvasive tests and conventional physical and clinical symptoms only after inflammation (pancreatitis) has occurred due to a massive necrotic cell death. With both exocrine and endocrine components in the pancreatic organ system, many patients with chronic pancreatitis also present overt diabetes mellitus as a manifestation of either exocrine or endocrine pancreatic insufficiencies, or both. It is possible to use combined biomarkers of endocrine and exocrine pancreatic injury to evaluate chronic and advanced stages of pancreatitis and fibrosis which involve both components of the gland. Therefore, identification and development of biomarkers of early stage pancreatic injury even before inflammation should have great translational and clinical benefits for interventional therapies. Like other key gastrointestinal organs, the exocrine pancreas is also metabolically an active organ, and expresses various phase I and phase II enzymes [oxidative, reductive, and conjugation enzymes including fatty acid ethyl ester (FAEE) synthase] (Kaphalia, 2011). Such metabolic organization of the gland might also bioactivate and/or biotransform drugs and chemicals damaging to the gland itself. Therefore, certain proteins and peptides specific to the exocrine pancreas secreted into the blood and/or excreted via urine during injury can be reliable and specific biomarkers of pancreatic injury/ toxicity/inflammation.
BIOMARKERS OF ACUTE AND CHRONIC PANCREATITIS Clinically, two types of biomarkers are described: (1) those specific and related to target organ function detected in the blood and excreted through urine, termed endogenous biomarkers, and (2) various chemical(s)/agent(s) and their metabolite(s) and infectious agents involved in initiation and progression of the disease, called exogenous biomarkers. Except for known cases of chemical poisoning and history of abuse (alcohol or drug) or occupational exposure, it is generally very difficult to pinpoint the agent responsible for the injury. In such cases, patient information could be critical. Global approaches such as proteomics, metabolomics, and genomics could be important for biomarker discovery, but such approaches are generally not cost effective and may require confirmation using RTPCR, LC-MS, and/or GC-MS analyses. A biomarker candidate should be measurable via noninvasive methods. Several matrices such as plasma,
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TABLE 16.1 Biomarkers of acute and chronic pancreatitis Matrix
Biomarkers and/or biomarker candidates
Serum/plasma
Indirect pancreatic functional biomarkers Amylase, isoamylase, and total amylase Lipase Carbohydrate-deficient transferrin Trypsinogen activation products G Trypsingen activation peptide (TAP) G Trypsin-1 and 2 and their conjugates with α-antitrypsin (trypsin-1-AAT and trypsin-2-AAT) C-reactive protein (CRP) Elastase-1 and phospholipase A2 Procalcitonin (precursor of calcitonin) Proteins (proteomics, 2-DGE and MALDI-TOF/TOF) Lipids (lipidomics, NMR, GC-MS, and LC-MS) Metabolites (small molecule metabolomics using LC-MS and GC-MS) Inflammatory cytokines and chemokines microRNAs (micro array) Peptides (RA1609 and RT2864) Direct analysis of suspected etiologic agent(s) Alcohol and alcohol metabolites (FAEEs), pesticides and metal poisoning and suicide cases, drugs and occupational exposure to chemicals and infectious agents Amylase, isoamylase, TAP, CAPAP, phospholipase A2 Elastase 1 Cathepsins B, L, and S (lysosomal hydrolases) Carboxypeptidase B activation peptide (CACAP) Differential expression of genes, proteins and microRNA, and altered metabolomics, inflammatory cytokines and chemokines, and growth factors Breath analysis for H2S, N2O and 66u substance
Urinary Fecal Pancreatic juice Pancreatic tissue specimens Breath analysis
Note: The biomarkers of acute pancreatitis may not be applicable for chronic pancreatitis, which can be confirmed by imaging techniques (ultrasound, CT, MRCP, and/or ERCP) in conjunction with clinical symptoms consistent with chronic pancreatitis.
serum, urine, saliva, hair, feces, or sweat can be used for identification and levels of the etiologic agent(s) and its metabolite(s). Several biomarker candidates identified in experimental acute pancreatitis models can be developed using translational research approaches. Physical examination, imaging (ultrasound, chest radiography, and barium sulfate X-rays), direct pancreatic function tests, and analysis of serum/plasma/urine for markers of pancreatic injury/pancreatitis should be gold standard for routine and less expensive diagnosis (Table 16.1).
relatively noninvasive investigations such as magnetic resonance cholangio-pancreatography (MRCP) and endoscopic ultrasound has meant that ERCP is now rarely performed without therapeutic intent. Therefore, patient history and imaging results should be meaningful, along with markers of pancreatic function tests in the plasma, urine, and/or saliva, keeping in view the acute and chronic nature of the disease.
Functional biomarkers of endocrine and exocrine pancreatitis Abdominal imaging techniques Abdominal ultrasound can be done in routine evaluation of acute pancreatitis; however, the severity of pancreatitis can rarely be ascertained by abdominal ultrasound. A less invasive endoscopic ultrasonography and computed tomography (CT) and more invasive retrograde cholangiopancreatography (ERCP) are better diagnostics for the gallstones in the common bile duct. ERCP is used primarily to diagnose and treat conditions of the bile ducts and main pancreatic duct, including gallstones, although the development of safer and
Most available biomarkers of pancreatic injury are limited to acute pancreatitis. Markers of chronic pancreatitis necessarily do not correspond to those identified for acute pancreatitis. Elevated titers of autoantibodies directed against amylase α-2A is suggested as a novel specific serologic biomarker to help identify patients at risk for autoimmune pancreatitis and fulminant type 1 diabetes, for early prevention and therapy (Wiley and Pietropaolo, 2009). However, low serum levels of CD44, CD44v6, and neopterin as indicators of immune dysfunction in chronic pancreatitis could not be specific or selective (Schlosser et al., 2001). Similarly, oxidative
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stress-related lipid peroxidation products proposed as biomarkers of acute pancreatitis may have limited success due to lack of specificity (Col et al., 2010). Correlation between lipid peroxidation products such as malondialdehyde in plasma/serum/urine and severity of acute pancreatitis needs to be established. A number of biomarkers of pancreatitis (acute or chronic) identified in the plasma/serum are described in the following sections.
Amylase All amylases (glycoside hydrolases) catalyze the breakdown of complex sugars (such as starch) and act at α-1,4-glycosidic bonds. The concentration of pancreatic and salivary amylase can be several orders of magnitude greater than that in other tissues/organs such as lungs, tears, sweat, and human milk. However, amylases can also be found as minor genetic variants. α-Amylase, faster acting than other isozymes, is the major form present in both human pancreas and salivary glands, with a half-life in the serum of B10 hr. The levels can return to normal within 24 hr after pancreatic injury is resolved and/or if enzyme is not being added to the serum (Nord et al., 1973). Amylase levels can show a variable response depending upon the etiologic agent(s) involved in the pancreatitis, and its lower levels have been reported on admission in patients with alcohol-induced acute pancreatitis. About 50% of the patients with abdominal pain and hyperlipidemia considered to have acute pancreatitis as confirmed by CT show no significant elevation of either serum or urinary amylase levels (Toskes, 1990). Therefore, the sensitivity of serum amylase estimation compromises the gold standard for the diagnosis of acute pancreatitis and pancreatic trauma (Lankisch and Banks, 1998; Moridani and Bromberg, 2003; Herman et al., 2011). Total serum amylase levels can be altered by changes in either pancreatic or salivary amylases (Tietz, 1988). Although measuring total amylase after inhibition of salivary amylase is sometimes suggested as being a more accurate diagnostic marker, the assay adds more steps and increases the cost of the test. Even reducing amylase ordering has been suggested for emergency departments (Volz et al., 2010). Macroamylasemia due to binding of amylase with serum globulin can also cause misleading amylase levels (Wilding et al., 1964). The precision of total amylase vs. pancreatic amylase appears to be identical (Moridani and Bromberg, 2003). Therefore, a straightforward approach of measuring total amylase is cost effective and saves time. Elevated urinary amylase is also a sensitive indicator of acute pancreatitis and is increased in almost 95% of patients with pancreatitis and remains elevated longer
than the serum amylase activity. An elevated urinary amylase ( .threefold the upper limit of normal) is a clear diagnosis of acute pancreatitis. However, elevated urine amylase can also be seen in salivary gland disease, bowel perforation, and ketoacidosis (Lankisch and Banks, 1998). An abnormal urinary amylase creatinine clearance ratio can be a proof of acute pancreatitis. Although urinary amylase has not been widely used, elevated urinary amylase as well as serum amylase can be considered as diagnostic biomarkers of acute pancreatitis.
Isoamylase Any of several isoenzymes of α-amylase catalyzes the hydrolysis of 1,6-α-glycosidic branch linkages in glycogen and amylopectin and their β-limit dextrins. The ratio of the amounts of pancreatic and salivary isoamylases in the urine is proposed as an index of insufficient exocrine pancreatic functions (Aw et al., 1967). However, urinary isoamylase levels have not been used by the clinics so far.
Lipase Unlike amylase, lipase mainly is of pancreatic origin; a small amount can also be produced in the liver, stomach, and tongue. Lipase catalyzes the breakdown and hydrolysis of fats and acts at a specific position on the glycerol backbone of the lipid substrate (A1, A2, or A3). Pancreatic lipase is the main enzyme that converts triglycerides to monoglycerides and fatty acids. As compared to serum amylase and other markers so far being used, serum lipase appears to be a sensitive marker for acute pancreatitis. Serum lipase is highly sensitive, can be detected for several days, and is sensitive even with normal amylase levels. Most hospitals use serum lipase for the diagnosis of acute pancreatitis and its specificity is considered to be excellent (Steinberg et al., 1985).
Carbohydrate-deficient transferrin Transferrin, a serum protein, is a polypeptide with two N-linked polysaccharide chains, which are branched with sialic acid (monosaccharide carbohydrate) residues. Transferrin carries iron through the bloodstream to the bone marrow, as well as to the liver and spleen. Correlation between carbohydrate-deficient transferrin (CDT) and mean corpuscular volume with excessive alcohol consumption has been reported statistically significant (Basterra et al., 2001; Aparicio et al., 2001; Sharpe, 2001). Elevated CDT found in the blood of chronic alcoholics can also be seen in various medical conditions (ARUP Laboratories, 2010). The CDT levels are shown to be correlated with alcohol consumption and, possibly, acute pancreatitis in alcoholic patients
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(Jaakkola et al., 1994). Overall, CDT appears to have some clinical utility, but could not be used by clinics for the diagnosis of acute or chronic pancreatitis, mainly due to specificity and cost of analysis reasons.
Activation by-products of trypsinogen and carboxypeptidase B Pro-enzyme trypsinogen occurs in two major isoenzymes in humans (trypsinogen 1, i.e. cationic trypsinogen form, and trypsinogen 2, i.e. anionic trypsinogen form), which are activated to trypsin by enterokinase (EK) in the gut for digestion. However, activation of trypsinogen within the acinar cell plays a key role in the pathogenesis of acute pancreatitis, because conversion of trypsinogen triggers a pancreatic enzyme cascade that activates the remaining zymogens, resulting in autodigestion of the surrounding tissue (Rinderknecht, 1986). Trypsin rapidly gets inactivated by 1-α-antitrypsin (AAT), a neutralization process. High serum concentrations of trypsin-1-AAT and trypsin-2-AAT have been reported in acute pancreatitis (Borgstro¨m and Ohlsson, 1978; Hedstro¨m et al., 1994). Trypsinogen activation peptide (TAP) is a by-product of the trypsinogen activation process. Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyzes proteins at the carboxyl side of the amino acids lysine or arginine. Trypsin produced due to premature trypsinogen activation in the pancreas ideally fulfills the criterion of organ specificity of a biomarker. The activation peptide at the amino terminus of vertebrate trypsinogen contains the sequence Asp-AspAsp-Asp-Lys (D4K, highly conserved during vertebrate evolution) as the carboxy-terminal moiety. These peptides are generally liberated in the proximal small intestine during digestion, after recognition and cleavage at the lysine carbonyl, by small gut EK. Intra-acinar activation of trypsinogen releases TAP into circulation, which is rapidly cleared by the kidneys for excretion in urine because of its small size. TAP can be quantitated by an enzyme-linked immunosorbent assay (ELISA) in the plasma and urine using a specific antibody to TAP (Wu et al., 2008). Only early acute or severe pancreatitis, as compared to mild acute pancreatitis, is correlated with serum and urinary levels of TAP, which are rapidly depleted to undetectable levels. Quick collection and analysis of samples is necessary and results may differ depending on the etiology of acute pancreatitis [e.g. alcohol- vs. gallstone-induced (Lempinen et al., 2003)]. Serum TAP appears to have close correlation with the severity of pancreatic injury and can be used as an early marker of acute pancreatitis. As compared to serum TAP, urinary TAP has a more consistent relationship with early acute pancreatitis. It has been reported that plasma TAP increased
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immediately after the induction of pancreatitis as compared to delayed excretion of TAP in the urine by several hours in an experimental pancreatitis model (Wang et al., 2001). About 30% of the patients with acute pancreatitis had normal TAP values on admission (Gudgeon et al., 1990). There are contradictory reports regarding the utility of serum and urinary TAP and these levels can remain undetected in mild cases of acute pancreatitis. Therefore, TAP levels only reflect severe acute pancreatitis (Kylanpa¨a¨-Ba¨ck et al., 2002; Johnson et al., 2004). However, the methods of analysis of trypsin and TAP are time-consuming and difficult and also not cost effective for routine clinical use. Like TAP, procarboxypeptidase B has an activation peptide, carboxypeptidase activation peptide (CAPAP), which is a larger peptide and more stable in the serum and urine than other peptides (Burgos et al., 1991; Abu Hilal et al., 2007). Serum and urinary levels of CAPAP were found to correlate well with severe acute pancreatitis based upon a study conducted in a small number of patients (Pezzelli et al., 2000; Appelros et al., 2001). Both TAP and CAPAP could serve as markers of early and severe forms of acute pancreatitis at the time of admission to hospital. Therefore, the utility of both markers needs to be tested in larger cohorts.
Elastase and phospholipase A2 Pancreatic elastase is a form of elastase and a subfamily of serine proteases (referring to the chymotrypsin-like elastase family), member 3B (CELA3B), that hydrolyze many proteins in addition to elastin. Human pancreatic elastase-1 (E1) is quite stable and remains undegraded during intestinal transit. Therefore its concentration in feces reflects exocrine pancreatic function. During an inflammation of the pancreas, E1 is released into the bloodstream. Thus the quantification of pancreatic elastase-1 in serum and feces allows diagnosis or exclusion of acute pancreatitis (Dominguez-Munoz et al., 2006; Naruse et al., 2006). Urinary excretion of elastase-1 has been shown to increase in all patients with chronic pancreatic disease regardless of the neoplastic or inflammatory nature of the disease (Fabris et al., 1989). Unfortunately, determination of elastase-1 does not provide any additional advantage over the combination of lipase and amylase for the diagnosis of acute pancreatitis and may not be a specific biomarker candidate. Phospholipase A2 specifically recognizes the sn-2 acyl bond of phospholipids and catalytically hydrolyzes the bond releasing arachidonic acid and lysophospholipids. However, phospholipase A2 can be found in several other organs and induced in chronic liver cirrhosis, thus lacking target-organ specificity (Vishwanath et al., 1996). Urinary excretion of phospholipase A2 in chronic pancreatic diseases particularly during relapse as well as in
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other physiological conditions has been reported (Fabris et al., 1992). Like trypsin and TAP, elastase and phospholipase A2 increase in serum and feces in acute pancreatitis, but could not be used as conventional diagnostic biomarkers for acute pancreatitis probably due to cost considerations and intricate analysis protocols.
Procalcitonin Procalcitonin is a precursor of calcitonin, and serum procalcitonin as measured using a chemoluminescent immunoassay was found to be a simple, promising biomarker with accuracy similar to acute physiology and chronic health examination (APACHE)-II score, in predicting severity of acute pancreatitis (Woo et al., 2011). However, precalcitonin can be useful only in predicting the severity of acute pancreatitis and is comparable to other biomarkers such as APACHE-II, C-reactive protein (CRP), lactate dehydrogenase, and urea.
Cathepsins Pancreatitis is shown to be associated with an increase in the active form of cathepsins B, L, and S, which regulate premature activation of trypsinogen within pancreatic acinar cells (Lyo et al., 2012). Cathepsins are lysosomal proteases and are postulated to play a role in the initiation of pancreatitis (van Acker et al., 2006). The levels of these cathepsins have been shown to increase in the pancreatic juice from patients with chronic pancreatitis. In the case of ethanol-induced pancreatic injury, cathepsins respond to FAEEs (nonoxidative metabolites of ethanol-induced pancreatic lysosomal fragility) (Haber et al., 1993). However, due to the lack of specificity, and analysis of cathepsins using mostly fluorimetrics, these are not cost effective alternatives as markers (Barrett, 1980).
Peptides Walgren et al. (2007) identified two peptide markers (RA1609 and RT2864) of pancreatic toxicity that are target-organ specific using a model pancreatic toxin cyanohydroxybutene. These markers were verified in the serum of patients diagnosed with pancreatitis, and changes in serum RA1609 and RT2864 were indicative of and specific to pancreatitis. The human sera analyzed in this study were from patients with pancreatitis, which was not defined as acute or chronic. Moreover, clinical use of such markers has yet to be established for a routine analysis.
C-reactive protein and pancreatitis-associated protein CRP, an acute phase protein primarily synthesized in the liver, is a class of proteins whose plasma
concentrations increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) in response to inflammation. Determination of the severity of acute pancreatitis is difficult in the early phase after onset, and often difficulties are encountered in making decisions to initiate intensive care during the early phase. CRP values increase significantly in early stages of necrotic pancreatitis. It is an important prognostic marker of pancreatic necrosis, with the highest sensitivity and negative prognostic value given the cut-off is 110 mg/L. The patients with CRP values below 110 mg/L are at low risk to develop pancreatic necrosis (Barauskas et al., 2004). Measurement of acute-phase proteins, especially CRP, is a useful marker of inflammation in both medical and veterinary clinical pathology. It correlates with the erythrocyte sedimentation rate. However, CRP is a marker of inflammation (proinflammatory cytokines) and higher CRP may also indicate liver failure (Ananian et al., 2005). Pancreatitis-associated protein (PAP), an acute phase protein like CRP, is overexpressed in acute pancreatitis and was detected in pancreatic juice from 49 patients with chronic pancreatitis. However, higher levels of PAP also detected in 15 control subjects discredited the significance of the PAP as a reliable biomarker of chronic pancreatitis (Motoo et al., 2001).
Inflammatory cytokines and chemokines Necrosis of exocrine pancreas, the primary cause of acute and chronic pancreatitis, can be identified by contrast-enhanced computed tomography and magnetic resonance imaging. Massive necrotic cell death follows strong inflammatory responses and infection. Proinflammatory cytokines and chemokines such as TNF, IL-1α, IL-1β, IL-6, IL-8, IL-12, and MCP-1 have been identified to serve as biomarkers of acute and chronic pancreatitis (Papachristou, 2008; Achur et al., 2010). However, the ability to predict which patients will develop severe disease is limited. Large prospective studies are still needed to address these questions by identifying risk factors for acute pancreatitis and serum biomarkers of severe disease conditions. Correlation of specific serum inflammatory cytokines and chemokines increasing or decreasing with severity of pancreatitis needs to be established before embarking on cytokines and chemokines as potential biomarkers of acute or chronic pancreatitis. Chronic pancreatitis is a chronic inflammatory disease. However, a caution should be exercised to use certain or a combination of inflammatory cytokines and chemokines, in view of broad inflammatory response in chronic diseases of diverse nature. Secondary target organ toxicity is also one of the potential interferences in such determinations.
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Non-oxidative lipid metabolites of ethanol Ethanol is one of the major etiologic agents for acute and chronic alcoholic pancreatitis after biliary duct disease (Lankisch and Banks, 1998; Kaphalia, 2011). Non-oxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) is the most prevalent means of ethanol metabolism in the pancreas, frequently damaged during chronic alcohol abuse (Laposata and Lange, 1986). These esters are lipophilic and have been shown to cause selective pancreatic acinar cell toxicity (Werner et al., 1997; Kaphalia and Ansari, 2001; Gukovskaya et al., 2002; Vonlaufen et al., 2007; Wu et al., 2008). The plasma/serum levels of FAEEs are generally high and correlate well with blood alcohol concentration particularly during chronic alcohol abuse (Laposata and Lange, 1986; Kaphalia et al., 2004). Our studies in a deer mouse model of chronic ethanol feeding have shown that increased levels of plasma FAEE correlate well with those in the pancreas (Kaphalia et al., 2010). However, an interrelationship between plasma/serum levels of FAEEs and severity of pancreatitis in patients with acute or chronic pancreatitis needs to be established. Other non-oxidative metabolites of ethanol which correlate with blood alcohol levels in the blood include phosphatidylethanol (PEt) formed by the phospholipase D catalyzed reaction (Aradottir et al., 2006). A good clinical efficiency of PEt and its pancreatic toxicity have not been demonstrated so far for detecting chronic heavy drinking (Viel et al., 2012).
Differentially altered proteins, metabolites, small molecules, and microRNA Direct functional tests can be conducted by identifying the proteins, metabolites (small molecule), and microRNAs differentially altered in the pancreatic juice and duodenal contents from proteins with acute or chronic pancreatitis. Omic and array research is a systemic global approach and provides a unique “fingerprint,” which varies with time or stresses, that a cell or organism undergoes. High resolution 1H-NMR analysis of plasma and urine samples and data analysis as determined by principal component analysis (PCA) using metabolomic modeling of L-arginine-induced exocrine pancreatitis suggest metabolomics is a valuable approach, which can distinguish patients with pancreatitis from healthy controls (Bohus et al., 2008; Lusczek et al., 2013). Lipid profiling of serum and pancreatic fluid conducted in chronic pancreatitis using an LC-ESI-MS/MS system in five controls and six patients of mild and five of severe chronic pancreatitis showed an increase in the levels of oxidized fatty acid products, suggesting their utility as biomarker candidates for the diagnosis of
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chronic pancreatitis (Stevens et al., 2012). However, the specificity of such biomarkers needs to be established. Identification of proteins has been done only in the pancreas in acute pancreatitis models (Fe´taud et al., 2008; Garcia-Herna´ndez et al., 2012; Zhang et al., 2012). Therefore, significant efforts should be directed at profiling both plasma and pancreatic proteins differentially, expressed in acute and chronic pancreatitis in animal models, followed by their validation in the plasma of patients with acute or chronic pancreatitis. Proteomic profiling using SELDI-TOF-MS has been done in the serum as a predictor of severity of acute pancreatitis from 21 patients with mild and 7 patients with severe acute pancreatitis (Papachristou et al., 2007). Various other proteomic studies (separation of proteins by twodimensional polyacrylamide gel electrophoresis and identification by MALDI-TOF/TOF) done on the pancreatic fluid, plasma, serum, and pancreatic tissue from patients with chronic pancreatitis provide significant leads for further pursuing studies in larger cohorts (Chen et al., 2007; Hartmann et al., 2007). While gene profiling studies can also identify biomarker candidates, studies are limited to the target organ and to use in experimental models. Pathway analysis and systems biology approaches, generally performed to infer relationships between genes, proteins, and small molecules (metabolomics), are important for identification of biomarkers and may be a future direction in the biomarker field. MicroRNAs (miRNAs) are functional, 22 nt, noncoding RNAs that negatively regulate gene expression and play a role in the initiation and progression of certain diseases. However, the identity of differentially altered target microRNA should be confirmed. Hierarchical clustering and principal component analysis of the data sets can distinguish pancreatic tissue of patients with pancreatitis from normal control tissue. Only a handful of studies are available to identify miRNAs as biomarkers in experimental rat models of acute or chronic pancreatitis, and in pancreatic cancer (Bloomston et al., 2007; Lee et al., 2007; Kong et al., 2010). Kong et al. (2010) identified plasma miR-216a as a potential marker of pancreatic injury in a rat model of acute pancreatitis. Like amylase and lipase, increased expression of miR-216a in the plasma was restricted to acute pancreatitis at 24 hr after the insult. Such miR-based markers need to be confirmed in patients with acute and chronic pancreatitis. In one study, two dozen miRNAs were shown to be differentially altered in the pancreatic tissue from patients with chronic pancreatitis (n 5 42) as compared to those in adjacent normal tissue as control (Bloomston et al., 2007). These studies are very preliminary and need further investigation to establish their utility as biomarker candidates in clinical diagnostics. Even if miRNA microarray technologies identify some promising biomarkers of disease/injury, cost and precision are other factors to be considered.
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Breath analysis Human exhaled breath contains many molecules either present as gases or occurring in a soluble form in the vapor of the breath. One can rapidly and easily assess the presence of volatile compounds (hydrogen sulfide, nitric oxide, and molecular mass 66 u substance) in the exhaled breath of chronic pancreatitis patients (Morselli-Labate et al., 2007). Although a study was conducted in 11 patients with chronic pancreatitis and 31 healthy subjects and the marker appears novel, no significant differences were found between patients and healthy controls.
Comparison of biomarkers between acute and chronic pancreatitis Basic laboratory tests for diagnosing acute and chronic pancreatitis are vastly different because the former is short-term, largely indicated by significant increases for amylase, isoamylase, lipase, TAP, trypsin, elastase-1, phospholipase A2, CDT, CRP, PAP, CAPAP, procalcitonin in serum, plasma, or urine, and other markers such as cathepsins in pancreatic juice. The indirect pancreatic function tests in serum or urine can be evident in early stages of chronic pancreatitis, but a number of biomarkers as mentioned above may be in the normal range or fluctuate depending upon the chronicity of the pancreatitis, because slow destruction of the exocrine gland results in exocrine pancreatic insufficiency. Therefore, testing exocrine pancreatic insufficiency is still recommended when chronic pancreatitis is suspected. Although several tests, including the secretin test, cerulein test and Lundh test, have been described in detail by Lankisch and Banks (1998), fecal tests for trypsin, chymotrypsin, and elastase-1 can also provide some clues for presence of chronic pancreatitis. Most of the methods used are either titrimetric estimation or ELISA. Moreover, fecal chymotrypsin estimation is not novel. Most of these tests are not even sensitive enough to diagnose mild to moderate exocrine pancreatic insufficiency. Fecal fat estimation, particularly when stimulated lipase and protease output falls below 10% of the normal, is a safe biomarker for steatorrhea (DiMagno et al., 1973). However, fecal fat contents were of no value for differential diagnosis of pancreatic and nonpancreatic steatorrhea (Roberts et al., 1986). Pancreas-specific chymotrypsin splits N-benzyl-L-tyrosyl-p-aminobenzoic acid to p-aminobenzoic acid (NTBPABA) in the duodenum. Estimation of p-aminobenzoic acid (PABA) cannot be accurate, since some gut bacteria also split NBT-PABA, causing a false normal test result. Chronic pancreatitis can be diagnosed by serum, urinary, and fecal biomarkers of pancreatic functions in
conjunction with patient history and imaging (CT, MRCP, ERCP) results. In view of available biomarkers and nature of pathology, a single biomarker should not be sufficient to diagnose chronic pancreatitis. Identification and development of metabolomic-, proteomic-, and miRNA-based biomarkers should be undertaken in experimental models applied to patients at various stages of acute and chronic pancreatitis.
BIOMARKERS OF ENDOCRINE AND AUTOIMMUNE PANCREATITIS Endocrine functional impairment with insulin resistance can be used as an index of endocrine pancreatitis (Wu et al., 2011). Type 1 diabetes mellitus (loss of insulinsecreting capacity) is now classified as autoimmune (type 1A) or idiopathic (type 1B). Presence or absence of glutamine acid decarboxylase antibodies can differentiate type 1 diabetes. Some patients with idiopathic type 1 diabetes may also have nonautoimmune and remarkably abrupt onset and high serum pancreatic enzyme levels (Imagawa et al., 2000). Presence of elevated titers of autoantibodies directed to amylase α-2A may represent a novel specific biomarker of autoimmune pancreatitis and fulminant type 1 diabetes. A correlation between urinary p-aminobenzoic acid (PABA, a split product of n-benzoyl-1-tyrosyl-p-aminobenzoic acid) excretion and plasma glucagon concentration suggest that in chronic pancreatitis there is collateral impairment of exocrine and endocrine functions (Keller et al., 1984).
BIOMARKERS FOR EARLY DETECTION OF PANCREATIC CANCER Although the focus of this chapter is biomarkers of pancreatitis (acute and chronic), early detection of pancreatic cancer is also critical for the success of interventional and surgical therapies. Pancreatic cancer is a most devastating and fatal disease, without any overt symptoms at initial stages. Most common exocrine pancreatic cancers are pancreatic ductal adenocarcinoma (PDAC) followed by adenosquamous carcinomas and broad-based pancreatic cystic neoplasma. However, pancreatic neuroendocrine tumors constitute a small number of cases, B1% of all pancreatic cancers. Endocrine pancreatic tumors have been variously called islet cell tumors, pancreas endocrine tumors (PETs), and pancreatic neuroendocrine tumors (PNETs).
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The annual clinically recognized incidence is low, about five per one million person/years. However, autopsy studies incidentally identify PETs in up to 1.5%, most of which would remain inert and asymptomatic. The majority of PNETs are usually categorized as benign but the definition of malignancy in pancreas endocrine tumors has been ambiguous. The more aggressive endocrine pancreatic cancers are known as pancreatic neuroendocrine carcinomas (PNEC). Similarly, there has likely been a degree of admixture of PNEC and extrapulmonary small cell carcinoma. Most of the biomarkers of pancreatic cancers, identified for PDAC, are carcinoembryonic (CEA), carbohydrate antigen (CA19-9) and mucin family (MUC), the latter being most commonly used along with circulating tumor cells; markers in pancreatic juice and signaling pathway have been extensively reviewed (Tanase et al., 2009; Chakraborty et al., 2011; Poruk et al., 2013). Early detection of pancreatic cancer offers the promise of improved mortality rates through surgical resection. Therefore, biomarkers should be determined for the early stages of pancreatic cancer to improve prognosis and mortality rates by using intervention therapies. For further details on biomarkers of pancreatic cancer, readers are referred to Chapter 45.
CONCLUDING REMARKS AND FUTURE DIRECTIONS Reliable and sensitive biomarkers of acute and chronic pancreatitis and pancreatic cancers are needed, as current available biomarkers have limitations and lack specificity. Serum total amylase and lipase in combination or individually remains the gold standard for functional clinical diagnostics of acute pancreatitis, if the test is conducted on admission of the patient to the hospital. Lipase assay has a definite advantage over amylase in clinical diagnostics and has more specificity for diagnosing acute pancreatitis. However, imaging technologies provide strong support for diagnosis of both acute and chronic pancreatitis. Identification and development of biomarkers of chronic pancreatitis remain as research challenges, because several indirect pancreatic functional biomarkers are inconclusive, primarily because of the impaired secretory component of exocrine pancreas due to the slow destruction of the gland in chronic pancreatitis, particularly at later stages. The focus of future research lies in identification of molecular biomarkers by utilizing proteomic, metabolomic, and miRNA array technologies, using very well-defined patient populations.
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16 Biomarkers of acute and chronic pancreatitis Bhupendra S. Kaphalia
INTRODUCTION
etiological agents, the sequence of pancreatic injury follows a similar pattern for most of the etiologies. Some patients with pancreatitis can also develop pancreatic cancer. Overall, chronic pancreatitis is a devastating disease with great social, economic, and psychological impact. The prognosis of pancreatitis and pancreatic cancer is very poor and many patients die before reaching the clinical stage of the disease. Therefore, identification of biomarkers for both pre- and post-pancreatitis stages could be important for clinical interventions.
Early detection and prevention of pancreatic injury, progressing to inflammation and fibrosis and possibly pancreatic cancer, should be critical for the success of both clinical and surgical interventional therapies. Although a significant literature available on biomarkers of pancreatic diseases is focused on various forms of pancreatic cancer, identification of biomarkers for early pancreatic injury and precancerous stages should be the key to early prevention and therapy. Therefore, summarizing reliable and probable biomarkers identified for acute and chronic pancreatic toxicities/pancreatitis rather than pancreatic cancer is the focus of this chapter. Both biliary duct disease and chronic alcohol abuse constitute 70 75% of the etiologies of acute and chronic pancreatitis; the remaining third may be idiopathic, including those caused by chemical exposure, pesticide and metal poisoning, use of drugs, infections, and autoimmune and anatomical conditions. Congenital abnormalities, generally rare and asymptomatic, are related to two important events during embryological development of the gland: rotation and fusion. Anatomical disorders, deposition of cholesterol and related substances, and obstruction of the pancreatic duct due to tumor and cyst formation could be rare (Lankisch and Banks, 1998; Kaphalia, 2011). Pancreatic toxicity is generally characterized by dysregulation of lipid metabolism and edema in early reversible stages, followed by massive necrosis resulting in inflammation, with or without fibrosis (scarring of the tissue) at the advanced stages. Irrespective of
R. Gupta (Ed): Biomarkers in Toxicology. ISBN: 978-0-12-404630-6
ANATOMICAL, PHYSIOLOGICAL, AND METABOLIC CONSIDERATIONS FOR PANCREATIC INJURY The pancreas is an important digestive and glandular organ in vertebrates, consisting of endocrine and exocrine components. About 85% of the gland consists of the exocrine pancreas populated by acinar cells and a few duct cells. While the endocrine pancreas secretes insulin, a hormone needed to control the body’s glucose, the exocrine pancreas synthesizes and stores zymogens (inactive forms of digestive enzymes such as trypsinogen) required for the digestion of food. The digestive enzymes are secreted in the upper part of the intestine and activated by a gut hormone (enterokinase) for the digestion of food, as detailed in several textbooks and reviews. An activation of zymogens within exocrine acinar cells in the gland triggers massive necrosis followed by inflammation with or without
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© 2014 Elsevier Inc. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-404630-6.00016-6
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fibrosis, commonly termed pancreatitis, and classified as acute pancreatitis and chronic pancreatitis.
Acute pancreatitis Clinically, acute pancreatitis is characterized by an acute abdominal pain accompanied by elevated pancreatic enzymes in the blood or urine, or both. Acute pancreatitis could be a single episode or it may reoccur; it is induced by the release of activated proteases from the pancreatic acinar cells and is often hemorrhagic. It occurs suddenly and lasts for a short period of time and usually resolves by itself in most of the cases. The severity of acute pancreatitis depends upon histologic evaluation and may vary from mild (fat necrosis with enlarged gland (edema)) to severe (large confluent foci fat necrosis around the pancreas, focal hemorrhage and acinar cell necrosis).
Chronic pancreatitis Chronic or calcifying pancreatitis is a continuing inflammatory response characterized by severe morphological changes (such as irregular sclerosis and permanent loss of exocrine parenchyma), which may be focal, segmental, or diffused. Clinically, chronic pancreatitis is characterized by recurrent or persisting abdominal pain, although chronic pancreatitis may also present without pain. Chronic pancreatitis does not resolve by itself and could progress to a slow destruction of the pancreatic gland. Irrespective of etiology, the clinical pattern of chronic pancreatitis is characterized by the recurrent episodes of acute pancreatitis in the early stages followed by pancreatic insufficiency, steatorrhea, pancreatic calcification and, maybe, diabetes mellitus at the chronic stage. However, an intra-acinar activation of zymogens in the gland itself is the primary cause of pancreatic injury and pancreatitis (Lankisch and Banks, 1998).
Biomolecular basis of pancreatitis A large number of digestive enzymes are synthesized and stored by the exocrine pancreas to be released into the upper part of the intestine for digestion. The metabolic composition of the pancreas, particularly the exocrine pancreas, has been previously detailed (Kaphalia, 2011). Activation of zymogens within the acinar cells is potentially damaging to the gland and causes autodigestion of exocrine pancreas and surrounding tissue. This process involves several extra-acinar cellular events, both in the pancreas and elsewhere in the body, due to the generation of inflammatory mediators such as
cytokines, chemokines, and growth factors. It is also generally known that lipid degradation by-products produced due to oxidative stress are involved in various target organ pathogeneses. Clinically, the disease can be evident by noninvasive tests and conventional physical and clinical symptoms only after inflammation (pancreatitis) has occurred due to a massive necrotic cell death. With both exocrine and endocrine components in the pancreatic organ system, many patients with chronic pancreatitis also present overt diabetes mellitus as a manifestation of either exocrine or endocrine pancreatic insufficiencies, or both. It is possible to use combined biomarkers of endocrine and exocrine pancreatic injury to evaluate chronic and advanced stages of pancreatitis and fibrosis which involve both components of the gland. Therefore, identification and development of biomarkers of early stage pancreatic injury even before inflammation should have great translational and clinical benefits for interventional therapies. Like other key gastrointestinal organs, the exocrine pancreas is also metabolically an active organ, and expresses various phase I and phase II enzymes [oxidative, reductive, and conjugation enzymes including fatty acid ethyl ester (FAEE) synthase] (Kaphalia, 2011). Such metabolic organization of the gland might also bioactivate and/or biotransform drugs and chemicals damaging to the gland itself. Therefore, certain proteins and peptides specific to the exocrine pancreas secreted into the blood and/or excreted via urine during injury can be reliable and specific biomarkers of pancreatic injury/ toxicity/inflammation.
BIOMARKERS OF ACUTE AND CHRONIC PANCREATITIS Clinically, two types of biomarkers are described: (1) those specific and related to target organ function detected in the blood and excreted through urine, termed endogenous biomarkers, and (2) various chemical(s)/agent(s) and their metabolite(s) and infectious agents involved in initiation and progression of the disease, called exogenous biomarkers. Except for known cases of chemical poisoning and history of abuse (alcohol or drug) or occupational exposure, it is generally very difficult to pinpoint the agent responsible for the injury. In such cases, patient information could be critical. Global approaches such as proteomics, metabolomics, and genomics could be important for biomarker discovery, but such approaches are generally not cost effective and may require confirmation using RTPCR, LC-MS, and/or GC-MS analyses. A biomarker candidate should be measurable via noninvasive methods. Several matrices such as plasma,
BIOMARKERS OF ACUTE AND CHRONIC PANCREATITIS
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TABLE 16.1 Biomarkers of acute and chronic pancreatitis Matrix
Biomarkers and/or biomarker candidates
Serum/plasma
Indirect pancreatic functional biomarkers Amylase, isoamylase, and total amylase Lipase Carbohydrate-deficient transferrin Trypsinogen activation products G Trypsingen activation peptide (TAP) G Trypsin-1 and 2 and their conjugates with α-antitrypsin (trypsin-1-AAT and trypsin-2-AAT) C-reactive protein (CRP) Elastase-1 and phospholipase A2 Procalcitonin (precursor of calcitonin) Proteins (proteomics, 2-DGE and MALDI-TOF/TOF) Lipids (lipidomics, NMR, GC-MS, and LC-MS) Metabolites (small molecule metabolomics using LC-MS and GC-MS) Inflammatory cytokines and chemokines microRNAs (micro array) Peptides (RA1609 and RT2864) Direct analysis of suspected etiologic agent(s) Alcohol and alcohol metabolites (FAEEs), pesticides and metal poisoning and suicide cases, drugs and occupational exposure to chemicals and infectious agents Amylase, isoamylase, TAP, CAPAP, phospholipase A2 Elastase 1 Cathepsins B, L, and S (lysosomal hydrolases) Carboxypeptidase B activation peptide (CACAP) Differential expression of genes, proteins and microRNA, and altered metabolomics, inflammatory cytokines and chemokines, and growth factors Breath analysis for H2S, N2O and 66u substance
Urinary Fecal Pancreatic juice Pancreatic tissue specimens Breath analysis
Note: The biomarkers of acute pancreatitis may not be applicable for chronic pancreatitis, which can be confirmed by imaging techniques (ultrasound, CT, MRCP, and/or ERCP) in conjunction with clinical symptoms consistent with chronic pancreatitis.
serum, urine, saliva, hair, feces, or sweat can be used for identification and levels of the etiologic agent(s) and its metabolite(s). Several biomarker candidates identified in experimental acute pancreatitis models can be developed using translational research approaches. Physical examination, imaging (ultrasound, chest radiography, and barium sulfate X-rays), direct pancreatic function tests, and analysis of serum/plasma/urine for markers of pancreatic injury/pancreatitis should be gold standard for routine and less expensive diagnosis (Table 16.1).
relatively noninvasive investigations such as magnetic resonance cholangio-pancreatography (MRCP) and endoscopic ultrasound has meant that ERCP is now rarely performed without therapeutic intent. Therefore, patient history and imaging results should be meaningful, along with markers of pancreatic function tests in the plasma, urine, and/or saliva, keeping in view the acute and chronic nature of the disease.
Functional biomarkers of endocrine and exocrine pancreatitis Abdominal imaging techniques Abdominal ultrasound can be done in routine evaluation of acute pancreatitis; however, the severity of pancreatitis can rarely be ascertained by abdominal ultrasound. A less invasive endoscopic ultrasonography and computed tomography (CT) and more invasive retrograde cholangiopancreatography (ERCP) are better diagnostics for the gallstones in the common bile duct. ERCP is used primarily to diagnose and treat conditions of the bile ducts and main pancreatic duct, including gallstones, although the development of safer and
Most available biomarkers of pancreatic injury are limited to acute pancreatitis. Markers of chronic pancreatitis necessarily do not correspond to those identified for acute pancreatitis. Elevated titers of autoantibodies directed against amylase α-2A is suggested as a novel specific serologic biomarker to help identify patients at risk for autoimmune pancreatitis and fulminant type 1 diabetes, for early prevention and therapy (Wiley and Pietropaolo, 2009). However, low serum levels of CD44, CD44v6, and neopterin as indicators of immune dysfunction in chronic pancreatitis could not be specific or selective (Schlosser et al., 2001). Similarly, oxidative
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stress-related lipid peroxidation products proposed as biomarkers of acute pancreatitis may have limited success due to lack of specificity (Col et al., 2010). Correlation between lipid peroxidation products such as malondialdehyde in plasma/serum/urine and severity of acute pancreatitis needs to be established. A number of biomarkers of pancreatitis (acute or chronic) identified in the plasma/serum are described in the following sections.
Amylase All amylases (glycoside hydrolases) catalyze the breakdown of complex sugars (such as starch) and act at α-1,4-glycosidic bonds. The concentration of pancreatic and salivary amylase can be several orders of magnitude greater than that in other tissues/organs such as lungs, tears, sweat, and human milk. However, amylases can also be found as minor genetic variants. α-Amylase, faster acting than other isozymes, is the major form present in both human pancreas and salivary glands, with a half-life in the serum of B10 hr. The levels can return to normal within 24 hr after pancreatic injury is resolved and/or if enzyme is not being added to the serum (Nord et al., 1973). Amylase levels can show a variable response depending upon the etiologic agent(s) involved in the pancreatitis, and its lower levels have been reported on admission in patients with alcohol-induced acute pancreatitis. About 50% of the patients with abdominal pain and hyperlipidemia considered to have acute pancreatitis as confirmed by CT show no significant elevation of either serum or urinary amylase levels (Toskes, 1990). Therefore, the sensitivity of serum amylase estimation compromises the gold standard for the diagnosis of acute pancreatitis and pancreatic trauma (Lankisch and Banks, 1998; Moridani and Bromberg, 2003; Herman et al., 2011). Total serum amylase levels can be altered by changes in either pancreatic or salivary amylases (Tietz, 1988). Although measuring total amylase after inhibition of salivary amylase is sometimes suggested as being a more accurate diagnostic marker, the assay adds more steps and increases the cost of the test. Even reducing amylase ordering has been suggested for emergency departments (Volz et al., 2010). Macroamylasemia due to binding of amylase with serum globulin can also cause misleading amylase levels (Wilding et al., 1964). The precision of total amylase vs. pancreatic amylase appears to be identical (Moridani and Bromberg, 2003). Therefore, a straightforward approach of measuring total amylase is cost effective and saves time. Elevated urinary amylase is also a sensitive indicator of acute pancreatitis and is increased in almost 95% of patients with pancreatitis and remains elevated longer
than the serum amylase activity. An elevated urinary amylase ( .threefold the upper limit of normal) is a clear diagnosis of acute pancreatitis. However, elevated urine amylase can also be seen in salivary gland disease, bowel perforation, and ketoacidosis (Lankisch and Banks, 1998). An abnormal urinary amylase creatinine clearance ratio can be a proof of acute pancreatitis. Although urinary amylase has not been widely used, elevated urinary amylase as well as serum amylase can be considered as diagnostic biomarkers of acute pancreatitis.
Isoamylase Any of several isoenzymes of α-amylase catalyzes the hydrolysis of 1,6-α-glycosidic branch linkages in glycogen and amylopectin and their β-limit dextrins. The ratio of the amounts of pancreatic and salivary isoamylases in the urine is proposed as an index of insufficient exocrine pancreatic functions (Aw et al., 1967). However, urinary isoamylase levels have not been used by the clinics so far.
Lipase Unlike amylase, lipase mainly is of pancreatic origin; a small amount can also be produced in the liver, stomach, and tongue. Lipase catalyzes the breakdown and hydrolysis of fats and acts at a specific position on the glycerol backbone of the lipid substrate (A1, A2, or A3). Pancreatic lipase is the main enzyme that converts triglycerides to monoglycerides and fatty acids. As compared to serum amylase and other markers so far being used, serum lipase appears to be a sensitive marker for acute pancreatitis. Serum lipase is highly sensitive, can be detected for several days, and is sensitive even with normal amylase levels. Most hospitals use serum lipase for the diagnosis of acute pancreatitis and its specificity is considered to be excellent (Steinberg et al., 1985).
Carbohydrate-deficient transferrin Transferrin, a serum protein, is a polypeptide with two N-linked polysaccharide chains, which are branched with sialic acid (monosaccharide carbohydrate) residues. Transferrin carries iron through the bloodstream to the bone marrow, as well as to the liver and spleen. Correlation between carbohydrate-deficient transferrin (CDT) and mean corpuscular volume with excessive alcohol consumption has been reported statistically significant (Basterra et al., 2001; Aparicio et al., 2001; Sharpe, 2001). Elevated CDT found in the blood of chronic alcoholics can also be seen in various medical conditions (ARUP Laboratories, 2010). The CDT levels are shown to be correlated with alcohol consumption and, possibly, acute pancreatitis in alcoholic patients
BIOMARKERS OF ACUTE AND CHRONIC PANCREATITIS
(Jaakkola et al., 1994). Overall, CDT appears to have some clinical utility, but could not be used by clinics for the diagnosis of acute or chronic pancreatitis, mainly due to specificity and cost of analysis reasons.
Activation by-products of trypsinogen and carboxypeptidase B Pro-enzyme trypsinogen occurs in two major isoenzymes in humans (trypsinogen 1, i.e. cationic trypsinogen form, and trypsinogen 2, i.e. anionic trypsinogen form), which are activated to trypsin by enterokinase (EK) in the gut for digestion. However, activation of trypsinogen within the acinar cell plays a key role in the pathogenesis of acute pancreatitis, because conversion of trypsinogen triggers a pancreatic enzyme cascade that activates the remaining zymogens, resulting in autodigestion of the surrounding tissue (Rinderknecht, 1986). Trypsin rapidly gets inactivated by 1-α-antitrypsin (AAT), a neutralization process. High serum concentrations of trypsin-1-AAT and trypsin-2-AAT have been reported in acute pancreatitis (Borgstro¨m and Ohlsson, 1978; Hedstro¨m et al., 1994). Trypsinogen activation peptide (TAP) is a by-product of the trypsinogen activation process. Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyzes proteins at the carboxyl side of the amino acids lysine or arginine. Trypsin produced due to premature trypsinogen activation in the pancreas ideally fulfills the criterion of organ specificity of a biomarker. The activation peptide at the amino terminus of vertebrate trypsinogen contains the sequence Asp-AspAsp-Asp-Lys (D4K, highly conserved during vertebrate evolution) as the carboxy-terminal moiety. These peptides are generally liberated in the proximal small intestine during digestion, after recognition and cleavage at the lysine carbonyl, by small gut EK. Intra-acinar activation of trypsinogen releases TAP into circulation, which is rapidly cleared by the kidneys for excretion in urine because of its small size. TAP can be quantitated by an enzyme-linked immunosorbent assay (ELISA) in the plasma and urine using a specific antibody to TAP (Wu et al., 2008). Only early acute or severe pancreatitis, as compared to mild acute pancreatitis, is correlated with serum and urinary levels of TAP, which are rapidly depleted to undetectable levels. Quick collection and analysis of samples is necessary and results may differ depending on the etiology of acute pancreatitis [e.g. alcohol- vs. gallstone-induced (Lempinen et al., 2003)]. Serum TAP appears to have close correlation with the severity of pancreatic injury and can be used as an early marker of acute pancreatitis. As compared to serum TAP, urinary TAP has a more consistent relationship with early acute pancreatitis. It has been reported that plasma TAP increased
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immediately after the induction of pancreatitis as compared to delayed excretion of TAP in the urine by several hours in an experimental pancreatitis model (Wang et al., 2001). About 30% of the patients with acute pancreatitis had normal TAP values on admission (Gudgeon et al., 1990). There are contradictory reports regarding the utility of serum and urinary TAP and these levels can remain undetected in mild cases of acute pancreatitis. Therefore, TAP levels only reflect severe acute pancreatitis (Kylanpa¨a¨-Ba¨ck et al., 2002; Johnson et al., 2004). However, the methods of analysis of trypsin and TAP are time-consuming and difficult and also not cost effective for routine clinical use. Like TAP, procarboxypeptidase B has an activation peptide, carboxypeptidase activation peptide (CAPAP), which is a larger peptide and more stable in the serum and urine than other peptides (Burgos et al., 1991; Abu Hilal et al., 2007). Serum and urinary levels of CAPAP were found to correlate well with severe acute pancreatitis based upon a study conducted in a small number of patients (Pezzelli et al., 2000; Appelros et al., 2001). Both TAP and CAPAP could serve as markers of early and severe forms of acute pancreatitis at the time of admission to hospital. Therefore, the utility of both markers needs to be tested in larger cohorts.
Elastase and phospholipase A2 Pancreatic elastase is a form of elastase and a subfamily of serine proteases (referring to the chymotrypsin-like elastase family), member 3B (CELA3B), that hydrolyze many proteins in addition to elastin. Human pancreatic elastase-1 (E1) is quite stable and remains undegraded during intestinal transit. Therefore its concentration in feces reflects exocrine pancreatic function. During an inflammation of the pancreas, E1 is released into the bloodstream. Thus the quantification of pancreatic elastase-1 in serum and feces allows diagnosis or exclusion of acute pancreatitis (Dominguez-Munoz et al., 2006; Naruse et al., 2006). Urinary excretion of elastase-1 has been shown to increase in all patients with chronic pancreatic disease regardless of the neoplastic or inflammatory nature of the disease (Fabris et al., 1989). Unfortunately, determination of elastase-1 does not provide any additional advantage over the combination of lipase and amylase for the diagnosis of acute pancreatitis and may not be a specific biomarker candidate. Phospholipase A2 specifically recognizes the sn-2 acyl bond of phospholipids and catalytically hydrolyzes the bond releasing arachidonic acid and lysophospholipids. However, phospholipase A2 can be found in several other organs and induced in chronic liver cirrhosis, thus lacking target-organ specificity (Vishwanath et al., 1996). Urinary excretion of phospholipase A2 in chronic pancreatic diseases particularly during relapse as well as in
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other physiological conditions has been reported (Fabris et al., 1992). Like trypsin and TAP, elastase and phospholipase A2 increase in serum and feces in acute pancreatitis, but could not be used as conventional diagnostic biomarkers for acute pancreatitis probably due to cost considerations and intricate analysis protocols.
Procalcitonin Procalcitonin is a precursor of calcitonin, and serum procalcitonin as measured using a chemoluminescent immunoassay was found to be a simple, promising biomarker with accuracy similar to acute physiology and chronic health examination (APACHE)-II score, in predicting severity of acute pancreatitis (Woo et al., 2011). However, precalcitonin can be useful only in predicting the severity of acute pancreatitis and is comparable to other biomarkers such as APACHE-II, C-reactive protein (CRP), lactate dehydrogenase, and urea.
Cathepsins Pancreatitis is shown to be associated with an increase in the active form of cathepsins B, L, and S, which regulate premature activation of trypsinogen within pancreatic acinar cells (Lyo et al., 2012). Cathepsins are lysosomal proteases and are postulated to play a role in the initiation of pancreatitis (van Acker et al., 2006). The levels of these cathepsins have been shown to increase in the pancreatic juice from patients with chronic pancreatitis. In the case of ethanol-induced pancreatic injury, cathepsins respond to FAEEs (nonoxidative metabolites of ethanol-induced pancreatic lysosomal fragility) (Haber et al., 1993). However, due to the lack of specificity, and analysis of cathepsins using mostly fluorimetrics, these are not cost effective alternatives as markers (Barrett, 1980).
Peptides Walgren et al. (2007) identified two peptide markers (RA1609 and RT2864) of pancreatic toxicity that are target-organ specific using a model pancreatic toxin cyanohydroxybutene. These markers were verified in the serum of patients diagnosed with pancreatitis, and changes in serum RA1609 and RT2864 were indicative of and specific to pancreatitis. The human sera analyzed in this study were from patients with pancreatitis, which was not defined as acute or chronic. Moreover, clinical use of such markers has yet to be established for a routine analysis.
C-reactive protein and pancreatitis-associated protein CRP, an acute phase protein primarily synthesized in the liver, is a class of proteins whose plasma
concentrations increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) in response to inflammation. Determination of the severity of acute pancreatitis is difficult in the early phase after onset, and often difficulties are encountered in making decisions to initiate intensive care during the early phase. CRP values increase significantly in early stages of necrotic pancreatitis. It is an important prognostic marker of pancreatic necrosis, with the highest sensitivity and negative prognostic value given the cut-off is 110 mg/L. The patients with CRP values below 110 mg/L are at low risk to develop pancreatic necrosis (Barauskas et al., 2004). Measurement of acute-phase proteins, especially CRP, is a useful marker of inflammation in both medical and veterinary clinical pathology. It correlates with the erythrocyte sedimentation rate. However, CRP is a marker of inflammation (proinflammatory cytokines) and higher CRP may also indicate liver failure (Ananian et al., 2005). Pancreatitis-associated protein (PAP), an acute phase protein like CRP, is overexpressed in acute pancreatitis and was detected in pancreatic juice from 49 patients with chronic pancreatitis. However, higher levels of PAP also detected in 15 control subjects discredited the significance of the PAP as a reliable biomarker of chronic pancreatitis (Motoo et al., 2001).
Inflammatory cytokines and chemokines Necrosis of exocrine pancreas, the primary cause of acute and chronic pancreatitis, can be identified by contrast-enhanced computed tomography and magnetic resonance imaging. Massive necrotic cell death follows strong inflammatory responses and infection. Proinflammatory cytokines and chemokines such as TNF, IL-1α, IL-1β, IL-6, IL-8, IL-12, and MCP-1 have been identified to serve as biomarkers of acute and chronic pancreatitis (Papachristou, 2008; Achur et al., 2010). However, the ability to predict which patients will develop severe disease is limited. Large prospective studies are still needed to address these questions by identifying risk factors for acute pancreatitis and serum biomarkers of severe disease conditions. Correlation of specific serum inflammatory cytokines and chemokines increasing or decreasing with severity of pancreatitis needs to be established before embarking on cytokines and chemokines as potential biomarkers of acute or chronic pancreatitis. Chronic pancreatitis is a chronic inflammatory disease. However, a caution should be exercised to use certain or a combination of inflammatory cytokines and chemokines, in view of broad inflammatory response in chronic diseases of diverse nature. Secondary target organ toxicity is also one of the potential interferences in such determinations.
BIOMARKERS OF ACUTE AND CHRONIC PANCREATITIS
Non-oxidative lipid metabolites of ethanol Ethanol is one of the major etiologic agents for acute and chronic alcoholic pancreatitis after biliary duct disease (Lankisch and Banks, 1998; Kaphalia, 2011). Non-oxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) is the most prevalent means of ethanol metabolism in the pancreas, frequently damaged during chronic alcohol abuse (Laposata and Lange, 1986). These esters are lipophilic and have been shown to cause selective pancreatic acinar cell toxicity (Werner et al., 1997; Kaphalia and Ansari, 2001; Gukovskaya et al., 2002; Vonlaufen et al., 2007; Wu et al., 2008). The plasma/serum levels of FAEEs are generally high and correlate well with blood alcohol concentration particularly during chronic alcohol abuse (Laposata and Lange, 1986; Kaphalia et al., 2004). Our studies in a deer mouse model of chronic ethanol feeding have shown that increased levels of plasma FAEE correlate well with those in the pancreas (Kaphalia et al., 2010). However, an interrelationship between plasma/serum levels of FAEEs and severity of pancreatitis in patients with acute or chronic pancreatitis needs to be established. Other non-oxidative metabolites of ethanol which correlate with blood alcohol levels in the blood include phosphatidylethanol (PEt) formed by the phospholipase D catalyzed reaction (Aradottir et al., 2006). A good clinical efficiency of PEt and its pancreatic toxicity have not been demonstrated so far for detecting chronic heavy drinking (Viel et al., 2012).
Differentially altered proteins, metabolites, small molecules, and microRNA Direct functional tests can be conducted by identifying the proteins, metabolites (small molecule), and microRNAs differentially altered in the pancreatic juice and duodenal contents from proteins with acute or chronic pancreatitis. Omic and array research is a systemic global approach and provides a unique “fingerprint,” which varies with time or stresses, that a cell or organism undergoes. High resolution 1H-NMR analysis of plasma and urine samples and data analysis as determined by principal component analysis (PCA) using metabolomic modeling of L-arginine-induced exocrine pancreatitis suggest metabolomics is a valuable approach, which can distinguish patients with pancreatitis from healthy controls (Bohus et al., 2008; Lusczek et al., 2013). Lipid profiling of serum and pancreatic fluid conducted in chronic pancreatitis using an LC-ESI-MS/MS system in five controls and six patients of mild and five of severe chronic pancreatitis showed an increase in the levels of oxidized fatty acid products, suggesting their utility as biomarker candidates for the diagnosis of
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chronic pancreatitis (Stevens et al., 2012). However, the specificity of such biomarkers needs to be established. Identification of proteins has been done only in the pancreas in acute pancreatitis models (Fe´taud et al., 2008; Garcia-Herna´ndez et al., 2012; Zhang et al., 2012). Therefore, significant efforts should be directed at profiling both plasma and pancreatic proteins differentially, expressed in acute and chronic pancreatitis in animal models, followed by their validation in the plasma of patients with acute or chronic pancreatitis. Proteomic profiling using SELDI-TOF-MS has been done in the serum as a predictor of severity of acute pancreatitis from 21 patients with mild and 7 patients with severe acute pancreatitis (Papachristou et al., 2007). Various other proteomic studies (separation of proteins by twodimensional polyacrylamide gel electrophoresis and identification by MALDI-TOF/TOF) done on the pancreatic fluid, plasma, serum, and pancreatic tissue from patients with chronic pancreatitis provide significant leads for further pursuing studies in larger cohorts (Chen et al., 2007; Hartmann et al., 2007). While gene profiling studies can also identify biomarker candidates, studies are limited to the target organ and to use in experimental models. Pathway analysis and systems biology approaches, generally performed to infer relationships between genes, proteins, and small molecules (metabolomics), are important for identification of biomarkers and may be a future direction in the biomarker field. MicroRNAs (miRNAs) are functional, 22 nt, noncoding RNAs that negatively regulate gene expression and play a role in the initiation and progression of certain diseases. However, the identity of differentially altered target microRNA should be confirmed. Hierarchical clustering and principal component analysis of the data sets can distinguish pancreatic tissue of patients with pancreatitis from normal control tissue. Only a handful of studies are available to identify miRNAs as biomarkers in experimental rat models of acute or chronic pancreatitis, and in pancreatic cancer (Bloomston et al., 2007; Lee et al., 2007; Kong et al., 2010). Kong et al. (2010) identified plasma miR-216a as a potential marker of pancreatic injury in a rat model of acute pancreatitis. Like amylase and lipase, increased expression of miR-216a in the plasma was restricted to acute pancreatitis at 24 hr after the insult. Such miR-based markers need to be confirmed in patients with acute and chronic pancreatitis. In one study, two dozen miRNAs were shown to be differentially altered in the pancreatic tissue from patients with chronic pancreatitis (n 5 42) as compared to those in adjacent normal tissue as control (Bloomston et al., 2007). These studies are very preliminary and need further investigation to establish their utility as biomarker candidates in clinical diagnostics. Even if miRNA microarray technologies identify some promising biomarkers of disease/injury, cost and precision are other factors to be considered.
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Breath analysis Human exhaled breath contains many molecules either present as gases or occurring in a soluble form in the vapor of the breath. One can rapidly and easily assess the presence of volatile compounds (hydrogen sulfide, nitric oxide, and molecular mass 66 u substance) in the exhaled breath of chronic pancreatitis patients (Morselli-Labate et al., 2007). Although a study was conducted in 11 patients with chronic pancreatitis and 31 healthy subjects and the marker appears novel, no significant differences were found between patients and healthy controls.
Comparison of biomarkers between acute and chronic pancreatitis Basic laboratory tests for diagnosing acute and chronic pancreatitis are vastly different because the former is short-term, largely indicated by significant increases for amylase, isoamylase, lipase, TAP, trypsin, elastase-1, phospholipase A2, CDT, CRP, PAP, CAPAP, procalcitonin in serum, plasma, or urine, and other markers such as cathepsins in pancreatic juice. The indirect pancreatic function tests in serum or urine can be evident in early stages of chronic pancreatitis, but a number of biomarkers as mentioned above may be in the normal range or fluctuate depending upon the chronicity of the pancreatitis, because slow destruction of the exocrine gland results in exocrine pancreatic insufficiency. Therefore, testing exocrine pancreatic insufficiency is still recommended when chronic pancreatitis is suspected. Although several tests, including the secretin test, cerulein test and Lundh test, have been described in detail by Lankisch and Banks (1998), fecal tests for trypsin, chymotrypsin, and elastase-1 can also provide some clues for presence of chronic pancreatitis. Most of the methods used are either titrimetric estimation or ELISA. Moreover, fecal chymotrypsin estimation is not novel. Most of these tests are not even sensitive enough to diagnose mild to moderate exocrine pancreatic insufficiency. Fecal fat estimation, particularly when stimulated lipase and protease output falls below 10% of the normal, is a safe biomarker for steatorrhea (DiMagno et al., 1973). However, fecal fat contents were of no value for differential diagnosis of pancreatic and nonpancreatic steatorrhea (Roberts et al., 1986). Pancreas-specific chymotrypsin splits N-benzyl-L-tyrosyl-p-aminobenzoic acid to p-aminobenzoic acid (NTBPABA) in the duodenum. Estimation of p-aminobenzoic acid (PABA) cannot be accurate, since some gut bacteria also split NBT-PABA, causing a false normal test result. Chronic pancreatitis can be diagnosed by serum, urinary, and fecal biomarkers of pancreatic functions in
conjunction with patient history and imaging (CT, MRCP, ERCP) results. In view of available biomarkers and nature of pathology, a single biomarker should not be sufficient to diagnose chronic pancreatitis. Identification and development of metabolomic-, proteomic-, and miRNA-based biomarkers should be undertaken in experimental models applied to patients at various stages of acute and chronic pancreatitis.
BIOMARKERS OF ENDOCRINE AND AUTOIMMUNE PANCREATITIS Endocrine functional impairment with insulin resistance can be used as an index of endocrine pancreatitis (Wu et al., 2011). Type 1 diabetes mellitus (loss of insulinsecreting capacity) is now classified as autoimmune (type 1A) or idiopathic (type 1B). Presence or absence of glutamine acid decarboxylase antibodies can differentiate type 1 diabetes. Some patients with idiopathic type 1 diabetes may also have nonautoimmune and remarkably abrupt onset and high serum pancreatic enzyme levels (Imagawa et al., 2000). Presence of elevated titers of autoantibodies directed to amylase α-2A may represent a novel specific biomarker of autoimmune pancreatitis and fulminant type 1 diabetes. A correlation between urinary p-aminobenzoic acid (PABA, a split product of n-benzoyl-1-tyrosyl-p-aminobenzoic acid) excretion and plasma glucagon concentration suggest that in chronic pancreatitis there is collateral impairment of exocrine and endocrine functions (Keller et al., 1984).
BIOMARKERS FOR EARLY DETECTION OF PANCREATIC CANCER Although the focus of this chapter is biomarkers of pancreatitis (acute and chronic), early detection of pancreatic cancer is also critical for the success of interventional and surgical therapies. Pancreatic cancer is a most devastating and fatal disease, without any overt symptoms at initial stages. Most common exocrine pancreatic cancers are pancreatic ductal adenocarcinoma (PDAC) followed by adenosquamous carcinomas and broad-based pancreatic cystic neoplasma. However, pancreatic neuroendocrine tumors constitute a small number of cases, B1% of all pancreatic cancers. Endocrine pancreatic tumors have been variously called islet cell tumors, pancreas endocrine tumors (PETs), and pancreatic neuroendocrine tumors (PNETs).
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REFERENCES
The annual clinically recognized incidence is low, about five per one million person/years. However, autopsy studies incidentally identify PETs in up to 1.5%, most of which would remain inert and asymptomatic. The majority of PNETs are usually categorized as benign but the definition of malignancy in pancreas endocrine tumors has been ambiguous. The more aggressive endocrine pancreatic cancers are known as pancreatic neuroendocrine carcinomas (PNEC). Similarly, there has likely been a degree of admixture of PNEC and extrapulmonary small cell carcinoma. Most of the biomarkers of pancreatic cancers, identified for PDAC, are carcinoembryonic (CEA), carbohydrate antigen (CA19-9) and mucin family (MUC), the latter being most commonly used along with circulating tumor cells; markers in pancreatic juice and signaling pathway have been extensively reviewed (Tanase et al., 2009; Chakraborty et al., 2011; Poruk et al., 2013). Early detection of pancreatic cancer offers the promise of improved mortality rates through surgical resection. Therefore, biomarkers should be determined for the early stages of pancreatic cancer to improve prognosis and mortality rates by using intervention therapies. For further details on biomarkers of pancreatic cancer, readers are referred to Chapter 45.
CONCLUDING REMARKS AND FUTURE DIRECTIONS Reliable and sensitive biomarkers of acute and chronic pancreatitis and pancreatic cancers are needed, as current available biomarkers have limitations and lack specificity. Serum total amylase and lipase in combination or individually remains the gold standard for functional clinical diagnostics of acute pancreatitis, if the test is conducted on admission of the patient to the hospital. Lipase assay has a definite advantage over amylase in clinical diagnostics and has more specificity for diagnosing acute pancreatitis. However, imaging technologies provide strong support for diagnosis of both acute and chronic pancreatitis. Identification and development of biomarkers of chronic pancreatitis remain as research challenges, because several indirect pancreatic functional biomarkers are inconclusive, primarily because of the impaired secretory component of exocrine pancreas due to the slow destruction of the gland in chronic pancreatitis, particularly at later stages. The focus of future research lies in identification of molecular biomarkers by utilizing proteomic, metabolomic, and miRNA array technologies, using very well-defined patient populations.
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