|Role of proteases and antiprotease in the etiology of chronic pancreatitis.|
|Jump to Full Text|
|PMID: 23150021 Owner: NLM Status: MEDLINE|
|BACKGROUND/AIM: Chronic pancreatitis (CP) is the progressive and irreversible destruction of the pancreas characterized by the permanent loss of endocrine and exocrine function. Trypsin, the most important digestive enzyme plays a central role in the regulation of all other digestive enzymes. Chymotrypsin, an endopeptidase hydrolyzes peptides at amino acids with aromatic side chains. Alpha-1-antitrypsin is a principal antiprotease which protects the mucosal tissue from the proteolytic effects of trypsin and chymotrypsin by the formation of molar complexes. The present study is aimed at examining the role of proteases (trypsin and chymotrypsin) and anti-protease (α1-anti-trypsin) in the etiopathogenesis of chronic pancreatitis.
PATIENTS AND METHODS: A total of 90 CP patients and 110 age and sex matched controls were considered for the study. Serum trypsin, chymotrypsin and α1-anti-trypsin levels were determined prospectively in CP patients and compared to healthy controls as described previously.
RESULTS: The mean activity of trypsin were found to be increased in CP patients (X ± SD = 0.82 ± 0.838) in comparison to normal control group (X ± SD = 0.55 ± 0.328), (P = 0.001). Chymotrypsin activity were also found to be elevated in CP patients (X ± SD = 0.63 ± 0.278) in comparison to control group (X ± SD = 0.39 ± 0.295), (P = 0.0001). The mean α-1-anti-trypsin activity were found to be lowered in CP patients (X ± SD = 0.42 ± 0.494) in comparison to control group (X ± SD = 0.67 ± 0.465), with the variation being significant (P = 0.0003).
CONCLUSION: The findings suggest an imbalance in the synthesis and degradation of proteolytic enzymes and antiprotease indicating an altered aggressive and defensive role in the pathogenesis of chronic pancreatitis.
|Srimanjari Kavutharapu; Balakrishna Nagalla; Vidyasagar Abbagani; Shravan K Porika; Jyothy Akka; Pratibha Nallari; Venkateshwari Ananthapur|
|Type: Comparative Study; Journal Article; Research Support, Non-U.S. Gov't|
|Title: Saudi journal of gastroenterology : official journal of the Saudi Gastroenterology Association Volume: 18 ISSN: 1998-4049 ISO Abbreviation: Saudi J Gastroenterol Publication Date: 2012 Nov-Dec|
|Created Date: 2012-11-15 Completed Date: 2013-05-07 Revised Date: 2013-07-11|
Medline Journal Info:
|Nlm Unique ID: 9516979 Medline TA: Saudi J Gastroenterol Country: India|
|Languages: eng Pagination: 364-8 Citation Subset: IM|
|Department of Cell Biology, Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad, India.|
|APA/MLA Format Download EndNote Download BibTex|
Biological Markers / blood
Pancreas / metabolism*
Pancreatitis, Chronic / blood*
Peptide Hydrolases / blood*
Trypsin / blood*
alpha 1-Antitrypsin / blood*
|0/Biological Markers; 0/alpha 1-Antitrypsin; EC 3.4.-/Peptide Hydrolases; EC 22.214.171.124/Trypsin|
Journal ID (nlm-ta): Saudi J Gastroenterol
Journal ID (iso-abbrev): Saudi J Gastroenterol
Journal ID (publisher-id): SJG
Publisher: Medknow Publications & Media Pvt Ltd, India
Copyright: © Saudi Journal of Gastroenterology
Received Day: 23 Month: 9 Year: 2011
Accepted Day: 07 Month: 7 Year: 2012
Print publication date: Season: Nov-Dec Year: 2012
Volume: 18 Issue: 6
First Page: 364 Last Page: 368
PubMed Id: 23150021
Publisher Id: SJG-18-364
|Role of Proteases and Antiprotease in the Etiology of Chronic Pancreatitis|
|Shravan K. Porika3|
|Department of Cell Biology, Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad, India
1Division of Biostatistics, National Institute of Nutrition, Hyderabad, India
2Department of Gastroenterology, Gandhi Hospital, Hyderabad, India
3Department of Genetics, Osmania University, Hyderabad, India
|Correspondence: Address for correspondence: Dr. Venkateshwari Ananthapur, Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad, India. E-mail: firstname.lastname@example.org
Chronic pancreatitis is the progressive and irreversible destruction of the pancreas, characterized by permanent loss of endocrine and exocrine function. The incidence of chronic pancreatitis (CP) ranges from two to ten per 100,000, and has an estimated prevalence of 25/100,000. Disease characteristics include inflammation, glandular atrophy, ductal changes, and fibrosis. It is presumed that exposure to toxins and oxidative stress, result in acute pancreatitis,[4, 5] which if continued leads to, early and late-phase inflammatory responses with the production of profibrotic cells and stellate cells, culminating in collagen deposition, periacinar fibrosis, and chronic pancreatitis.
Pancreatic acinar cell secretion results in three major categories of enzymes: Amylolytic, lipolytic and proteolytic enzymes which are capable of using carbohydrates, fats and proteins respectively. Acinar cell secretion is primarily induced by the ingestion of food, which initiates activation of multiple endocrine, neurocrine and paracrine pathways, regulating the release of appropriate amounts of acinar digestive enzymes. The proteolytic class of pancreatic enzymes secreted as inactive precursors into the duodenum consists of trypsin, chymotrypsin and carboxypolypeptides wherein the trypsin, chymotrypsin and elastase are endopeptidases of the serine protease family of enzymes.
Trypsin, unlike amylase and lipase, is only produced by the pancreas. Trypsinogen is one of the most important digestive enzymes as it plays a central role in the regulation of all the other digestive enzymes. Trypsin makes up to 19% of the protein in pancreatic juice, and is the most abundant of all pancreatic digestive enzymes. Chymotrypsinogen is the second most abundant serine protease in pancreatic secretion and makes up to 9% of total pancreatic juice protein. Chymotrypsin is an endopeptidase that hydrolyzes peptides at amino acids with aromatic side chains, including phenylalanine, tyrosine, and tryptophan. Alpha-1-antitrypsin (α-1AT) is a principal serum protease inhibitor which protects the mucosal tissue from proteolytic enzymes like trypsin and chymotrypsin by the formation of molar complexes. The enzymes that are inhibited by alpha-1-antitrypsin are particularly the serine proteases, which are characterized by a serine residue at their active sites. Thus, the present study is aimed at evaluating the role of proteases (trypsin and chymotrypsin) and anti-protease (α-1AT) in the etiopathogenesis of chronic pancreatitis.
A total of 90 radiologically and endoscopically confirmed chronic pancreatitis patients referred to the Gastroenterology unit of Osmania General Hospital and Gandhi Hospital, Hyderabad are included in the present study. A total of 110 sex matched healthy control subjects referred to our Institute for regular health check were also considered for comparative studies.
Demographic details such as age, sex, habits like smoking and alcohol consumption, duration of the disease, were obtained with the help of a standard proforma from all the subjects. The study was also approved by the Institutional Ethical committee. Blood samples were collected in plain vacutainers from all the subjects. Serum was separated and stored frozen until further use.
Estimation of serum trypsin activity was carried out in serum samples of all the subjects following the method of Varley, (1991). The acid liberated due to the hydrolysis of p-Toluene-Sulfonyl-2-arginine-Methyl ester (TAME) by the action of trypsin was measured spectrophotometrically at 300nm against a blank. 2.6 ml of Tris-HCl buffer and 0.3ml of TAME are taken in a test tube. The tubes were incubated for 5 minutes, to which 100 μl of serum was added and the OD was measured against a blank.
Estimation of serum chymotrypsin activity was carried out in serum samples of all the subjects according to the method of Varley, (1991). The acid liberated due to the hydrolysis of Acetyl-L-Tyrosine Ethyl Ester (ATEE) by action of chymotrypsin was measured spectrophotometrically at 300nm against a blank. To each tube 1.5ml of Tris-HCl and 1.4ml of ATEE solution were added. Later 100μl of serum was added to the test sample and 100μl of distilled water to the blank tube and the absorbance was read at 300nm against a reagent blank.
Serum Alpha-1-anti-trypsin activity was carried out in serum samples of all the subjects according to the method of Ahmed et al., (1979). The (α-1AT) activity in the serum was measured spectrophotometrically by the amount of tyrosine liberated on action of trypsin with hemoglobin by measuring the trypsin inhibiting capacity. To the test tube labeled as test, 1ml of 0.05M phosphate buffer, 1ml of trypsin (0.08mg/1ml of 0.001N HCl) and 1 ml of hemoglobin solution were mixed to which 40μl of serum was added and incubated for one hour at 37°C. The control reaction was carried out in the same way with the addition of serum after incubation. This was followed by addition of 2ml of 20% Trichloro acetic acid to both test and control tubes which were incubated for 10 minutes at room temperature. The solution was centrifuged till no precipitate was left suspended. Later, 250μl of the clear supernatant was taken in separate tubes labeled as test and control to which 250μl of 0.6N Folin's Phenol reagent and 1.5 ml of sodium carbonate solution was added to both test and control tubes and incubated at room temperature for 20 minutes till the solution turned blue. The absorbance was then read against water blank at 680nm in a spectrophotometer.
Statistical analysis was carried out to interpret the variation of serum levels of trypsin, chymotrypsin and alpha 1 antitrypsin with regard to demographic parameters using IBM, SPSS version 17 software. The Pearson's χ2 test was also carried out to examine the differences between the case and the control groups with respect to age, sex, smoking, and alcoholism. Odds ratio (ORs) and 95% confidence intervals for the factors under consideration were computed by logistic regression analysis.
A total of 110 controls and 90 chronic pancreatitis patients were included in the present study. The demographic data of the samples under study are presented in Table 1. The proportion of smokers in CP patients (52.2%) was significantly higher compared to healthy controls (19.1%) [χ2 = 24.21, P < 0.0001]. Similarly, the proportion of patients above the age of 35 years in CP patients (61.1%) was significantly higher than that in healthy controls (12.7%) [χ2 = 51.3, P < 0.0001]. However, no significant difference was found with respect to gender and alcoholism on comparison of chronic pancreatitis patients with healthy controls (P > 0.05).
The mean activity of pancreatic enzymes in controls and chronic pancreatitis patients is presented in Table 2. The mean activity of trypsin was found to be increased in chronic pancreatitis patients (X ± SD = 0.82 ± 0.838) in comparison to normal control group (X ± SD = 0.55 ± 0.328), (t = 2.945, P = 0.001), indicating its possible role in the tissue damage and fibrosis of the pancreas. Chymotrypsin activity was also found to be elevated in chronic pancreatitis patients (X ± SD = 0.63 ± 0.278) in comparison to control group (X ± SD = 0.39 ± 0.295), ( t = 5.884, P<0.0001), suggesting its role in the tissue damage. The mean α-1-anti-trypsin activity was found to be lowered in chronic pancreatitis patients (X ± SD = 0.42 ± 0.494) in comparison to control group (X ± SD = 0.67 ± 0.465), with the variation being significant (t = 3.619, P = 0.0003). It indicates lowered defensive role of alpha-1-anti trypsin against proteolytic enzymes in the pancreas.
Correlation coefficients of the mean levels of trypsin, chymotrypsin and alpha 1 antitrypsin also revealed increased proteolytic enzymes and lowered alpha 1 antitrypsin which may further aggravate the pancreatic damage, and progression of the disease [Table 3].
Logistic regression analysis revealed a significant association of demographic variables like age, smoking and protease chymotrypsin and alpha 1 antitrypsin in the etiopathology of CP was observed, further strengthening the role of these factors [Table 4].
Chronic pancreatitis is an inflammatory disease of the pancreas. The digestive juice/enzymes secreted into the duodenum by the pancreatic duct include trypsin, chymotrypsin and α-1-anti-trypsin which are zymogenic and get activated on reaching the small intestine. In an inflamed pancreas, the enzymes directly attack the pancreatic tissue, resulting in an imbalance in the synthesis of proteolytic enzymes and antiproteases and autodigestion of the pancreatic tissue culminating as CP.
Trypsin activity is properly suppressed in the pancreatic acinar cells under normal conditions, as small amount of trypsinogen is converted to active trypsin and inactivated by pancreatic secretory trypsin inhibitor (PSTI), thereby preventing damage to pancreatic acinar cells as a first line of defense. Serum trypsin may be normal or subnormal in chronic pancreatitis (Elias et al., 1977). Previous studies have revealed that exocrine insufficiency is invariably associated with a low trypsin concentration,[15, 16] but a normal concentration has been reported. However, increased levels observed in the present study in CP compared to controls suggests that proteolytic activity of trypsin on pancreas may result in the cascade of events which further results in the activation of other proteases with subsequent cell damage. Chymotrypsin activity was also found to be significantly elevated in chronic pancreatitis patients when compared to controls. Elevated levels of chymotrypsin may result in further inflammation and damage of the pancreatic tissue.
Alpha-1 antitrypsin (α-1AT) is a known protease inhibitor, of trypsin and chymotrypsin. These proteases when left unchecked by the protease inhibitor can cause destruction of normal tissues. Thus, the low serum activity of this glycoprotein results in an imbalance between circulating proteases as aggressive enzymes are thought to play a role in the development of chronic pancreatitis. Studies by Haber et al., (1991), have also shown that mortality of experimental pancreatitis is increased with reduced concentrations of circulating α-1AT. The decrease in the antiprotease may result in the activation of proteases that release into the blood stream leading to membrane damage.
An infiltration of inflammatory cells including neutrophils, monocytes, and macrophages, play a critical role in tipping the protease-antiprotease balance toward protease excess. The α-1AT protein is involved in processes which could be important for the maintenance of normal pancreatic function, like regulation of reactive oxygen species toxicity, cell-mediated immunity/tolerance, neutrophil and endotoxin mediated inflammation, endothelial function and apoptosis. A deficiency of this enzyme may lead to an excess release of inflammatory cytokines and free enzyme that in turn drives inflammatory responses, which further activates the pancreatic stellate cells resulting in fibrosis.
When α-1AT concentration and isozymes phenotyping are taken into account, the conflicting data revealed the involvement of the α-1AT variants in chronic pancreatitis. Some findings indicated increased prevalence of PiS and PiZ in chronic alcoholic patients, while others indicated no difference.[18, 22, 23] Serum α-1AT concentrations were greater in patients with pancreatitis who were sampled shortly after an acute attack of pancreatitis compared to pancreatitis patients sampled at other times, or to alcoholic controls. Increased α-1AT concentration was found in pancreatic juice of patients with chronic pancreatitis by Miszczuk-Jamska et al.(1983) It was speculated that this could alter the balance in pancreatic protease-antiprotease system and subsequently lead to chronic pancreatitis.
Correlation coefficient studies revealed heterogeneity of the disease with regard to protease and antiprotease. A significantly elevated level of trypsin, resulting in pancreatic tissue damage was observed in a section of subjects while the other group of individuals exhibited an increased levels of chymotrypsin and lowered levels of α-l-antitrypsin resulting in an increased pancreatic damage with elevated proteolytic enzymes and lowered anti-proteases. The second group revealed lowered action of α-l-antitrypsin on the regulation of proteolytic enzymes like trypsin and chymotrypsin in the patients. Thus, the present study highlights the possible genetic heterogeneity of chronic pancreatitis with respect to the levels of trypsin, chymotrypsin and α-1-anti trypsin activity in the disease.
Overall, the aggressive proteolytic enzymes trypsin and chymotrypsin were found to be elevated while the defensive alpha-1 anti-trypsin was lowered, further confirming the tilt in homeostasis of proteolytic and anti-proteolytic enzyme secretion. If proteinase inhibitors like alpha-1-anti-trypsin alter their normal function, limiting of unwanted proteolysis and prevention of tissue damage cannot be achieved appropriately. The lowered alpha-1-anti-trypsin can justify the altered immune and defense in CP, resulting in an increase in trypsin and chymotrypsin which later promotes progression of the disease with a history of bad prognosis.
In conclusion, our results indicate a significant increase in the activity of trypsin and chymotrypsin activity and a decrease in α-1-anti trypsin activity which activates further and initiate subsequent cascade of events resulting in the pancreatic acinar cell destruction and chronic pancreatitis.
Source of Support: University Grants Commission (UGC), New Delhi [File No: 36.185/2008 (SR)]
Conflict of Interest: None declared.
The authors acknowledge financial support from University Grants Commission (UGC), New Delhi [File No: 36-185/2008 (SR)].
|1.||Nair RJ,Lawler L,Miller MR. Chronic pancreatitisAm Fam PhysicianYear: 20077616798818092710|
|2.||Stephen TA. Chronic pancreatitisCurr Treat Options GastroenterolYear: 199924018|
|3.||Schneider A,Whitcomb DC. Hereditary pancreatitis: A model for inflammatory diseases of the pancreasBest Pract Res Clin GastroenterolYear: 2002163476312079262|
|4.||Ahmad SA,Wray C,Rilo HL,Choe KA,Gelrud A,Howington JA,et al. Chronic pancreatitis: Recent advances and ongoing challengesCurr Probl SurgYear: 20064312723816530053|
|5.||Keiles S,Kammesheidt A. Identification of CFTR, PRSS1, and SPINK1 mutations in 381 patients with pancreatitisPancreasYear: 200633221717003641|
|6.||Etemad B,Whitcomb DC. Chronic pancreatitis: Diagnosis, classification, and new genetic developmentsGastroenterologyYear: 200112068270711179244|
|7.||Elias E,Redshaw M,Wood T. Diagnostic importance of changes in circulating concentrations of immunoreactive trypsinLancetYear: 1977266869150|
|8.||Rinderknecht H,Renner IG,Carmack C. Trypsinogen variants in pancreatic juice of healthy volunteers, chronic alcoholics, and patients with pancreatitis and cancer of the pancreasGutYear: 19792088691533700|
|9.||Carrere J,Figarella C,Guy O,Thouvenot JP. Human pancreatic chymotrypsinogen A: A non-competitive enzyme immunoassay, and molecular forms in serum and amniotic fluidBiochim Biophys ActaYear: 198688346533524693|
|10.||Schultze HE,Heide K,Haupt H. Alpha-1-antitrypsin from human serumKlin WochenschrYear: 196240427913909528|
|11.||Travis J,Salveson GS. Human plasma proteinase inhibitorsAnnu Rev BiochemYear: 198352665709|
|12.||Varley H,Gowenlock AH,Bell M. Practical Clinical BiochemistryYear: 199115th edLondonWilliam Heinemann Books Ltd548|
|13.||Miles EW,Bauerle R,Ahmed SA. Tryptophan synthase from Escherichia coli and Salmonella typhimuriumMethods EnzymolYear: 19871423984143298982|
|14.||Etemad B,Whitcomb DC. Chronic pancreatitis: Diagnosis, classification, and new genetic developmentsGastroenterologyYear: 200112068270711179244|
|15.||Lake-Bakaar G,McKavanagh S,Redshaw M,Wood T,Summerfield JA,Elias E. Serum immunoreactive trypsin concentration after a Lundh mealJ Clin PatholYear: 19793210038521492|
|16.||Adrian TE,Besterman HS,Mallinson CN,Pera A,Redshaw MR,Wood TP,et al. Plasma trypsin in chronic pancreatitis and pancreatic adenocarcinomaClin Chim ActaYear: 19799720512487604|
|17.||Koop H,Lankisch PG,Stockmann F,Arnold K. Trypsin radioimmunoassay in the diagnosis of chronic pancreatitisDigestionYear: 19802015167390044|
|18.||Rabassa AA,Schwartz MR,Ertan A. Alpha 1-antitrypsin deficiency and chronic pancreatitisDig Dis SciYear: 199540199720017555455|
|19.||Haber PS,Wilson JS,McGarity BH,Hall W,Thomas MC,Pirola RC. Alpha 1-antitrypsin phenotypes and alcoholic pancreatitisGutYear: 19913294581885078|
|20.||Hinman LM,Stevens CA,Matthay RA,Gee JB. Elastase and lysozyme activities in human alveolar macrophages.Effects of cigarette smokingAm Rev Respir DisYear: 1980121263716899736|
|21.||Sandstrom CS,Ohlsson B,Melander O,Westin U,Mahadeva R,Janciauskiene S. An association between type 2 diabetes and alpha-1-antitrypsinDiabet MedYear: 2008251370319046232|
|22.||Braxel CJ,Versieck J,Lemey G,Vanballenberghe L,Barbier F. Alpha 1-antitrypsin in pancreatitisDigestionYear: 1982239366980154|
|23.||Edmunds SE,Wilkinson ML. Alpha 1-antitrypsin deficiency and pancreatitis in a juvenileAust N Z J MedYear: 19912134571953516|
|24.||Miszczuk-Jamska B,Guy O,Figarella C. Alpha 1-proteinase inhibitor in pure human pancreatic juice. Characterization of a complexed form in patients with chronic calcifying pancreatitis and its significanceHoppe Seylers Z Physiol ChemYear: 19836415976016607205|
Distribution of demographic details of controls and chronic pancreatitis patients
Mean levels of pancreatic enzymes (trypsin, chymotrypsin, α1-anti trypsin) in controls and chronic pancreatitis groups
Keywords: Alpha 1 anti-trypsin, chronic pancreatitis, chymotrypsin, trypsin.
Previous Document: Efficacy of endoscopic ultrasound guided fine needle aspiration in patients with solid pancreatic ne...
Next Document: Correlation of serology with morphological changes in gastric biopsy in Helicobacter pylori infectio...