Serum Trypsinogen Research Articles

Covering the Cover

Covering the Cover

WS1.2 Rate of decline in serum trypsinogen as a validation for the PIP score

WS1.2 Rate of decline in serum trypsinogen as a validation for the PIP score

Neutrophil-derived matrix metalloproteinase-9 is a potent activator of trypsinogen in acinar cells in acute pancreatitis

MMPs are generally considered to regulate degradation and remodeling of the ECM. Convincing data also implicate a role for MMPs in inflammatory conditions, such as AP, although the mechanisms are not known. The aim of this study was to define the role of MMPs in regulating activation of trypsinogen and tissue damage in AP, which was induced by infusion of taurocholate into the pancreatic duct in mice. A broad-spectrum MMP inhibitor (BB-94) and MMP-9 gene-deficient mice were used. Neutrophil secretions and rMMP-9 were used to stimulate trypsinogen activation in isolated acinar cells. Taurocholate challenge increased serum amylase, neutrophil infiltration, MIP-2 (CXCL2) formation, trypsinogen activation, and tissue damage in the pancreas. Treatment with the broad-spectrum inhibitor of MMPs, BB-94, markedly reduced activation of trypsinogen, levels of CXCL2, infiltration of neutrophils, and tissue damage in AP. Taurocholate challenge increased serum levels of MMP-9 but not MMP-2. Taurocholate-induced amylase levels, neutrophil accumulation, production of CXCL2, trypsinogen activation, and tissue damage in the pancreas were abolished in MMP-9-deficient mice. Moreover, secretions from activated neutrophils isolated from WT but not from MMP-9-deficient animals stimulated trypsinogen activation in acinar cells. Notably, rMMP-9 greatly enhanced activation of trypsinogen in acinar cells. These findings demonstrate that neutrophil-derived MMP-9 is a potent activator of trypsinogen in acinar cells and regulates pathological inflammation and tissue damage in AP.

Assessment of Chronic Pancreatitis

OBJECTIVE: We aimed to compare perfusion computed tomography (CTP) characteristics of the normal pancreas with those of chronic pancreatitis (CP) and to examine the possibility of evaluating pancreatic exocrine function with CTP. METHODS: Thirty-two patients (control group, n = 18; CP group, n = 14) who completed the whole pancreas CT perfusion examination with 256-slice CT were studied. Four parameters, including perfusion (PF), peak enhancement intensity (PEI), time-to-peak (TTP), and blood volume (BV), were measured and compared between the control and CP groups, and between patients with and without exocrine pancreatic insufficiency (EPI) in the CP group. Pancreatic exocrine function was determined via serum trypsinogen. RESULTS: There was no significant difference between the distribution of PF, PEI, and BV in different pancreas regions, namely, the head, body, and tail (P > 0.05). PF, PEI, and BV of the CP group were significantly decreased, and TTP was significantly increased compared with the control group (P < 0.05). A significant decrease of PF, PEI, and BV and increase of TTP were observed in patients with EPI than in patients without EPI (P < 0.05). CONCLUSIONS: Perfusion CT is an appropriate imaging technique to diagnose CP and may be useful as a screening test to rule out early EPI.

Specific Immunoassay Reveals Increased Serum Trypsinogen 3 in Acute Pancreatitis

Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin 1 and 2, trypsin 3 degrades pancreatic secretory trypsin inhibitor, which may lead to an excess of active trypsin and acute pancreatitis (AP). We developed an immunoassay for trypsinogen 3 and studied whether an assay of serum trypsinogen 3 is of clinical utility in the diagnosis of AP. Monoclonal antibodies were generated using recombinant human trypsinogen 3 as the antigen and used to establish a sandwich-type immunoassay. We analyzed serum trypsinogen 3 concentrations in 82 patients with AP and 63 patients with upper abdominal pain (controls). The reference interval was determined using serum samples from 172 apparently healthy individuals. The measuring range of the trypsinogen 3 assay was 1.0-250 μg/L. Intra- and interassay CVs were <11%, and cross-reactivity with other trypsinogen isoenzymes was <0.1%. The median trypsinogen 3 concentration in serum from healthy individuals was <1.0 μg/L, and the upper reference limit was 4.4 μg/L. We observed increased trypsinogen 3 concentrations in patients with mild (median 9.5 μg/L) and severe (15.0 μg/L) AP; in both groups, the concentrations were significantly higher than in controls (median <1.0 μg/L) (P < 0.0001). In ROC analysis, the area under the curve of trypsinogen 3 for separation between AP and controls was 0.90 (P < 0.0001). We established for the first time a specific immunoassay for trypsinogen 3 using monoclonal antibodies. Patients with AP were found to have increased serum concentrations of trypsinogen 3. The availability of this assay will be useful for studies of the clinical utility of trypsinogen 3.

Lymphocyte function antigen‐1 regulates neutrophil recruitment and tissue damage in acute pancreatitis

Leucocyte infiltration is a rate-limiting step in the pathophysiology of acute pancreatitis (AP) although the adhesive mechanisms supporting leucocyte-endothelium interactions in the pancreas remain elusive. The aim of this study was to define the role of lymphocyte function antigen-1 (LFA-1) in regulating neutrophil-endothelium interactions and tissue damage in severe AP. Pancreatitis was induced by retrograde infusion of sodium taurocholate into the pancreatic duct in mice. LFA-1 gene-targeted mice and an antibody directed against LFA-1 were used to define the role of LFA-1. Taurocholate challenge caused a clear-cut increase in serum amylase, neutrophil infiltration, CXCL2 (macrophage inflammatory protein-2) formation, trypsinogen activation and tissue damage in the pancreas. Inhibition of LFA-1 function markedly reduced taurocholate-induced amylase levels, accumulation of neutrophils, production of CXC chemokines and tissue damage in the pancreas. Notably, intravital microscopy revealed that inhibition of LFA-1 abolished taurocholate-induced leucocyte adhesion in postcapillary venules of the pancreas. In addition, pulmonary infiltration of neutrophils was attenuated by inhibition of LFA-1 in mice challenged with taurocholate. However, interference with LFA-1 had no effect on taurocholate-induced activation of trypsinogen in the pancreas. Our novel data suggest that LFA-1 plays a key role in regulating neutrophil recruitment, CXCL2 formation and tissue injury in the pancreas. Moreover, these results suggest that LFA-1-mediated inflammation is a downstream component of trypsinogen activation in the pathophysiology of AP. Thus, we conclude that targeting LFA-1 may be a useful approach to protect against pathological inflammation in the pancreas.

Bone Marrow Failure Syndromes

Laboratory diagnosis of inherited bone marrow failure syndromes includes general evaluations, such as blood counts, examination of the peripheral blood smear for morphology, and bone marrow aspirates and biopsies, which may help the clinician classify the patient, particularly if there are no characteristic physical anomalies. Specific diagnoses require unique tests that are only available for a few of the diagnoses. The most useful is chromosome breakage in the diagnosis of FA, with gene mutation analysis or mapping about to become the gold standard when all of the FA genes have been cloned. The diagnosis of DC remains clinical at this time, although linkage to Xq28 and skewed maternal X inactivation may be helpful in some families. Laboratory proof of SD may be provided by decreased serum trypsinogen or other evidence of exocrine pancreatic insufficiency. CHH is substantiated when absent central pigment in hair is found and when it is mapped to 9p21-p13. The only mitochondrial syndrome, PS, is proved with demonstration of deleted mitochondrial DNA. RD is diagnosed from blood and marrow studies that demonstrate lack of lymphoid as well as myeloid activity. Amega requires absent or abnormal marrow megakaryocytes; if radii are also absent, the diagnosis is TAR. DBA usually has elevated red-cell ADA, and the DBA locus may map to 19q13. KS is diagnosed in patients who have congenital nonimmune severe neutropenia. Clinical suspicion of particular diagnoses can often be substantiated by laboratory tests of varying specificity.

Shwachman-Diamond syndrome (SDS)

Intermittent neutropenia is the most common haematological finding (85-100%); in addition aplastic anemia (80%), increased hemoglobin F levels (80%), thrombocytopenia (25-85%) and impaired neutrophil chemotaxis, Band T-cell defects can be found. Fluctuating or persistent exocrine pancreatic dysfunction (with low serum amylase in 50-75%, low serum trypsinogen in 70-98% and abnormal pancreatic stimulation test in nearly 100%). Growth retardation (shortness 60%, weight 50%, microcephalus <50%). Other manifestations include delayed puberty, rib and thoracic bone abnormalities 30-50%, metaphyseal dysostosis 50-75%, dental dyplasia, hepatomegaly 1060%, elevated liver transaminases 50-75%, Ichtyosis severe and recurrent viral, bacterial and fungal infections 50-75% and developmental delay.

Management of pancreatic exocrine insufficiency: Australasian Pancreatic Club recommendations

Pancreatic exocrine insufficiency (PEI) occurs when the amounts of enzymes secreted into the duodenum in response to a meal are insufficient to maintain normal digestive processes. The main clinical consequence of PEI is fat maldigestion and malabsorption, resulting in steatorrhoea. Pancreatic exocrine function is commonly assessed by conducting a 3-day faecal fat test and by measuring levels of faecal elastase-1 and serum trypsinogen. Pancreatic enzyme replacement therapy is the mainstay of treatment for PEI. In adults, the initial recommended dose of pancreatic enzymes is 25,000 units of lipase per meal, titrating up to a maximum of 80,000 units of lipase per meal. In infants and children, the initial recommended dose of pancreatic enzymes is 500 units of lipase per gram of dietary fat; the maximum daily dose should not exceed 10,000 units of lipase per kilogram of bodyweight. Oral pancreatic enzymes should be taken with meals to ensure adequate mixing with the chyme. Adjunct therapy with acid-suppressing agents may be useful in patients who continue to experience symptoms of PEI despite high-dose enzyme therapy. A dietitian experienced in treating PEI should be involved in patient management. Dietary fat restriction is not recommended for patients with PEI. Patients with PEI should be encouraged to consume small, frequent meals and to abstain from alcohol. Medium-chain triglycerides do not provide any clear nutritional advantage over long-chain triglycerides, but can be trialled in patients who fail to gain or to maintain adequate bodyweight in order to increase energy intake.

Letters to the Editor

1 July 2009 Dear Editor, PERSISTENT ELEVATED TISSUE-TRANSGLUTAMINASE IN CYSTIC FIBROSIS I read with interest the article by Hasosah et al. regarding persistent tissue-transglutaminase in cystic fibrosis (CF).1 I do not wish to comment on the gastroenterological aspects of the case but do wish to draw to your attention to the fact that the extended delay in the diagnosis of cystic fibrosis (until age 7) is extremely unlikely to occur in Australia because of newborn screening (5% of babies with CF are missed).2 Almost all babies with CF will have an elevated serum trypsinogen in the first few weeks of life, measured as immunoreactive trypsinogen (IRT). Babies with an elevated IRT are screened for the common CFTR gene mutations (this varies by state) that include p.F508del.3 The presence of screening does not seem to have resulted in paediatricians forgetting about a clinical diagnosis of CF, and the mean age at diagnosis of babies with pancreatic insufficient CF missed by newborn screening was 4 months in Victoria.4 Newborn screening for CF has been shown in a randomised controlled trial to be associated with better nutritional and cognitive outcomes, and a retrospective cohort study to be associated with better pulmonary function.5, 6 The other benefit of newborn screening for CF is the facility to offer genetic counselling to parents (and their extended family) and offer reproductive choices regarding their next child.7 The case report did not indicate whether siblings had been born after the index case, but the tragedy of this case would be compounded by the birth of a second child with cystic fibrosis. I was also intrigued by the additional diagnosis of congenital hypothyroidism, another condition that can be detected by newborn screening.

Serum and Urine Trypsinogen Activation Peptide in Assessing Post-Endoscopic Retrograde Cholangiopancreatography Pancreatitis

Pezzilli, Raffaele MD1; Mariani, Alberto MD2; Gabbrielli, Armando MD3; Morselli-Labate, Antonio M. PhD1; Barassi, Alessandra MD4 Author Information

Regulation of HSP60 and the role of MK2 in a new model of severe experimental pancreatitis

The objective of this study was to investigate the role of MAPKAP kinase 2 (MK2) and heat shock protein (HSP) HSP60 in the pathogenesis of a new model of severe acute pancreatitis (AP). MK2 plays a significant role in the regulation of cytokines. It has been shown that induction and expression of several HSPs can protect against experimental pancreatitis. Interplay between both systems seems of high interest. Mice with a homozygous deletion of the MK2 gene were used. Severe AP was induced by combined intraperitoneal injections of cerulein with lipopolysaccharide (LPS). Severity of AP was assessed by biochemical markers and histology. The serum IL-6 and lung myeloperoxidase (MPO) levels were determined for assessing the extent of systemic inflammatory response. Expression of HSP25, HSP60, HSP70, and HSP90 was analyzed by Western blotting. Repeated injections of cerulein alone or cerulein plus LPS (Cer+LPS) resulted in local inflammatory responses in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the Cer+LPS group. Compared with the C57Bl wild-type mice, the MK2-/- mice presented with significant milder pancreatitis and attenuated responses of serum amylase and trypsinogen activity. Furthermore, serum IL-6 was decreased as well as lung MPO activity. Injection of LPS alone displayed neither pancreatic inflammatory responses nor alterations of pancreatic enzyme activities but evidently elevated serum IL-6 levels and increased lung MPO activity. In contrast hereto, in the MK2-/- mice, these changes were much milder. Increased expression of HSP25 and HSP60 occurred after induction of AP. Especially, HSP60 was robustly elevated after Cer+LPS treatment, in both MK2-/- and wild-type mice. Thus the homozygous deletion of the MK2 gene ameliorates the severity of acute pancreatitis and accompanying systemic inflammatory reactions in a new model of severe acute pancreatitis. Our data support the hypothesis that MK2 participates in the multifactorial regulation of early inflammatory responses in AP, independently of the regulation of stress proteins like HSP25 and HSP60 and most likely due to its effect on cytokine regulation.

Structural changes induced in proteins by therapeutic ultrasounds

The structural effect induced by therapeutic ultrasound on proteins in aqueous solution has been investigated with FTIR spectroscopy, UV–VIS spectroscopy, circular dichroism and light scattering. Six proteins (cytochrome, lysozyme, myoglobin, bovine serum albumin, trypsinogen, and α-chymotrypsinogen A) with different molecular weight and secondary structure have been studied. The experiment has been performed using an ultrasound source at resonant frequency of 1 MHz and sonication times of 10, 20, 30, 40, 50, and 60 min. A different behaviour of proteins under sonication depends on the dominant secondary structure type (α-helix or β-sheets) and on the grade of the ordered structure. The results suggest that the free radicals, produced by water sonolysis, have an important role in the changes of structural order.

The Effect of Renal Replacement Therapies on Serum Gastrointestinal System Hormones

Background. The kidney is a major site for the inactivation, degradation, and clearance of a variety of peptide hormones. It has been shown that the uremia increases or decreases gastrointestinal system (GIS) hormones. Moreover, studies investigating the serum GIS hormones levels in chronic renal failure (CRF) were conducted mainly in a particular period of the renal replacement therapy, and the changes caused by continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD) could not be fully demonstrated. In this study, we investigated the effect of CAPD and HD on serum GIS hormones (amylase, lipase, trypsinogen, and gastrin) levels in CRF patients who were diagnosed for the first time. Methods. Serum amylase, lipase, trypsinogen, and gastrin levels were measured in 36 patients who were just diagnosed with CRF, 22 patients with CAPD and 14 patients with HD. GIS hormones of these patients were measured before treatment and three months from the beginning of CAPD and HD treatment. As the control group, 20 normal healthy cases with well-matched age and gender were used. Results. The mean serum amylase, lipase, secretin, and gastrin levels were found meaningfully decreased according to the beginning values at third months of the CAPD and HD treatment. However, they were higher than control group. Conclusion. In patients receiving CAPD or HD as renal replacement therapy, GIS hormone levels were found to be lower, albeit higher than the healthy control group.

DETERMINATION OF THE DIAGNOSTIC ACCURACY OF SERUM TRYPSINOGEN AS A SCREENING TEST FOR THE EVALUATION OF PANCREAS EXOCRINE INSUFFICIENCY

Background: The diagnosis of early pancreas exocrine insufficiency remains difficult to determine. The validity and clinical efficacy of current serologic and radiologic studies have not been determined from a gold standard. For example, serum trypsinogen is a commonly used serologic marker to screen for pancreatic disease by non-gastroenterologist. The endoscopic pancreatic function test affords clinical investigators a less cumbersome, "non-histologic" gold standard for comparison studies to determine diagnostic accuracy of screening tests. Aim: To determine the accuracy of serum trypsinogen by comparison to the "gold standard" synthetic secretin stimulation test Methods: The Pancreas Clinic database was queried for patients with serum trypsinogen and 1 hour pancreatic function test studies. Serum trypsinogen was measured as trypsin-like immunoreactivity (abnormal: ≤ 40 ng/mL, normal: > 40 ng/ml) Peak duodenal fluid bicarbonate was measured with an autoanlyzer after synthetic secretin stimulation. (abnormal: ≤ 80 meq/L, normal: > 80meq/L). Data analysis: Microsoft Excel statistical software package. Results: A total of 48 patients were identified: 20 males, mean age 46 years. A 2 × 2 table was constructed to determine the validity and clinical efficacy of serum trypsinogen when compared to peak bicarbonate concentration. There were a significant number of false negatives (n = 15) and false positives (n = 6) which greatly effected the validity of the test. Sensitivity and specificity were 53% and 63%, respectively. Furthermore, the clinical efficacy was also poor, with positive and negative predictive values of 74% and 40, respectively.Table 1: 2×2 Table - Trypsinogen versus BicarbonateConclusions: 1) Accuracy of serum trypsinogen for pancreas exocrine insufficiency is poor. 2) Serum trypsinogen should not be used to screen for exocrine pancreas function. Clinical Implication: The endoscopic pancreatic function test can be used by clinical investigators to determine the validity and clinical efficacy of commonly used diagnostic procedures in the evaluation of pancreas exocrine insufficiency.

Fluorimetric studies and noncovalent labeling of protein with the near‐infrared dye HITCI for analysis by CE‐LIF

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.

Serum Trypsinogen Activation Peptide in the Assessment of the Diagnosis and Severity of Acute Pancreatic Damage

To evaluate the clinical value of a new direct and competitive immunoassay for trypsinogen activation peptide (TAP) determination in acute pancreatitis (AP). The subjects were 34 patients with AP (22 mild, 12 severe), 12 patients with nonpancreatic acute abdominal pain (AA), 11 healthy subjects (HS), and 16 consecutive patients who underwent therapeutic ERCP (ERCP). Serum TAP, amylase, and lipase levels were determined in AP, AA, and HS at their initial observation; the AP patients were also studied for 6 consecutive days after admission. In the ERCP patients, serum TAP, amylase, and lipase levels, as well as urine TAP and amylase levels, were determined before and 6 hours after endoscopy. Serum TAP levels on admission were 0.35 +/- 1.60 OD (mean +/- SD) in AP patients and 0.005 +/- 0.001 OD in AA patients, while HS patients had no detectable serum TAP levels. ERCP patients had no detectable serum TAP levels before and 6 hours after the ERCP, whereas urine TAP concentrations were 1.72 +/- 3.43 OD and 0.75 +/- 1.49 OD before and 6 hours after the execution of the endoscopy, respectively (P = 0.249). The sensitivities and specificities of serum TAP, amylase, and lipase levels in discriminating between AP and AA were 23.5% and 91.7%, 94.1% and 100%, and 97.1% and 100%, respectively, while those used in the assessment of the severity of AP were 29.9% and 73.5%, 38.8% and 81.2%, and 28.4% and 83.6%, respectively. TAP is of limited value in assessing the diagnosis and the severity of acute pancreatic damage.

Symmetric and asymmetric squarylium dyes as noncovalent protein labels: a study by fluorimetry and capillary electrophoresis

Noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emissions of which have been shown to be enhanced upon complexation with proteins such as bovine serum albumin (BSA), human serum albumin (HSA), β-lactoglobulin A, and trypsinogen. Although these dyes are poorly soluble in aqueous solution, they can be dissolved first in methanol followed by dilution with aqueous buffer without precipitation, and are then suitable for use as fluorescent labels in protein determination studies. The nature of interactions between these dyes and proteins was studied using a variety of buffer systems, and it was found that electrostatic interactions are involved but not dominant. Dye/protein stoichiometries in the noncovalent complexes were found to be 1:1 for SQ-3, although various possible stoichiometries were found for NN127 depending upon pH and protein. Association constants on the order of 10 5 and 10 7 were found for noncovalent complexes of SQ-3 and NN127, respectively, with HSA, indicating stronger interactions of the symmetric dye with proteins. Finally, HSA complexes with NN127 were determined by capillary electrophoresis with laser-induced fluorescence detection (CE–LIF). In particular, NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE–LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.

Medical therapy for chronic pancreatitis pain.

Chronic pancreatitis should be considered in all patients with unexplained abdominal pain. Management of abdominal pain in these patients continues to pose a formidable challenge. The importance of small duct disease without radiographic abnormalities is now a well-established concept. It is meaningful to determine whether patients with chronic pancreatitis have small duct or large duct disease because this distinction has therapeutic implications. Diagnostic evaluation should begin with simple noninvasive and inexpensive tests like serum trypsinogen and fecal elastase, to be followed where appropriate by more complicated measures such as the secretin hormone stimulation test, especially in patients with suspected small duct disease. No universal causal treatment is available. Non-enteric-coated enzyme preparations are useful for treatment of pain, whereas enteric-coated enzyme preparations are preferred for steatorrhea. Octreotide is used increasingly for abdominal pain that is unresponsive to pancreatic enzyme therapy. When medical therapy for chronic pancreatitis pain has failed, endoscopic therapy, endoscopic ultrasound-guided celiac plexus block, and thoracoscopic splanchnicectomy, performed by experts, may be considered for a highly selected patient population. Surgical ductal decompression is appropriate in patients with considerable pancreatic ductal dilation. The role and efficacy of cholecystokinin-receptor antagonists, antioxidants, and antidepressant drugs remain to be defined.

Assessing the severity of canine pancreatitis

The objective of this study was to determine whether laboratory testing currently available is able to provide prognostic information in canine pancreatitis. A prospective study of dogs with naturally occurring pancreatitis was undertaken. Twenty-two cases with histologically confirmed pancreatic inflammation were included in the study. Each dog had routine haematology parameters, serum biochemistry (including lipase and amylase), serum trypsin-like immunoreactivity and trypsinogen activation peptides (TAP) in urine and plasma measured. Twelve of the dogs were classified as having severe disease. These dogs had statistically significant increases in urinary TAP–creatinine ratio (UTCR) measurement, serum lipase, serum phosphate and serum creatinine concentrations. Additionally dogs with severe pancreatitis had significantly decreased urine specific gravity levels. The most sensitive and specific test to assess the severity of pancreatitis was the measurement of UTCR.