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Miklós Sahin-tóth - One of the best experts on this subject based on the ideXlab platform.
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Novel Pathogenic PRSS1 Variant p.Glu190Lys in a Case of Chronic Pancreatitis
Frontiers Media S.A., 2019Co-Authors: Zsanett Jancso, Grzegorz Oracz, Aleksandra Anna Kujko, Eliwira Kolodziejczyk, Evette S. Radisky, Agnieszka Magdalena Rygiel, Miklós Sahin-tóthAbstract:Mutations in the PRSS1 (serine protease 1) gene encoding human cationic Trypsinogen cause hereditary pancreatitis or may be associated with sporadic chronic pancreatitis. The mutations exert their pathogenic effect either by increasing intra-pancreatic Trypsinogen activation (trypsin pathway) or by causing proenzyme misfolding and endoplasmic reticulum stress (misfolding pathway). Here we report a novel heterozygous c.568G>A (p.Glu190Lys) variant identified in a case with chronic pancreatitis. The parents of the index patient had no history of pancreatitis but were unavailable for genetic testing. Functional characterization revealed 2.5-fold increased autoactivation of the mutant Trypsinogen relative to wild type. Unlike many other clinically relevant PRSS1 mutations, p.Glu190Lys did not alter the chymotrypsin C (CTRC)-dependent degradation of Trypsinogen nor did it increase CTRC-mediated processing of the Trypsinogen activation peptide. Cellular secretion of the mutant protein was unchanged indicating normal folding behavior. Based on the genetic and functional evidence, we classify the p.Glu190Lys PRSS1 variant as likely pathogenic, which stimulates autoactivation of cationic Trypsinogen independently of CTRC
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Trypsinogen isoforms in the ferret pancreas.
Scientific reports, 2018Co-Authors: Eszter Hegyi, Miklós Sahin-tóthAbstract:The domestic ferret (Mustela putorius furo) recently emerged as a novel model for human pancreatic diseases. To investigate whether the ferret would be appropriate to study hereditary pancreatitis associated with increased Trypsinogen autoactivation, we purified and cloned the Trypsinogen isoforms from the ferret pancreas and studied their functional properties. We found two highly expressed isoforms, anionic and cationic Trypsinogen. When compared to human cationic Trypsinogen (PRSS1), ferret anionic Trypsinogen autoactivated only in the presence of high calcium concentrations but not in millimolar calcium, which prevails in the secretory pathway. Ferret cationic Trypsinogen was completely defective in autoactivation under all conditions tested. However, both isoforms were readily activated by enteropeptidase and cathepsin B. We conclude that ferret Trypsinogens do not autoactivate as their human paralogs and cannot be used to model the effects of Trypsinogen mutations associated with human hereditary pancreatitis. Intra-pancreatic Trypsinogen activation by cathepsin B can occur in ferrets, which might trigger pancreatitis even in the absence of Trypsinogen autoactivation.
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Evolution of Trypsinogen Activation Peptides
2013Co-Authors: Jian-min Chen, Zoltan Kukor, Miklos Toth, Cédric Le Maréchal, À Laurent Tsakiris, Odile Raguénès, Jj Claude Férec, Miklós Sahin-tóthAbstract:The activation peptide of mammalian Trypsinogens contains a highly conserved tetra-aspartate sequence (D19-D20-D21-D22) preceding the K23-I24 scissile peptide bond, which is hydrolyzed as the first step in the activation process. Here, we examined the evolution and function of Trypsinogen activation peptides through integrating functional characterization of disease-associated mutations with comparative genomic analysis. Activation properties of three chronic pancreatitis-associated activation peptide mutants (the novel D19A and the previously reported D22G and K23R) were simultaneously analyzed, for the first time, in the context of recombinant human cationic Trypsinogen. A dramatic increase in autoactivation of cationic Trypsinogen was observed in all three mutants, with D22G and K23R exhibiting the most marked increases. The physiological activator enteropeptidase activated the D19A mutant normally, activated the D22G mutant very poorly, and stimulated activation of the K23R mutant. The biochemical and structural data, taken together with a comprehensive sequence comparison, indicates that the tetra-aspartate sequence in mammalian Trypsinogen activation peptides has evolved not only for optimal enteropeptidase recognition in the duodenum but also for efficient inhibition of Trypsinogen autoactivation within the pancreas. Moreover, the use of lysine instead of arginine at the P1 position of activation peptides also has an advantageous effect against Trypsinogen autoactivation. Finally, fixed substitutions in the key residues of the Trypsinogen activation peptide may suggest the evolution of new function
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Autoactivation of mouse Trypsinogens is regulated by chymotrypsin C via cleavage of the autolysis loop.
The Journal of biological chemistry, 2013Co-Authors: Balázs Németh, Thomas Wartmann, Walter Halangk, Miklós Sahin-tóthAbstract:Chymotrypsin C (CTRC) is a proteolytic regulator of Trypsinogen autoactivation in humans. CTRC cleavage of the Trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes Trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic Trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse Trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four Trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse Trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150–Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149–Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in Trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse Trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the Trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic Trypsinogen activation.
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A common African polymorphism abolishes tyrosine sulfation of human anionic Trypsinogen (PRSS2).
The Biochemical journal, 2009Co-Authors: Zsolt Rónai, Heiko Witt, Olga Rickards, Giovanni Destro-bisol, Andrew Bradbury, Miklós Sahin-tóthAbstract:Human pancreatic Trypsinogens undergo post-translational sulfation on Tyr154, catalyzed by the Golgi-resident enzyme tyrosylprotein sulfotransferase 2. Sequence alignments suggest that sulfation of Tyr154 is facilitated by a unique sequence context characteristically found in primate Trypsinogens. In search for genetic variants that might alter this sulfation motif, we identified a single nucleotide polymorphism (c.457G>C) in the human anionic Trypsinogen gene (PRSS2), which changed Asp153 to His (p.D153H). The p.D153H variant is common in subjects of African origin, with a minor allele frequency of 9.2%, whereas it is absent in subjects of European descent. We demonstrate that Asp153 is the main determinant of tyrosine sulfation in anionic Trypsinogen, as both the natural p.D153H variation and the p.D153N mutation result in complete loss of Trypsinogen sulfation. In contrast, mutation of Asp156 and Glu157 only slightly decrease tyrosine sulfation, whereas mutation of Gly151 and Pro155 are without consequence. With respect to the biological relevance of the p.D153H variant, we found that tyrosine sulfation had no significant effect on the activation of anionic Trypsinogen or the catalytic activity and inhibitor sensitivity of anionic trypsin. Taken together with previous studies, the observations suggest that the primary role of Trypsinogen sulfation in humans is to stimulate autoactivation of cationic Trypsinogen (PRSS1), whereas sulfation of anionic Trypsinogen is unimportant for normal digestive physiology. As a result, the p.D153H polymorphism which eliminates this modification could become widespread in a healthy population.
Ulf-håkan Stenman - One of the best experts on this subject based on the ideXlab platform.
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Reference Intervals for and Validation of Recalibrated Immunoassays for Trypsinogen-1 and Trypsinogen-2
Clinical chemistry, 2012Co-Authors: Outi Itkonen, Leena Kylanpaa, Wan-ming Zhang, Ulf-håkan StenmanAbstract:To the Editor: Serum Trypsinogen assays are used as diagnostic and prognostic tools for cystic fibrosis and acute pancreatitis (AP) (1, 2). Calibrator stability is a challenge in these assays, because Trypsinogen readily autoactivates and subsequently autodegrades. Furthermore, the reference intervals described thus far have been based on samples from a limited number of individuals (1, 3). We recalibrated a new immunoassay for Trypsinogen-2 and a previously described assay for Trypsinogen-1 (1) with stable calibrators (2, 4) and report reference intervals for these 2 analytes in serum. We have produced new monoclonal antibodies and developed a time-resolved immunofluorometric assay for Trypsinogen-2, as previously described (1). Monoclonal antibodies F87–9E6 and F88–8F7 were used as capture antibody and tracer antibody, respectively. To prevent activation, we produced stable mutated (Lys23Gln) Trypsinogen-1 and Trypsinogen-2 by recombinant technology (2, 4) and used them to recalibrate both the previously described Trypsinogen-1 assay (1) and the new Trypsinogen-2 assay. In this assay, the calibrators covered the concentration interval of 1.0 to 1000 μg/L, the limit of detection was 0.24 μg/L, and …
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specific immunoassay reveals increased serum Trypsinogen 3 in acute pancreatitis
Clinical Chemistry, 2011Co-Authors: Jani Oiva, Esko Kemppainen, Pauli Puolakkainen, Ulf-håkan Stenman, Outi Itkonen, Riitta Koistinen, Kristina Hotakainen, Wangming Zhang, Leena Kylanpaa, Hannu KoistinenAbstract:BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
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Specific Immunoassay Reveals Increased Serum Trypsinogen 3 in Acute Pancreatitis
Clinical chemistry, 2011Co-Authors: Jani Oiva, Esko Kemppainen, Pauli Puolakkainen, Ulf-håkan Stenman, Outi Itkonen, Riitta Koistinen, Kristina Hotakainen, Wangming Zhang, Leena Kylanpaa, Hannu KoistinenAbstract:BACKGROUND: 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. METHODS: 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. RESULTS: The measuring range of the Trypsinogen 3 assay was 1.0–250 μg/L. Intra- and interassay CVs were
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Trypsinogen-2 and Trypsinogen activation peptide (TAP) in urine of patients with acute pancreatitis
The Journal of surgical research, 2003Co-Authors: Marko Lempinen, Pauli Puolakkainen, Reijo Haapiainen, Ulf-håkan Stenman, Patrik Finne, Esko KemppainenAbstract:Abstract Background and aims. There is an obvious clinical need for a simple test that can identify patients at risk of developing severe acute pancreatitis. In this work we compared urinary Trypsinogen-2 with urinary Trypsinogen activation peptide (TAP) and serum C-reactive protein (CRP) for early differentiation between mild and severe acute pancreatitis. Patients and methods. The study population consisted of 127 consecutive patients with acute pancreatitis of whom 29 had severe disease. Urinary Trypsinogen-2 was measured by a quantitative immunofluorometric assay and TAP by a competitive immunoassay. Serum CRP was determined by immunoturbidimetry. Results. The sensitivity and specificity to identify severe acute pancreatitis on admission was 72% and 81% for urinary Trypsinogen-2, 64% and 82% for urinary TAP, and 29% and 93% for serum CRP, respectively. At 24 h after admission, the values were 82% and 78% for urinary Trypsinogen-2, 52% and 92% for urinary TAP, and 84% and 72% for serum CRP, respectively. Receiver-operating characteristics curve analysis showed that the area under the curve was larger for urinary Trypsinogen-2 than for urinary TAP and serum CRP on admission and 24 h after admission. On admission the positive likelihood ration for urinary trypsiongen-2 was 3.7, for urinary TAP 3.6, and 4.3 for serum CRP, respectively. The corresponding negative likelihood ratios were 0.34, 0.43, and 0.76, respectively. Conclusion. Urinary Trypsinogen-2 was superior to serum CRP and as god as or even better than urinary TAP and in the early prediction of disease severity in acute pancreatitis. These results suggest that it could be a valuable adjunct in the early assessment of the severity of acute pancreatitis.
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Down-regulation of Trypsinogen-2 expression by chemically modified tetracyclines: association with reduced cancer cell migration.
International journal of cancer, 2000Co-Authors: Annukka Lukkonen, Timo Sorsa, Tuula Salo, Taina Tervahartiala, Erkki Koivunen, Lorne M. Golub, Sanford R. Simon, Ulf-håkan StenmanAbstract:Many types of human tumor express Trypsinogen-2, which may be a significant factor in the activation of pro-MMPs and the invasiveness of tumors. Prevention of Trypsinogen-2 expression in cancer cells might be of benefit in cancer therapy. We describe here chemicals capable of down-regulating the expression of Trypsinogen-2. Doxycycline (DOXY) and chemically modified tetracyclines (CMTs), previously known as inhibitors of the matrix metalloproteinase (MMP)-dependent proteinase cascade, down-regulated the mRNA and protein expression of Trypsinogen-2 by COLO-205 human colon adenocarcinoma cells at therapeutically attainable concentrations (0. 1 to 1.0 microM). DOXY specifically inhibited the activation of pro-MMP-9 and cell migration induced by enteropeptidase, a specific activator of Trypsinogen. Pro-MMP-9 activation and cell migration were also inhibited by tumor-associated trypsin inhibitor (TATI), which is a highly specific inhibitor of trypsin. CMT-3 as well as CMT-5 also inhibited cell migration, but an effect on the enteropeptidase-enhanced activation of pro-MMP-9 was not observed. Our results indicate that CMTs, DOXY and TATI inhibit cancer cell migration by down-regulating Trypsinogen-2 expression or activity. Inhibition of Trypsinogen-2 expression may represent a mechanism contributing to the ability of CMTs to suppress the pericellular proteolytic activity of some tumors.
Esko Kemppainen - One of the best experts on this subject based on the ideXlab platform.
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specific immunoassay reveals increased serum Trypsinogen 3 in acute pancreatitis
Clinical Chemistry, 2011Co-Authors: Jani Oiva, Esko Kemppainen, Pauli Puolakkainen, Ulf-håkan Stenman, Outi Itkonen, Riitta Koistinen, Kristina Hotakainen, Wangming Zhang, Leena Kylanpaa, Hannu KoistinenAbstract:BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
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Specific Immunoassay Reveals Increased Serum Trypsinogen 3 in Acute Pancreatitis
Clinical chemistry, 2011Co-Authors: Jani Oiva, Esko Kemppainen, Pauli Puolakkainen, Ulf-håkan Stenman, Outi Itkonen, Riitta Koistinen, Kristina Hotakainen, Wangming Zhang, Leena Kylanpaa, Hannu KoistinenAbstract:BACKGROUND: 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. METHODS: 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. RESULTS: The measuring range of the Trypsinogen 3 assay was 1.0–250 μg/L. Intra- and interassay CVs were
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Trypsinogen-2 and Trypsinogen activation peptide (TAP) in urine of patients with acute pancreatitis
The Journal of surgical research, 2003Co-Authors: Marko Lempinen, Pauli Puolakkainen, Reijo Haapiainen, Ulf-håkan Stenman, Patrik Finne, Esko KemppainenAbstract:Abstract Background and aims. There is an obvious clinical need for a simple test that can identify patients at risk of developing severe acute pancreatitis. In this work we compared urinary Trypsinogen-2 with urinary Trypsinogen activation peptide (TAP) and serum C-reactive protein (CRP) for early differentiation between mild and severe acute pancreatitis. Patients and methods. The study population consisted of 127 consecutive patients with acute pancreatitis of whom 29 had severe disease. Urinary Trypsinogen-2 was measured by a quantitative immunofluorometric assay and TAP by a competitive immunoassay. Serum CRP was determined by immunoturbidimetry. Results. The sensitivity and specificity to identify severe acute pancreatitis on admission was 72% and 81% for urinary Trypsinogen-2, 64% and 82% for urinary TAP, and 29% and 93% for serum CRP, respectively. At 24 h after admission, the values were 82% and 78% for urinary Trypsinogen-2, 52% and 92% for urinary TAP, and 84% and 72% for serum CRP, respectively. Receiver-operating characteristics curve analysis showed that the area under the curve was larger for urinary Trypsinogen-2 than for urinary TAP and serum CRP on admission and 24 h after admission. On admission the positive likelihood ration for urinary trypsiongen-2 was 3.7, for urinary TAP 3.6, and 4.3 for serum CRP, respectively. The corresponding negative likelihood ratios were 0.34, 0.43, and 0.76, respectively. Conclusion. Urinary Trypsinogen-2 was superior to serum CRP and as god as or even better than urinary TAP and in the early prediction of disease severity in acute pancreatitis. These results suggest that it could be a valuable adjunct in the early assessment of the severity of acute pancreatitis.
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Sequential changes in pancreatic markers in acute pancreatitis.
Scandinavian journal of gastroenterology, 2003Co-Authors: Marko Lempinen, Pauli Puolakkainen, U H Stenman, A. Hietaranta, Reijo Haapiainen, Esko KemppainenAbstract:Background: Trypsinogen activation within acinar cells plays a crucial role in the pathogenesis of acute pancreatitis (AP). Our aim was to characterize temporal changes of Trypsinogen-1, Trypsinogen-2, complexes of trypsin-1- ! 1 -antitrypsin (T1-AAT) and trypsin-2- ! 1 -antitrypsin (T2-AAT), Trypsinogen activation peptide (TAP) and pancreatic secretory trypsin inhibitor (PSTI) in patients with AP. Methods: The study comprised 64 consecutive patients with AP (19 with severe disease) and 32 controls. The concentrations of Trypsinogen-1 and -2, PSTI, T1-AAT and T2-AAT were determined by time-resolved immunofluorometric assays (IFMA), and TAP was measured using a competitive enzyme immunoassay from serum and urine. Results: The concentrations of Trypsinogen-1 and -2 in serum reflected similar patterns, but excretion of Trypsinogen-1 into urine was markedly lower than that of Trypsinogen-2, the concentration of which correlated strongly with disease severity. The concentrations of T1-AAT were no higher ...
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Urine Trypsinogen-2 as marker of acute pancreatitis.
Clinical chemistry, 1996Co-Authors: Johan Hedström, Esko Kemppainen, Pauli Puolakkainen, Reijo Haapiainen, V Sainio, Eero Kivilaakso, K O Schauman, Ulf-håkan StenmanAbstract:We examined the clinical utility of urine Trypsinogen-2 as a marker of acute pancreatitis (AP). Fifty-nine patients with AP, 42 with acute abdominal diseases of extrapancreatic origin, and 63 without evidence of acute abdominal disease were studied. Urine Trypsinogen-2 was determined by a time-resolved immunofluorometric assay. As reference methods we used serum Trypsinogen-2, urine amylase, and serum amylase. The diagnostic accuracy of the markers was evaluated by receiver-operating characteristic (ROC) analysis. At admission, urine Trypsinogen-2 differentiated patients with AP from controls with high accuracy. The area under the ROC curve (AUC) was 0.978, which was equal to that of serum Trypsinogen-2 (0.998) and serum amylase (0.969) and significantly larger than that of urine amylase. For differentiation between severe and mild AP, urine Trypsinogen-2 (0.730) was equal to serum Trypsinogen-2 (0.721), and clearly better than amylase in serum and urine. These results suggest that determination of urine Trypsinogen-2 is a useful test to detect AP and to evaluate disease severity.
Outi Itkonen - One of the best experts on this subject based on the ideXlab platform.
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Reference Intervals for and Validation of Recalibrated Immunoassays for Trypsinogen-1 and Trypsinogen-2
Clinical chemistry, 2012Co-Authors: Outi Itkonen, Leena Kylanpaa, Wan-ming Zhang, Ulf-håkan StenmanAbstract:To the Editor: Serum Trypsinogen assays are used as diagnostic and prognostic tools for cystic fibrosis and acute pancreatitis (AP) (1, 2). Calibrator stability is a challenge in these assays, because Trypsinogen readily autoactivates and subsequently autodegrades. Furthermore, the reference intervals described thus far have been based on samples from a limited number of individuals (1, 3). We recalibrated a new immunoassay for Trypsinogen-2 and a previously described assay for Trypsinogen-1 (1) with stable calibrators (2, 4) and report reference intervals for these 2 analytes in serum. We have produced new monoclonal antibodies and developed a time-resolved immunofluorometric assay for Trypsinogen-2, as previously described (1). Monoclonal antibodies F87–9E6 and F88–8F7 were used as capture antibody and tracer antibody, respectively. To prevent activation, we produced stable mutated (Lys23Gln) Trypsinogen-1 and Trypsinogen-2 by recombinant technology (2, 4) and used them to recalibrate both the previously described Trypsinogen-1 assay (1) and the new Trypsinogen-2 assay. In this assay, the calibrators covered the concentration interval of 1.0 to 1000 μg/L, the limit of detection was 0.24 μg/L, and …
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specific immunoassay reveals increased serum Trypsinogen 3 in acute pancreatitis
Clinical Chemistry, 2011Co-Authors: Jani Oiva, Esko Kemppainen, Pauli Puolakkainen, Ulf-håkan Stenman, Outi Itkonen, Riitta Koistinen, Kristina Hotakainen, Wangming Zhang, Leena Kylanpaa, Hannu KoistinenAbstract:BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
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Specific Immunoassay Reveals Increased Serum Trypsinogen 3 in Acute Pancreatitis
Clinical chemistry, 2011Co-Authors: Jani Oiva, Esko Kemppainen, Pauli Puolakkainen, Ulf-håkan Stenman, Outi Itkonen, Riitta Koistinen, Kristina Hotakainen, Wangming Zhang, Leena Kylanpaa, Hannu KoistinenAbstract:BACKGROUND: 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. METHODS: 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. RESULTS: The measuring range of the Trypsinogen 3 assay was 1.0–250 μg/L. Intra- and interassay CVs were
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Human Trypsinogens in the pancreas and in cancer
Scandinavian journal of clinical and laboratory investigation, 2010Co-Authors: Outi ItkonenAbstract:This study led to the development of monoclonal antibodies and time-resolved immunofluorometric methods recognizing human Trypsinogen-1 and -2, respectively. Using these methods in normal sera the concentration of Trypsinogen-1 was found to be higher than that of Trypsinogen-2. However, in acute pancreatitis the concentration of serum Trypsinogen-2 was 50-fold higher than in controls, whereas the difference in Trypsinogen-1 concentration was only 15-fold. Serum samples from patients who had undergone pancreatoduodenectomy contained Trypsinogen-2, while Trypsinogen-1 was detected in only one of nine samples. Furthermore, in human ovarian cyst fluids tumor-associated Trypsinogen-2 (TAT-2) is the predominant isoenzyme and in mucinous cyst fluids the levels of TAT-2 were associated with malignancy. These results suggest that (i) Trypsinogen-2 could be used as a diagnostic marker for acute pancreatitis, (ii) its expression is not restricted to the pancreas, and (iii) TAT could be involved in ovarian tumor dissemination and breakage of tissue barriers. In ion exchange chromatography, isoelectric variants of the Trypsinogen isoenzymes were seen. Mass spectrometric analysis of these revealed that pancreatic Trypsinogens are sulfated at tyrosine 154 (Tyr154), whereas TAT-2 from a colon carcinoma cell line is not. Tyr154 is located within the primary substrate binding pocket of trypsin. Thus, Tyr154 sulfation is likely to influence substrate binding. The previously known differences in charge and substrate binding between pancreatic and tumor-associated Trypsinogens are suggested to be caused by sulfation of Tyr154 in pancreatic Trypsinogens.
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Serum samples from pancreatectomized patients contain Trypsinogen immunoreactivity
The Journal of laboratory and clinical medicine, 1996Co-Authors: Outi Itkonen, Erkki Koivunen, Ulf-håkan Stenman, Hannu Halila, Sirpa Osman, T. SchröderAbstract:The concentrations of Trypsinogen-1 and -2 in serum samples from patients who have undergone pancreatectomy were measured by highly sensitive and specific time-resolved immunofluorometric assays. The isoenzyme pattern was determined by ion-exchange chromatography and determination of immunoreactivity in the fractions. All samples contained Trypsinogen-2, the mean level being one fifth of that in healthy controls. Trypsinogen-1 was detected in one of nine samples. In addition to the main Trypsinogen isoenzymes, we observed in normal serum two Trypsinogen isoenzymes previously found in mucinous ovarian cyst fluid. Our results suggest that Trypsinogen is not exclusively expressed by the pancreas and certain tumors but that it also may be produced by normal extrapancreatic tissues. This should be considered when an assay of Trypsinogen in serum is used for clinical purposes.
Pauli Puolakkainen - One of the best experts on this subject based on the ideXlab platform.
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specific immunoassay reveals increased serum Trypsinogen 3 in acute pancreatitis
Clinical Chemistry, 2011Co-Authors: Jani Oiva, Esko Kemppainen, Pauli Puolakkainen, Ulf-håkan Stenman, Outi Itkonen, Riitta Koistinen, Kristina Hotakainen, Wangming Zhang, Leena Kylanpaa, Hannu KoistinenAbstract:BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
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Specific Immunoassay Reveals Increased Serum Trypsinogen 3 in Acute Pancreatitis
Clinical chemistry, 2011Co-Authors: Jani Oiva, Esko Kemppainen, Pauli Puolakkainen, Ulf-håkan Stenman, Outi Itkonen, Riitta Koistinen, Kristina Hotakainen, Wangming Zhang, Leena Kylanpaa, Hannu KoistinenAbstract:BACKGROUND: 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. METHODS: 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. RESULTS: The measuring range of the Trypsinogen 3 assay was 1.0–250 μg/L. Intra- and interassay CVs were
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Trypsinogen-2 and Trypsinogen activation peptide (TAP) in urine of patients with acute pancreatitis
The Journal of surgical research, 2003Co-Authors: Marko Lempinen, Pauli Puolakkainen, Reijo Haapiainen, Ulf-håkan Stenman, Patrik Finne, Esko KemppainenAbstract:Abstract Background and aims. There is an obvious clinical need for a simple test that can identify patients at risk of developing severe acute pancreatitis. In this work we compared urinary Trypsinogen-2 with urinary Trypsinogen activation peptide (TAP) and serum C-reactive protein (CRP) for early differentiation between mild and severe acute pancreatitis. Patients and methods. The study population consisted of 127 consecutive patients with acute pancreatitis of whom 29 had severe disease. Urinary Trypsinogen-2 was measured by a quantitative immunofluorometric assay and TAP by a competitive immunoassay. Serum CRP was determined by immunoturbidimetry. Results. The sensitivity and specificity to identify severe acute pancreatitis on admission was 72% and 81% for urinary Trypsinogen-2, 64% and 82% for urinary TAP, and 29% and 93% for serum CRP, respectively. At 24 h after admission, the values were 82% and 78% for urinary Trypsinogen-2, 52% and 92% for urinary TAP, and 84% and 72% for serum CRP, respectively. Receiver-operating characteristics curve analysis showed that the area under the curve was larger for urinary Trypsinogen-2 than for urinary TAP and serum CRP on admission and 24 h after admission. On admission the positive likelihood ration for urinary trypsiongen-2 was 3.7, for urinary TAP 3.6, and 4.3 for serum CRP, respectively. The corresponding negative likelihood ratios were 0.34, 0.43, and 0.76, respectively. Conclusion. Urinary Trypsinogen-2 was superior to serum CRP and as god as or even better than urinary TAP and in the early prediction of disease severity in acute pancreatitis. These results suggest that it could be a valuable adjunct in the early assessment of the severity of acute pancreatitis.
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Sequential changes in pancreatic markers in acute pancreatitis.
Scandinavian journal of gastroenterology, 2003Co-Authors: Marko Lempinen, Pauli Puolakkainen, U H Stenman, A. Hietaranta, Reijo Haapiainen, Esko KemppainenAbstract:Background: Trypsinogen activation within acinar cells plays a crucial role in the pathogenesis of acute pancreatitis (AP). Our aim was to characterize temporal changes of Trypsinogen-1, Trypsinogen-2, complexes of trypsin-1- ! 1 -antitrypsin (T1-AAT) and trypsin-2- ! 1 -antitrypsin (T2-AAT), Trypsinogen activation peptide (TAP) and pancreatic secretory trypsin inhibitor (PSTI) in patients with AP. Methods: The study comprised 64 consecutive patients with AP (19 with severe disease) and 32 controls. The concentrations of Trypsinogen-1 and -2, PSTI, T1-AAT and T2-AAT were determined by time-resolved immunofluorometric assays (IFMA), and TAP was measured using a competitive enzyme immunoassay from serum and urine. Results: The concentrations of Trypsinogen-1 and -2 in serum reflected similar patterns, but excretion of Trypsinogen-1 into urine was markedly lower than that of Trypsinogen-2, the concentration of which correlated strongly with disease severity. The concentrations of T1-AAT were no higher ...
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Urine Trypsinogen-2 as marker of acute pancreatitis.
Clinical chemistry, 1996Co-Authors: Johan Hedström, Esko Kemppainen, Pauli Puolakkainen, Reijo Haapiainen, V Sainio, Eero Kivilaakso, K O Schauman, Ulf-håkan StenmanAbstract:We examined the clinical utility of urine Trypsinogen-2 as a marker of acute pancreatitis (AP). Fifty-nine patients with AP, 42 with acute abdominal diseases of extrapancreatic origin, and 63 without evidence of acute abdominal disease were studied. Urine Trypsinogen-2 was determined by a time-resolved immunofluorometric assay. As reference methods we used serum Trypsinogen-2, urine amylase, and serum amylase. The diagnostic accuracy of the markers was evaluated by receiver-operating characteristic (ROC) analysis. At admission, urine Trypsinogen-2 differentiated patients with AP from controls with high accuracy. The area under the ROC curve (AUC) was 0.978, which was equal to that of serum Trypsinogen-2 (0.998) and serum amylase (0.969) and significantly larger than that of urine amylase. For differentiation between severe and mild AP, urine Trypsinogen-2 (0.730) was equal to serum Trypsinogen-2 (0.721), and clearly better than amylase in serum and urine. These results suggest that determination of urine Trypsinogen-2 is a useful test to detect AP and to evaluate disease severity.
