Role of pancreatic enzymes in the development of multiple organ failure after shock

Malinoski, Darren; Barrios, Cristobol; Kim, Hubert D.; Acosta, José A.; Schmid‐Schönbein, Geert W.; Hugli, Tony E.; Coimbra, Raúl; Hoyt, David B.

doi:10.1080/17471060801925288

Cited by 6 publications

(3 citation statements)

References 47 publications

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“…Proteases play an important role in the initiation and progression of many important diseases and medical conditions including cancer, − physiological shock, , diabetes, , hypertension, , acute coronary syndrome, pancreatitis, ,, and inflammatory bowel disease . A number of proteases have utility as potential diagnostic biomarkers and as therapeutic drug targets.…”

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confidence: 99%

Whole Blood Assay for Elastase, Chymotrypsin, Matrix Metalloproteinase-2, and Matrix Metalloproteinase-9 Activity

2010

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The ability to measure protease activity in the blood is important for the development of future diagnostics and for biomedical research. Presently, protease assays require sample preparation, making them time-consuming, costly, less accurate, and unsuitable for point-of-care (POC) diagnostics. Recently, we demonstrated a unique method for measuring clinically relevant levels of trypsin activity in only a few microliters of whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target protease. Using a simple electrophoretic format, the fragments could be rapidly separated, concentrated, and detected directly from a whole blood sample. We now report on the development of new protease substrates for the measurement of elastase, chymotrypsin, matrix metalloproteinase (MMP)-2, and MMP-9 activity in whole blood. In these studies, detection limits ranging from 1 to 40 pg in 6 μL of 1× phosphate-buffered saline (PBS) (0.2-6 ng/mL) were achieved after a only 1 h reaction of enzyme and substrate. In subsequent experiments measuring spiked protease in whole blood (with endogenous protease present), detection limits ranging from 100 to 200 ng/mL were achieved after a 1 h reaction. Thus, these new substrates demonstrate broad applicability toward clinically relevant detection of important disease-relevant proteases.

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confidence: 99%

Whole Blood Assay for Elastase, Chymotrypsin, Matrix Metalloproteinase-2, and Matrix Metalloproteinase-9 Activity

2010

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“…A large number of degradative enzymes such as proteases, lipases, amylases, nucleases and matrix metalloproteases play an important role in the initiation and progression of serious diseases and medical conditions, which include physiological shock [1], pancreatitis [2,3], inflammatory bowel disease [4], hypertension [5], acute coronary syndrome [6], diabetes [7,8], pancreatic cancer [2] and many other types of cancer [9,10]. Unfortunately, current techniques for measuring degradative enzyme activity require considerable sample preparation, thereby making the assays timeconsuming, costly and error-prone.…”

Section: Introductionmentioning

confidence: 99%

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

Lefkowitz

Marciniak

et al. 2010

Electrophoresis

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In biomedical research and clinical diagnostics, it is a major challenge to measure disease-related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time-consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates. Charge-changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic alpha-chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both alpha-chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross-reactivity with the trypsin-like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin-specific), a detection limit of about 10-20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point-of-care diagnostics.

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“…Proteases play an important role in the initiation and progression of many diseases and medical conditions such as cancer [1][2][3][4][5][6][7], physiological shock [8], diabetes [9,10], hypertension [11], acute coronary syndrome [12], pancreatitis [5,13], and inflammatory bowel disease [14]. In the particular case of the pancreatic protease trypsin or its inactive precursor trypsinogen, abnormal serum levels have been observed in both acute and chronic pancreatitis [5,13,15,16] and in various forms of cancer, including pancreatic, gastric, ovarian, and biliary tract cancers [15].…”

Section: Introductionmentioning

confidence: 99%

Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis

2010

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The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

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Role of pancreatic enzymes in the development of multiple organ failure after shock

Cited by 6 publications

References 47 publications

Whole Blood Assay for Elastase, Chymotrypsin, Matrix Metalloproteinase-2, and Matrix Metalloproteinase-9 Activity

Whole Blood Assay for Elastase, Chymotrypsin, Matrix Metalloproteinase-2, and Matrix Metalloproteinase-9 Activity

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis

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