From bedside to bench: evaluating the effect of different storage and extraction techniques on protease activity analysis in inflammatory bowel disease patients
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Proteolytic enzymes (proteases) are integral to multiple physiological processes, and increased protease activity is observed in inflammatory bowel disease (IBD) and other gastrointestinal diseases. Therefore, protease activity has been explored for potential diagnostic biomarkers and therapeutic targets. Faecal samples are commonly used to measure protease activities associated with gastrointestinal conditions as they allow non-invasive access to the diseased environment. However, faecal sample storage and protein extraction methods are not standardised for the measurement of faecal protease (FP) activity. In addition to being of scientific importance, the evaluation of the viability of FP activity as a potential biomarker for IBD management is also of clinical interest. In this study, the effect of different sample storage conditions and extraction techniques on FP activity were measured, on samples from IBD patients, using fluorescent substrates highly selective for serine proteases. FP activity was found to be stable for up to 72 hours at both 4°C and room temperature. Activity was found to decline more rapidly when samples were stored in extraction buffer rather than as crude samples. Regarding extraction techniques, FP activity was highest when extracted in Tris based buffers at pH 7.5 to 8; bead-beating of samples during extraction was found to have no significant effect on activity; and processing the extracted sample on ice was shown to minimise loss of FP activity. These results provide a framework for standardised evaluation of FP activity and suggest that protease activities have the potential to be a viable future biomarker.
Date Issued
2025-09-15
Date Acceptance
2025-04-24
Citation
Journal of Pharmaceutical and Biomedical Analysis, 2025, 263
ISSN
0731-7085
Publisher
Elsevier BV
Journal / Book Title
Journal of Pharmaceutical and Biomedical Analysis
Volume
263
Copyright Statement
© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
License URL
Identifier
10.1016/j.jpba.2025.116924
Subjects
Inflammatory Bowel Disease
Crohns disease
Ulcerative Colitis
protease analysis
calprotectin
faecal biomarker
sample processing
Publication Status
Published
Article Number
116924
Date Publish Online
2025-04-26