Dipeptidyl peptidase 4 (DPP4) in fecal samples: validation of the extraction methodology and stability in short-term storage conditions

Sandra F. Gomes; Francisco Jorge Melo; Rita Silva; Mafalda Santiago; Maria Manuela Estevinho; Sandra Dias; Cláudia Camila Dias; Fernando Magro

doi:10.1515/cclm-2023-0139

Article

Dipeptidyl peptidase 4 (DPP4) in fecal samples: validation of the extraction methodology and stability in short-term storage conditions

Sandra F. Gomes , Francisco Jorge Melo , Rita Silva , Mafalda Santiago , Maria Manuela Estevinho , Sandra Dias , Cláudia Camila Dias and Fernando Magro

Published/Copyright: April 25, 2023

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 61 Issue 9

Abstract

Objectives

This study assesses the clinical relevance of dipeptidyl peptidase 4 (DPP4) membrane exopeptidase as a biomarker of inflammatory bowel disease (IBD). A spike-and-recovery approach of DPP4 in fecal samples was used to compare two different methods for protein extraction, followed by a stability assessment.

Methods

Fecal samples of healthy volunteers spiked with known concentrations of recombinant DPP4 were processed using a standard manual extraction protocol and the CALEX^® protocol. The two methods were compared by quantification of fecal DPP4 by ELISA, followed by Bland-Altman analysis. For the stability assays DPP4 was extracted from fecal samples and stored under different conditions of temperature and time after collection.

Results

In general, the levels of spiked DPP4 in stool samples were lower with the manual protocol than in those obtained with the CALEX^® method; this trend was corroborated by Bland-Altman analysis. Nonetheless, variability was within the acceptable limits for both protocols. In the stability assessment, no statistically significant differences were found between the results obtained under the different storage conditions.

Conclusions

Both manual and CALEX^® protocols provided equal extraction ability of DPP4 from stool samples. In addition, DPP4 provided flexibility in terms of sample storage enabling the accurate assessment of samples delivered up to a week before analysis.

Keywords: dipeptidyl peptidase 4; extraction; fecal samples; inflammatory bowel disease; stability

Corresponding author: Sandra F. Gomes, Department of Biomedicine, Unit of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal; Department of Public Health and Forensic Sciences and Medical Education, Faculty of Medicine, University of Porto, Porto, Portugal; and Center for Drug Discovery and Innovative Medicines (MedInUP), University of Porto, Porto, Portugal, Phone: +351 915963501, E-mail: sfferreiragomes@gmail.com

Sandra F. Gomes and Francisco Jorge Melo contributed equally to this work.

Acknowledgments

The authors thank Paula Pinto, PharmD, PhD (PMA – Pharmaceutical Medicine Academy) for providing medical writing and editorial assistance.

Research funding: This work was supported by the Portuguese Study Group of Inflammatory Bowel Disease (GEDII). Sandra F. Gomes is supported by funding from the Portuguese FCT under the grant UI/BD/150826/2021 (“The modulation of cytokines, toll-like receptors and adipose-derived cell secretome on intestinal epithelial cells and adipocytes upon dipeptidyl peptidase-4”). Francisco Jorge Melo is supported by funding from the Portuguese FCT under the grant 2021.06912.BD (“Looking 4WARD: The role of dipeptidyl peptidase 4 (DPP-4) in inflammatory bowel disease (IBD) as a novel biomarker for predicting disease activity and monitoring response to therapy in IBD patients”). The funding organizations played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: Fernando Magro served as a speaker and received honoraria from Merck Sharp & Dohme, Abbvie, Vifor, Falk, Laboratórios Vitória, Ferring, Hospira, and Biogen; the other authors have no conflict of interests to disclose.
Informed consent: Informed consent was obtained from all individuals included in this study.
Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by the authors’ Institutional Review Board (Ethics Committee of University Hospital Center of São João, Porto, Portugal; approval number 492/2020).

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2023-0139).

Received: 2023-02-06

Accepted: 2023-03-23

Published Online: 2023-04-25

Published in Print: 2023-08-28

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https://doi.org/10.1515/cclm-2023-0139

Keywords for this article

dipeptidyl peptidase 4; extraction; fecal samples; inflammatory bowel disease; stability