MSACL 2019 EU Abstract

Multiplex Assay for Absolute Quantification of Inflammatory Proteins in 134 Meconium and First Stool Swabs from CELSPAC-TNG Birth Cohort Study

Veronika Vidová (1), Eliška Stuchlíková (1), Vojtěch Thon (1), Ivo Borek (2), Petr Janků (3), Jana Klánová (1), Zdeněk Spáčil (1)
(1) RECETOX Centre, Faculty of Science, Masaryk University, Brno, Czech Republic (2) Department of Pediatrics, University Hospital Brno and Masaryk University Medical School, Brno, Czech Republic (3) Department of Gynecology and Obstetrics, University Hospital Brno and Masaryk University Medical School, Brno, Czech Republic

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Eliška Stuchlíková (Presenter) Masaryk University

Relevant Financial Disclosures (within past 24 months) Grant/Research Support Grant Agency of the Czech Republic, The Ministry of Education, Youth and Sports Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Introduction: The early-life development of the immune system and a specific low-grade inflammatory response may be monitored via quantification of inflammatory protein markers. Activated neutrophils are associated with calprotectins (CAL1 and 2) and myeloperoxidase (MPO), thus elevated abundance of those proteins in stool indicates the response against pathogens or auto-antigens. Similarly, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are released into the intestinal lumen from eosinophils activated by food allergens. In this study, we have developed multiplex protein assay for the absolute quantification of inflammatory proteins (ECP, EDN, CAL1, CAL2, MPO, and A1AT) and IgA in meconium/first stool samples from CELSPAC-TNG birth cohort study.

Methods: Swabs were extracted into 50 mM ammonium bicarbonate buffer with 5 mg/ml sodium deoxycholate, proteins were reduced and alkylated, spiked with isotopically labelled peptides with trypsin cleavable tag, enzymatically digested with trypsin and desalted using SPE. Peptides were analysed by UHPLC/SRM-MS in positive ion mode.

Results: The multiplex assay was applied to 134 samples of meconium/stool swabs from neonates. Fecal levels of IgA2 were used to distinguish between samples of meconium (IgA2 levels < LOQ) and first stool (levels up to 493.0 µg of peptide/mg of total protein). Median of concentration of EDN was 52.4 ng of peptide/mg of total protein with the highest concentration 644.3 ng/mg and median of concentration of ECP was 24.9 ng/mg with the highest concentration 735.0 ng/mg. A positive correlation was observed between EDN and ECP (0.767) and between MPO and ECP (0.779) and EDN (0.667).
Samples were grouped according to the mode of delivery (i.e. vaginal delivery and Caesarean section) and levels of inflammatory proteins were compared. Levels of CAL1 and CAL2 were significantly higher in neonates delivered via Caesarean section. On the other hand, protein MPO was significantly higher in naturally born neonates. The different protein levels most likely reflect the diverse colonization dependent on the mode of delivery and the distinct development of mucosal gut homeostasis.

Conclusion: In this study, we have developed a multiplex assay for absolute quantification of inflammatory proteins in feces and applied the assay to 134 samples from CELSPAC-TNG birth cohort study. We have compared levels of inflammatory proteins in samples from newborns delivered via Caesarean section and by vaginal delivery and we have found significantly different abundances of MPO, CAL1 and CAL2.

Acknowledgement: This work was funded by Czech Science Foundation (GACR, 17-24592Y), CETOCOEN PLUS (MEYS, CZ.02.1.01/0.0/0.0/15_003/0000469) and RECETOX research infrastructure (MEYS, LM2015051 and CZ.02.1.01/0.0/0.0/16_013/0001761).