Faecal inflammatory protein markers in children with autism spectrum disorder are comparable to their healthy siblings

Osredkar, Joško (author), Finderle, Petra (author), Godnov, Uroš (author), Jekovec-Vrhovšek, Maja (author), Vidova, Veronika (author), Elliott, James Price (author), Fabjan, Teja (author), Avguštin, Gorazd (author), Osredkar, Damjan (author), Kumer, Kristina (author)

Background: Autism spectrum disorder (ASD) is a complex neurodevelopmental condition often accompanied by gastrointestinal (GI) symptoms. Inflammatory proteins in stool have been proposed as potential biomarkers, but evidence remains inconsistent. We compared fecal levels of a1-antitrypsin (A1AT), immunoglobulin A (IgA), and calprotectin (Cal) in 57 children with ASD and 57 biological siblings without ASD. Sibling designs are now preferred to disentangle ASD-specific biology from shared environmental and microbiome factors. Participants were carefully screened to exclude recent antibiotic use, digestive problems, gastrointestinal infections, and abnormal dietary patterns, thereby controlling for major factors known to influence gut inflammatory markers. Methods: Stool samples were thawed, freeze-dried, and proteins extracted using ammonium bicarbonate buffer with sodium deoxycholate. After BCA quantification, samples were reduced, alkylated, spiked with stable isotope– labelled peptides, and digested with trypsin. Peptides were purified and analyzed by UHPLC–MS/MS (Agilent 6495A) in dynamic SRM mode. Quantification used internal standards and normalization to total protein. Ratios of IgA1/IgA2 and S100A8/S100A9 were calculated. ASD severity was evaluated using the Childhood Autism Rating Scale (CARS). Results: Children with ASD showed trends toward higher IgA and calprotectin and lower a1-antitrypsin compared with siblings, but differences were not statistically significant. Subgroup analysis suggested different distribution patterns in moderate versus severe ASD, including higher IgA in the moderate group and altered S100A8/S100A9 ratio in the severe group. These subgroup findings were exploratory, derived from critically underpowered post-hoc analyses (severe subgroup: n = 11 pairs, ~18% power for medium effects), and should be considered hypothesis-generating only, pending validation in adequately powered pre-registered studies. Conclusions: The results are consistent with recent meta-analyses reporting no consistent evidence of gut inflammation in ASD. Larger, sex-matched studies with full assay validation are needed to clarify the role of stool proteins in ASD.

• Language: English
• Keywords: autism, inflammation, protein biomarkers
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy; MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: 17 str.
• Numbering: Vol. 17, art. 1792801
• PID: 20.500.12556/RUL-185151
• UDC: 616.896
• ISSN on article: 1664-0640
• DOI: 10.3389/fpsyt.2026.1792801
• COBISS.SI-ID: 275381251
• Publication date in RUL: 24.07.2026

Record is a part of a journal

• Title: Frontiers in psychiatry
• Shortened title: Front. psychiatry
• Publisher: Frontiers Research Foundation
• ISSN: 1664-0640
• COBISS.SI-ID: 54153314

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: avtizem, vnetje, proteinski biološki označevalci

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: J3-1756
• Name: Okoljski in genetski dejavniki pri motnjah avtističnega spektra

• Funder: ARRS - Slovenian Research Agency
• Project number: P3-0124
• Name: Metabolni in prirojeni dejavniki reproduktivnega zdravja, porod III