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

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 α1-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: 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.