Stool Metabolomics Reveals Catecholamine and Tryptophan-NAD+ Pathway Alterations in Autism Spectrum Disorder

Abstract

Gut-brain axis dysregulation and microbiome-linked metabolic disturbances have been implicated in autism spectrum disorder (ASD), yet gut-derived neuroactive metabolites remain incompletely characterized. We applied targeted liquid chromatography-tandem mass spectrometry in a cross-sectional case-control study to quantify 18 stool metabolites related to catecholamine synthesis, GABAergic signaling, and tryptophan (Trp) and NAD+ metabolism in 59 participants (32 ASD, 27 non-ASD controls). Group differences were evaluated using log2 fold change and covariate-adjusted linear models. Random forest classifiers with five-fold cross-validation assessed multivariate discriminative performance, and within-group Spearman correlations examined metabolic network organization. Norepinephrine exhibited the largest elevation in ASD, while dopamine and tetrahydrobiopterin showed nominal group effects consistent with altered BH4-dependent catecholamine metabolism. Among individual metabolites, tetrahydrobiopterin, GABA, and kynurenine were the most informative, and their combination achieved an area under the receiver operating characteristic curve of 0.737 (95% confidence interval, 0.603 to 0.871). Correlation analyses revealed conserved lithocholic acid-deoxycholic acid coupling in both groups. In controls, Trp correlated with kynurenine, whereas ASD showed expanded Trp associations with GABA, norepinephrine, anandamide, and nicotinic acid, consistent with reorganization of Trp-NAD+ precursor pathways. These findings identify coordinated, gut-linked metabolic differences in ASD and define a metabolite signature reflecting altered catecholaminergic and Trp-related network structure. ImportanceAutism spectrum disorder is currently diagnosed using behavioral assessments, and objective biological measures are limited. The gut microbiome produces many small molecules that can influence brain development and function, but the specific metabolites involved in ASD remain unclear. Using targeted analysis of stool samples, we identified changes in metabolites related to neurotransmitter synthesis and tryptophan metabolism and showed that their relationships form distinct metabolic networks in individuals with ASD. A small panel of metabolites provided moderate ability to distinguish ASD from controls. These results suggest that gut-derived metabolites capture biologically meaningful variation in ASD and may help guide future studies aimed at understanding gut-brain communication and developing noninvasive biomarkers.