Early childhood serum and urine metabolite signatures of internalizing, externalizing, and ADHD traits and their trajectories to adolescence: the Rhea mother–child cohort

Early-life emotional and behavioral difficulties increase vulnerability to later psychopathology, but it remains unclear whether a biological footprint is detectable in early childhood. We aimed to identify serum- and urine-based metabolite signatures of internalizing, externalizing, and attention-deficit/hyperactivity disorder (ADHD) traits in 6-year-old children and examine their associations with traits through adolescence. We analyzed 196 participants of the Rhea cohort (Greece), with serum and urine metabolomics at age 6 and internalizing, externalizing, and ADHD traits assessed at ages 6, 11, and 15. Trait- and biofluid-specific metabolite signatures were identified using elastic net regression with cross-validation. Associations with subsequent traits at repeated time points were evaluated using confounder-adjusted linear mixed-effects models. Metabolite signatures comprising amino acids, acylcarnitines, phosphatidylcholines, and gut microbiota-related metabolites were identified for internalizing, externalizing, and ADHD traits. Serum metabolite scores were associated with increased internalizing (β = 0.40; 95% CI 0.14–0.68) and externalizing traits (β = 0.83; 95% CI 0.56–1.1). Urine metabolite scores were associated with internalizing (β = 0.42; 95% CI 0.16–0.68) and externalizing traits (β = 0.69; 95% CI 0.43–0.95), while the association with ADHD traits was nominal (β = 0.25; 95% CI: 0.006–0.502; nominal p = 0.045; BH-FDR p = 0.054). Associations were stable over time for internalizing and ADHD but weakened with age for externalizing traits. This proof-of-concept study shows that complementary serum and urine metabolomic profiles in early childhood are associated with subsequent internalizing and externalizing trait trajectories, while a nominal longitudinal association was observed between the urine metabolite profile and ADHD traits. These findings warrant evaluation in larger independent studies.