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

INTRODUCTION: Early-life emotional and behavioral difficulties increase vulnerability to later psychopathology, but it remains unclear whether a biological footprint is detectable in early childhood. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.