Nutritional and metabolic signatures in pediatric phenylketonuria and hyperphenylalaninemia: Insights from untargeted urinary metabolomics

• Urinary metabolomics discriminated PKU, BH4, HPA, and controls. • Variability reflected endogenous, dietary, and microbiota-related metabolism. • BH4 individuals showed the greatest heterogeneity in metabolomic fingerprints. • Microbiota- and diet-related metabolites were reduced in PKU. • Muscle-related metabolites (methylhistidines, creatine, BCAAs) were lower in PKU Dietary interventions are essential for managing phenylketonuria (PKU) and may influence metabolic regulation beyond phenylalanine control. We characterized the urinary metabolomic fingerprint of pediatric participants (n = 82) recruited into clinical phenotypes: PKU, PKU with response to tetrahydrobiopterin (BH4), and hyperphenylalaninemia (HPA), as well as sex- and age-matched healthy children controls. Untargeted metabolomics (HPLC-Q-TOF-MS/MS) revealed 59 discriminant metabolites across multiple biochemical pathways, including phenylalanine, tryptophan, and caffeine metabolisms, as well as metabolites related to dietary exposure or gut microbial metabolism. Distinct urinary signatures were described across phenotypes. phenylalanine-related pathways predominated in PKU, accompanied by increased excretion of vitamin derivatives, consistent with protein substitute supplementation. In contrast, reduced levels of non-phenylalanine amino acid derivatives, methylhistidines, creatine, and branched-chain amino acid-related metabolites were observed in PKU, suggesting alterations in muscle metabolism or natural protein intake. Microbiota-derived metabolites were also less represented in PKU, indicating potential effects of dietary restrictions on gut–host metabolic interactions. HPA individuals showed a urinary fingerprint closer to controls, whereas the BH4 subgroup exhibited the greatest metabolic heterogeneity, reflecting variability in dietary and pharmacological treatment responses. These findings reveal metabolic diversity within the pediatric PKU spectrum driven by clinical phenotype and nutritional management. Urinary metabolomics may support more precise monitoring of metabolic health status and guide precision nutrition strategies in PKU and HPA from early life.