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ABSTRACT

Summary Background Saliva is an emerging but understudied biofluid in metabolomics, offering a similar window into biological information as blood, while enabling repetitive and non-invasive sampling. Methods In this observational study, we comprehensively analysed the salivary metabolome of 965 children and adolescents (7–17y) from two exploratory cohorts in Europe, using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry. Preprocessed data were assessed via (partial) Spearman correlation, Mummichog-based pathway enrichment, and univariate (Welsh t-test, one-way ANOVA, Wilcoxon or Kruskal–Wallis rank-sum test) analyses. All p-values were corrected using the Benjamini-Hochberg procedure. Associations with age, anthropometrics, mental wellbeing, lifestyle, dietary intake and microbiome were explored. Key metabolite associations were validated in two independent cohorts (8–18y, n = 471), yielding a total study population of 1436 participants. Findings Over 3500 untargeted features and 188 targeted metabolites were consistently detected in the pooled quality control saliva of the exploratory cohorts. Weight status (|ρ| < 0.255) and age (|ρ| < 0.341) reflected the salivary metabolome best, compared to mental wellbeing (|ρ| < 0.227) and lifestyle factors (|ρ| < 0.185). Multiple metabolites, especially (acetylated) amino acids and derivatives were elevated in participants with overweight or obesity and those experiencing high psychological stress, mirroring blood-based studies. In addition, these metabolites correlated with Bacteroidota, Pseudomonadota, Actinomycetota and Bacillota (|ρ| < 0.423) phyla, suggesting oral microbiome disturbances. Notably, the artificial sweetener acesulfame K, detected for the first time in children and adolescents’ saliva, was associated with excess weight (|ρ| < 0.243), psychological stress (|ρ| < 0.148), specific food and/or beverage consumption (|ρ| < 0.348) and Bacillota spp. (|ρ| < 0.196). Interpretation Our findings reinforce and expand upon previous blood-based studies, while revealing novel salivary biomarkers and, as such, provide a high-quality repository of the paediatric salivary metabolome for future studies exploring its association with children and adolescent's health. Funding FAME and OPERA were supported by the Research Foundation Flanders, Ghent University, and the European Research Council. PANIC was funded by Finnish national agencies and foundations. Fin-HIT was funded by the Folkhälsan Research Foundation and the Päivikki and Sakari Sohlberg Foundation.

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