Integrated absolute-quantitative gut microbiota profiling and targeted metabolomics reveal microbial and metabolic alterations in children with juvenile idiopathic arthritis

Gut microbiota alterations have been implicated in juvenile idiopathic arthritis (JIA), but most previous studies relied on relative abundance profiling and lacked direct metabolite validation. This study aimed to characterise quantitative gut microbiota and short-chain fatty acid (SCFA)/organic acid profiles and identify candidate biomarkers in children with JIA. Absolute quantification identified significant differences in α diversity between children with JIA and controls, including Pielou’s evenness, Shannon index, and Simpson index, whereas relative quantification detected none. Bray–Curtis dissimilarity, a β-diversity index, also differed significantly between the two groups (PERMANOVA, P < 0.001). At the genus level, Escherichia-Shigella , Flavonifractor , and Methyloversatilis were significantly enriched in JIA patients. Targeted metabolomics revealed divergent faecal and serum metabolic patterns. In faeces, succinate was significantly reduced in JIA patients (fold change = 0.30, adjusted P = 0.005), whereas other faecal metabolites did not reach significance. In serum, acetate, propionate, butyrate, and 2,3-dihydroxy-3-methylbutanoate were significantly elevated in JIA patients, while lactate, malonate, isobutyrate, pentanoate, glutarate, and hexanoate were significantly decreased. Notably, in the full cohort, serum butyrate was markedly elevated in JIA patients compared with controls (median 1543.71 ng/mL, IQR 1237.52–1874.68 vs. 96.98 ng/mL, IQR 78.68–109.65), with a 15.9-fold elevation. In treatment-stratified sensitivity analyses, serum butyrate remained significantly elevated in both drug-naïve patients ( n = 15; median 1733.74 ng/mL, IQR 1461.95–2042.70; fold change = 17.9 vs. controls) and drug-exposed patients ( n = 34; median 1499.60 ng/mL, IQR 1175.45–1755.05; fold change = 15.5 vs. controls), with no significant difference between the two subgroups. Serum butyrate showed strong discriminatory potential for distinguishing JIA patients from controls, with an optimal cut-off value of 511.94 ng/mL. This study identified gut microbiota and metabolite alterations in children with JIA, and demonstrated that absolute quantification can unmask subtle differences undetectable by relative abundance profiling. The core microbial and metabolic features of JIA are largely disease-intrinsic, yet pharmacotherapy can modulate these features. Serum butyrate shows potential as a biomarker for JIA. This study was registered on April 11, 2023, at the Chinese Clinical Trial Registry (registration number ChiCTR2300070403) and at the China National Health Security Information Platform / Medical Research Registration and Filing Information System (registration number MR3323015522).