Fluoride exposure disrupts creatinine homeostasis and undermines the reliability of urinary metabolomics...

Urinary creatinine is widely used to adjust for dilution in studies of urinary biomarkers and metabolomics, yet its stability under environmental exposures is insufficiently evaluated. We analyzed data from U.S. adolescents (NHANES, n = 2378) and a cross-sectional sample of older Chinese adults (n = 475) to assess whether fluoride exposure disrupts urinary creatinine homeostasis and compromises its effectiveness as a normalization factor. In NHANES, participants with missing urinary fluoride measurements, abnormal urine albumin-to-creatinine ratios, or extreme fluoride values were excluded. In both populations, urinary fluoride demonstrated a nonlinear inverted L-shaped association with creatinine, characterized by a steep increase at lower exposure levels followed by a plateau. To quantify the downstream implications for metabolomic analyses, we compared results from unadjusted data with three dilution-control strategies: creatinine adjustment, specific gravity adjustment, and osmolality adjustment. Creatinine normalization markedly altered metabolite identification and pathway enrichment outcomes: 47.34 % of differential metabolites reversed their expression direction, and 24.72 % changed statistical significance. Enriched pathways shifted as well-from amino acid metabolism and circadian rhythm in the unadjusted analysis to lysine degradation and pyrimidine metabolism after creatinine adjustment. In contrast, specific gravity- and osmolality-based normalization produced results highly concordant with the unadjusted data in both metabolite directionality and pathway structure. Together, these findings indicate that fluoride exposure perturbs creatinine homeostasis and, consequently, the use of creatinine as a normalization factor can introduce substantial bias in urinary metabolomics. Caution is therefore warranted when applying creatinine adjustment in environmental epidemiology, particularly in populations with fluoride exposure.