Dietary Vitamin B-9 and B-12 Deficiencies Alter Gut Homeostasis in Sprague–Dawley Rats

BACKGROUND: B vitamins, particularly B-9 and B-12, are essential cofactors in metabolic reactions, including one-carbon (1C) metabolism, which supports growth and overall metabolic function. Although gut bacteria synthesize some B vitamins, this is insufficient to meet host requirements, making dietary intake crucial. Despite their established biochemical roles, their effects on gut homeostasis remain poorly understood. OBJECTIVES: The study investigated the effects of dietary vitamin B-9 and B-12 deficiencies on gut morphology, microbial composition, and their metabolites. METHODS: A total of 48 Sprague-Dawley rats (4-5 wk old, equal sexes) were fed an AIN-93G control diet (n = 16), a vitamin B-9-deficient diet (LB9, n = 16), or a vitamin B-12-deficient diet (LB12, n = 16) for 6 wk. Blood, tissue, and fecal samples were analyzed for serum vitamins, targeted 1C metabolites, histology, gut microbiota, and fecal metabolomics. Data were analyzed using 1-way or 2-way analysis of variance, with significance set at P ≤ 0.05. RESULTS: Sex-specific differences were observed in body, liver, and brain weights, although no treatment-related effects were detected for these parameters. Serum vitamin B-9 and B-12 concentrations were significantly reduced, with a more pronounced decrease in males in LB9 (9.4 nmol/L) and LB12 (169.6 pmol/L) compared with controls (116.1 nmol/L and 590.0 pmol/L, respectively, P < 0.001). No significant differences were detected in serum 1C metabolites. Histological analysis revealed increased mucosal height in LB12 rats (P = 0.006) and a 68% reduction in acidic mucin content in both deficient groups (P = 0.02). Gut microbial composition and fecal metabolite profiles varied by sex, with notable alterations in bacterial genera including Streptococcus, Lactobacillus, Blautia, Lachnochlostridium, Colidextribacter, and Dorea, as well as metabolites such as hydrocinnamic acid, tryptophan, and N-acetylneuraminic acid. CONCLUSIONS: Dietary vitamin B-9 and B-12 deficiencies induce sex-dependent alterations in gut morphology, microbiota, and metabolite profiles, emphasizing their essential roles in maintaining gastrointestinal and metabolic homeostasis.