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Vitamin B6 plays a crucial role as a cofactor in various enzymatic reactions but bacteria-produced vitamin B6 is not sufficient to meet host requirements. Our objective was to assess the impact of diet-derived vitamin B6 on gut microbiota and host serum metabolomics. Sprague–Dawley rats (<i>n</i> = 47) were fed a control, low B6 (LB6) or high B6 (HB6) diet for six weeks. Serum and cecal samples were collected for biochemical, metabolomics and gut microbiota profiling. There was a significant sex effect for gut microbiota and several metabolic markers. Bodyweight and percent body fat were significantly reduced in LB6 compared to control and HB6 rats. Microbial beta-diversity differed significantly between LB6 and the control and HB6 rats in both sexes. <i>Lachnospiraceae</i>_NK4A136_group and <i>Bacteroides</i> were the primary taxa driving the difference between LB6 and control. There was a significant separation of cecal and serum metabolites of LB6 compared to control and HB6 rats. In the cecum, arginine biosynthesis was impaired, while vitamin B6 metabolism, lysine degradation and nicotinate and nicotinamide metabolism were impaired in serum metabolite profiles. Cecal propionate and butyrate were significantly reduced in LB6 rats irrespective of sex. Host vitamin B6 deficiency but not excess significantly alters gut microbial composition and its metabolites.