Oral vitamin B12 supplement is delivered to the distal gut, altering the corrinoid profile and selectively depleting Bacteroides in C57BL/6 mice

Abstract

Vitamin B₁₂ is a critical nutrient for humans as well as microbes. Due to saturable uptake, high dose oral B₁₂ supplements are largely unabsorbed and reach the distal gut where they are available to interact with the microbiota. The aim of this study was to determine if oral B₁₂ supplementation in mice alters 1) the concentration of B₁₂ and related corrinoids in the distal gut, 2) the fecal microbiome, 3) short chain fatty acids (SCFA), and 4) susceptibility to experimental colitis. C57BL/6 mice (up to 24 animals/group) were supplemented with oral 3.94 µg/ml cyanocobalamin (B₁₂), a dose selected to approximate a single 5 mg supplement for a human. Active vitamin B₁₂ (cobalamin), and four B₁₂-analogues ([ADE]CN-Cba, [2Me-ADE]CN-Cba, [2MeS-ADE]CN-Cba, CN-Cbi) were analyzed in cecal and fecal contents using liquid chromatography/mass spectrometry (LC/MS), in parallel with evaluation of fecal microbiota, cecal SCFA, and susceptibility to dextran sodium sulfate (DSS) colitis. At baseline, active B₁₂ was a minor constituent of overall cecal (0.86%) and fecal (0.44%) corrinoid. Oral B₁₂ supplementation increased active B₁₂ at distal sites by >130-fold (cecal B₁₂ increased from 0.08 to 10.60 ng/mg, fecal B₁₂ increased from 0.06 to 7.81 ng/ml) and reduced microbe-derived fecal corrinoid analogues ([ADE]CN-Cba, [2Me-ADE]CN-Cba, [2MeS-ADE]CN-Cba). Oral B₁₂ had no effect on cecal SCFA. Microbial diversity was unaffected by this intervention, however a selective decrease in Bacteroides was observed with B₁₂ treatment. Lastly, no difference in markers of DSS-induced colitis were detected with B₁₂ treatment.

Keywords: Inflammatory bowel disease; colitis; microbiota; vitamin B12.

Figure 1.

B₁₂ supplement alters the corrinoid profile in the cecum and feces. (a) Base moieties of the most abundant corrinoids. Cobinamide lacks the base as well as ribose and phosphate moieties present in other corrinoids. (b) Cecal corrinoid profile overall and (c) by individual corrinoid in control (n = 5) and B₁₂ treated (n = 4) mice by unpaired two-tailed Student’s t-test. (d) Fecal corrinoid profile overall and (e) by individual corrinoid at baseline and after 16-day B₁₂ supplementation (ADE n = 16/group, others n = 22/group, paired two-tailed t-test).

Figure 2.

Influence of oral B₁₂ supplement on the fecal microbiome. Beta diversity analysis of (a) phylum and (b) genus level (n = 12 mice/time point) at baseline and following 16-day B₁₂ supplementation. *p < 0.05. (c) Significant reduction in the relative abundance of Bacteroides following B₁₂ supplementation (paired two-tailed t-test). (d) Cecal short-chain fatty acids in animals administered H₂O (Control) and experimental colitis (DSS) with and without B₁₂. (Control n = 15, B₁₂ n = 15, DSS n = 20, DSS/B₁₂ n = 18; paired two-tailed t-test (c) and ANOVA with Tukey’s post-test (d).

Figure 3.

B₁₂ supplement in DSS colitis. (a) Vitamin B₁₂ had no effect on weight loss in DSS colitis (sum of three replicate experiments, Control (n = 15), B₁₂ (n = 15), DSS (n = 23), DSS/B₁₂ (n = 24) by ANOVA and Dunnett’s multiple comparisons (Control vs DSS or DSS/B₁₂: p < 0.0001, but DSS vs DSS/B₁₂: p = ns) or measures of disease including (b) colon length, Control (n = 15), B₁₂ (n = 15), DSS (n = 20), DSS/B₁₂ (n = 19) by ANOVA and Tukey’s multiple comparisons, or (c) enteral administered FITC-dextran detected in circulation of Control (n = 14), B₁₂ (n = 15), DSS (n = 20), DSS/B₁₂ (n = 19) by ANOVA and Tukey’s multiple comparisons. (d) Colon tissue cytokines were not significantly different comparing DSS vs DSS/B₁₂ by ANOVA and Tukey’s multiple comparisons. Unique letter represents p < 0.05.