Pilot Trial of Vitamin D3 and Calcifediol in Healthy Vitamin D Deficient Adults: Does It Change the Fecal Microbiome?

Abstract

Context: Experimental studies suggest that vitamin D receptor signaling may benefit the gut microbiome. In humans, whether vitamin D supplementation directly alters the gut microbiome is not well studied.

Objective: To determine whether correcting vitamin D deficiency with cholecalciferol (vitamin D3, D3) or calcifediol (25-hydroxyvitamin D3, 25(OH)D3) changes gut microbiome composition.

Methods: 18 adults with vitamin D deficiency (25-hydroxyvitamin D [25(OH)D] <20 ng/mL) received 60 µg/day of D3 or 20 µg/day of 25(OH)D3 for 8 weeks. Changes in serum 25(OH)D, 1,25-diydroxyvitamin D (1,25(OH)2D), and 24,25-dihydroxyvitamin D (24,25(OH)2D) were assessed. We characterized composition of the fecal microbiota using 16S rRNA gene sequencing, and examined changes in α-diversity (Chao 1, Faith's Phylogenetic Diversity, Shannon Index), β-diversity (DEICODE), and genus-level abundances (DESeq2).

Results: Vitamin D3 and 25(OH)D3 groups were similar. After 8 weeks of vitamin D3, mean 25(OH)D and 24,25(OH)2D increased significantly, but 1,25(OH)2D did not (25(OH)D: 17.8-30.1 ng/mL, P = .002; 24,25(OH)2D: 1.1 to 2.7 ng/mL, P =0.003; 1,25(OH)2D: 49.5-53.0 pg/mL, P = .9). After 8 weeks of 25(OH)D3, mean 25(OH)D, 24,25(OH)2D, and 1,25(OH)2D increased significantly (25(OH)D: 16.7-50.6 ng/mL, P < .0001; 24,25(OH)2D: 1.3-6.2 ng/mL, P = .0001; 1,25(OH)2D: 56.5-74.2 pg/mL, P = .05). Fecal microbial α-diversity and β-diversity did not change with D3 or 25D3 supplementation. Mean relative abundance of Firmicutes increased and mean relative abundance of Bacterioidetes decreased from baseline to 4 weeks, but returned to baseline by study completion. DESeq2 analysis did not confirm any statistically significant taxonomic changes.

Conclusion: In a small sample of healthy adults with vitamin D deficiency, restoration of vitamin D sufficiency with vitamin D3 or 25(OH)D3 did not lead to lasting changes in the fecal microbiota.

Trial registration: ClinicalTrials.gov NCT02091219.

Keywords: Cholecalciferol; calcifediol; gut microbiome.

© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Figures

Figure 1.

Study flow chart.

Figure 2.

Changes in fecal microbiota composition following 4 and 8 weeks of cholecalciferol (D3) supplementation. (A) α-Diversity measured by Chao1, Faith’s PD, and Shannon Index. (B) PCoA plot of DEICODE distances for the fecal microbiome. Significance of changes in β-diversity by timepoint was calculated using PERMANOVA adjusted for subject. (C) Genus level taxonomic profiles at baseline, week 4 and week 8 after D3 supplementation.

Figure 3.

Changes in fecal microbiota composition following 4 and 8 weeks of calcifediol (25D3) supplementation. (A) α-Diversity measured by Chao1, Faith’s PD, and Shannon Index. (B) PCoA plot of DEICODE distances for the fecal microbiome. Significance of changes in β-diversity by timepoint was calculated using PERMANOVA, adjusted for subject. (C) Genus level taxonomic profiles at baseline, week 4 and week 8 after 25D3 supplementation.

Figure 4.

Changes in fecal microbiota composition following 4 and 8 weeks of cholecalciferol (D3) or calcifediol (25D3) supplementation. (A) α-diversity measured by species richness (Chao1), phylogenetic diversity (Faith’s PD), and species evenness (Shannon Index). (B) Principle coordinates analysis (PCoA) plots of DEICODE distances for the fecal microbiome. The P value for differences in β-diversity by timepoint was calculated using PERMANOVA adjusted for participant. (C) Supervised multilevel analysis, adjusted for participant specific effects using sPLS-DA. (D) Genus, (E) family, (F) order, and (G) phylum level taxonomic profiles at baseline, week 4 and week 8 after 25D3 or D3 supplementation.

Figure 5.

Associations of vitamin D metabolite and metabolite ratios with microbial α-diversity measured by Faith’s PD. Associations assessed by multivariable linear regression adjusted for participant. Results reflect within-individual differences in Faith’s PD relative to vitamin D metabolite or vitamin D metabolite ratios.

Figure 6.

Associations of vitamin D metabolite and metabolite ratios with microbial β-diversity. Significance of associations was assessed by PERMANOVA adjusted for participant. Results reflect within-individual differences in β-diversity relative to vitamin D metabolite or vitamin D metabolite ratios.