Vitamin D metabolites and the gut microbiome in older men

The vitamin D receptor (VDR) is highly expressed in the gastrointestinal tract, suggesting a role for vitamin D in gut health. While some studies have shown associations between vitamin D levels and various health outcomes, others have found no such associations. The primary circulating form of vitamin D, 25(OH)D, reflects total body stores, while the active form, 1,25(OH)₂D, directly interacts with the VDR. Ratios of 1,25(OH)₂D to 25(OH)D (activation ratio) and 24,25(OH)₂D to 25(OH)D (catabolism ratio) may better reflect vitamin D metabolic activity. This study aimed to investigate the association between serum vitamin D metabolites (25(OH)D, 1,25(OH)₂D, and 24,25(OH)₂D) and the gut microbiome composition in a large cohort of older men. The hypothesis was that levels of active vitamin D metabolites and associated metabolic ratios, rather than total vitamin D stores, would be strongly associated with the composition and diversity of the gut microbiome. This investigation is crucial because of the significant clinical interest in understanding the interplay between vitamin D and gut health in relation to various diseases, such as osteoporosis, obesity, inflammatory bowel disease, diabetes, cardiovascular disease, cancer, and autoimmune diseases. The lack of conclusive evidence from previous studies necessitates further exploration into the complex relationship between vitamin D metabolism, gut microbiota, and overall health. The potential for a pathogenic cascade involving vitamin D deficiency and dysbiosis needs to be clarified. This study addresses this gap in knowledge using a large and well-characterized population to allow for robust conclusions.

Several studies suggest a link between gut microbiota, intestinal vitamin D metabolism (VDM), and circulating vitamin D levels. Probiotic supplements have been shown to influence circulating vitamin D levels. However, the results regarding the associations between low 25(OH)D levels and disease are inconsistent, with some studies showing associations and others showing none. A large randomized controlled trial showed no benefit of vitamin D supplementation in preventing cardiovascular events or cancer. While 25(OH)D is the preferred clinical measure for vitamin D sufficiency, 1,25(OH)₂D is the active form that interacts with the VDR and regulates gene expression. Ratios of vitamin D activation and catabolism may be better predictors of clinically important outcomes than 25(OH)D alone. Existing literature also points towards a potential for dysbiosis in mice with disrupted vitamin D metabolism and suggests that manipulation of the gut microbiome or VDM might have favorable health impacts. Previous human studies using high-dose vitamin D supplementation or probiotic introduction show some positive effects, but more research is needed to elucidate the interplay between vitamin D signaling and the microbiome.

This cross-sectional study utilized data from the Osteoporotic Fractures in Men (MrOS) study, specifically focusing on 567 older men who provided stool samples and had serum vitamin D metabolite data available. Stool samples were collected using the OMNIgene-GUT kit, and the V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Sequence data were processed using QIIME2, including deblurring to minimize sequencing errors. Serum concentrations of 25(OH)D, 1,25(OH)₂D, and 24,25(OH)₂D were measured using liquid chromatography coupled to tandem mass spectrometry (LC-MSMS). Microbial α-diversity was assessed using Faith's phylogenetic diversity (PD), and β-diversity was analyzed using unweighted UniFrac. Redundancy analysis (RDA) was used to identify non-redundant covariates explaining variation in α- and β-diversity. Multiple linear regression (MLR) was used to examine the relationship between PD and vitamin D metabolites, adjusting for confounders. Random forest classification with nested cross-validation was used to identify sOTUs associated with vitamin D metabolites. Specific details on sample collection, processing, sequencing, data analysis, and statistical methods are available in the supplementary information. The LC-MSMS methods used for the measurement of the three vitamin D metabolites are extensively described. The methods were rigorously validated to ensure accuracy and reliability. The study included a large number of covariates such as age, race, body mass index, smoking status, alcohol consumption, physical activity level, season of sample collection, and various medication use. Dietary intake of resistant starch was also estimated.

Redundancy analyses revealed that serum 1,25(OH)₂D explained the highest proportion of variance in α-diversity (5%) and β-diversity (2%). Multiple linear regression analyses, adjusting for confounders, showed a positive association between 1,25(OH)₂D levels and α-diversity (p = 7.23 × 10⁻⁷). Higher levels of 24,25(OH)₂D, activation ratio, and catabolism ratio were also positively associated with α-diversity. Antibiotic use was negatively associated with α-diversity. PERMANOVA tests on β-diversity confirmed the significant associations of 1,25(OH)₂D, 24,25(OH)₂D, activation, and catabolism ratios. Random forest analysis identified 12 sOTUs associated with vitamin D metabolism; 11 were from the Firmicutes phylum, many of which were butyrate-producing bacteria positively associated with 1,25(OH)₂D and the activation ratio. Men with higher 1,25(OH)₂D and activation ratios, but not 25(OH)D, had a higher likelihood of possessing butyrate-producing bacteria. Vitamin D deficiency was associated with a higher activation ratio, and vitamin D adequacy with a higher catabolism ratio. The random forest models showed high predictive accuracy (AUC > 0.7) indicating a strong relationship between gut microbiome composition and vitamin D metabolism.

This study provides strong evidence for the interaction between host vitamin D signaling and gut microbiome health in older men. The positive association between active vitamin D metabolites (1,25(OH)₂D) and favorable gut microbiome composition, particularly the presence of butyrate-producing bacteria, supports the hypothesis that vitamin D plays a significant role in shaping the gut microbiome. The finding that 1,25(OH)2D and the activation ratio, but not 25(OH)D itself, were the strongest correlates of microbiome diversity, suggests that the active form of Vitamin D and its rate of production may be particularly crucial in regulating gut microbiota. Butyrate is known to have beneficial effects on gut health, and its association with higher levels of 1,25(OH)₂D highlights the potential for a positive feedback loop between vitamin D and gut microbial health. The study's findings suggest potential targets for interventions aimed at improving gut health through dietary modifications or vitamin D supplementation in appropriate populations. However, the cross-sectional design prevents the determination of causality. Future longitudinal studies are needed to investigate the temporal relationship between vitamin D metabolism and changes in gut microbiome composition.

This large cross-sectional study demonstrates a significant association between active vitamin D metabolites and a favorable gut microbiome profile in older men. Higher levels of 1,25(OH)₂D and the activation ratio are associated with increased α-diversity and the presence of butyrate-producing bacteria. These findings suggest a potential role for vitamin D in maintaining gut microbial health and highlight the potential for interventions targeting vitamin D metabolism to improve gut health. Future research should focus on longitudinal studies to establish causality and investigate the mechanisms underlying this relationship.

The cross-sectional design of this study limits the ability to establish causality between vitamin D metabolites and gut microbiome composition. The use of 16S rRNA gene sequencing, rather than shotgun metagenomics, limits the resolution of taxonomic classifications. The study population consisted primarily of older, predominantly white men, limiting the generalizability of findings to other populations. The metabolomic data for the clinical measures of vitamin D metabolites are not publicly available due to their generation in a large clinical laboratory, however, other data are available through the MrOS data online repository with restrictions.