The potential role of vitamin D supplementation as a gut microbiota modifier in healthy individuals

Vitamin D, essential for skeletal health, electrolyte balance, and immune regulation, is deficient in a significant portion of the global population, particularly in Arab countries (up to 85%). This deficiency is associated with various diseases, including cancer, cardiovascular disease, diabetes, obesity, and inflammatory bowel disease. A bidirectional relationship exists between vitamin D, gut microbiota, and inflammation, suggesting that vitamin D influences gut microbial composition and vice-versa. While studies have explored vitamin D supplementation's impact on microbiota in individuals with chronic diseases, its effect on healthy, deficient individuals remains unclear. Furthermore, the role of gut microbiota in the variable response to vitamin D supplementation is unknown. This study aimed to address these gaps by investigating the effects of vitamin D supplementation on the gut microbiota of healthy vitamin D-deficient women and determining whether specific microbial signatures predict the response to supplementation.

Prior research has primarily focused on the impact of vitamin D supplementation on the gut microbiota in individuals with chronic diseases such as cystic fibrosis and multiple sclerosis. These studies demonstrated that vitamin D supplementation altered gut microbiota composition, often increasing beneficial bacteria like *Akkermansia* and butyrate-producing bacteria. In pre-diabetic individuals, increased serum 25(OH)D levels were associated with altered ratios of Firmicutes and Bacteroidetes. However, studies on healthy individuals are limited and show conflicting results. A small study reported increases in *Bacteroides* and *Akkermansia* with vitamin D supplementation, but larger studies evaluating the lower gastrointestinal tract are lacking. Inter-individual variability in serum 25(OH)D response to supplementation is substantial, with around 25% of individuals showing minimal increase. While factors like dose, supplement type, age, and baseline 25(OH)D levels contribute, a significant portion of the variability remains unexplained. This study hypothesized that the gut microbiota composition may influence the response to vitamin D supplementation.

This study enrolled 100 healthy, vitamin D-deficient women (80 completed both phases). Participants were given a weekly oral dose of 50,000 IU vitamin D3 for 12 weeks. Blood and stool samples were collected at baseline (pre-supplementation) and after 12 weeks (post-supplementation). Serum 25(OH)D levels, calcium, creatinine, BUN, AST, and ALT were measured. Gut microbiota composition was characterized using 16S rRNA gene sequencing on the Illumina MiSeq platform. Data processing in QIIME2 involved denoising with DADA2, generating amplicon sequence variants (ASVs), and taxonomic classification using Greengenes database. Alpha and beta diversity were assessed using various indices (Observed OTUs, Chao1, Shannon, InvSimpson, weighted UniFrac). Statistical analyses included paired Wilcoxon tests, mixed models (lme4), PERMANOVA, and DESeq2 for differential abundance analysis. PICRUSt was used for predicted functional profiling. Participants were categorized as responders (serum 25(OH)D > 20 ng/ml post-supplementation) or non-responders.

Vitamin D supplementation significantly increased average serum 25(OH)D levels (from 11.03 ± 0.51 ng/ml to 34.37 ± 1.47 ng/ml, p=5.1e-14). It also increased blood calcium levels and improved blood markers of kidney and liver function. Gut microbiota analysis revealed a significant increase in overall microbial diversity post-supplementation (increased observed OTUs and Chao1 indices, p<0.001). The Bacteroidetes to Firmicutes ratio increased significantly (p=0.0579). At the genus level, *Akkermansia* and *Bifidobacterium* abundance increased significantly (p<0.05). A shift towards a *Bacteroides*-dominated enterotype was observed (increased Bacteroides/Prevotella ratio, p=0.0057). Comparing responders and non-responders, responders exhibited more pronounced changes in phylum abundance, including significant increases in Bacteroidetes, Actinobacteria, Proteobacteria, and Lentisphaeraea, and a significant decrease in Firmicutes (p<0.05). Non-responders showed a significant decrease in *Bacteroides acidifaciens* post-supplementation (p<0.01). PICRUSt analysis predicted increased genes involved in folate biosynthesis, glycine metabolism, and lipid metabolism post-supplementation.

This study demonstrates that vitamin D supplementation positively impacts the gut microbiota of healthy, vitamin D-deficient women, increasing diversity and shifting the microbial composition towards a potentially more beneficial state (higher Bacteroidetes/Firmicutes ratio, increased *Akkermansia* and *Bifidobacterium*). The observed changes align with previous findings linking altered gut microbiota with various diseases. The increased abundance of bacteria involved in lipid metabolism may enhance vitamin D bioavailability. The identification of *Bacteroides acidifaciens* as a potential biomarker for vitamin D response warrants further investigation. The mechanisms underlying vitamin D's modulation of the gut microbiota are likely multifactorial, involving both systemic effects (VDR signaling) and potential direct effects of unabsorbed vitamin D in the distal gut. This warrants further research exploring the direct and indirect mechanisms involved.

This study provides evidence that vitamin D supplementation beneficially alters gut microbiota composition and diversity in healthy, deficient women, improving biomarkers for kidney and liver function. The association between specific microbial signatures and vitamin D response highlights the potential for personalized approaches to vitamin D supplementation. Future research should focus on larger cohorts, mechanistic studies, and multi-omics approaches to elucidate the complex interplay between vitamin D, gut microbiota, and individual response.

The study's limitations include the lack of vitamin D-sufficient controls and a placebo group. The female-only cohort limits generalizability to males. A larger cohort with sufficient responders/non-responders is needed to validate the findings. Furthermore, the study relied on 16S rRNA sequencing, which provides limited resolution at the species level compared to metagenomic approaches.