ABSTRACT Owing to the adverse impact of vitamin D deficiency on the skeleton and risk of skin cancer associated with prolonged cutaneous exposure to sunlight, oral supplementation with vitamin D3 or D2 has been widely accepted and promoted as the mode of achieving and maintaining calcium homeostasis in children and adults. Given recent appreciation of the profound affect that the gut microbiome has on host human health and disease, with support of this supplement we will be performing the first pilot study to compare and contrast the impact of the most widely used oral vitamin D supplement (vitamin D3) versus the more rapidly acting, experimental 25-hydroxylated metabolite, 25D3, on the gut-residing microbes that have the first opportunity to encounter and respond to those vitamin D metabolites. With support from this supplement, we wish to take advantage of the still-recruiting, multi-ethnic vitamin D replacement trial comparing oral administration of vitamin D3 and 25-hydroxyvitamin D3 (25D3) in vitamin D deficient subjects (total serum 25D ≤20 ng/ml) set forth in Specific Aim 3 of the parent grant R01AR063910. This application includes a plan for supplemental analysis of the gut microbiome in those subjects before and after normalization of total and free serum 25D. We propose that i) there will exist predictable changes in the taxomic 16S rRNA-sequencing profiles of the stool microbiota and 2) the impact of that altered bacterial microbiome on the host's skeletal and immune homeostasis that will be conditioned by the form of vitamin D supplementation and the host's ethnicity (White, Black, Hispanic, Asian). These new analyses will i) compliment the already-funded program's novel assay technology for determining serum free 25D and 1,25D and ii) further advance our definition of `personalized' vitamin D health in terms of both classical (bone) and non-classical (immune) actions of vitamin D. !

RELEVANCE TO PUBLIC HEALTH This supplemental R21 application challenges current research and clinical practice paradigms which do not consider the potential effects of orally administered, bone-active agents on the gut microbiota nor how this might explain inter-individual differences in response to a “standardized dose” of the oral agent. This project has great potential to fill important knowledge gaps regarding whether i) drugs previously considered to target only the human host also have a functional impact on the gut microbiome, ii) orally-administered vitamin D3 versus 25-hydroxyvitamin D3 will have such an effect and iii) that effect has the potential to alter (e.g. enhance) the expected skeletal response to these two metabolites. Expedient support of this supplemental request will permit us to take immediate advantage of still-to-be-recruited subjects in our ongoing clinical experiment under the auspices of R01AR063910. !