The main purpose of this research study is to learn about the effects of supplemental vitamin D3 and vitamin K2 on blood levels and the gut microbial community, called the gut microbiome. There are many influences on blood levels of vitamin D and K to include your dietary intake, lifestyle behaviors (physical activity, sleep), genetic alterations in metabolism of these vitamins, and the gut microbial abundance and diversity. You will be asked to take one capsule containing vitamin D3 (125 mcg), vitamin K2 (180 mcg), a combination of vitamins D3 and K2 (same doses as previous), or a placebo powder for 90 days. You will also be asked to complete questionnaires, undergo body composition and blood pressure measurements, provide blood samples and a stool swab, all on 2 occasions about 3 - 4 months apart. By conducting this study, we aim to identify the frequency of common genetic alterations in vitamins D and K metabolism and establish the impact on the gut microbiome for the first time. Your participation in this study will last about 4 - 6 months and include up to 3 visits.

Health promotion efforts for all branches of Service aim to ensure the health and readiness of the Force. This timely, relevant study supports this effort as well as the TriService Nursing Research Program priorities of Fit and Ready Force, Care for the Caregiver, and Translate Research into Practice. In 2023, the Defense Health Agency unveiled its strategic plan through 2028 which included these two priorities, 1) 'achieving new levels of excellence by fully leveraging emerging scientific and technological advancements to optimize the health and care experience of our beneficiaries', and 2) 'creating a workplace where people are the primary focus, and the principles of high reliability nurture and energize each individual'. The previous nutrigenomics work by this research team, the expertise and enthusiasm of our new members, and the choice of diverse cohorts for this study, presents an exciting and potentially impactful extension of nutrigenomics with metagenomics of the gut microbiome to provide insight into complex nutrient pathways and specifically, the response of the gut microbial community to select micronutrients. Vitamin D and vitamin K2 are vital micronutrients that support immune, bone, gut, and cardiometabolic health with insufficiencies contributing to musculoskeletal injuries (MSKi), metabolic syndrome, cardiovascular disease, and gut dysbiosis. This Follow-On research application advances the science linking vitamin D, lifestyle behaviors, and chronic disease prevention in healthy adults by probing unexplored areas such as a key symbiotic nutrient, vitamin K2, with vitamin D3, next generation sequencing (NGS) of polymorphisms involved in vitamin metabolism pathways as well as metagenomics to assess the response of the gut microbial community to dietary and supplemental nutrients. The Specific Aims and research questions for our study include:

Specific Aim 1. Evaluate effect of vitamin D3 and K2 dietary intake and supplementation groups vs placebo group on the gut microbiome; Research Question (RQ)1.a. Will gut microbiome composition, diversity, and richness respond differently to each treatment group - vitamin D3, vitamin K2, D3+K2 versus the control group? RQ1.b. What effect will vitamin D3 and/or K2 metabolism pathway single nucleotide polymorphisms (SNPs) have on gut microbiome response?

Specific Aim 2. Assess response to vitamin supplementation with serum 25-hydroxyvitamin D [25(OH)D] and osteocalcin levels pre and post supplement; RQ2. Will selected doses, based on clinical experience and published data, achieve designated endpoints for sufficiency; serum 25(OH)D greater than 30 ng/mL and undercarboxylated osteocalcin (ucOC) within normal limits?

Specific Aim 3. Assess the influence of vitamin D and K metabolism pathway SNPs on serum 25(OH)D and osteocalcin to determine vitamin D3, K2, and D3 + K2 supplementation efficacy; RQ3. To what extent does the presence of vitamin D and/or K metabolism pathway SNPs affect supplement efficacy when comparing treatment vs control group response measured by serum biomarkers?

Specific Aim 4. Explore relationships between diet/Dietary Inflammatory Index (DII), lifestyle behaviors (steps and sleep), body composition, metabolic and inflammatory biomarkers, SNPs, and the gut microbiome from Baseline to 3 months; RQ4.a. What factors need consideration when exploring genetic and non-genetic contributions to the gut microbiome in the treatment vs control group? RQ4.b. What role do age, gender, body mass index, inflammation status, and lipid profile play in the gut microbiome composition?

Hypothesis 1: Genotypic alterations in vitamin D3 and/or K2 metabolism pathways will influence gut microbiome composition in response to supplementation.

Hypothesis 2: Genotypic alterations in vitamin D3 and/or K2 metabolism pathways will influence serum response to supplementation.

Inclusion Criteria:

• able to read and understand English
• 25 OHD serum level equal to or less than 30 ng/mL
• willing to discontinue vitamin D3 and/or K2 supplements for 4.5 months
• military beneficiary (Active Duty, Retired, Dependent)
• remaining employed at workplace for 6 months

Exclusion Criteria:

• Diagnosed medical condition that prevents nutrient absorption from food (e.g. Inflammatory Bowel Disease, Celiac Disease, post-Roux-en-Y/Gastric sleeve surgery)
• Pregnant or breastfeeding
• Current antibiotic use or within past 3 months
• Current anticoagulant use or within past 3 months
• Current weight loss or fat malabsorption medication use (e.g. Glucagon-Like Peptide 1 agonists, Orlistat)
• Allergies or sensitivities to methylcellulose, vitamin D (D3, D2), or vitamin K (K2, Menaquinone-4, Menaquinone-7)

A data sharing agreement has been developed and submitted to the Defense Health Agency; approval has been received.

Supporting information will be available upon request after completion of protocol, for the following 2 years.

Contact principal investigator at mary.s.mccarthy1.civ@health.mil