Background and objectives: Lower vitamin status has been linked to cognitive deficits, pending mechanistic elucidation. Serum 25-hydroxyvitamin D [25(OH)D], folate and cobalamin were explored against brain volumes and white matter integrity (WMI).Methods: Three prospective waves from Healthy Aging in Neighborhoods of Diversity Across the Life Span (HANDLS) study were used [Baltimore, City, MD, 2004–2015, N = 183–240 urban adults (Agev1: 30–64 years)]. Serum vitamin 25-hydroxyvitamin D [25(OH)D], folate and cobalamin concentrations were measured at visits 1 (v1: 2004–2009) and 2 (v2: 2009–2013), while structural and diffusion Magnetic Resonance Imaging (sMRI/dMRI) outcomes were measured at vscan: 2011–2015. Top 10 ranked adjusted associations were corrected for multiple testing using familywise Bonferroni (FWER 50 years: +205 ± 54]; parietal WM; [overall: +251 ± 77, males: +486 ± 129 and Agev1 > 50 years: +393 ± 108] and left occipital pole volume [overall: +15.70 ± 3.83 and above poverty: 19.0 ± 4.3], findings replicated for 25(OH)D (v2-v1) annualized exposure, which was also linked with greater WMI (fractional anisotropy, FA) in the anterior limb of the internal capsule (ALIC); FWER < 0.05 [Overall: +0.0020 ± 0.0004; Whites: +0.0024 ± 0.0004] and in the cingulum (hippocampus) [Overall: +0.0016 ± 0.0004]. Only trends were detected for cobalamin exposures (q < 0.10), while serum folate (v1) was associated with lower mean diffusivity (MD) in ALIC, reflecting greater WMI, overall.Conclusions: Among urban adults, serum 25(OH)D status and increase were consistently linked to larger occipital and parietal WM volumes and greater region-specific WMI. Pending longitudinal replication of our findings, randomized controlled trials of vitamin D supplementation should be conducted against brain marker outcomes.

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