Vitamin D and Caudal Primary Motor Cortex: A Magnetic Resonance Spectroscopy Study

Background Vitamin D is involved in brain physiology and lower-extremity function. We investigated spectroscopy in a cohort of older adults to explore the hypothesis that lower vitamin D status was associated with impaired neuronal function in caudal primary motor cortex (cPMC) measured by proton magnetic resonance spectroscopic imaging. Methods Twenty Caucasian community-dwellers (mean±standard deviation, 74.6±6.2 years; 35.0% female) from the ‘Gait and Brain Study’ were included in this analysis. Ratio of N-acetyl-aspartate to creatine (NAA/Cr), a marker of neuronal function, was calculated in cPMC. Participants were categorized according to mean NAA/Cr. Lower vitamin D status was defined as serum 25-hydroxyvitamin D (25OHD) concentration <75 nmol/L. Age, gender, number of comorbidities, vascular risk, cognition, gait performance, vitamin D supplements, undernourishment, cPMC thickness, white matter hyperintensities grade, serum parathyroid hormone concentration, and season of evaluation were used as potential confounders. Results Compared to participants with high NAA/Cr (n = 11), those with low NAA/Cr (i.e., reduced neuronal function) had lower serum 25OHD concentration (P = 0.044) and more frequently lower vitamin D status (P = 0.038). Lower vitamin D status was cross-sectionally associated with a decrease in NAA/Cr after adjustment for clinical characteristics (β = −0.41, P = 0.047), neuroimaging measures (β = −0.47, P = 0.032) and serum measures (β = −0.45, P = 0.046). Conclusions Lower vitamin D status was associated with reduced neuronal function in cPMC. These novel findings need to be replicated in larger and preferably longitudinal cohorts. They contribute to explain the pathophysiology of gait disorders in older adults with lower vitamin D status, and provide a scientific base for vitamin D replacement trials.


Introduction
Beyond its long-known involvement in bone health, vitamin D has also emerged as a secosteroid hormone with non-skeletal effects [1,2]. In particular, vitamin D is involved in neurophysiology [3,4] and lower vitamin D status has been associated with whole-brain atrophy in older adults [5]. However, the specific brain regions that are altered with lower serum 25-hydroxyvitamin D (25OHD) concentrations remain unknown [6]. Based on the repeated findings that lower vitamin D status is associated with poor lower-extremity function in older adults, such as gait and balance disorders [7] with consequent falls and fractures [8], it appears likely that lower vitamin D status may negatively affect the brain structures involved in lower-limb motor control. Since the primary motor cortex (PMC) is the final integrator of motor control via a somatotopic organization [9,10), we hypothesized that lower vitamin D status in older adults could directly affect the subregion of the PMC responsible for lower-limb motricity (i.e., caudal PMC, cPMC). Our objective was to determine whether lower vitamin D status in older adults was associated with impaired neuronal function in cPMC measured by proton magnetic resonance spectroscopy ( 1 H-MRS).

Participants
Baseline data from 20 consecutive participants recruited in the 'Gait and Brain Study' from September 2011 to March 2012 was used for this analysis. This cohort is being followed to prospectively evaluate the mobility declines in older adults with prodromal cognitive decline. The sampling and data collection procedures have been described elsewhere [11].
Anatomical images guided the placement of a 20 mm isotropic voxel on leg and foot regions of right PMC. Both water suppressed (averages = 192) and unsuppressed (averages = 8) spectra were localized by point resolved spectroscopy (PRESS, repetition time/ echo time = 2000/135 ms, voxel size = 8 cm 3 ). Data processing included lineshape correction and removal of residual unsuppressed water signal [10,12]. Resultant metabolite spectra were fitted in the time domain incorporating a template of prior knowledge of metabolite lineshapes including N-acetylaspartate (NAA) and creatine (Cr). NAA level is linked to the functional status of neuronal mitochondria and is considered a marker of neuronal function [13). Cr provides a measure of oxidative energy stores [14]. Therefore, NAA/Cr ratio provides a reproducible and sensitive measurement of neuronal integrity that also reduces quantification errors associated with differences in tissue partial volume between voxels. In our sample, participants were separated into two groups using a threshold NAA/Cr ratio of the mean value (i.e., NAA/Cr = 1.17).

Serum Vitamin D Concentration
Venous blood was collected from resting participants at the time of brain assessments. Serum 25OHD concentration, an effective indicator of vitamin D status, was measured by radioimmunoassay (DiaSorin, IncstarCorp, Stillwater, MN). The intra-and interassay precisions were 5.2% and 11.3% respectively. Lower vitamin D status was defined as 25OHD concentrations ,75 nmol/L (to convert to ng/mL, divide by 2.496) [2,15]. All measurements were performed at the University of Western Ontario, London, ON.

Confounders
Age, gender, number of comorbidities, vascular risk, global cognitive performance, high-level gait performance, use of vitamin D supplements, undernourishment, cPMC thickness, white matter hyperintensities (WMH) grade, serum parathyroid hormone (PTH) concentration, and season of evaluation were used as potential confounders.
Comorbidities were defined as diseases lasting at least 3 months or running a course with minimal changes. Vascular risk was assessed using the 7-point Vascular Factors Index [16]. Global cognition was assessed using the Montreal Cognitive Assessment (MoCA) [17]. High-level gait performance was estimated from stride time variability (STV), a valid marker of cortical control of gait [7], and measured with a computerized walkway (GAITRiteH, CIR Systems, Havertown, PA) while steady-state walking and counting aloud backwards by 7 starting from 100 [11]. The use of vitamin D supplements was reported by direct inquiry, whatever the dosage schedule or route of administration, and regardless of the date of commencement. Undernourishment was defined as body mass index ,21 kg/m 2 [18]. The average cPMC thickness, calculated as the average distance between gray matter/white matter boundary and gray matter/cerebrospinal fluid boundary within the cPMC, was obtained from 3D T 1 -weighted MP-RAGE images using FreeSurfer (v5.1.0), a set of tools that automatically segments, labels and quantifies brain tissue volumes [19]. WMH grade was measured using a semiquantitative visual rating scale of 0-9 (worst) [20] applied to T 2 -weighted FLAIR images (acquisition matrix = 2566232, reconstructed to 5126464 matrix, FOV = 220 mm6200 mm, thickness = 4 mm, gap = 0.5 mm, 41 slices, TR = 8 s, TE = 120 ms, TI = 2400 ms, flip angle = 130 degrees, averages = 1). Serum concentration of intact PTH was measured by enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH). Finally, the season of evaluation was recorded as follows: spring from March 21 to June 20, summer from June 21 to September 20, fall from September 21 to December 20, winter from December 21 to March 20.

Statistical Analysis
Comparisons between participants separated into two groups based on NAA/Cr in cPMC (#1.17 or .1.17) were performed using two-sided Mann-Whitney U-test or Chi-square test, as appropriate. Multivariate linear regressions were used to examine the association between lower vitamin D status (independent variable) and NAA/Cr in cPMC (dependent variable), while adjusting for clinical characteristics, neuroimaging measures, and serum measures. P-values,0.05 were considered significant. All statistics were performed using SPSS (v19.0, IBM Corporation, Chicago, IL).

Ethics Statement
Participants in the study were included after having given their written informed consent for research. The study was conducted in accordance with the ethical standards set forth in the Helsinki Declaration (1983). The project was approved by the University of Western Ontario Research Ethics Board for Health Sciences Research Involving Human Subjects.
Multivariate linear regression models showed that lower vitamin D status was associated with a decrease in NAA/Cr in cPMC (b = 20.45, P = 0.046) ( Table 2) and with MoCA score (b = 20.08, P = 0.047).

Discussion
To our knowledge, this study provides the first evidence of an association between lower vitamin D status and reduced cPMC function illustrated by decreased NAA/Cr.
The PMC, located in the precentral gyrus of the cerebral cortex (Brodmann area 4), is the final integrator of all brain processes involved in gait control before the descending corticospinal tract [9,10]. Thus, finding that lower vitamin D status was associated with decreased NAA/Cr in cPMC has potential clinical implications. Locomotion disorders accompanying lower vitamin D status have been explained mainly by an adverse impact on striated muscles [21]; however, a recent meta-analysis highlighted that such muscular effects of vitamin D remain uncertain [8], paving the way for new working hypotheses. We propose that lower vitamin D status could influence lower-extremity function by affecting brain structures responsible for higher-level motor control. In fact, this hypothesis is consistent with previous studies reporting that transgenic mice lacking functional vitamin D receptors (VDRs) in the brain exhibited uncoordinated lower-limb movements [22].
The mechanism linking vitamin D with cPMC function is not firmly established. Vitamin D is a lipophilic molecule that crosses the blood-brain barrier and exerts action through VDRs present in cortical neurons [2][3][4]. Vitamin D regulates the gene expression of several neurotrophins and protects neural networks by controlling mitosis rate and neuronal growth [4,5]. Animal experiments have also shown that vitamin D regulates intra-neuronal calcium homeostasis, and oxidative and inflammatory changes in the brain [3,4], promoting neuron viability and function. However, causality cannot be determined from our cross-sectional study. It may be argued that, contrary to our hypothesis, reduced neuronal function in cPMC could result in lower vitamin D status due to lower-extremity dysfunction with poor mobility and low sun exposure. However, this hypothesis should be mitigated by the high functionality of our sample, all scoring 6 out of 6 on Katz's Activities of Daily Living score. Moreover, although we were able to control for important characteristics that could modify the association, residual potential confounders such as serum concentrations of calcium and phosphorus might still be present.
We also found a significant inverse association, although of low magnitude, between MoCA score and NAA/Cr in the cPMC. This result is consistent with prior transcranial magnetic stimulation/electroencephalogram studies reporting a link between cognition and PMC, specifically that cognitively preparing a movement results in an anticipatory reduction of PMC excitability [23].

Conclusions
In conclusion, we report an association between lower vitamin D status and decreased NAA/Cr -indicative of reduced neuronal function-in the cPMC. Even if these novel findings need to be replicated in larger and preferably longitudinal cohorts, they contribute to explain the pathophysiology of higher-level gait disorders in older adults with lower vitamin D status.