Vitamin B12 deficiency is commonly associated with lack of its intake through food as well as the poor absorption of the vitamin B12 by the intestine¹. Other causes of this deficiency include undergoing certain types of bariatric surgery, bacterial overgrowth that competes for vitamin B12, and alcohol consumption. It is believed that the manifestations of vitamin B12 deficiency are irreversible if left untreated. It has been shown in clinical studies that vitamin B12 affects the functional brain; however this effect has not been studied in detail. The objective of our study was to investigate the functional alterations using regional homogeneity analysis², in different regions of the brain due to the depletion of vitamin B12, and further study the reversibility of these functional changes following vitamin B12 replacement therapy, if any. To the best of our knowledge, this is the first study that shows the functional changes in brain due to vitamin B12 deficiency.

Dataset: In this study, 13 treatment naïve vitamin B12 deficient right-handed patients (males 9, females 4, mean age 33.31 ± 7.9 years; age range, 22–54 years) diagnosed on the basis of biochemical evidence and neurological symptoms were studied with resting state (rsfMRI). Age and sex matched 15 healthy right-handed controls were also included in the study (males 11, females 4, mean age 30.07 ± 8.19 years; age range, 18–53 years). Six of these 13 patients were agreed for a repeat MRI study after 6 weeks of replacement therapy. The study was approved by the institutional ethics committee. Informed and written consent was obtained from each subject.

Image processing: Using SPM8 software, the functional images were slice-time and motion corrected, co-registered to the anatomical template and smoothed with a kernel of 8 mm (full-width-at-half-maximum). Any signal drifts were corrected by removing the very low frequency components (<0.01 Hz). To correct for physiological fluctuations, the time-series from the cerebrospinal fluid (CSF) and white matter were included as co-variates in the linear regression analysis. Gray matter, white matter, and CSF voxels were segmented from the T1-weighted images using Freesurfer. For regional analysis, regions in default mode network (DMN), frontal-parietal network (FPN) and cingulu-orbital network (CON) areas/regions were also included in this study.

Regional Homogeneity: The time series in our rsfMRI were band pass filtered in the frequency range of 0.01-0.08Hz. Kendall’s coefficient of concordance (KCC)² was used to measure ReHo of the time series of a given voxel with its nearest 26 neighboring voxels. The KCC-ReHo among given voxels ranged from 0 to 1. ReHo measures the regional homogeneity (i.e. similarity in contiguous voxels) of time-series and was also averaged over all gray matter voxels. This method is based on observations that meaningful BOLD fluctuations are more likely to occur in clusters of several contiguous voxels than in a single voxel.

Analysis of rsfMRI data reveals that ReHo in patients with Vitamin B12 deficiencies was significantly lower than controls in the entire cerebrum (p<0.05). Pre-frontal cortex areas in the three brain networks associated with cognition control (figure 1) showed significantly decreased ReHo. In this study, ReHo was also correlated with NPT of each patient and control showing significant correlation with picture completion, digit symbol, block design and figure connection tests A and B. Six vitamin B12 deficient patients were followed up after therapy showed increased ReHo after 6 weeks of appropriate therapy. Post therapy improvement of NPT scores were reported for of all the patients. Brain regions that showed significant improvement after replacement therapy include pre-frontal cortex areas of the three brain networks and hippocampus (figure 2). ReHo was found to be lower in patients in the prefrontal cortex part of all the three brain networks i.e. medial prefrontal, anterior prefrontal and dorsal lateral prefrontal; and hippocampus. Findings in patients with vitamin B12 deficiency are in sync with neuro-cognition studies that suggest that alterations in prefrontal cortex and hippocampus affect cognitive ability in brain³. ReHo in prefrontal cortex regions was found to increase in patients with vitamin B12 deficiency after six weeks of therapy suggesting that changes in pre-frontal cortex areas in DMN, FPN and CON are reversible. The reversibility in functional damage was associated with improvement in cognition and clinical symptoms in these patients. Improvement in the NPT and its correlation with ReHo in the described cognitive network further confirms the objectivity of functional reversibility seen in ReHo derived from rsfMRI. Rate of increase in ReHo was found to be variable in different patients, suggesting that time required for improvement in cognition vary from patient to patient.