Neurovascular Correlates of Cobalamin,Folate,and Homocysteine in Dementia

INTRODUCTION

Cobalamin (Cbl, also known as vitamin B12) and folate (also known as vitamin B9) are two micronutrients with critical roles in the human central nervous system [1]. Previous studies suggest that these micronutrients have neurotrophic effects [2–4] and their deficiencies may cause detrimental alterations to cerebral function [2, 5] and structures [5–7]. Specifically, both Cbl and folate facilitate the remethylation of homocysteine (Hcy), a non-proteinogenic amino acid byproduct formed in the metabolism process of methionine. An elevated level of Hcy has been noted as a modifiable risk factor of dementia [8] and other neurological diseases such as stroke and cerebral small vessel disease [9], presumably due to the build-up of free radicals [1] alongside endothelial dysfunction [9] and accumulation of extracellular matrix [3].

Cbl and folate are commonly prescribed as supplements to prevent the progression of dementia and cognitive decline, although mixed results have been reported in regard to their efficacy [10, 11]. The inconsistencies/controversies could stem from the heterogeneous pathophysiological substrates of dementia. Take vascular dementia (VaD) and Alzheimer’s disease (AD) for example, two most prevalent types of dementia that account for 20–30% and 60–70% of dementia cases, respectively [12, 13]. While VaD, as the name suggests, is caused by impaired cerebral blood flow (CBF), AD is generally associated with proteinopathy such as amyloid and tau in the brain. Hcy may thus affect VaD and AD through different pathways which in turn are differentially modulated by Cbl and folate.

Previous studies evaluated the effects of Cbl, folate, and Hcy on the brain separately [2, 15]. The effects had been primarily assumed on CBF [2, 5], blood-brain barrier [3], and white matter [5, 15]. However, it remains unclear whether their effects are interrelated and/or concentration dependent, and the neural correlates are not fully understood. Therefore, this study had two aims. First, we explored the association between the serum levels of Cbl/folate/Hcy and regional CBF and white matter integrity. Second, we tested the hypothesis that the effect of Cbl/folate/Hcy is differential in subcortical ischemic vascular dementia (SIVD, the most common subtype of VaD [16]) and AD.

MATERIALS AND METHODS

The framework of this study is summarized in Fig. 1.

Clinical registry

This is a cross-sectional observational study approved by the institutional review board. From an outpatient clinic, a total of 124 patients with clinically-diagnosed dementia according to published guidelines were prospectively recruited: 57 with SIVD [17], 67 with AD [18]. All patients received a serology test and a brain magnetic resonance (MR) imaging exam. Demographic data and cognitive status evaluated by the Mini-Mental State Examination (MMSE) and the clinical dementia rating were recorded. All patients provided written informed consent. None of the patients had significant artery stenosis or occlusion according to MR images (see below).

OPEN IN VIEWER

Fig. 1

Study framework. A) The first part of study was to explore neurovascular correlates of cobalamin (Cbl), folate, and homocysteine (Hcy) in dementia. B) The second part of study was to investigate the differential neurovascular effects of Cbl/folate/Hcy in Alzheimer’s disease (AD) and subcortical ischemic vascular disease (SIVD). ASL, arterial spin labeling; DTI, diffusion tensor imaging; FLAIR, fluid-attenuated inversion recovery; ROI, region of interest.

RESULTS

Table 1 summarizes the demographic data of the participants included in the following analyses. None of the items was statistically different between SIVD and AD patients (p = 0.053 –0.854).

Dependence of CBF and WMHs on the status of Cbl/folate/Hcy

Based on Cbl tertiles, significant group difference was found in CBF within the left lateral prefrontal cortex (p = 0.039), but not in WMHs. The results of post-hoc comparisons are shown in Fig. 2A. Based on folate tertiles, significant group difference was found in CBF within the right parietal cortex (p = 0.034) and left medial temporal cortex (p = 0.018), but not in WMHs. The results of post-hoc comparisons are shown in Fig. 2B and 2C. Based on Hcy tertiles, significant group difference was found in WMHs (p = 0.003), but not in CBF. The results of post-hoc comparisons are shown in Fig. 2D.

OPEN IN VIEWER

Table 1

Demographics of the participants entered in statistical analysis (N = 124)

		Mean	Range
Age	(y)	75.7	45–85
Gender	(N; male/female)	42/82
Dementia subtype	(N; SIVD/AD)	57/67
Educational level	(y)	5.3	0–16
Creatinine	(mg/dL)	1.3	0.5–11
Cbl	(pg/mL)	755.2	85–7500
Folate	(ng/mL)	14.1	4–85
Hcy	(μmol/L)	13.1	6–36
Clinical Dementia Rating Scale Sum of Boxes		4.9	0.5–14
Mini-Mental State Examination		18.9	6–26

SIVD, subcortical ischemic vascular disease; AD, Alzheimer’s disease; Cbl, cobalamin; Hcy, homocysteine.

OPEN IN VIEWER

Fig. 2

Association between serology metrics and neurovascular image metrics. A) Cerebral blood flow (CBF) comparisons among the groups defined by the tertiles of cobalamin (Cbl) level. Range of Cbl (pg/mL) [Case number] of Low = 85–342 [41]; Median = 348–685 [41]; High = 717–3094 [40]. B, C) CBF comparisons among the groups defined by the tertiles of folate level. Range of folate (ng/mL) [Case number] of Low = 4–9 [41]; Median = 9–15 [41]; High = 15–41 [41]. D) White matter hyperintensity (WMH) volume comparisons among groups defined by the tertiles of homocysteine (Hcy) level. Range of Hcy (μmol/L) [Case number] of Low = 5.58–10.20 [41]; Median = 10.21–14.08 [41]; High = 14.31–36.05 [39]. Two-way analysis of variance showed no significant interaction between dementia subtype and Cbl level (p = 0.319–0.407) or folate level (p = 0.514–0.669) on CBF, and no significant interaction between dementia subtype and homocysteine level on WMH volume (p = 0.545).

CBF moderation by Cbl in SIVD

After noting the widespread correlation between serum levels of Cbl and CBF in SIVD patients, we explored the potential moderating role of Cbl in the fronto-subcortical circuits that were previously shown to interconnect cognitive hubs in SIVD [24, 25]. Among the frontal ROIs listed in Table 2, only the medial prefrontal cortex exhibited CBF correlation with the ipsilateral basal ganglia and was entered in moderation analysis. As summarized in Fig. 3, the analysis revealed a bilateral moderation effect of Cbl on the CBF coupling between the basal ganglia and ipsilateral medial prefrontal cortex (left/right: p = 0.039/0.014, 0.049/0.009 after controlling for Hcy level, and 0.015/0.051 after controlling for folate level).

OPEN IN VIEWER

Fig. 3

Breakdown of the moderation effect of cobalamin on the cerebral blood flow coupling between basal ganglia and medial prefrontal cortex in subcortical ischemic vascular disease. Moderation effects were found both before (subplots A and B for the left and right hemispheres, respectively) and after (subplots C and D for the left and right hemispheres, respectively) controlling for the effect of homocysteine. Cbl, cobalamin; BG, basal ganglia; MPFC, medial prefrontal cortex; L, left; R, right; CI, confidence interval.

DISCUSSION

In this study, we have explored and identified the neurovascular correlates of Cbl, folate, and Hcy from a cohort of dementia patients. Specifically, Cbl and folate were found to primarily associate with CBF, while Hcy exclusively associated with WMHs. When dementia types were considered, SIVD exhibited more noticeable associations between CBF/WMHs and Cbl/folate/Hcy than AD did. As far as the CBF association was concerned, Cbl had a widespread effect in SIVD but no significant effect in AD, while folate had a local effect in both SIVD and AD. Within the fronto-subcortical circuits, we identified Hcy-dependent white matter impairment and a Cbl-moderated CBF coupling in SIVD. These findings may advance current understanding of the neurotrophic roles of Cbl/folate and whereby improve the formulation of Cbl/folate treatment for dementia.

Few studies have addressed the neural or vascular correlates of Cbl, folate, and Hcy in humans[2, 5]. Some animal studies described Cbl/folate-related CBF alteration, albeit often along with an elevated Hcy level and comorbid conditions such as malnutrition, low body weight, and anemia [26–28]. Based on structural MR imaging, most previous studies reported Hcy-related brain volume reduction [15] at a macroscopic scale, while our study focused specifically on white matter lesion and microstructural changes of neuronal fibers. Accumulating evidence suggests that hyperhomocysteinemia could trigger and promote atherogenesis and thrombosis [29]. When small vessels are damaged, Hcy-related microangiopathy could cause WMH formation [30] and brain volume loss [15]. In line with published studies, our results confirmed the adverse effect of serum Hcy on white matter integrity, both macroscopically (i.e., in terms of WMH volume) and microscopically (i.e., in terms of DTI metrics). Furthermore, when compared with AD patients, the neurotoxic effect of Hcy on white matter was more noticeable in SIVD patients, impacting both axon and myelin sheath. Specifically, an elevated serum level of Hcy was found to associate with increased RD (positive correlation) and decreased FA (negative correlation), which together suggested myelin injury/degeneration (i.e., more pronounced diffusion perpendicular to fiber tracts along with diminished directional dependence of diffusion). On the other hand, an elevated level of plasma Hcy was found to associate with increased AxD (positive correlation), which had been shown to reflect axonal injury (e.g., greater extracellular water content as a consequence of white matter atrophy) [31].

Our results showed that both Cbl and folate correlated with CBF, in patterns distinguishable between SIVD and AD. The Cbl-CBF correlation was widespread (involving both cortical and subcortical areas and multiple lobes) in SIVD but absent in AD. By contrast, the folate-CBF correlation was focal in both SIVD and AD but involved different areas (left lateral prefrontal cortex and left medial prefrontal cortex, respectively). These findings raise the possibility that Cbl and folate may have differential effects on endothelial function and in turn vascular reactivity and/or neurovascular coupling. Specifically, the global Cbl-CBF correlation mirrors the clinical observation of CBF restoration after Cbl supplement [2, 5]. On the other hand, the focal folate-CBF correlation could be partly explained by a specialized folate transport system within the endothelial wall of cerebral capillaries. The dedicated endothelium-dependent response yields relative resilience to the stress/damage imposed by folate deficiency. Furthermore, SIVD and AD presenting different patterns of Cbl/folate-CBF correlation could be because vascular dysfunction/damage is the primary cause in the former but widely considered secondary in the latter [21]. The brain areas where CBF appears to be modulated by Cbl/folate may derive biomarkers useful for the planning and assessment of supplemental Cbl/folate treatment for dementia.

Our results showed that in SIVD patients, CBF was correlated between medial prefrontal cortex and basal ganglia in both hemispheres, and the coupling was positively moderated by Cbl beyond the effect of Hcy. The finding coincides with previous reports that fronto-subcortical circuits are accountable for the cognitive deficit in SIVD [24, 32] and vulnerable to Cbl deficiency [2]. Basal ganglia and medial prefrontal cortex are known for their critical roles in cognitive processes such as motivation and goal-directed behavior [25]. Since both Cbl deficiency [33] and SIVD [24] can lead to executive dysfunction, Cbl supplement may be of clinical interest to improve dysexecutive syndrome among those affected.

On the other hand, we found that the elevated serum level of Hcy was associated with increased microstructural injury along the white matter tracts connecting basal ganglia and medial prefrontal cortex, and the tract impairment was uncorrelated with Cbl. Taken together, these findings suggest that Cbl has a protective and moderating effect on the fronto-subcortical circuits via a neurovascular substrate dissociable from the nocuous effect of Hcy on white matter microstructure. This echoes the reported dissociation between functional/hemodynamic connectivity and structural connectivity [5].

Our data showed that the serum level of folate inversely correlated with white matter damage in both AD and SIVD. The folate-CBF correlation, however, was positive within the left medial prefrontal cortex in AD but negative within the left lateral prefrontal cortex in SIVD. This finding implies that the rescue effect of folate could reverse when microvasculature has been critically compromised. The paradoxical folate-CBF association could also stem from the obscure Cbl-folate interaction regarding which contradictory findings had been published. For example, high serum folate with a low Cbl status was reported to be cognitively nocuous among the elderly [34], neutral in young adult [35], or of positive effect on cognition [36]. An elevated level of folate was associated with both smaller brain volume [37] and a marginal effect on cerebral microstructural integrity [4]. Our results suggested that the folate status had a limited part in the moderation of Cbl on the fronto-subcortical CBF coupling. It is worth noting that anemia could give rise to CBF alteration according to animal studies that reported perturbed CBF in rats subjected to folate deprivation and with concomitant low red blood cell counts [26, 27]. However, a longitudinal observation among rats fed a folate-deficient diet later showed that anemia could not fully explain the observed CBF perturbation [27]. We acknowledge that the effect of anemia cannot be ruled out in this study, although the effect should be small considering that only approximately one-third of our subjects were classified to be anemic. Alternatively, this paradoxical folate-CBF association could be contributed by the U-shape relationship between folate and Hcy [38], or other biomarkers of the methionine cycle and transsulfuration pathway which are beyond the scope of the current research.

A few limitations of this study should be noted. First, our data did not include genetic variants regarding Cbl or folate metabolism (e.g., methylenetetrahydrofolate reductase) [39]. Second, we were unable to entirely exclude the effect of lifestyle behaviors. Dietary supplement use and malabsorption issues might vary among our elderly subjects. Given that recent diet and fortified supplement could change serum levels of Cbl and folate, the measured serology values might not be equivalent to biological availability (e.g., folate level in serum and red blood cells [40]; Cbl and holotranscobalamin [41]) or indicative of a long-term effect [40]. Third, this study lacked a healthy control group and thus the contribution of normal aging to the neurovascular correlates found in this study cannot be ruled out. Nonetheless, disease-related neurodegeneration appeared to play a major role given that statistical significance remained after age was controlled for in a large part of the partial correlation analyses and that the correlates exhibited different patterns in AD and SIVD (i.e., the patterns would be similar if normal aging was the dominant effect). Forth, causal inferences cannot be made based on the cross-sectional design of the present study, which warrants further research with a longitudinal and randomized design.

In summary, by using multimodal MR imaging, we identified the neurovascular correlates of previously reported neurotrophic effect of Cbl/folate and neurotoxic effect of Hcy in dementia. Mainly associated with CBF, Cbl had a widespread effect, involving both cortical and subcortical areas and multiple lobes, whereas the effect of folate was relatively focal. An elevated serum level of Hcy was associated with both macroscopic and microscopic white matter abnormalities. The correlates exhibited distinct patterns between AD and SIVD. Our results provided radiological/neurological insights to the roles of Cbl/folate/Hcy in dementia, which may help resolving the mixed findings with regard to Cbl/folate treatment efficacy and further improving the planning/assessment of supplemental Cbl/folate treatment for dementia.