Abstract
Background:
Homocysteine has been identified as a potential risk factor for stroke, cerebral small-vessel diseases (SVD), and dementia.
Objective:
The present study aimed to investigate the predictive value of homocysteine levels on incident dementia while simultaneously controlling for MRI findings and vascular risk factors.
Methods:
Within a Japanese cohort of participants with vascular risk factors in an observational study, we evaluated the association between baseline total homocysteine (tHcy) levels (per 1 μmol/L and the tertile of tHcy), the prevalence of MRI-findings at baseline, and incident all-cause dementia. Baseline brain MRI was used to determine SVD (lacunas, white matter hyperintensities, and cerebral microbleeds [CMBs]) and atrophy (medial-temporal lobe atrophy and bicaudate ratio). Logistic regression analyses were used to estimate the cross-sectional association between tHcy and each of MRI findings. Cox proportional hazards analyses were performed to estimate the longitudinal association between tHcy and dementia.
Results:
In the 643 subjects (age: 67.2 ± 8.4 years, male: 59% ; education: 12.9 ± 2.6 years), multivariable analyses adjusted for several potential confounders, including estimated glomerular filtration rate (eGFR) and intima-media thickness, showed that highest tHcy tertile was associated with lacunas, CMBs, and strictly deep CMBs. During the mean 7.3-year follow-up (range: 2–13), 47 patients were diagnosed with dementia (Alzheimer’s disease: 24; vascular dementia: 18; mixed-type: 3; other: 2). After adjusting for age, gender, APOE ɛ4, education, BMI, MMSE, hypertension, cerebrovascular events, eGFR, and MRI-findings, tHcy level (hazard ratios [HR]: 1.08, p = 0.043) and the highest tertile of tHcy (HR: 2.50, p = 0.047) for all-cause dementia remained significant.
Conclusions:
Our results provide additional evidence of tHcy that contributes to increased susceptibility to dementia risk.
Keywords
INTRODUCTION
Increased plasma total homocysteine (tHcy) levels have been associated with vascular injury [1]. Epidemiological studies have shown that higher tHcy levels are independently associated with not only cardiovascular diseases and stroke but also cerebral small-vessel disease (SVD) [2, 3]. Also, both stroke and SVD are generally considered robust risk factors of cognitive impairment and dementia [4]. Therefore, it has been proposed that increased tHcy levels may be involved in predicting dementia [5]. Indeed, numerous observational studies have reported that increased tHcy levels were positively associated with cognitive impairment and dementia [5–8]. However, conflicting results have also been reported [9, 10].
Underlying cognitive impairment in the elderly is often a mixed pathology of Alzheimer’s disease (AD) and cerebral microangiopathy [11], which may heighten the risk of dementia [4]. In this context, SVD is of particular interest because it frequently occurs in the elderly population and even in patients with AD dementia [12]. Previously, we have reported that both chronic kidney disease and cerebral microbleeds (CMBs) (particularly, mixed, or multiple CMBs) are independently related to an increased risk of dementia [13, 14]. Given the association between tHcy levels and vascular injuries, such as SVD or renal impairment [3, 15], it is plausible that the relationship of tHcy levels and dementia risk could be mediated through these effects, but it remains to be investigated whether tHcy levels are independently associated with a higher risk of dementia. The aim of this study was to evaluate the predictive value of baseline tHcy levels on incident dementia in patients with vascular risk factors who are initially withoutdementia.
MATERIALS AND METHODS
Participants
The current participants originated from the Osaka Follow-up Study for Carotid Atherosclerosis, Part 2 (OSACA2) study, an ongoing longitudinal study in which physicians control risk factors in high-risk patients for the primary and secondary prevention of cardiovascular disease [16]. The detailed study design has been described elsewhere [13, 16]. Briefly, outpatients who visited the Department of Neurology and Stroke Center at Osaka University Hospital, aged >40 years with more than 1 vascular risk factor, including hypertension, diabetes, dyslipidemia, smoking history, established arteriosclerosis documented as transient ischemic attack, stroke, coronary heart disease, or peripheral artery disease, were enrolled. Patients were excluded from the study if they had experienced a symptomatic vascular event during the previous 3 months. Between January 2001 and December 2011, 901 outpatients who had been enrolled in this study underwent baseline examinations, including brain MRI and a clinical assessment that included medical history, medication inquiry, physical and neurological examination, blood sampling, and carotid ultrasound. MRI was generally performed to examine lesions in cases with stroke histories or suspicious neurological symptoms (e.g., headache, vertigo, dizziness, numbness, syncope, or subjective memory impairment) [17]. The Mini-Mental State Examination (MMSE) [18] and the Clinical Dementia Rating Scale (CDR) [19] were used to screen suspected cases of cognitive decline in all patients. Cognitive tests were administered within one month from the MRI examination. Entry inclusion criteria included a MMSE score ≥24 and 0 on the CDR. Each CDR score was based on interviews with the participant as well as a collateral source familiar with the participant. All patients were examined by neurologists. We identified 717 possible participants after excluding those with incomplete baseline examinations, including no baseline tHcy or MMSE (n = 95) and the absence of gradient-recalled echo (GRE) T2*-weighted MRI (n = 89). Additionally, we excluded patients with suspected cases of previous cognitive impairment (MMSE <24) (n = 74). Finally, all analyses were based on 643 patients with complete baseline data (Fig. 1). This study was approved by the local ethical review board, and all patients provided written informedconsent.
MRI protocol and assessment
MRI protocols were described previously [17]. The images were analyzed centrally, and all ratings were made by a single experienced rater blinded to the clinical information. CMBs were defined as punctuate hypointense lesions <10 mm on GRE. The locations of CMBs were classified as: lobar (cortex, subcortex, and white matter), or deep (basal ganglia, thalamus, brain stem, and cerebellum) [17]. Lacuna infarction (LI) was defined as a focal lesion >3 mm and <15 mm, with a hypointense lesion and hyperintense rim on FLAIR-images when located supratentorially according to the corresponding hyper- and hypointensity on T2- and T1-weighted images, respectively [20]. Silent lacunar infarction (SLI) was defined as LI on MRI without any clinical history of cerebrovascular disease. White matter hyperintensities (WMH) were defined as hyperintense signal abnormalities surrounding the ventricles (periventricular hyperintensities [PVH]) and in the deep white matter (deep white matter hyperintensities [DWMH]) on FLAIR-images. The degree of WMH was visually rated with the Scheltens scale with slight modifications: scores of 0 to 6 (0 = absent, 1 = <3 mm in ≤5, 2 = <3 mm in ≥6, 3 = 4 to 10 mm in ≤5, 4 = 4 to 10 mm in ≥6, 5 = <11 mm in >1, 6 = confluent) were given for DWMH (frontal, temporal, parietal, occipital) (range, 0–24) and scores of 0 to 2 (0 = absent, 1 =≤5 mm, 2 = >5 mm and <10 mm) were given for PVH (frontal caps, lateral bands, occipital caps) (range, 0–6) [21]. The sum of ratings was used as a global WMH (range, 0–30). The areas of hyperintensity on T2-weighted images around infarctions and lacunes were not included. We assessed medial-temporal lobe atrophy (MTA) at baseline on T1-weighed images [22]. Briefly, it was evaluated using a 4-point scale (0 = none, 1 = questionable, 2 = apparent, 3 = severe) (range, 0–3). In case of asymmetry, the side with more severe atrophy was used for rating. As an estimate of subcortical atrophy, the bicaudate ratio (BCR) was calculated as the minimum intercaudate distance divided by brain width along the same line [23]. Increased BCR is best explained by frontal horn ventricular enlargement due to atrophy of deep frontal subcortical white matter and also associations with global brainatrophy [23].
Homocysteine measurements
On baseline visit, overnight fasting venous blood samples were taken in the morning and collected in tubes containing EDTA, centrifuged at 3000 rpm at 4°C for 15 min; and stored at –80°C until assayed. Plasma tHcy levels were determined by the use of high-performance liquid chromatography with fluorometric detection at the different laboratory.
Potential risk factors
Analyses were adjusted for variables that have frequently been associated with increased risk of dementia, including age, gender, education, body mass index (BMI), APOE ɛ4 allele status (having at least one ɛ4 allele versus none), SVD and atrophy, and vascular risk factors (hypertension, diabetes, dyslipidemia, smoking, intima-media thickness [IMT], and a history of cerebrovascular events). Hypertension was defined as blood pressure ≥140/90 mmHg [24] on measurements taken at least twice or anti-hypertensive medications. Diabetes was defined as fasting plasma glucose levels ≥126 mg/dL, HbA1c level ≥6.5% [25], or antidiabetic therapies. Dyslipidemia was defined as a low-density lipoprotein cholesterol level ≥140 mg/dL, total cholesterol level ≥220 mg/dL, triglyceride level ≥150 mg/dL [26], or cholesterol lowering therapies. Smoking was evaluated based on current habits. Baseline kidney function was estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Disease formulafor Japanese [27]. We calculated the mean IMTas an estimate of large-vessel by averaging the thickness at 12 sites: the near and far walls of the rightand left distal common carotid arteries, bifurcation, and the internal carotid artery [28]. Patients werecategorized as having a history of cerebrovascular events if they had had a stroke or undergonesurgical or endovascular treatment [16]. Incident cerebrovascular events during the follow-up were also assessed [16].
Diagnosis of dementia
Cognitive status was assessed prospectively using the MMSE and CDR by a neurologist; the protocol has been published elsewhere [13]. Briefly, subjects visited outpatient clinic settings to control risk factors regularly for prevention of stroke [16]. Changes in subjects’ general medical conditions were obtained yearly through medical records and interviews. Furthermore, several aspects of everyday cognitively driven functioning to rate participants on the CDR were assessed in every clinical visit. Thus, annual evaluations were performed by trained neurologists and included a medical history, CDR score determination, andstandardized neurologic examination. And also, MMSE scores obtained at baseline and every 2 years thereafter, followed by annual or biannual neurologic and neuropsychological assessment of subjects with suspected cognitive impairment. The final follow-up data were collected in June 2013. Briefly, clinically significant cognitive impairment was defined as MMSE score <24 or a decline ≥1.5SD of change scores [13]. On the MMSE, this corresponded to a decline ≥3 points. Additionally, subjects were considered to be suspected cases of dementia if they had two consecutive semiannual CDR scores ≥1 and did not revert back to normal cognition. Then, suspected cases of dementia and cognitive decline (MMSE score <24, MMSE decline ≥3 or CDR ≥1) underwent additional neurpsychological testing [29]. Additionally, we continuously covered all the medical records at our clinic or other clinics to acquire information the diagnosis of dementia, cerebrovascular events, or death. Furthermore, for those patients who could not attend the clinical visit, a phone interview collecting clinical data was performed with the patient and the carer, whenever possible. Final diagnosis of dementia was based on a consensus by a panel that consisted of experienced neurologists with use of the DSM-III-R and established clinical criteria [30, 31]. Diagnoses were also supported by brain MRI examinations when available. The criteria for mixed dementia were achieved when the investigator considered that the clinical picture presented both aspects of Alzheimer’s disease (AD dementia) and vascular dementia (VaD) [32]. Time to dementia was defined as the time between the baseline visit and the date of dementia diagnosis. Additionally, subjects were followed until death or refusal of further participation. Those who did not progress to dementia were censored at their last visit.
Statistical analyses
The Shapiro-Wilk test showed that several variables deviated from normal distribution. For group comparisons between subjects with high tHcy levels at baseline (above the median) and those with low tHcy levels (below the median), we used χ2 tests (for categorical variables), Student’s t tests (for continuous normally distributed variables), or the Mann-Whitney U tests (for continuous, nonnormally distributed variables). For cross-sectional analyses of prevalent MRI-findings, logistic regression analysis was used to estimate the association between tHcy levels (per 1 μmol/L increase) and the tertile of tHcy (highest tertile versus lowest [reference]) and each SVD (CMBs [strictly lobar, mixed (lobar and deep), strictly deep], LI, PVH, DWMH) and atrophy (MTA, BCR): adjusted for age, gender, BMI, baseline MMSE score, smoking, hypertension, previous cerebrovascular events, eGFR and IMT, which were associated with increased tHcy levels at baseline (Table 1), and clinical utility. The comparison of WMH and atrophy was divided into the highest 25% versus the lower 75% .
The Kaplan-Meier method with log-rank tests was used to compare dementia-free survival for high tHcy (above the median) versus low tHcy (below the median). For longitudinal analyses of incident dementia, the hazard ratios (HR) in the Cox proportional hazards analyses was used to estimate the risk of dementia (subtypes) for known risk factor individually, tHcy level (per 1 μmol/L increase) and tHcy level tertile (highest tertile versus lowest [reference]) in unadjusted models, and in models controlling for demographic variables, including age, gender, education level, and APOE ɛ4 carrier. In addition to demographic variables, the multivariable analysis included all significant variables from prior analyses (using p < 0.05 as cutoff for significance) (Table 3) and clinical utility. Therefore, we considered the following variables for all-cause dementia as the outcome: age, gender, education level, APOE ɛ4, BMI, MMSE score, hypertension, eGFR, previous cerebrovascular events, atrophy (MTA), and SVD (mix CMBs and WMH) (Table 4). Because CMBs and WMH are strongly associated with each other, we repeated the analyses with WMH as SVD instead of the presence of CMBs. VaD and mixed dementia were pooled in a single category. Data were analyzed using SAS statistical software (version 9.2, SASInstitute).
RESULTS
Population characteristics
All patient baseline characteristics are summarized in Table 1. A total of 643 patients were included (age: 67.2 ± 8.4 [range: 45–86] years, male: 59% , education: 12.9 ± 2.6 years). The mean tHcy level was 10.0 ± 3.8 μmol/L (tertile 1: ≤8.2; tertile 2: 8.3–10.7; tertile 3: ≥10.8 μmol/L). Compared to the main study sample (n = 643), the rest of the cohort (n = 258) had less diabetes, lower eGFR, lower baseline MMSE scores, lower mean-IMT, severer PVH and BCR (data not shown). In the main cohort, 79% had hypertension, 60% had dyslipidemia, and 24.3% had previous cerebrovascular events.
The baseline characteristics of patients with higher tHcy levels (above the median of 9.2 μmol/L) versus those with lower tHcy levels (below the median of 9.2 μmol/L) also shown in Table 1. Several characteristics were comparable, but patients with higher tHcy levels were significantly older, more often male and current smokers, and had a baseline history of hypertension, cerebrovascular events, higher serum creatinine levels, lower eGFR, higher IMT, and more severe MRI-findings (LI [number, presence], CMBs [number, presence (except for strictly lobar CMBs)], PVH, MTA, and BCR).
Homocysteine and baseline MRI-findings
Table 2 shows the association between tHcy levels and MRI-findings. Hcy levels (per1 μmol/L increase) were associated with CMBs, strictly deep CMBs, LI, PVH, and DWMH when the results were adjusted for age and gender only (model 1). After further adjusting for age, gender, BMI, MMSE, smoking, hypertension, previous cerebrovascular events, eGFR, and IMT (model 2), higher tHcy levels were associated with LI (odds ratio [OR]: 1.06, p = 0.04), and strictly deep CMBs (OR: 1.09, p = 0.021), and borderline associated with CMBs (OR: 1.06, p = 0.066). For the highest tHcy tertile, OR [95% CI] was 1.79 [1.08–2.97] for LI, 1.99 [1.12–3.62] for CMBs, and 3.06 [1.30–7.74] for strictly deep CMBs, compared to the lowest tertile. No significant association was found between tHcy levels and atrophy.
Homocysteine and dementia
By the end of the follow-up period (mean, 7.3 ± 3.2 years ; range, 2–13 years), all-cause dementia was diagnosed in 47 patients (AD, 24; VaD, 18; mixed-type, 3; other, 2). Table 3 shows the hazard ratios (HR) for the association between baseline status and dementia. In the unadjusted analysis (model 1) and that adjusted for demographics variables (age, gender, education, and APOEɛ4) (model 2), age, previous cerebrovascular events, eGFR (1SD decrease), mix CMBs, WMH, and MTA significantly increased the risk of all-cause dementia (Table 3).
The survival analyses of the dementia-free rate curves for baseline tHcy levels are shown in Fig. 2. Patients with the higher tHcy levels at baseline were significantly more likely to progress to all-cause dementia (Log-rank test: p = 0.0001).
Table 4 shows the HR for the association between baseline tHcy level or the highest tertile and dementia. In the unadjusted analysis (model1) and that adjusted for age, gender, education, APOE ɛ4, BMI, and MMSE (model 2), tHcy levels and highest tertile were associated with incident dementia. After further adjusting for hypertension, previous cerebrovascular events, eGFR, mix CMBs (or WMH), and MTA (model 3), tHcy levels (HR: 1.08, p = 0.043) and highest tertile (HR: 2.50, p = 0.047) for all-cause dementia risk remained significant.
Homocysteine and dementia subtypes
To explore associations for the risk of dementia subtypes, we included only the demographic variables,as each incidence was small. Table 3 shows that age, APOE ɛ4 status, eGFR, MMSE, and MTA predicted AD dementia, while age, male, MRI-findings (LI, WMH [PVH, DWMH], mix CMBs, MTA), and cerebrovascular events were related to VaD.
After additional adjusting BMI, and MMSE (Table 4), highest tertile (HR: 3.31, p = 0.081) showed a borderline association with AD dementia, while higher tHcy levels (HR: 1.14, p = 0.011) and the highest tertile (HR: 6.29, p = 0.037) were significantly associated with VaD.
Homocysteine, incident cerebrovascular events, and mortality
In addition, 66 (10.6% ) patients experienced incident cerebrovascular events (ischemic stroke, 49; hemorrhagic stroke, 6; surgical or endovascular treatments after TIA, 11), and 44 (6.8% ) died (Table 1). In unadjusted models via Cox regression analysis, higher tHcy levels showed a borderline association with mortality (HR: 1.06 [0.98–1.12], p = 0.08) and the risk of incident cerebrovascular events (1.06 [0.99–1.11], p = 0.063), while the highest tertile was significantly associated with mortality (2.66 [1.33–5.59], p = 0.006) and the risk of incident cerebrovascular events (2.13 [1.15–4.07], p = 0.042), respectively. After adjusting age, gender, and previous cerebrovascular events, each estimate of association was not statistically significant.
DISCUSSION
Our results add to the accumulating evidence for clinically relevant association that tHcy significantly contributes to the incident dementia in patients with vascular risk factors, independent of APOE ɛ4 status, vascular risk factors, renal impairment, and MRI-findings (SVD and MTA), which are known to be predictors of dementia. We also took account of MRI-findings (SVD, atrophy) at baseline. Higher tHcy levels were also associated with CMBs (especially, strictly deep CMBs), and lacunas, independent of vascular risk factors, IMT, and renal impairment.
Several observational studies have investigated the relationship of tHcy levels with incident dementia and cognitive impairment, whereas these results were inconsistent [5–10]. Indeed, our findings were in line with several prospective studies, regarding the predictive value of tHcy levels for incident dementia [5–8]. However, tHcy levels correlate with aging [33], vascular burden [1], and renal impairment [15]. Thus, relationships between tHcy levels and dementia risk are confounded by these factors, and the results may not be valid unless these confoundings are considered. One notable finding of this study is therefore the significant effect of baseline tHcy levels on dementia incidence that is not explained by cerebral SVD, brain atrophy and renal impairment.
This observational finding is strengthened by several biological mechanisms that have consistently been linked to the pathological role of tHcy for both vascular and AD dementia. Elevated tHcy levels increase vascular burden. Hcy induces endothelial dysfunction and smooth muscle cell proliferation [34], increases hypercoagulability [35] and oxidative stress [36], and may result in the increased permeability of blood-brain barrier, leading to SVD as well as cognitive decline. A direct relationship between tHcy levels and cardiovascular events has been observed in the general population [2], although randomized controlled trials of tHcy lowering have failed to reduce the burden of cardiovascular disease [37]. However, subgroup analyses of theVITAmins TO Prevent Stroke trial might reduce the risk reduction for composite primary endpoint (stroke, myocardial infarction, or vascular death), after a 3-year vitamin B supplementation period, in patients with SVD [38]. Moreover, elevated tHcy levels were associated with greater WMH progression and reduced eGFR, suggesting that tHcys levels contribute to generalized SVD [39]. Indeed, vascular risk factors and AD pathology are correlated with each other in the elderly, making it difficult to tease out the individual effects of each [4]. In addition, associations between Hcy and brain atrophy have also been suggested in some [40–42] but not all [43] previous studies using in MRI. However, the lack of association between tHcy and atrophy in our study may be due to differencesin the use of MRI measures and in populationcharacteristics. MRI was performed at enrollment and used for visual ratings of the degree of MTA. We demonstrated an effect of tHcy levels on brain atrophy in participants not having dementia at baseline. Our sample had relatively lower tHcy levels and was younger as compared with earlier studies [40–42], which may have made it more difficult to find significant associations.
Additionally, several potential mechanisms underlying the deleterious effects of Hcy in AD pathology have been proposed. Diet-induced increases in tHcy levels modulated the pathology of all three major AD phenotypes in a transgenic mouse model: not only amyloid-β accumulation but also tau pathology and memory deficits [44]. In a recent autopsy population-based study, high baseline tHcy levels may contribute particularly to increased neurofibrillary tangle burden, which seems to be more pronounced in the presence of cerebrovascular pathology despite the lack of a direct association between cerebrovascular pathology and Hcy, suggesting a direct association between Hcy and neurofibrillary formation [42]. Furthermore, a meta-analysis of genetic association studies of the effects of the methylenetetrahydrofolate reductase gene (MTHFR) polymorphism on AD dementia found a small but significant association supporting the causal susceptibility between Hcy and dementia [45]. Taken together, these findings indicate that the association between high tHcy and dementia is biologicallyplausible.
Recent meta-analysis of randomized trials that addressed the effects of homocysteine-lowering treatment with B vitamins on cognitive change, it did not appear to improve cognitive function, as measured by the MMSE, during mean 5 years follow-up [46]. However, no trials have attempted to prevent incident dementia as an endpoint by reducing tHcy, thus limiting comparisons. Also, differences of outcome measure, target population (e.g., baseline tHcy levels), study duration, or implementing the study after mandatory folic acid fortification make the result difficult to interpret. AD pathology, such as the neurofibrillary tangle and amyloid-β accumulation, as well as subclinical vascular injury is long-term process and large trials with an extended period of time are needed to determine the potential efficiency.
The strengths of our study are the longitudinal study design, the use of MRI data in all subjects, and the complete follow-up of all subjects. This study also has several limitations. First, the small number of incident dementia led to weak statistical power for evaluating dementia subtype. Although the lack of association in multivariate analysis between tHcy, incident cerebrovascular events and mortality observed in our study, attrition as a result of death may have contributed to underestimation of the relation between tHcy and dementia risk. Second, it may indicate the use of MRI measures are saturating, i.e., ceiling effects due to visual ratings. Third, we seemingly identified stronger associations of Hcy with VaD. Our study was limited to a cohort of Japanese individuals with vascular profiles and concomitant SVD burden because MRI was performed for examination of suspected brain disease; thus, our results may not be generalizable to other races and cohorts. Finally, we do not have data on tHcy trends due to the single blood sampling or on vitamin concentrations (such as vitamin B12 and folate), or on medication change during follow-up period.
The deleterious effects of higher tHcy levels remain consistent irrespective of the MRI-findings, thus Hcy may be a potential target in the prevention of dementia. Despite an intensive vascular risk management in our neurology and stroke clinics, tHcy levels at baseline were independently related to dementia risk, thereby highlighting the unique susceptibility of patients to incident dementia risk. Our findings support the idea that modulating tHcy might be beneficial for reducing any dementia risk.
