Key Points

Question Is methylation status (ie, methionine to homocysteine ratio) associated with incident dementia and structural brain changes in older adults?

Findings In this cohort study of longitudinal data from 2570 elderly individuals who were dementia free at baseline, a higher methionine to homocysteine ratio was observed in participants with better B 12 or folate status and was associated with decreased risk of incident dementia and Alzheimer disease. A higher methionine to homocysteine ratio was associated with a decreased rate of total brain tissue volume loss during 6 years.

Meanings Markers of methylation status were associated with dementia development and structural brain changes during 6 years, suggesting that a higher methionine to homocysteine ratio may be important in reducing the rate of brain atrophy and decreasing the risk of dementia in older adults.

Abstract

Importance Impairment of methylation status (ie, methionine to homocysteine ratio) may be a modifiable risk factor for structural brain changes and incident dementia.

Objective To investigate the association of serum markers of methylation status and sulfur amino acids with risk of incident dementia, Alzheimer disease (AD), and the rate of total brain tissue volume loss during 6 years.

Design, Setting, and Participants This population-based longitudinal study was performed from March 21, 2001, to October 10, 2010, in a sample of 2570 individuals aged 60 to 102 years from the Swedish Study on Aging and Care in Kungsholmen who were dementia free at baseline and underwent comprehensive examinations and structural brain magnetic resonance imaging (MRI) on 2 to 3 occasions during 6 years. Data analysis was performed from March 1, 2018, to October 1, 2018.

Main Outcomes and Measures Incident dementia, AD, and the rate of total brain volume loss.

Results This study included 2570 individuals (mean [SD] age, 73.1 [10.4] years; 1331 [56.5%] female). The methionine to homocysteine ratio was higher in individuals who consumed vitamin supplements (median, 1.9; interquartile range [IQR], 1.5–2.6) compared with those who did not (median, 1.8; IQR, 1.3–2.3; P < .001) and increased per each quartile increase of vitamin B 12 or folate. In the multiadjusted model, an elevated baseline serum total homocysteine level was associated with an increased risk of dementia and AD during 6 years: for the highest homocysteine quartile compared with the lowest, the hazard ratios (HRs) were 1.60 (95% CI, 1.01-2.55) for dementia and 2.33 (95% CI, 1.26-4.30) for AD. In contrast, elevated concentrations of methionine were associated with a decreased risk of dementia (HR, 0.54; 95% CI, 0.36-0.81) for the highest quartile compared with the lowest. Higher values of the methionine to homocysteine ratio were significantly associated with lower risk of dementia and AD: for the fourth methionine-homocysteine quartile compared with the first quartile, the HR was 0.44 (95% CI, 0.27-0.71) for incident dementia and 0.43 (95% CI, 0.23-0.80) for AD. In the multiadjusted linear mixed models, a higher methionine to homocysteine ratio was associated with a decreased rate of total brain tissue volume loss during the study period (β [SE] per 1-SD increase, 0.038 [0.014]; P = .007).

Conclusions and Relevance The methionine to homocysteine status was associated with dementia development and structural brain changes during the 6-year study period, suggesting that a higher methionine to homocysteine ratio may be important in reducing the rate of brain atrophy and decreasing the risk of dementia in older adults.

Introduction

Vitamin B 12 and folate are essential vitamins for the remethylation of homocysteine to methionine and the subsequent formation of S-adenosylmethionine (SAM), the primary methyl donor for many biochemical reactions involved in normal brain functions.1-4 Interference with this process may lead to impairment in the formation of methionine and an unfavorable methylation status and may result in the accumulation of serum total homocysteine (tHcy), which has been associated with several cerebrovascular and cardiovascular conditions.5 Elevated tHcy levels may further impair the methylation status by converting to S-adenosyl homocysteine (SAH), a potent competitive inhibitor of several methyl transferases.2,6

Whereas several studies7-9 have reported an association between increased tHcy values and dementia or structural brain changes, only a few cross-sectional studies10-12 have investigated the associations between methylation status (ie, methionine to homocysteine ratio) and cognitive impairment or dementia with mixed results. Furthermore, the effects of sulfur amino acids other than tHcy on dementia have rarely been investigated.13,14

The potential association of sulfur amino acids with dementia is important because they are modifiable risk factors and thus a potential target in preventive interventions. We previously reported that the rate of total brain volume loss in older adults was associated with tHcy and vitamin B 12 status 6 years earlier.15 However, the methionine to homocysteine status was not investigated in relation to brain magnetic resonance imaging (MRI) measures in that report.15 The aim of the current study was to investigate the associations of methionine to homocysteine status, other sulfur amino acids, vitamin B 12 , and red blood cell (RBC) folate with the risk of incident dementia during 6 years in a population-based cohort of older adults without mandatory folic acid fortification. We proposed that methylation status may be reflected by the serum methionine to homocysteine status. In a supplementary analysis, we examined the association between methionine to homocysteine ratio and the rate of total brain volume loss in a subsample with available brain MRI data.

Methods

Study Population

The study population was derived from the Swedish National Study on Aging and Care in Kungsholmen (SNAC-K), a population-based, prospective study conducted in the Kungsholmen area of central Stockholm, Sweden. SNAC-K involved a random sample of persons 60 years or older who live at home or in an institution. Because of more rapid changes in health and a higher attrition rate among older age groups, sampling was stratified by age cohort. Assessments took place at 6-year intervals for younger cohorts (60, 66, 72, and 78 years of age) and at 3-year intervals for older cohorts (81, 84, 87, 90, 93, 96, and ≥99 years of age). From March 21, 2001, to August 30, 2004, of the 4590 living and eligible individuals randomly selected for SNAC-K, 3363 (73.3%) participated in the baseline examination; the end of follow-up was October 10, 2010.16,17 The Ethics Committee at Karolinska Institutet and the Regional Ethical Review Board in Stockholm approved the protocols of each phase of SNAC-K and approved this study, and written informed consent was provided by all participants.

At baseline and each follow-up, the SNAC-K participants underwent a thorough clinical examination, interview, and assessments by a physician, a registered nurse, and a psychologist. Data on sociodemographic characteristics, medical history, drug use, and cognitive function were collected according to a structured protocol, and the diagnoses of dementia and Alzheimer disease (AD) were made according to DSM-IV criteria18 in which a validated 3-step diagnostic procedure was used as previously reported.19 In brief, 2 examining physicians independently made a preliminary diagnosis, and in the case of disagreement, a third opinion was sought to reach a consensus diagnosis. For the deceased participants, the diagnosis of dementia was made by 2 physicians through reviewing the medical records and death certificates.

Data on vitamin supplement use were collected from study participants and verified by inspecting drug prescriptions and containers. Systolic blood pressure (SBP) was measured twice using the participant’s left arm after the patient had been sitting for 5 minutes, and the mean of the measurements was calculated.

Blood samples obtained after clinical examination were routinely analyzed for RBC folate levels. Of the initial sample, participants who were diagnosed with prevalent dementia (DSM-IV criteria, n = 311) and those who did not have blood samples obtained (n = 271) were excluded, leaving 2903 participants with available RBC folate values at baseline. Of these individuals, 333 refused to participate in the follow-up examination or had moved before examination (213 individuals from the younger age group and 120 individuals from the older age group). Therefore, the study population for the current analysis consisted of 2570 individuals without dementia at baseline. Of these individuals, 501 underwent MRI on a 1.5-T magnetic resonance scanner (eAppendix in the Supplement) at baseline and every 3 years thereafter for the older cohort (ie, those ≥78 years of age at baseline; n = 92 at 3-year follow-up) and every 6 years thereafter for the whole cohort (n = 260; n = 53 in the older cohort and n = 207 in the younger cohort). Characteristics of the MRI subsample and MRI procedures are described in detail elsewhere.15

Compared with the rest of the SNAC-K sample, the study population was younger (mean [SD] age, 85.3 [10.7] years vs 73.1 [10.4] years; P < .001), was less likely to be female (357 [77.6%] vs 1825 [62.9%]; P < .001), had a higher educational level (mean [SD] years of schooling, 9.7 [3.4] vs 12.1 [0.4]; P < .001), and had a better Mini-Mental State Examination total score (mean [SD], 17.0 [10.3] vs 28.7 [1.8]; P < .001). Furthermore, the study population had a lower percentage of cardiovascular conditions (ie, atrial fibrillation, coronary heart disease, and heart failure) (1021 [35.2%] vs 254 [55.2%]), smoked more often (1555 [53.8%] vs 153 [41.0%]), and consumed fewer vitamin supplements (644 [22.2%] vs 173 [37.6%]) compared with nonparticipants. The levels of RBC folate, methionine, and glutathione were higher and the levels of homocysteine, cysteine, and cystathionine were lower in study participants compared with non-participants.

Biochemical Analyses

At baseline, venous blood samples were taken while the participant was not fasting, and routine analyses, including RBC folate assessment, were performed within 2 hours using chemiluminescence microparticle folate-binding protein assay at Sabbatsberg Hospital, Stockholm, Sweden (results available for 2570 participants). The coefficient of variation was 4.8% at 147 ng/mL and 6.1% at 238 ng/mL (to convert folate to nanomoles per liter, multiply by 2.266). Serum specimens were stored at –80° for 10 to 12 years. Batches were transferred thereafter on dry ice to the University of Oxford, Oxford, United Kingdom. Because there was sufficient demand for blood samples to be used for a variety of other biochemical assays in SNAC-K, sufficient serum volumes were not available for 215 participants. In the other 2355 participants, vitamin B 12 and holotranscobalamin levels were measured by microbiological methods, as described previously.20 The coefficient of variation for both assays was 5%. The levels of sulfur amino acids (tHcy, methionine, cystathionine, total cysteine, and total glutathione) were measured using tandem mass spectrometry after treatment of serum with a reducing agent, as described previously.21 Interassay coefficients of variation were between 5% and 10%. Three individuals with a tHcy value greater than 9.46 mg/dL (to convert to micromoles per liter, multiply by 7.397) were excluded. Genotyping of APOE was performed as described previously.22

Statistical Analysis

Data analysis was performed from March 1, 2018, to October 1, 2018. Baseline characteristics of individuals were compared according to incident dementia status using χ2 tests for the proportions and t test or Mann-Whitney test for continuous variables, when appropriate. Continuous variables are presented as mean (SD) or median (interquartile range [IQR]), whereas categorical variables are presented as number (percentage). In addition to investigating the association between methionine to homocysteine status with the outcomes, we considered the association of cystathionine to homocysteine ratio as a possible indicator of the transsulfuration pathway because cystathionine represents a strong marker of flux through transsulfuration.23,24

Cox proportional hazards regression models were used to estimate the hazard ratio (HR) and 95% CI of incident dementia and AD in association with vitamin B 12 , holotranscobalamin, RBC folate, and sulfur amino acids categorized into quartiles, with the lowest quartile as the reference category.

For participants without dementia, the follow-up time was calculated from the date of the baseline interview to the date of the last follow-up examination. For participants with incident dementia, the follow-up time was estimated as the time during which participants were free of dementia plus half of the follow-up time during which dementia developed because of its insidious onset. Participants who died or did not return to the next follow-up were censored as of the time of the last evaluation. The proportional hazards assumption was confirmed by graphs and tests based on Schoenfeld residuals.

Models were adjusted for age, sex, and educational level and then additionally for other potential confounding or mediating factors, including SBP, creatinine concentration, use of vitamin supplements, smoking, history of cardiovascular conditions (ie, atrial fibrillation, coronary heart disease, and heart failure) and stroke, and plasma albumin level. Because data on APOEε4 were not available for all participants, we ran additional analyses adjusting for APOE-ε4 status.

In addition to examining the associations with incident dementia and AD, we sought to investigate the association of methionine to homocysteine ratio (as a continuous variable) with structural brain changes using linear mixed models for repeated measures. In the linear mixed models, the β coefficient for the methionine to homocysteine status represents the cross-sectional association with the baseline brain volume. The β coefficient for the interaction between the methionine to homocysteine status and time represents the association of these biomarkers with the rate of change in brain volume per year. A positive β coefficient indicates that an improvement in the methionine to homocysteine status was associated with a decreased rate of brain volume loss over time. We analyzed the data using Stata statistical software, version 15 (StataCorp). A 2-tailed P < .05 was considered to be statistically significant.

Results

This study included 2570 individuals (mean [SD] age, 73.1 [10.4] years; 1331 [56.5%] female). During the study period, 203 patients with incident dementia (129 with AD) were identified in the older age group and 38 (20 with AD) in the younger age group. Individuals with incident dementia were older at baseline, were more likely to be female, were less educated, were more likely to use vitamin supplements, smoked less often, and had higher frequency of cardiovascular conditions, stroke, and APOE-ε4 allele (Table 1). The baseline levels of tHcy, cystathionine, and cysteine were higher, and the levels of albumin, methionine, and glutathione were lower in patients who developed dementia compared with those who did not.

The methionine to homocysteine ratio was higher in individuals who consumed vitamin supplements (median, 1.9; IQR, 1.5–2.6) compared with those who did not (median, 1.8; IQR, 1.3–2.3; P < .001) and increased per each quartile increase of vitamin B 12 or folate (Table 2). Cross-correlations among B 12 , folate, and different sulfur amino acids are given in Table 3.

After all study covariates were adjusted for, the HR for dementia was 0.54 (95% CI, 0.36-0.81) for individuals in the highest methionine quartile compared with the lowest quartile (Table 4). Additional adjustment for duration of fasting, tHcy, cysteine, vitamin B 12 , or folate did not influence the association (eTable 1 and eTable 2 in the Supplement). In contrast, higher tHcy values were associated with increased risk of dementia and AD: for the highest tHcy quartile, the HR was 1.60 (95% CI, 1.01-2.55) for dementia and 2.33 (95% CI, 1.26-4.30) for AD (Table 5). Adding the APOE-ε4 allele into the models did not change the association between methionine and dementia or tHcy and dementia.

Higher methionine to tHcy ratios were significantly associated with the lower risk of dementia and AD: the HR for incident dementia was 0.53 (95% CI, 0.35-0.80) for the third methionine to tHcy quartile and 0.44 (95% CI, 0.27-0.71) for the fourth quartile, after controlling for all study covariates (Table 2). Compared with the first quartile, the third quartile of baseline cystathionine was significantly associated with increased risk of dementia (HR, 1.71; 95% CI, 1.07-2.72). No significant associations were observed for RBC folate, vitamin B 12 , other sulfur amino acids, or cystathionine to tHcy ratio in relation to the risk of dementia.

After adjusting for age, sex, and educational level, a higher methionine to homocysteine ratio was associated with total brain tissue volume (β per 1-SD increase, 0.446; SE, 0.162; P = .006). Additional adjustment for other study covariates did not influence the results. In the longitudinal analysis during 6 years, a higher methionine to homocysteine ratio was associated with a decreased rate of total brain tissue volume loss (β for each 1-SD increase, 0.038; SE, 0.014; P = .007; ie, 0.038% less atrophy per year) (Table 3). Furthermore, a higher methionine to homocysteine ratio was associated with a decreased rate of gray matter volume loss (β, 0.034; SE, 0.012; P = .003) but not white matter volume loss (β, 0.007; SE, 0.012; P = .548) or white matter hyperintensity volume (β, −0.000001; SE, 0.000001; P = .32) in the longitudinal analysis.

Discussion

In this longitudinal population-based study of older adults, higher levels of methionine, lower levels of tHcy, and higher methionine to homocysteine ratios were associated with a decreased risk of dementia during 6 years. Furthermore, a higher methionine to homocysteine ratio was associated with a decreased rate of brain volume loss during follow-up. The observed associations were independent of several potential confounders, including common sociodemographic and vascular risk factors or markers of the transsulfuration pathway.

Similar to our findings, several prospective studies7,8 reported an association between increased baseline tHcy values and risk of incident dementia. In contrast, the association of methylation status with dementia risk has been investigated only in a few case-control studies,11,25 which reported lower values of SAM/SAH ratio (another indicator of methylation capacity) in patients with AD compared with controls. Another study12 that additionally examined the association with cerebrospinal fluid biomarkers of AD did not find any differences in the mean SAM/SAH ratio between patients with AD and controls, although decreased SAM/SAH ratios were associated with elevated cerebrospinal fluid phospho-tau values. None of these studies directly investigated the association between methionine and dementia but have considered SAM as a surrogate marker of methionine status.

Methionine is an essential amino acid involved in several metabolic processes, such as protein synthesis and polyamine metabolism, and decreased plasma methionine concentrations have been observed in a number of cardiovascular and neurologic conditions.26-29 Methionine status is closely associated with dietary intake as well as B 12 and folate status, its biosynthesis is closely associated with the transmethylation and transsulfuration pathways,30 and it serves as the precursor for the production of several other amino acids and SAM, which is the primary methyl donor for many biochemical reactions involved in normal brain functions, including the production of phosphatidylcholine (important for cell membrane structure and synaptic function, monoaminergic neurotransmitters, and nucleic acids).7 Impairment in methylation status is reportedly associated with white matter damage, AD-type neuropathology, and brain atrophy, factors associated with cognitive decline and dementia.4,7,15 A higher methionine to homocysteine ratio was associated with a decreased rate of brain volume loss, notably gray matter in our study, which is consistent with findings from the experimental studies.1,3,5,7 In contrast, an increased tHcy level may be associated with an increased loss of gray matter and of total brain volume.15,31 As discussed in detail elsewhere,7 increased tHcy values may be associated with increased risk of dementia through several plausible mechanisms, such as the potentiation of amyloid-β generation or its neurotoxicity, formation of neurofibrillary tangles, or cerebrovascular pathologic processes.

Although we previously reported an association between higher levels of vitamin B 12 or holotranscobalamin and decreased rate of brain tissue volume loss,15 no associations with incident dementia or AD were found in the current study. Notably, vitamin B 12 and holotranscobalamin were associated only longitudinally and not cross-sectionally with brain atrophy. Perhaps vitamin B 12 status needs a longer time to influence brain structure, and the effects first manifest after several years of follow-up. This may explain the lack of association with dementia in the current study because substantial cerebral atrophy usually presents before clinical manifestation of dementia32,33; a longer follow-up period might have been needed to detect the association of vitamin B 12 and clinical dementia in our population. An association between B 12 or holotranscobalamin and dementia or cognitive decline was reported mainly in studies5,7 with longer follow-up periods than our study.

In our study, the moderate cystathionine level (the third quartile), but not the highest level or cystathionine to homocysteine ratio, was associated with an increased risk of dementia. Cystathionine is produced from homocysteine during transsulfuration, and increased cystathionine values have been associated with atherosclerosis and a subsequent increase in the risk of various vascular conditions, which may increase the risk of dementia.34,35 Although our findings need to be confirmed in other settings, a possible explanation for the lack of association in the top quartile could be that people with higher cystathionine values are at higher risk of dying of cardiovascular diseases34 and are no longer at risk of dementia. Notably, participants with cardiovascular conditions had higher cystathionine values compared with those without such conditions in our study.

Because of the observational design of our study, a causal interpretation of our findings cannot be made. Future studies will need to investigate in more detail possible underlying mechanisms and identify the role of the optimal methionine to homocysteine ratio and its possible interaction with other biochemical pathways in individuals who are at increased risk of structural brain changes and dementia in the context of clinical trials. High tHcy and low B 12 and folate levels are surprisingly common conditions in older adults,5,7 and our results indicated a better methionine to homocysteine profile in participants with adequate B 12 or folate values. If the association is found to be causal, supplementation with B vitamins may be effective for prevention of brain damage and dementia risk because of increased tHcy and impaired methylation reactions. The Homocysteine and B Vitamins in Cognitive Impairment (VITACOG) trial has already shown that B vitamin treatment in people with mild cognitive impairment who have increased tHcy levels markedly slows regional gray matter atrophy and cognitive decline.7,31 Further adequately timed and powered randomized clinical trials are needed to determine causation.

Strengths and Limitations

The main strengths of this study are the relatively large number of community-dwelling older adults with available data on a large number of potential confounders, the availability of MRI during 6 years, and the evaluation of B 12 , folate, and sulfur amino acids simultaneously in association with the outcome. Although a few studies2,7 have investigated the association of SAM/SAH ratio or methionine status with dementia risk, none had a longitudinal design. The relatively long follow-up period, the comprehensive evaluation and diagnostic protocol at each examination, and recruitment of dementia-free individuals at baseline (mean [SD] Mini-Mental State Examination score, 28.7 [1.8]) make our results less prone to the influence of reverse causality (ie, the effects of preclinical dementia on methionine to homocysteine status).

The limitations of our study include the measurement of sulfur amino acids, vitamin B 12 , and RBC folate at only 1 time point, which may underestimate their associations because of regression dilution.15 Selective survival may also have contributed to an underestimation of the associations because high levels of tHcy and low levels of vitamin B 12 or folate have been associated with increased mortality in previous studies.1,6,36 Blood samples were collected in a nonfasting state, and methionine concentrations are known to increase postprandially.23 Although adjusting for hours of fasting did not change any of the associations substantially, this may not fully account for the measurement of sulfur amino acids in a nonfasting state. Although study participants were in general healthier than nonparticipants in our study, any nonresponse bias may have led to underestimation of the associations.15

Conclusions

Our findings suggest that interrelated but distinct processes that result in elevation of serum tHcy and serum methionine levels may be associated with brain atrophy and dementia risk in older adults. Thus, a better methylation status, reflected by a higher methionine to homocysteine ratio, may be beneficial for the structure and functioning of the brain.

Back to top Article Information

Accepted for Publication: April 29, 2019.

Corresponding Author: Babak Hooshmand, MD, PhD, MPH, Aging Research Center, Karolinska Institute, Tomtebodavägen 18A, Plan 9, Solna 171 65, Sweden (babak.hooshmand@ki.se).

Published Online: July 24, 2019. doi:10.1001/jamapsychiatry.2019.1694

Author Contributions: Dr Hooshmand had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Concept and design: Hooshmand, Refsum, Smith, Mangialasche, Kivipelto, Fratiglioni.

Acquisition, analysis, or interpretation of data: Hooshmand, Refsum, Kalpouzos, von Arnim, Kareholt, Kivipelto, Fratiglioni.

Drafting of the manuscript: Hooshmand, Kivipelto, Fratiglioni.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Hooshmand, Kalpouzos.

Obtained funding: Kivipelto, Fratiglioni.

Administrative, technical, or material support: Refsum, Kalpouzos.

Supervision: Hooshmand, Kivipelto, Fratiglioni.

Conflict of Interest Disclosures: Dr Smith reported receiving personal fees from P&G Health Care International, Aprofol AG, and Mylan Norge outside the submitted work and having a patent to US6008221 issued, a patent to US6127370, issued, a patent to US9364497B2 issued, and a patent to PCT/GB2015/050786 pending. Dr von Arnim reported receiving personal fees from Lilly Deutschland GmbH, Biogen, and Roche outside the submitted work. Dr Fratiglioni reported receiving grants from VR and Forte during the conduct of the study. No other disclosures were reported.

Funding/Support: The Swedish National Study on Aging and Care in Kungsholmen (SNAC-K) is supported by the Swedish Ministry of Health and Social Affairs, Stockholm County Council, and Stockholm municipality. This study was supported by Avtal om läkarutbildning och forskning grants 20130507 and 20150589, a Vetenskapsrådet grant, a Forte grant, Alzheimerfonden (Sweden), Center for Innovative Medicine at Karolinska Institute South Campus, Knut and Alice Wallenberg Foundation (Sweden), Stiftelsen Stockholms Sjukhem (Sweden), The Norwegian Research Council (Norway), Charles Wolfson Charitable Trust (United Kingdom), Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse, and Fredrik och Ingrid Thurings Stiftelse (Sweden).

Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and the decision to submit the manuscript for publication.

Additional Contributions: The SNAC-K participants and colleagues in the SNAC-K group collaborated in data collection and management. Ying Shang, MSc, Karolinska Institute, Stockholm, Sweden, Cynthia Prendergast, PhD, Cheryl Turner, MSc, and Fredrik Jerneren, PhD, Department of Pharmacology, University of Oxford, United Kingdom, performed assays of B 12 , holotranscobalamin, and sulfur amino acids and helped with part of the data collection. All were compensated for their work.