Association of Methionine to Homocysteine Status with Brain Magnetic Resonance Imaging Measures and Risk of Dementia

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JAMA Psychiatry | Original Investigation Association of Methionine to Homocysteine Status With Brain Magnetic Resonance Imaging Measures and Risk of Dementia

Babak Hooshmand, MD, PhD, MPH; Helga Refsum, MD, PhD; A. David Smith, DPhil; Grégoria Kalpouzos, PhD; Francesca Mangialasche, MD, PhD; Christine A. F. von Arnim, MD; Ingemar Kåreholt, PhD; Miia Kivipelto, MD, PhD; Laura Fratiglioni, MD, PhD

Supplemental content 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 B12 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.

Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Author: Babak Hooshmand, MD, PhD, MPH, Aging Research Center, Karolinska Institute, Tomtebodavägen 18A, JAMA Psychiatry. 2019;76(11):1198-1205. doi:10.1001/jamapsychiatry.2019.1694 Plan 9, Solna 171 65, Sweden Published online July 24, 2019. ([email protected]).

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itamin B12 and folate are essential vitamins for the re- methylation of homocysteine to methionine and the Key Points subsequent formation of S-adenosylmethionine (SAM), V Question Is methylation status (ie, methionine to homocysteine the primary methyl donor for many biochemical reactions in- ratio) associated with incident dementia and structural brain volved in normal brain functions.1-4 Interference with this pro- changes in older adults? cess may lead to impairment in the formation of methionine Findings In this cohort study of longitudinal data from 2570 and an unfavorable methylation status and may result in the elderly individuals who were dementia free at baseline, a higher accumulation of serum total homocysteine (tHcy), which has methionine to homocysteine ratio was observed in participants

been associated with several cerebrovascular and cardiovas- with better B12 or folate status and was associated with decreased cular conditions.5 Elevated tHcy levels may further impair the risk of incident dementia and Alzheimer disease. A higher methylation status by converting to S-adenosyl homocyste- methionine to homocysteine ratio was associated with a ine (SAH), a potent competitive inhibitor of several methyl decreased rate of total brain tissue volume loss during 6 years. transferases.2,6 Meanings Markers of methylation status were associated with Whereas several studies7-9 have reported an association dementia development and structural brain changes during between increased tHcy values and dementia or structural 6 years, suggesting that a higher methionine to homocysteine brain changes, only a few cross-sectional studies10-12 have ratio may be important in reducing the rate of brain atrophy and decreasing the risk of dementia in older adults. investigated the associations between methylation status (ie, methionine to homocysteine ratio) and cognitive impairment or dementia with mixed results. Furthermore, the ef- The Ethics Committee at Karolinska Institutet and the Re- fects of sulfur amino acids other than tHcy on dementia have gional Ethical Review Board in Stockholm approved the pro- rarely been investigated.13,14 tocols of each phase of SNAC-K and approved this study, and The potential association of sulfur amino acids with de- written informed consent was provided by all participants. mentia is important because they are modifiable risk factors At baseline and each follow-up, the SNAC-K participants and thus a potential target in preventive interventions. We pre- underwent a thorough clinical examination, interview, and as- viously reported that the rate of total brain volume loss in older sessments by a physician, a registered nurse, and a psycholo-

adults was associated with tHcy and vitamin B12 status 6 years gist. Data on sociodemographic characteristics, medical his- earlier.15 However, the methionine to homocysteine status was tory, drug use, and cognitive function were collected according not investigated in relation to brain magnetic resonance to a structured protocol, and the diagnoses of dementia and imaging (MRI) measures in that report.15 The aim of the cur- Alzheimer disease (AD) were made according to DSM-IV rent study was to investigate the associations of methionine criteria18 in which a validated 3-step diagnostic procedure was 19 to homocysteine status, other sulfur amino acids, vitamin B12, used as previously reported. In brief, 2 examining physi- and red blood cell (RBC) folate with the risk of incident de- cians independently made a preliminary diagnosis, and in the mentia during 6 years in a population-based cohort of older case of disagreement, a third opinion was sought to reach a con- adults without mandatory folic acid fortification. We pro- sensus diagnosis. For the deceased participants, the diagno- posed that methylation status may be reflected by the serum sis of dementia was made by 2 physicians through reviewing methionine to homocysteine status. In a supplementary analy- the medical records and death certificates. sis, we examined the association between methionine to Data on vitamin supplement use were collected from study homocysteine ratio and the rate of total brain volume loss in participants and verified by inspecting drug prescriptions and a subsample with available brain MRI data. 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 Methods calculated. Blood samples obtained after clinical examination were Study Population routinely analyzed for RBC folate levels. Of the initial sample, The study population was derived from the Swedish National participants who were diagnosed with prevalent dementia Study on Aging and Care in Kungsholmen (SNAC-K), a popu- (DSM-IV criteria, n = 311) and those who did not have blood lation-based, prospective study conducted in the Kungshol- samples obtained (n = 271) were excluded, leaving 2903 par- men area of central Stockholm, Sweden. SNAC-K involved a ticipants with available RBC folate values at baseline. Of these random sample of persons 60 years or older who live at home individuals, 333 refused to participate in the follow-up exami- or in an institution. Because of more rapid changes in health nation or had moved before examination (213 individuals from and a higher attrition rate among older age groups, sampling the younger age group and 120 individuals from the older age was stratified by age cohort. Assessments took place at 6-year group). Therefore, the study population for the current analy- intervals for younger cohorts (60, 66, 72, and 78 years of age) sis consisted of 2570 individuals without dementia at base- and at 3-year intervals for older cohorts (81, 84, 87, 90, 93, 96, line. Of these individuals, 501 underwent MRI on a 1.5-T mag- and ≥99 years of age). From March 21, 2001, to August 30, netic resonance scanner (eAppendix in the Supplement)at 2004, of the 4590 living and eligible individuals randomly se- baseline and every 3 years thereafter for the older cohort (ie, lected for SNAC-K, 3363 (73.3%) participated in the baseline those ≥78 years of age at baseline; n = 92 at 3-year follow-up) examination; the end of follow-up was October 10, 2010.16,17 and every 6 years thereafter for the whole cohort (n = 260;

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n = 53 in the older cohort and n = 207 in the younger cohort). homocysteine ratio as a possible indicator of the transsulfu- Characteristics of the MRI subsample and MRI procedures are ration pathway because cystathionine represents a strong described in detail elsewhere.15 marker of flux through transsulfuration.23,24 Compared with the rest of the SNAC-K sample, the study Cox proportional hazards regression models were used to population was younger (mean [SD] age, 85.3 [10.7] years vs estimate the hazard ratio (HR) and 95% CI of incident demen-

73.1 [10.4] years; P < .001), was less likely to be female (357 tia and AD in association with vitamin B12, holotranscobala- [77.6%] vs 1825 [62.9%]; P < .001), had a higher educational min, RBC folate, and sulfur amino acids categorized into quar- level (mean [SD] years of schooling, 9.7 [3.4] vs 12.1 [0.4]; tiles, with the lowest quartile as the reference category. P < .001), and had a better Mini-Mental State Examination total For participants without dementia, the follow-up time score (mean [SD], 17.0 [10.3] vs 28.7 [1.8]; P < .001). Further- was calculated from the date of the baseline interview to the more, the study population had a lower percentage of cardio- date of the last follow-up examination. For participants with vascular conditions (ie, atrial fibrillation, coronary heart dis- incident dementia, the follow-up time was estimated as the ease, and heart failure) (1021 [35.2%] vs 254 [55.2%]), smoked time during which participants were free of dementia plus more often (1555 [53.8%] vs 153 [41.0%]), and consumed fewer half of the follow-up time during which dementia developed vitamin supplements (644 [22.2%] vs 173 [37.6%]) compared because of its insidious onset. Participants who died or did with nonparticipants. The levels of RBC folate, methionine, and not return to the next follow-up were censored as of the time glutathione were higher and the levels of homocysteine, cys- of the last evaluation. The proportional hazards assumption teine, and cystathionine were lower in study participants com- was confirmed by graphs and tests based on Schoenfeld pared with non-participants. residuals. Models were adjusted for age, sex, and educational level Biochemical Analyses and then additionally for other potential confounding or me- At baseline, venous blood samples were taken while the par- diating factors, including SBP, creatinine concentration, use ticipant was not fasting, and routine analyses, including RBC of vitamin supplements, smoking, history of cardiovascular folate assessment, were performed within 2 hours using che- conditions (ie, atrial fibrillation, coronary heart disease, and miluminescence microparticle folate-binding protein assay at heart failure) and stroke, and plasma albumin level. Because Sabbatsberg Hospital, Stockholm, Sweden (results available for data on APOEε4 were not available for all participants, we ran 2570 participants). The coefficient of variation was 4.8% at 147 additional analyses adjusting for APOE-ε4 status. ng/mL and 6.1% at 238 ng/mL (to convert folate to nanomoles In addition to examining the associations with incident de- per liter, multiply by 2.266). Serum specimens were stored at mentia and AD, we sought to investigate the association of me- –80° for 10 to 12 years. Batches were transferred thereafter on thionine to homocysteine ratio (as a continuous variable) with dry ice to the University of Oxford, Oxford, United Kingdom. structural brain changes using linear mixed models for re- Because there was sufficient demand for blood samples to be peated measures. In the linear mixed models, the β coeffi- used for a variety of other biochemical assays in SNAC-K, suf- cient for the methionine to homocysteine status represents the ficient serum volumes were not available for 215 partici- cross-sectional association with the baseline brain volume. The

pants. In the other 2355 participants, vitamin B12 and holo- β coefficient for the interaction between the methionine to ho- transcobalamin levels were measured by microbiological mocysteine status and time represents the association of these methods, as described previously.20 The coefficient of varia- biomarkers with the rate of change in brain volume per year. tion for both assays was 5%. The levels of sulfur amino acids A positive β coefficient indicates that an improvement in the (tHcy, methionine, cystathionine, total cysteine, and total glu- methionine to homocysteine status was associated with a de- tathione) were measured using tandem mass spectrometry af- creased rate of brain volume loss over time. We analyzed the ter treatment of serum with a reducing agent, as described data using Stata statistical software, version 15 (StataCorp). A previously.21 Interassay coefficients of variation were be- 2-tailed P < .05 was considered to be statistically significant. tween 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 Results as described previously.22 This study included 2570 individuals (mean [SD] age, 73.1 [10.4] Statistical Analysis years; 1331 [56.5%] female). During the study period, 203 pa- Data analysis was performed from March 1, 2018, to October tients with incident dementia (129 with AD) were identified 1, 2018. Baseline characteristics of individuals were com- in the older age group and 38 (20 with AD) in the younger age pared according to incident dementia status using χ2 tests for group. Individuals with incident dementia were older at base- the proportions and t test or Mann-Whitney test for continu- line, were more likely to be female, were less educated, were ous variables, when appropriate. Continuous variables are pre- more likely to use vitamin supplements, smoked less often, sented as mean (SD) or median (interquartile range [IQR]), and had higher frequency of cardiovascular conditions, stroke, whereas categorical variables are presented as number (per- and APOE-ε4 allele (Table 1). The baseline levels of tHcy, cys- centage). In addition to investigating the association be- tathionine, and cysteine were higher, and the levels of albu- tween methionine to homocysteine status with the out- min, methionine, and glutathione were lower in patients who comes, we considered the association of cystathionine to developed dementia compared with those who did not.

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Table 1. Baseline Characteristics of the Study Population

No Dementia Incident Dementia Abbreviations: IQR, interquartile Characteristic (n = 2329) (n = 241) P Value range; MMSE, Mini-Mental State Age, mean (SD), y 72.2 (10.1) 82.8 (7.6) <.001 Examination; RBC, red blood cell. Women, No. (%) 1440 (61.8) 178 (73.9) <.001 SI conversion factors: to convert Educational level, mean (SD), y 12.3 (4.5) 10.7 (6.2) <.001 creatinine to micromoles per liter, multiply by 88.4; albumin to grams Use of vitamin supplements, No. (%) 498 (21.4) 73 (30.3) .002 per liter, multiply by 10; RBC folate Systolic blood pressure, mean (SD), mm Hg 143.9 (19.6) 145.6 (22.6) .26 to nanomoles per liter, multiply by

Ever smoked, No. (%) 1272 (54.9) 99 (41.4) <.001 2.266; vitamin B12 to picomoles per liter, multiply by 0.7378; History of cardiovascular conditions, No. (%) 799 (34.3) 119 (49.4) <.001 methionine to micromoles per liter, History of stroke, No. (%) 106 (4.6) 29 (12.0) <.001 multiply by 67.02. Plasma creatinine level, mean (SD), mg/dL 1.01 (0.25) 1.01 (0.21) .99 a Mann-Whitney test was used. Plasma albumin level, mean (SD), g/dL 4.16 (0.34) 4.07 (0.33) <.001 b Determination of RBC folate values MMSE score, median (IQR)a 29 (28-30) 28 (26-29) <.001 was routinely performed for all participants (available in 2570 RBC folate level, median (IQR), ng/mLa,b 103.7 (82.5-138.7) 95.8 (73.9-147.0) .08 individuals), but the additional a Vitamin B12 level, median (IQR), pg/mL 470.3 (364.6-615.3) 471.0 (351.7-703.2) .86 markers were not routinely tested. Holotranscobalamin level, median (IQR), pmol/La 61.0 (43.0-88.0) 60.5 (41.0-98.0) .78 Thus, these values reflect those of the 2355 participants clinically Homocysteine, median (IQR), μmol/La 12.7 (10.4-15.9) 14.6 (11.6-18.1) <.001 evaluated with available blood for a Methionine level, median (IQR), mg/dL 0.34 (0.29-0.40) 0.31 (0.27-0.37) <.001 further analyses. Of these 2355 Cystathionine level, median (IQR), nmol/La 286 (203-434) 334 (242-484) <.001 individuals, 214 developed dementia during 6 years of Cysteine level, mean (SD), μmol/Lb 325.1 (54.4) 349.4 (55.8) <.001 follow-up. Glutathione level, median (IQR), μmol/La 3.5 (2.8-4.2) 3.2 (2.7-4.1) .04 c For APOEε4, data were available in a Methionine to homocysteine ratio 1.8 (1.4-2.4) 1.5 (1.2-1.9) .02 2364 individuals from the original Cystathionine to homocysteine ratioa 0.023 (0.016-0.032) 0.024 (0.018-0.034) .22 2570, of whom 217 individuals developed dementia during 6 years APOEε4 allele, No. (%)c 588 (27.4) 86 (39.6) <.001 of follow-up.

The methionine to homocysteine ratio was higher in indi- Table 2. Methionine to Homocysteine Status According to Quartiles viduals who consumed vitamin supplements (median, 1.9; of Folate and Vitamin B12 IQR, 1.5–2.6) compared with those who did not (median, 1.8; IQR, Methionine to 1.3–2.3; P < .001) and increased per each quartile increase of Homocysteine Status, Variable Median (IQR) P Valuea vitamin B12 or folate (Table 2). Cross-correlations among B12, Folate folate, and different sulfur amino acids are given in Table 3. After all study covariates were adjusted for, the HR for de- Quartile 1 (<82.5 ng/mL) 1.4 (1.00-1.9) Quartile 2 (82.6-104.1 ng/mL) 1.8 (1.4-2.2) mentia was 0.54 (95% CI, 0.36-0.81) for individuals in the high- <.001 est methionine quartile compared with the lowest quartile Quartile 3 (104.2-138.7 ng/mL) 1.9 (1.5-2.5) (Table 4). Additional adjustment for duration of fasting, tHcy, Quartile 4 (>138.7 ng/mL) 2.1 (1.6-2.7) Vitamin B cysteine, vitamin B12, or folate did not influence the associa- 12 tion (eTable 1 and eTable 2 in the Supplement). In contrast, Quartile 1 (<364.6 pg/mL) 1.6 (1.2-2.0) higher tHcy values were associated with increased risk of de- Quartile 2 (364.7-470.3 pg/mL) 1.7 (1.3-2.2) <.001 mentia and AD: for the highest tHcy quartile, the HR was 1.60 Quartile 3 (470.4-618.0 pg/mL) 1.9 (1.5-2.4) (95% CI, 1.01-2.55) for dementia and 2.33 (95% CI, 1.26-4.30) Quartile 4 (>618.0 pg/mL) 2.1 (1.5-2.7) forAD(Table 5). Adding the APOE-ε4 allele into the models Abbreviation: IQR, interquartile range. did not change the association between methionine and de- a Comparisons were performed using the Kruskal-Wallis test. mentia or tHcy and dementia. Higher methionine to tHcy ratios were significantly associated with the lower risk of dementia and AD: the HR for in- brain tissue volume (β per 1-SD increase, 0.446; SE, 0.162; cident dementia was 0.53 (95% CI, 0.35-0.80) for the third me- P = .006). Additional adjustment for other study covariates did thionine to tHcy quartile and 0.44 (95% CI, 0.27-0.71) for the not influence the results. In the longitudinal analysis during fourth quartile, after controlling for all study covariates 6 years, a higher methionine to homocysteine ratio was asso- (Table 2). Compared with the first quartile, the third quartile ciated with a decreased rate of total brain tissue volume loss of baseline cystathionine was significantly associated with (β for each 1-SD increase, 0.038; SE, 0.014; P = .007; ie, 0.038% increased risk of dementia (HR, 1.71; 95% CI, 1.07-2.72). No less atrophy per year) (Table 3). Furthermore, a higher methio- significant associations were observed for RBC folate, vita- nine to homocysteine ratio was associated with a decreased rate

min B12, other sulfur amino acids, or cystathionine to tHcy of gray matter volume loss (β, 0.034; SE, 0.012; P = .003) but ratio in relation to the risk of dementia. not white matter volume loss (β, 0.007; SE, 0.012; P =.548)or After adjusting for age, sex, and educational level, a higher white matter hyperintensity volume (β, −0.000001; SE, methionine to homocysteine ratio was associated with total 0.000001; P = .32) in the longitudinal analysis.

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a,b Table 3. Cross-Correlation Coefficients for Vitamin B12, RBC Folate, and Sulfur Amino Acids Methionine to Holotrans- Homocysteine Variable Folate Vitamin B12 cobalamin Homocysteine Methionine Cystathionine Cysteine Glutathione Status Supplements RBC folate NA 0.273c 0.293c −0.465c −0.002 −0.219c −0.23 0.053c 0.361c 0.195c c c c c d c c Vitamin B12 0.273 NA 0.724 −0.304 0.053 −0.030 0.051 −0.004 0.266 0.294 Holotranscobalamin 0.293c 0.724c NA −0.322c 0.072c −0.035e 0.107c 0.014 0.291c 0.326c Homocysteine −0.465c −0.304c −0.322c NA −0.038e 0.406c 0.485c −0.014 −0.792c −0.126c Methionine −0.002 0.053c 0.072c −0.038e NA 0.228c −0.044d −0.024 0.592c 0.000 Cystathionine −0.219c −0.030 −0.035e 0.406c 0.228c NA 0.311c −0.102c −0.181c −0.028 Cysteine −0.023 0.051d 0.107c 0.485c −0.044d 0.311c NA −0.065c −0.002 −0.026 Glutathione 0.053c −0.004 0.014 −0.014 −0.024 −0.102c −0.065c NA −0.002 −0.026 Methionine to 0.361c 0.266c 0.291c −0.792c 0.592c −0.181c −0.405c −0.002 NA 0.096c homocysteine ratio Supplements 0.195c 0.294c 0.326c −0.126c 0.000 −0.028 0.100c −0.026 0.096c NA Abbreviations: NA, not applicable; RBC, red blood cell. for further analyses. a Spearman rank-order correlations were used. c P < .01. b Determination of RBC folate values was routinely performed for all d P < .05. participants (available in 2570 individuals), but the additional markers were e P < .10. not routinely tested. Thus, values for other vitamins and sulfur amino acids reflect those of the 2355 participants clinically evaluated with available blood

tion of several other amino acids and SAM, which is the primary Discussion methyl donor for many biochemical reactions involved in normal brain functions, including the production of phosphatidyl- In this longitudinal population-based study of older adults, choline (important for cell membrane structure and synaptic higher levels of methionine, lower levels of tHcy, and higher function, monoaminergic neurotransmitters, and nucleic acids).7 methionine to homocysteine ratios were associated with a de- Impairment in methylation status is reportedly associated with creased risk of dementia during 6 years. Furthermore, a higher white matter damage, AD-type neuropathology, and brain atro- methionine to homocysteine ratio was associated with a de- phy,factors associated with cognitive decline and dementia.4,7,15 creased rate of brain volume loss during follow-up. The ob- A higher methionine to homocysteine ratio was associated served associations were independent of several potential con- with a decreased rate of brain volume loss, notably gray matter founders, including common sociodemographic and vascular in our study, which is consistent with findings from the experi- risk factors or markers of the transsulfuration pathway. mental studies.1,3,5,7 In contrast, an increased tHcy level may Similar to our findings, several prospective studies7,8 be associated with an increased loss of gray matter and of total reported an association between increased baseline tHcy val- brain volume.15,31 As discussed in detail elsewhere,7 increased ues and risk of incident dementia. In contrast, the associa- tHcy values may be associated with increased risk of dementia tion of methylation status with dementia risk has been inves- through several plausible mechanisms, such as the potentiation tigated only in a few case-control studies,11,25 which reported of amyloid-β generation or its neurotoxicity, formation of neu- lower values of SAM/SAH ratio (another indicator of methyla- rofibrillary tangles, or cerebrovascular pathologic processes. tion capacity) in patients with AD compared with controls. An- Although we previously reported an association between 12 other study that additionally examined the association with higher levels of vitamin B12 or holotranscobalamin and de- cerebrospinal fluid biomarkers of AD did not find any differ- creased rate of brain tissue volume loss,15 no associations with ences in the mean SAM/SAH ratio between patients with AD incident dementia or AD were found in the current study.

and controls, although decreased SAM/SAH ratios were asso- Notably, vitamin B12 and holotranscobalamin were associ- ciated with elevated cerebrospinal fluid phospho-tau values. ated only longitudinally and not cross-sectionally with brain

None of these studies directly investigated the association atrophy. Perhaps vitamin B12 status needs a longer time to in- between methionine and dementia but have considered SAM fluence brain structure, and the effects first manifest after sev- as a surrogate marker of methionine status. eral years of follow-up. This may explain the lack of associa- Methionine is an essential amino acid involved in several tion with dementia in the current study because substantial metabolic processes, such as protein synthesis and polyamine cerebral atrophy usually presents before clinical manifesta- metabolism, and decreased plasma methionine concentrations tion of dementia32,33; a longer follow-up period might have been

have been observed in a number of cardiovascular and neuro- needed to detect the association of vitamin B12 and clinical 26-29 logic conditions. Methionine status is closely associated with dementia in our population. An association between B12 or

dietary intake as well as B12 and folate status, its biosynthesis is holotranscobalamin and dementia or cognitive decline was closely associated with the transmethylation and transsulfura- reported mainly in studies5,7 with longer follow-up periods than tion pathways,30 and it serves as the precursor for the produc- our study.

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Table 4. Hazard Ratios (95% CIs) Examining the Associations of RBC Folate, Vitamin B12, and Sulfur Amino Acids Using Quartiles (With Quartile 1 as Reference) With Incident Dementia and Incident Alzheimer Diseasea

Incident Dementia Incident Alzheimer Disease Variable Quartile 2 Quartile 3 Quartile 4 Quartile 2 Quartile 3 Quartile 4 RBC Folate Multiadjusted HRb 1.03 (0.71-1.48) 0.92 (0.63-1.36) 1.01 (0.72-1.43) 0.76 (0.48-1.22) 0.75 (0.46-1.22) 0.76 (0.49-1.18) Adjusting also for APOEε4 statusc 0.90 (0.61-1.33) 0.97 (0.64-1.44) 1.06 (0.74-1.52) 0.73 (0.45-1.18) 0.77 (0.46-1.29) 0.75 (0.47-1.20)

Vitamin B12 Multiadjusted HRb 0.88 (0.60-1.31) 0.98 (0.66-1.46) 0.93 (0.62-1.40) 0.97 (0.59-1.59) 1.14 (0.69-1.89) 0.81 (0.47-1.38) Adjusting also for APOEε4 statusc 0.97 (0.65-1.46) 1.00 (0.66-1.51) 0.96 (0.63-1.45) 1.06 (0.63-1.78) 1.24 (0.74-2.06) 0.85 (0.49-1.49) Holotranscobalamin Multiadjusted HRb 0.95 (0.64-1.40) 0.74 (0.49-1.12) 0.89 (0.60-1.34) 1.08 (0.66-1.77) 0.91 (0.54-1.52) 0.84 (0.49-1.44) Adjusting also for APOEε4 statusc 1.00 (0.67-1.49) 0.74 (0.48-1.14) 0.92 (0.60-1.40) 1.09 (0.65-1.81) 0.90 (0.53-1.53) 0.87 (0.48-1.48) Homocysteine Multiadjusted HRb 1.04 (0.65-1.65) 1.50 (0.97-2.32)d 1.60 (1.01-2.55)d 1.56 (0.60-2.21) 1.90 (1.05-3.46)d 2.33 (1.26-4.30)d Adjusting also for APOEε4 statusc 1.15 (0.71-1.88) 1.52 (0.95-2.42)e 1.67 (1.02-2.72)d 1.30 (0.66-2.58)d 1.92 (1.01-3.66)d 2.42 (1.25-4.68)d Methionine Multiadjusted HRb 0.80 (0.57-1.13) 0.58 (0.39-0.86)d 0.54 (0.36-0.81)d 1.13 (0.73-1.73) 0.74 (0.45-1.22) 0.62 (0.36-1.06)e Adjusting also for APOEε4 statusc 0.81 (0.57-1.16) 0.58 (0.38-0.86)d 0.53 (0.34-0.80)d 1.14 (0.73-1.79) 0.70 (0.41-1.18) 0.61 (0.35-1.06)e Cystathionine Multiadjusted HRb 1.50 (0.93-2.42) 1.71 (1.07-2.72)c 1.37 (0.83-2.45) 1.52 (0.79-2.91) 1.94 (1.05-3.60)c 1.57 (0.82-3.02) Adjusting also for APOEε4 statusc 1.63 (0.99-2.71)d 1.84 (1.13-2.99)c 1.45 (0.86-2.44) 1.85 (0.91-3.78)d 2.37 (1.20-4.66)c 1.89 (0.93-3.86)d Cysteine Multiadjusted HRb 0.93 (0.54-1.60) 1.47 (0.89-2.42) 1.51 (0.89-2.55) 0.84 (0.41-1.71) 1.41 (0.74-2.69) 1.58 (0.81-3.07) Adjusting also for APOEε4 statusc 0.99 (0.56-1.76) 1.48 (0.87-2.53) 1.60 (0.92-2.78) 0.89 (0.42-1.88) 1.37 (0.68-2.73) 1.62 (0.80-3.27) Glutathione Multiadjusted HRb 1.07 (0.74-1.53) 0.81 (0.54-1.22) 1.17 (0.79-1.73) 1.07 (0.68-1.66) 0.65 (0.38-1.11) 1.07 (0.65-1.76) Adjusting also for APOEε4 statusc 1.10 (0.76-1.61) 0.81 (0.53-1.23) 1.14 (0.76-1.72) 1.07 (0.67-1.70) 0.71 (0.41-1.22) 1.03 (0.61-1.72) Methionine to Homocysteine Ratio Multiadjusted HRb 0.77 (0.55-1.09) 0.53 (0.35-0.80)c 0.44 (0.27-0.71)c 0.88 (0.58-1.36) 0.44 (0.25-0.77)c 0.43 (0.23-0.80)c Adjusting also for APOEε4 statusc 0.72 (0.50-1.03)d 0.51 (0.34-0.77)c 0.41 (9.25-0.67)c 0.85 (0.54-1.32) 0.44 (0.25-0.77)c 0.41 (0.21-0.80)c Cystathionine to Homocysteine Ratio Multiadjusted HRb 1.07 (0.72-1.61) 0.83 (0.55-1.28) 0.98 (0.65-1.48) 1.16 (0.70-1.91) 0.66 (0.38-1.16) 0.91 (0.54-1.52) Adjusting also for APOEε4 statusc 1.13 (0.74-1.71) 0.84 (0.54-1.30) 0.98 (0.64-1.49) 1.21 (0.72-2.04) 0.70 (0.39-1.25) 0.98 (0.58-1.68) Abbreviations: HR, hazard ratio; RBC, red blood cell. 1.35 or less, 1.36 to 1.79, 1.80 to 2.35, and 2.35 or greater; and for cystathionine a The first to fourth quartile categories were, respectively, as follows: for red to total homocysteine ratio: 0.016 or less, 0.017-0.023, 9.924 to 0.032, and blood cell folate: 82.5 ng/mL or less, 82.6 to 104.1 ng/mL, 104.02 to 138.7 greater than 0.032. b ng/mL, and greater than 138.7 ng/mL; for vitamin B12: 364.6 pg/mL or less, The multiadjusted model was adjusted for age, sex, educational level, 364.7 to 470.3 pg/mL, 470.4 to 618.1 pg/mL, and greater than 618.2 pg/mL; creatinine level, systolic blood pressure, use of vitamins, albumin level, for holotranscobalamin: 43.0 pmol/L or less, 43.1 to 61.0 pmol/L, 61.1 to 89.0 smoking status, and history of cardiovascular conditions and stroke. pmol/L, and greater than 89.0 pmol/L; for total homocysteine: 10.5 μmol/L or c Data on APOEε4 status were available in 2364 individuals from the original less, 10.6 to 12.8 μmol/L, 12.9 to 16.0 μmol/L, and greater than 16.0 μmol/L; for 2570 who had available data on RBC folate. For the rest, data on APOE-ε4 methionine: 19.5 μmol/L or less, 19.6 to 22.9 μmol/L, 23.0 to 26.8 μmol/L, and status were available in 2237 participants of the original 2355 with available greater than 26.8 μmol/L; for cystathionine: 205 nmol/L or less, 206 to 288 data on vitamin B12 and sulfur amino acid status. nmol/L, 289 to 434 nmol/L, and greater than 434 nmol/L; for cysteine: 290 d P < .05. μmol/L or less, 291 to 320 μmol/L, 321 to 358 μmol/L, and greater than 358 μmol/L; for glutathione: 2.75 μmol/L or less, 2.76 to 3.44 μmol/L, 3.45 to 4.20 e P < .10. μmol/L, and greater than 4.20; for methionine to total homocysteine ratio:

In our study, the moderate cystathionine level (the third with higher cystathionine values are at higher risk of dying of quartile), but not the highest level or cystathionine to homo- cardiovascular diseases34 and are no longer at risk of demen- cysteine ratio, was associated with an increased risk of de- tia. Notably, participants with cardiovascular conditions had mentia. Cystathionine is produced from homocysteine dur- higher cystathionine values compared with those without such ing transsulfuration, and increased cystathionine values have conditions in our study. been associated with atherosclerosis and a subsequent in- Because of the observational design of our study,a causal increase in the risk of various vascular conditions, which may terpretation of our findings cannot be made. Future studies will increase the risk of dementia.34,35 Although our findings need need to investigate in more detail possible underlying mecha- to be confirmed in other settings, a possible explanation for nisms and identify the role of the optimal methionine to homo- the lack of association in the top quartile could be that people cysteine ratio and its possible interaction with other biochemi-

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Table 5. Associations of Markers of Methylation Status and Transsulfuration Status With Brain Volumes at Baseline and Change in Brain Tissue Volumes During 6 Years

Total Brain Tissue Volune Gray Matter Volume White Matter Volume White Matter Hyperintensity Volumes β (SE) for the β (SE) for the β (SE) for the β (SE) for the Variable Multiadjusted Modela P Value Multiadjusted Modela P Value Multiadjusted Modela P Value Multiadjusted Modela P Value Methionine to Homocysteine Ratio Cross-sectionalb 0.477 (0.167) .004 0.187 (0.125) .14 0.340 (0.164) .04 −0.00002 (0.0001) .74 Longitudinal 0.038 (0.014) .007 0.034 (0.012) .003 0.007 (0.012) .55 −0.000001 (0.000001) .32 analysis Cystathionine to Homocysteine Ratio Cross-sectionalb 0.202 (0.137) .13 −0.070 (0.103) .497 0.269 (0.137) .051 0.00007 (0.00005) .16 Longitudinal −0.008 (0.012) .51 0.003 (0.010) .78 −0.004 (0.010) .65 −0.000001 (0.000005) .65 analysis a Adjusted for age, sex, educational level, creatinine level, systolic blood b For cross-sectional analysis, the sample size was 470; for longitudinal analysis, pressure, use of vitamins, albumin level, smoking status, history of the sample size was 281 for those with available follow-up magnetic resonance cardiovascular conditions, and APOE-ε4 status. imaging.

cal pathways in individuals who are at increased risk of structural tion score, 28.7 [1.8]) make our results less prone to the influ- brain changes and dementia in the context of clinical trials. High ence of reverse causality (ie, the effects of preclinical dementia

tHcy and low B12 and folate levels are surprisingly common con- on methionine to homocysteine status). ditions in older adults,5,7 and our results indicated a better me- The limitations of our study include the measurement of

thionine to homocysteine profile in participants with adequate sulfur amino acids, vitamin B12, and RBC folate at only 1 time

B12 or folate values. If the association is found to be causal, supple- point, which may underestimate their associations because of mentation with B vitamins may be effective for prevention of regression dilution.15 Selective survival may also have con- brain damage and dementia risk because of increased tHcy and tributed to an underestimation of the associations because high

impaired methylation reactions. The Homocysteine and B Vita- levels of tHcy and low levels of vitamin B12 or folate have been mins in Cognitive Impairment (VITACOG) trial has already shown associated with increased mortality in previous studies.1,6,36 that B vitamin treatment in people with mild cognitive impair- Blood samples were collected in a nonfasting state, and me- ment who have increased tHcy levels markedly slows regional thionine concentrations are known to increase postprandially.23 gray matter atrophy and cognitive decline.7,31 Further adequately Although adjusting for hours of fasting did not change any of timed and powered randomized clinical trials are needed to the associations substantially, this may not fully account for determine causation. the measurement of sulfur amino acids in a nonfasting state. Although study participants were in general healthier than non- Strengths and Limitations participants in our study, any nonresponse bias may have led The main strengths of this study are the relatively large num- to underestimation of the associations.15 ber 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 , folate, and sulfur 12 Conclusions amino acids simultaneously in association with the outcome. Although a few studies2,7 have investigated the association of Our findings suggest that interrelated but distinct processes SAM/SAH ratio or methionine status with dementia risk, none that result in elevation of serum tHcy and serum methionine had a longitudinal design. The relatively long follow-up pe- levels may be associated with brain atrophy and dementia risk riod, the comprehensive evaluation and diagnostic protocol in older adults. Thus, a better methylation status, reflected by at each examination, and recruitment of dementia-free indi- a higher methionine to homocysteine ratio, may be benefi- viduals at baseline (mean [SD] Mini-Mental State Examina- cial for the structure and functioning of the brain.

ARTICLE INFORMATION Karolinska Institutet, Stockholm, Sweden Mangialasche, Kivipelto, Fratiglioni. Accepted for Publication: April 29, 2019. (Kivipelto); Theme Aging, Karolinska University Acquisition, analysis, or interpretation of data: Hospital, Stockholm, Sweden (Kivipelto); Hooshmand, Refsum, Kalpouzos, von Arnim, Published Online: July 24, 2019. Stockholms Sjukhem, Research & Development Kareholt, Kivipelto, Fratiglioni. doi:10.1001/jamapsychiatry.2019.1694 Unit, Stockholm, Sweden (Kivipelto); Drafting of the manuscript: Hooshmand, Kivipelto, Author Affiliations: Aging Research Center, Neuroepidemiology and Ageing Research Unit, Fratiglioni. Karolinska Institute, Stockholm, Sweden School of Public Health, Imperial College London, Critical revision of the manuscript for important (Hooshmand, Kalpouzos, Mangialasche, Kåreholt, London, United Kingdom (Kivipelto); Department intellectual content: All authors. Fratiglioni); Department of Neurology, Ulm of Neurology, University of Eastern Finland, Kuopio, Statistical analysis: Hooshmand, Kalpouzos. University Hospital, Ulm, Germany (Hooshmand, Finland (Kivipelto). Obtained funding: Kivipelto, Fratiglioni. von Arnim); Department of Pharmacology, Author Contributions: Dr Hooshmand had full Administrative, technical, or material support: University of Oxford, Oxford, United Kingdom access to all the data in the study and takes Refsum, Kalpouzos. (Refsum, Smith); Institute of Nutrition, University of responsibility for the integrity of the data and the Supervision: Hooshmand, Kivipelto, Fratiglioni. Oslo, Oslo, Norway (Refsum); Division of Clinical accuracy of the data analysis. Conflict of Interest Disclosures: Dr Smith reported Geriatrics, Center for Alzheimer Research, Concept and design: Hooshmand, Refsum, Smith, receiving personal fees from P&G Health Care

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International, Aprofol AG, and Mylan Norge outside 8. Smith AD, Refsum H, Bottiglieri T, et al. One. 2015;10(8):e0134766. doi:10.1371/journal. the submitted work and having a patent to Homocysteine and dementia: an international pone.0134766 US6008221 issued, a patent to US6127370, issued, consensus statement. J Alzheimers Dis. 2018;62(2): 23. Guttormsen AB, Solheim E, Refsum H. Variation a patent to US9364497B2 issued, and a patent to 561-570. doi:10.3233/JAD-171042 in plasma cystathionine and its relation to changes PCT/GB2015/050786 pending. Dr von Arnim 9. Clarke R, Smith AD, Jobst KA, Refsum H, in plasma concentrations of homocysteine and

reported receiving personal fees from Lilly Sutton L, Ueland PM. Folate, vitamin B12, and serum methionine in healthy subjects during a 24-h Deutschland GmbH, Biogen, and Roche outside the total homocysteine levels in confirmed Alzheimer observation period. Am J Clin Nutr. 2004;79(1):76- submitted work. Dr Fratiglioni reported receiving disease. Arch Neurol. 1998;55(11):1449-1455. doi:10. 79. doi:10.1093/ajcn/79.1.76 grants from VR and Forte during the conduct of 1001/archneur.55.11.1449 24. Bleie Ø, Refsum H, Ueland PM, et al. Changes the study. No other disclosures were reported. 10. Dayon L, Guiraud SP, Corthésy J, et al. in basal and postmethionine load concentrations Funding/Support: The Swedish National Study One-carbon metabolism, cognitive impairment of total homocysteine and cystathionine after B on Aging and Care in Kungsholmen (SNAC-K) is and CSF measures of Alzheimer pathology: vitamin intervention. Am J Clin Nutr. 2004;80(3): supported by the Swedish Ministry of Health and homocysteine and beyond. Alzheimers Res Ther. 641-648. doi:10.1093/ajcn/80.3.641 Social Affairs, Stockholm County Council, and 2017;9(1):43. doi:10.1186/s13195-017-0270-x 25. Selley ML. A metabolic link between Stockholm municipality. This study was supported 11. Linnebank M, Popp J, Smulders Y, et al. S-adenosylhomocysteine and polyunsaturated fatty by Avtal om läkarutbildning och forskning grants S-adenosylmethionine is decreased in the acid metabolism in Alzheimer’s disease. Neurobiol 20130507 and 20150589, a Vetenskapsrådet grant, cerebrospinal fluid of patients with Alzheimer’s Aging. 2007;28(12):1834-1839. doi:10.1016/j. a Forte grant, Alzheimerfonden (Sweden), Center disease. Neurodegener Dis. 2010;7(6):373-378. neurobiolaging.2006.08.003 for Innovative Medicine at Karolinska Institute doi:10.1159/000309657 26. Singhal NK, Freeman E, Arning E, et al. South Campus, Knut and Alice Wallenberg 12. Popp J, Lewczuk P, Linnebank M, et al. Dysregulation of methionine metabolism in Foundation (Sweden), Stiftelsen Stockholms Homocysteine metabolism and cerebrospinal fluid multiple sclerosis. Neurochem Int. 2018;112:1-4. Sjukhem (Sweden), The Norwegian Research markers for Alzheimer’s disease. J Alzheimers Dis. doi:10.1016/j.neuint.2017.10.011 Council (Norway), Charles Wolfson Charitable Trust 2009;18(4):819-828. doi:10.3233/JAD-2009-1187 27. Dhar I, Lysne V, Seifert R, Svingen GFT, (United Kingdom), Konung Gustaf V:s och Drottning 13. Luchsinger JA, Tang MX, Shea S, Miller J, Green Ueland PM, Nygård OK. Plasma methionine and risk Victorias Frimurarestiftelse, and Fredrik och Ingrid R, Mayeux R. Plasma homocysteine levels and risk of acute myocardial infarction: Effect modification Thurings Stiftelse (Sweden). of Alzheimer disease. Neurology. 2004;62(11):1972- by established risk factors. 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