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A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients
Clinical Nutrition xxx (xxxx) xxx
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Randomized Control Trials
A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients Timothy Kwok a, *, Yuanyuan Wu a, Jenny Lee b, Ruby Lee c, Cho Yiu Yung d, Grace Choi e, Vivian Lee f, John Harrison g, Linda Lam h, Vincent Mok a a
Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong Department of Medicine, Alice Ho Mui Ming Nethersole Hospital, Taipo, Hong Kong Department of Health, Hong Kong d Department of Medicine & Geriatrics, United Christian Hospital, Kowloon, Hong Kong e Department of Occupational Therapy, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong f School of Pharmacy, The Chinese University of Hong Kong, Hong Kong g Alzheimer Center, VU Medical Center, Amsterdam, the Netherlands h Department of Psychiatry, The Chinese University of Hong Kong, Taipo Hospital, Taipo, Hong Kong b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 20 May 2019 Accepted 5 November 2019
Background & aims: Mild cognitive impairment (MCI) patients are at risk of cognitive decline, while elevated serum homocysteine is also associated with cognitive impairment. Thus, older people with MCI and hyperhomocysteinemia may be under greater risk of cognitive decline. We therefore performed a randomized trial of homocysteine-lowering by B vitamins supplementation to prevent cognitive decline in older MCI patients with elevated serum homocysteine. Methods: 279 MCI outpatients aged 65 years with serum homocysteine 10.0 mmol/L were randomly assigned to take either methylcobalamin 500 mg and folic acid 400 mg once daily, or two placebo tablets for 24 months. All subjects were followed up at 12 monthly intervals. The primary outcome was cognitive decline as defined by an increase in clinical dementia rating scale (CDR) sum of boxes (CDR_SOB). The secondary outcomes were global CDR, memory Z score, executive function Z score and Hamilton depression rating scale (HDRS) score. Results: The clinical characteristics between two groups were well matched, except that the supplement group had better executive function. The supplement effectively lowered serum homocysteine (mean 13.9 ± sd 3.5 mmol at baseline to 9.3 ± 2.4 mmol/L at month 24). At month 24, there was no significant group difference in CDR_SOB or any secondary outcomes (mean changes in CDR_SOB 0.36 versus 0.22 in supplement and placebo groups respectively). At month 12, the supplement group significantly improved in executive function and had lower HDRS score (P ¼ 0.004 and 0.012 respectively). Group difference was significant for HDRS, but borderline significant for executive function. (P ¼ 0.01; 0.06 respectively) These effects were not significant at month 24. Subgroup analysis showed that aspirin use had significant interaction with B supplements in CDR_SOB at month 24 (Beta 0.189, P ¼ 0.005). Conclusions: Vitamin B12 and folic acid supplementation did not reduce cognitive decline in older people with MCI and elevated serum homocysteine, though the cognitive decline over two years in placebo group was small. The supplement led to a significant reduction in depressive symptoms at month 12, though this effect was not sustained. Aspirin use had a negative interaction effect on cognitive functioning with B supplements. Clinical trial registration: Centre for Clinical Research and Biostatistics (CCRB) Clinical Trials Registry: CUHK_CCT00373. © 2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Folic acid Vitamin B12 Mild cognitive impairment (MCI) Elderly Randomized trial
* Corresponding author. Fax: þ852 2637 3852. E-mail addresses: [email protected] (T. Kwok), [email protected] (Y. Wu), [email protected] (J. Lee), [email protected] (R. Lee), [email protected] (C.Y. Yung), [email protected] (G. Choi), [email protected] (V. Lee), [email protected] (J. Harrison), [email protected] (L. Lam), [email protected] (V. Mok). https://doi.org/10.1016/j.clnu.2019.11.005 0261-5614/© 2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
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T. Kwok et al. / Clinical Nutrition xxx (xxxx) xxx
1. Introduction Preventing cognitive decline in older people is a public health priority. Mild cognitive impairment (MCI) is a transitional clinical state between normal aging related cognitive decline and dementia, a person with MCI is at an increased risk of developing dementia [1,2]. So far, there is no effective drug treatment to prevent cognitive decline in older people with MCI. Elevated plasma concentrations of homocysteine have been shown to be associated with poor cognitive function, cognitive decline and dementia [3,4]. High plasma homocysteine concentration has been associated with silent brain infarcts and atherosclerosis causing cognitive impairment [5,6], and activation of Nmethyl-D-aspartate receptors resulting in neuronal cell damage and hippocampal atrophy [6]. In addition, homocysteine in excess lowers hypomethylation potential, leading to impairment in synthesis of neurotransmitters, myelin, and an increase in amyloid b deposits [6]. Randomized clinical trials (RCTs) and observational studies have shown that B vitamins (folate and vitamin B12) can significantly decrease serum/plasma homocysteine concentration, especially in older people with suboptimal folate and vitamin B12 status [7]. This is because homocysteine is remethylated to methionine by methionine synthase which is a vitamin B12 and folate-dependent enzyme [7]. Randomized trials of B vitamins have shown mixed results in improving cognitive function in older people with or without dementia. A notable VITACOG trial showed that vitamin B12, folic acid and vitamin B6 was effective in slowing down the rate of brain atrophy in MCI patients, especially for subjects with plasma homocysteine 11 mmol/L [8]. There was also a mild improvement in executive function [9]. Interestingly, in subgroup analysis, aspirin use had a negative interaction effect of borderline significance on the protective effect of B vitamins against brain atrophy [8]. However, the VITACOG trial had limited sample size. Additional clinical trials are required to validate its findings. Therefore, we carried out another randomized trial of B vitamins to prevent cognitive decline in older people with MCI. In this trial, we used a lower than usual dose (400 mg) of folic acid, because recent metaanalysis suggested cancer risk may be increased by folic acid use [10]. In addition, we planned to perform subgroup analysis based on aspirin use. 2. Subjects and methods Between April 2013 and July 2016, people aged 65 years or more in the specialist medical outpatient clinics in Prince of Wales Hospital (a publicly funded teaching hospital in Hong Kong) were screened for cognitive impairment by research assistants (RAs) by the Montreal cognitive assessment (MoCA) test (Hong Kong version), after written informed consent. Those who had MoCA score lower than 22, which suggested mild cognitive impairment (MCI) [11], had fasting blood taken for serum homocysteine analysis (see below). Those with serum homocysteine less than 10 mmol/L were excluded because a previous randomized trial showed negative results of B vitamin supplementation in subjects with homocysteine less than 10 mmol/L [12], while another clinical trial (VITACOG) suggested that, B vitamins were effective in slowing down the rate of brain atrophy only in those with plasma homocysteine 11 mmol/L, [8]. Those with elevated serum homocysteine (10 mmol/L) were further clinically assessed in research clinic by a trained geriatrician who took a detailed history of cognitive impairment and coexisting illnesses, and also performed a neurological examination. Clinical diagnosis of MCI was made according to clinical criteria by
Petersen and Morris [13]. Those with dementia or peripheral neuropathy, or those with clinical depression, renal failure, anemia, disabling stroke and those who were receiving centrally acting medications or B vitamins supplementation, as well as those who did not have a reliable caregiver or a family member who was able to regularly inform on cognitive functioning (at least one personal contact each week) were excluded from this study. The trial was approved by the medical ethics committee of Chinese University of Hong Kong and New Territories East Cluster of Hospital Authority of Hong Kong, and was registered at the Clinical Trials Registry of the Centre for Clinical Research and Biostatistics (CCRB) (CUHK_CCT00373). After obtaining another written consent from the eligible subjects, the following tests were administered by a trained RA: 1. Demographic and clinical information including level of education, medical diagnoses and drug history. 2. Chinese version of the Mini-Mental State Examination (MMSE) e this Chinese version has been validated locally. In a previous local study, the optimal cutoff values were 22 or below for people with over 2 years of schooling, 20 for people with 1e2 years of schooling, and 18 for illiterate people [14]. 3. Clinical Dementia Rating (CDR) Scale - It was obtained from the subjects and the informants by a semi-structured interview, covering six domains of functioning: memory, orientation, judgment and problem solving, community affairs, home and hobbies, and personal care. In addition to a patient interview, a caregiver who could have personal contact regularly with the subject was asked to accomplish the interview CDR rating ranges from 0 (refers to normal) to 3 (refers to severe dementia). Its Chinese version has also been validated locally [15]. The global score, determined by an algorithm, ranges from 0 to 3, 0.5 indicating an intermediate state between normal cognitive function and dementia, labeled as ‘Questionable dementia’, 1 or more indicating clinical dementia. The CDR sum of boxes score ranging from 0 to 18 was obtained by summing each of the domain box scores. All RAs administering CDR had accomplished the online training courses for CDR administration. 4. Neurocognitive Test Battery (NTB) - We have compiled a small battery of neurocognitive tests which have been successfully used in drug trials of Alzheimer's Disease (AD), and are more sensitive to changes in early AD [16]. Some of them have been computerized and administered by a touch-screen portable computer. They have been shown to have high reproducibility and sensitivity, and are neutral to practice, education level and cultural differences [17]. The tests included International Shopping List Test (ISLT) [18] and Continuous Paired Associates Learning (CPAL, touch screen computer assisted) for memory, and Controlled Oral Word Association Test (COWAT) and Category Fluency Test (CFT) for executive function. 5. Based on the mean value and standard deviation (SD) of baseline data of all subjects, the z-scores were calculated for each of the individual test scores, with higher scores indicating better performance. For CPAL where higher scores representing poorer performance, the sign was reversed when doing the calculation. The memory and executive domain z-scores were calculated based on the average of the two tests in each domain. For subjects who could not complete a test satisfactorily, the results were regarded as missing. 6. Hamilton Depression Rating Scale (HDRS) - It is widely used to measure severity of depressive symptoms in clinical trials. It has 17 items, rating the severity of symptoms such as low mood, insomnia, agitation, anxiety and weight loss. Its Chinese version has been validated [19].
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
T. Kwok et al. / Clinical Nutrition xxx (xxxx) xxx
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7. Fasting blood was taken for serum folate, active vitamin B12 (holotranscobalamin), creatinine, complete blood count and thyroid function test. The blood samples were stored in an icebox and transported to the laboratory of the Department of Medicine & Therapeutics, Prince of Wales Hospital (PWH) within 2 h. The serum was then separated and kept under 80 C. The frozen samples were later thawed and analyzed for total L-homocysteine by a chemiluminescent microparticle immunoassay with total CV <7% (ARCHITECT Homocysteine assay) at a local accredited chemical pathology laboratory. The other biochemical analysis was performed at the Chemical Pathology Department of PWH. Active vitamin B12 and folate were measured by immunoassays. Active B12 24 pmol/L detected vitamin B12 deficiency with a specificity of 95%. Reference range of serum folate was 8.8e60.8 nmol/L Creatinine was measured by spectrophotometry. Those with serum homocysteine <10 mmol/L, active vitamin B12 < 28 pmol/L, folate <9.5 nmol/L, creatinine >200 mmol/L and hypothyroidism were excluded from the trial.
outcomes was performed using multiple linear regression models by multiplying the variable aspirin use (or not) with group assignment, with adjustment for age, sex, education level and baseline values. All statistical tests were two-sided and p-value < 0.05 was considered statistically significant for primary outcome. For secondary cognitive or mood outcomes, only p-value 0.01 was considered significant because of multiple comparisons.
The eligible subjects were randomly allocated to supplement and placebo group according to a randomization table compiled by the staff at the Clinical Research Pharmacy, who was blinded to subjects’ identity. The pharmacist did not have any contact or previous knowledge of the enrolled subjects. The pharmacist packaged the trial tablets (B vitamins or placebo) for each month into plain bottles labeled with the name and number of according subject. The pharmacist concealed the allocation of supplement and placebo group from both the investigators and RAs participating in this clinical trial. The subjects, RAs and clinicians were all kept blinded to the group assignment. The supplement group subjects took two active tablets (methylcobalamin 500 mg and folic acid 400 mg respectively) once daily for 24 months, and the placebo group subjects took two similar looking placebo tablets once daily. Active and identical placebo tablets were both placed in medicine bottles. The finished products complied with the microbiological standard in international pharmacopoeia by a testing laboratory. The drug supply was replenished once every three months and a pill count were performed by RA. All subjects were reviewed by a geriatrician in research clinic at twelve monthly intervals for 24 months. At each follow-up visit, adverse events were enquired; medication and new diagnosis were recorded. CDR, NTB and HDRS were repeated by the trained RAs as in baseline. Fasting blood were taken at month 12 and 24 for serum homocysteine, folate and active vitamin B12. All subjects were allowed to take any medication, except vitamin B12, B6 and folic acid. The use of vitamins was specifically enquired at each followup visit. All subjects who dropped out for whatever reasons were invited to continue with the month 12 and 24 follow-up visits. If the subjects were clinically diagnosed to have developed dementia at follow-up, use of AD drugs was allowed. The primary outcome was CDR_SOB. The secondary outcomes were CDR global score, memory Z score, executive function Z score, HDRS score and homocysteine.
Between April 2013 and July 2016, a total of 975 subjects aged 65 years or more were screened for cognitive impairment by the Chinese MoCA test, and 352 subjects (36.1%) with MCI and elevated levels of serum homocysteine were eligible for the randomized trial. Out of these, 279 subjects (79.3%) participated in the trial. The numbers of subjects screened, randomized and followed-up at different time points (month 12, 24) were summarized in Fig. 1. 138 subjects (supplement group) were randomly assigned to take methylcobalamin 500 mg and folic acid 400 mg once daily, and 141 subjects (placebo group) took two placebo tablets once daily. The participation rates at month 12 and month 24 were 88.2%, 85.3% respectively. The reasons for dropout included: withdrew consent (N ¼ 35), death (N ¼ 3) and terminal illness (N ¼ 3). The pill count indicated that the median percentages of trial tablets taken were 97% or more in both trial groups at both follow-up's. The baseline clinical characteristics and cognitive scores of the participants were shown in Table 1. The supplement and placebo group subjects were well matched except that the supplement group had better executive function (average z score 0.13 versus 0.13 in placebo group), and they were slightly younger (76.9 vs. 78.0). There were no significant differences in cognitive function and baseline characteristics between aspirin users and non-users, except that aspirin users had significantly higher serum creatinine (mean 94.6 versus 87.8 mmol/L), lower low density lipoprotein (1.98 vs 2.75 mmol/L), higher prevalence of diabetes mellitus (43.8% vs 27.4%), hypertension (76.6% vs 61.9%), stroke (20.3% vs 2.3%) and statin use (82.8% vs 26.5%). The serum homocysteine, folate and vitamin B12 before and after intervention were shown in Table 2. The supplement lowered serum homocysteine significantly (from 13.83 ± 3.41 at baseline to 9.37 ± 2.21 at month 12, and 9.32 ± 2.39 at month 24 respectively). The changes in cognitive test scores and HDRS at month 12 and 24 were shown in Table 3. Three placebo group subjects and three supplement group subjects took AD drugs for more than three months before either follow-up. Because they were small in number and their exclusion did not alter the results significantly, they were included in the analysis. The mean changes in CDR_SOB over 24 months were 0.36 (95%CI 0.15 to 0.57) versus 0.22 (0.05e0.39) in supplement and placebo groups respectively. There was no significant group difference in CDR_SOB, memory and executive Z scores at month 24. At month 12, the supplement group had a significant increase in executive function Z score and lower HDRS score from baseline (P ¼ 0.004; 0.012 respectively). The group differences were significant in HDRS (P ¼ 0.010), but not in executive function (P ¼ 0.06). These differences were not significant at month 24. The
2.1. Statistical analysis Intention-to-treat analyses were adopted. Paired t-test was used to examine the effects of B vitamin supplement or placebo at follow-up's, except that Wilcoxon signed rank test was used for HDRS as the data was skewed. The group difference in the changes in CDR_SOB and CDR global score was compared by Student t test and Chi-square test respectively. The interaction effects of concomitant use of aspirin on the effect of B vitamins supplement in cognitive functioning and secondary cognitive or mood
2.2. Sample size calculation In a local prospective clinical trial of older people with diabetes mellitus [20], among 50 control group subjects with CDR global score of 0.5 (questionable dementia), the average change in CDR_SOB at 27 months was 1.42 (SD 0.92). Based on this, a group sample size of 105 had 80% power to detect a 25% decrease in CDR SOB. Assuming a dropout rate of 20%, the group sample size required is 126. 3. Results
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
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T. Kwok et al. / Clinical Nutrition xxx (xxxx) xxx
975 screened for eligibility 696 excluded Reasons: Refusal 133; No carer 13; Memory well (high MMSE score) 30; Vascular cognitive impairment 1; Dementia 19; Depression 12; Parkinson’s disease 1; Creatinine > 150 5; Homocysteine < 10 315; MoCA >= 26 or too low 105; B12 deficiency 13; On vitamin B 30; Hypothyroidism 6; Having CA 1; Join other study 5; Too frail 4; Eye problem 2; Others 1
Randomized (N=279)
Placebo (N=141)
Active (N=138) Dropout: 17 Patient withdraw: 17
12 Months (N=121)
Dropout: 16 Patient withdraw: 13; Died: 2; Terminal illness: 1
12 Months (N=125)
Dropout: 3 Patient withdraw: 1; Died: 1; Terminal illness: 1
24 Months (N=118)
Dropout: 5 Patient withdraw: 4; Terminal illness: 1
24 Months (N=120) Fig. 1. Consort diagram of randomization.
changes in CDR global score were shown in Table 4. There was no significant group difference in the overall changes or an increase in CDR global score at both follow-up's. But supplement group subjects were more likely to have improved CDR global score at month 12 (P ¼ 0.027, Chi-square test). On subgroup analysis, supplement group had significantly higher CDR_SOB at month 12 and 24 among aspirin users after adjustment for confounders and baseline values (Beta ¼ 0.174, 0.292, P ¼ 0.022, 0.013 at month 12 and 24 respectively). The interaction effect of aspirin on CDR_SOB with supplement was significant at month 24 (beta 0.189; P ¼ 0.005). At month 12 only, supplement group had significantly better executive function among aspirin users, (Beta 0.217; P ¼ 0.007) but not in non aspirin users. This interaction effect was borderline significant (P ¼ 0.040). Post hoc subgroup analysis was also performed in those with higher serum homocysteine (15 mmol/L), lowest quartile of serum active vitamin B12 (58.5 pmol/L) and lowest quartile serum folate (21.9 nmol/L) at baseline. No significant interaction effect was observed in any of the cognitive or
mood outcomes at month 12 or 24, except that the supplement group had significantly lower HDRS score at month 24 among those with low baseline serum active vitamin B12 (interaction effect: Beta ¼ 0.191; P ¼ 0.018). 4. Discussion This trial showed that vitamin B12 and folic acid supplement did not significantly attenuate cognitive decline in older people with MCI over two years. But the observed cognitive decline was small. There was significant reduction in depressive symptoms at month 12 but this was not sustained at month 24. The trial groups were well matched except that subjects in the intervention group had better executive function, probably because these subjects tended to be younger. Compared to MCI subjects in previous trials [9,12], our subjects had relatively high vascular burden as reflected by the prevalence of diabetes mellitus (DM), hypertension, and statin and aspirin use. The education level of the subjects was generally low, which is consistent with that of the
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
T. Kwok et al. / Clinical Nutrition xxx (xxxx) xxx Table 1 Clinical characteristics of trial subjects at baseline. Placebo (N ¼ 141)
Supplement (N ¼ 138)
Number (%)
Number (%)
Female DM HBP CVA Aspirin use Statin use CDR ¼ 0 Education < 3 years
62 (43.9%) 43 (30.5%) 88 (62.4%) 9 (6.4%) 33 (23.4%) 58 (41.1%) 20 (14.2%) 38 (27.0%)
51 (36.9%) 44 (31.9%) 94 (68.1%) 9 (6.5%) 31 (22.5%) 54 (39.1%) 19 (13.8%) 26 (18.8%)
Mean (SD)
Mean (SD)
P
Age (yrs) BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) Glucose fasting (mmol/l) Creatinine (umol/L) Triglycerides (g/L) Total cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Non-HDL (mmol/L) MMSE (max 30) HDRS NTBa Memory z-score Executive function z-score
78.0 (5.3) 24.8 (3.2) 139.4 (16.6) 79.0 (8.3) 6.03 (1.63)
76.9 (5.4) 24.6 (3.4) 139.6 (16.0) 79.2 (9.2) 6.05 (1.51)
0.080 0.724 0.908 0.852 0.889
90.0 (24.3) 1.35 (0.73) 4.62 (1.04)
88.6 (20.5) 1.42 (1.30) 4.73 (1.04)
0.615 0.560 0.360
1.51 2.55 3.18 25.7 2.94
1.56 2.60 3.20 26.0 2.92
(0.47) (0.86) (0.93) (3.1) (3.01)
0.412 0.635 0.858 0.354 0.623
0.04 (0.88) 0.13 (0.07)
0.436 0.011
(0.44) (0.86) (0.92) (3.0) (3.03)
0.04 (0.77) 0.13 (0.07)
P
0.233 0.802 0.317 0.962 0.852 0.733 0.588 0.107
Abbreviations: DM - diabetes mellitus; HBP - high blood pressure; CVA e cerebrovascular accident; MMSE - Mini mental state examination; LDL - Low density lipoprotein; HDL - high density lipoprotein; NTB - Neurocognitive tests; CDR Clinical Dementia Rating scale; HDRS - Hamilton depression rating scale. a z-scores as compared with mean of all subjects; higher score indicating better performance.
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current cohort of older population in Hong Kong. Their average baseline serum homocysteine was more above 13 mmol/L because of the exclusion of subjects with low serum homocysteine, and the lack of local folate supplementation of flour. The compliance of the trial tablets was satisfactory, and over 85% of the subjects completed the trial. The supplements were effective in lowering serum homocysteine. The changes in CDR_SOB were however less than expected in the placebo group, thus limiting the power of the trial in detecting an effect from B vitamins supplementation. Nevertheless, we observed a borderline trend of improvement in executive function at month 12. This is consistent with the results of a recent meta-analysis of a small number of randomized trials of B vitamins in older people with MCI [21]. Unfortunately, the improvement was not sustained at month 24. This may be explained by correction of subclinical vitamin B12 or folate deficiency. However, higher serum homocysteine or lower serum vitamin B12 or folate at baseline showed no significant interaction effect on response in cognitive function. The supplement group had significant reduction in depressive symptoms at month 12, even though subjects with clinical depression were excluded at baseline. There is evidence that folate deficiency is associated with depression, and folic acid supplement is effective in treating depression [22,23], though the doses used in previous trials have been suggested to be 2 mg once daily. Few of the trials have been more than one year. This trial suggested that lower doses of folic acid may also be effective in reducing depressive symptoms, though the effect may not last more than one year. The relatively importance of vitamin B12 and folate status in depressive symptoms has been inconclusive [23e26]. It is noteworthy that B supplements had significant effects on depressive symptoms at month 24 only in those with lower baseline serum active vitamin B12. More trials on the potential effect of vitamin B12 with or without folic acid supplement on depression in those with mild vitamin B12 deficiency are warranted.
Table 2 Serum homocysteine and B vitamin concentrations during trial.
Month Number Homocysteine (mmol/L) Folate (nmol/L) Active vit. B12 (pmol/L)
Placebo
Supplement
Mean (SD)
Mean (SD)
0 141 13.8 (3.0) 29.4 (8.6) 85.8 (32.9)
12 125 13.5 (3.6) 28.9 (8.3) 86.8 (31.8)
24 120 14.0 (4.1) 27.8 (9.8) 85.8 (34.0)
0 138 13.8 (3.4) 27.8 (8.0) 85.5 (29.8)
12 121 9.4 (2.2)* 43.3 (4.6)* 125.2 (9.9)*
24 118 9.3 (2.4)* 48.0 (12.6)* 123.6 (13.6)*
*P < 0.0001, paired t test.
Table 3 Mean change from baseline in cognitive test and depressive symptom scores at follow-upb. Month
CDR_SOB Executive Z Memory Z HDRSa
12 24 12 24 12 24 12 24
Placebo
Supplement
Difference due to supplement
N
Mean change (95% CI)
N
Mean change (95% CI)
(95% CI)
126 122 126 122 122 112 126 122
0.05 (0.07, 0.16) 0.22 (0.05, 0.39)y 0.03 (0.08, 0.13) 0.03 (0.08, 0.14) 0.06 (0.05, 0.16) 0.01 (0.15, 0.14) 0.30 (0.23, 0.84) 0.24 (0.30, 0.77)
123 119 123 119 119 110 123 119
0.13 (0.01, 0.26) 0.36 (0.15, 0.57)z 0.18 (0.06, 0.30)z 0.00 (0.12, 0.12) 0.03 (0.09, 0.14) 0.00 (0.13, 0.12) 0.59 (1.01, 0.18)y 0.08 (0.63, 0.46)
0.08 (0.25, 0.10) 0.14 (0.40, 0.13) 0.15 (0.01, 0.31) 0.03 (0.19, 0.13) 0.03 (0.19, 0.13) 0.00 (0.19, 0.19) 0.90 (1.57, 0.22)z 0.32 (1.08, 0.44)
Abbreviations: CDR_SOB e Clinical dementia rating scale sum of boxes; HDRS e Hamilton depression rating scale; Z e Z score. yp < 0.05; zp 0.01. a Wilcoxon signed rank test used to compare baseline and follow-up values. b Follow-up value minus baseline value.
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
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significant negative interaction effect between aspirin and B vitamins in cognitive functioning.
Table 4 Changes in CDR global score.
DCDR 0.5 0.0 0.5 Total
Placebo
Supplement
Month 12
Month 24
Month 12
Month 24
3 (2.4%) 113 (91.1%) 8 (6.5%) 124
9 (7.9%) 96 (84.2%) 9 (7.9%) 114
11 (8.9%)* 105 (85.4%) 7 (5.7%) 123
17 (14.7%) 88 (75.9%) 11 (9.5%) 116
Conflict of interest The authors declare no conflict of interest. Acknowledgments
DCDR e clinical dementia rating global score at follow-up minus CDR at baseline.
* more likely to improve in CDR global score P ¼ 0.027, Chi-square test.
This study was supported by the General Research Fund of the Research Grants Council of Hong Kong (RGC Reference 466612).
The subgroup analysis of aspirin use was motivated by an incidental finding of a negative interaction effect of aspirin use on the protective effect of B vitamins against brain atrophy among MCI subjects in the VITACOG trial [8]. Our results showed complex interactions between aspirin and B vitamins. On one hand, aspirin had negative interaction with B vitamins in cognitive functioning at month 24. On the other hand, aspirin had borderline positive interaction effect on executive function at month 12. The negative interaction effect of aspirin on CDR_SOB was consistent with the negative interaction effect of aspirin on brain atrophy rate in the VITACOG trial. A possible explanation is that aspirin was interfering with the cerebral effects of folic acid. Folic acid is a synthetic form of folate and requires metabolism into dihydrofolate and then to tetrahydrofolate by dihydrofolate reductase (DHFR). Because DHFR expression in liver is low and variable, there have been concerns about the potential harm of unmetabolized folic acid. Older people in Ireland where there was no folic acid fortification of flour policy have been found to have higher fasting serum concentrations of unmetabolized folic acid with dietary folic acid supplement alone [27]. The concern is the unmetabolized folic acid may impair the active transport of active folates into the brain, though so far the harm of unmetabolized folic acid is yet to be demonstrated. Aspirin has been shown to inhibit the expression of a protein “E2F-1” which regulates DHFR expression [28]. It is therefore possible that aspirin inhibits the upregulation of DHFR which normally occurs with folic acid supplementation [29], resulting in higher serum concentrations of unmetabolized folic acid. A cross sectional study found that individuals with genetically defective DHFR had cognitive impairment when their serum folate concentrations were high [30]. It is intriguing to observe that the use of aspirin had a borderline positive interaction effect on the improvement of executive function at month 12. One of the proposed mechanisms whereby homocysteine contributes to atherosclerosis is the activation of platelets in response to damaged endothelium [31]. It is plausible that homocysteine lowering by B vitamins may work in synergy with aspirin in inhibiting platelet activation in those with vascular diseases. As executive function is primarily impaired in vascular cognitive impairment [32], it is plausible the combined effects of B vitamins and aspirin may enhance executive function in those with cerebrovascular diseases. But why this beneficial effect waned at month 24 remains to be explained. This trial had limitations. The sample size was insufficient to detect a small difference in cognitive function. The high vascular burden and the associated drug use may confound the outcomes of B vitamin supplementation. There was lack of brain imaging to measure brain atrophy rate, which is a more sensitive marker of AD. In conclusion, we did not observe significant effects of vitamin B12 and folic acid supplementation in attenuating cognitive decline in older people with mild cognitive impairment over a two year period, though the trial was underpowered because of limited cognitive decline in placebo group. On the other hand, B vitamins had a short term beneficial effect in mood. Lastly, we found a
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despite the lack of a mandatory fortification program. Am J Clin Nutr 2012;96: 613e21. Yan K-H, Lee L-M, Hsieh M-C, Yan M-D, Yao C-J, Chang P-Y, et al. Aspirin antagonizes the cytotoxic effect of methotrexate in lung cancer cells. Oncol Rep 2013;30:1497e505. Pentieva K, Selhub J, Paul L, Molloy AM, McNulty B, Ward M, et al. Evidence from a randomized trial that exposure to supplemental folic acid at recommended levels during pregnancy does not lead to increased unmetabolized folic acid concentrations in maternal or cord blood. J Nutr 2016;146: 494e500. Philip D, Buch A, Moorthy D, Scott TM, Parnell LD, Lai C-Q, et al. Dihydrofolate reductase 19-bp deletion polymorphism modifies the association of folate status with memory in a cross-sectional multi-ethnic study of adults. Am J Clin Nutr 2015;102:1279e88. E, Plicner D, Zielinski L, Tracz W. Elevated total homocysUndas A, Ste˛ pien teine is associated with increased platelet activation at the site of microvascular injury: effects of folic acid administration. J Thromb Haemost 2007;5: 1070e2. Graham N, Emery T, Hodges J. Distinctive cognitive profiles in Alzheimer's disease and subcortical vascular dementia. J Neurol Neurosurg Psychiatry 2004;75:61e71.
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Contents lists available at ScienceDirect
Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Randomized Control Trials
A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients Timothy Kwok a, *, Yuanyuan Wu a, Jenny Lee b, Ruby Lee c, Cho Yiu Yung d, Grace Choi e, Vivian Lee f, John Harrison g, Linda Lam h, Vincent Mok a a
Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong Department of Medicine, Alice Ho Mui Ming Nethersole Hospital, Taipo, Hong Kong Department of Health, Hong Kong d Department of Medicine & Geriatrics, United Christian Hospital, Kowloon, Hong Kong e Department of Occupational Therapy, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong f School of Pharmacy, The Chinese University of Hong Kong, Hong Kong g Alzheimer Center, VU Medical Center, Amsterdam, the Netherlands h Department of Psychiatry, The Chinese University of Hong Kong, Taipo Hospital, Taipo, Hong Kong b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 20 May 2019 Accepted 5 November 2019
Background & aims: Mild cognitive impairment (MCI) patients are at risk of cognitive decline, while elevated serum homocysteine is also associated with cognitive impairment. Thus, older people with MCI and hyperhomocysteinemia may be under greater risk of cognitive decline. We therefore performed a randomized trial of homocysteine-lowering by B vitamins supplementation to prevent cognitive decline in older MCI patients with elevated serum homocysteine. Methods: 279 MCI outpatients aged 65 years with serum homocysteine 10.0 mmol/L were randomly assigned to take either methylcobalamin 500 mg and folic acid 400 mg once daily, or two placebo tablets for 24 months. All subjects were followed up at 12 monthly intervals. The primary outcome was cognitive decline as defined by an increase in clinical dementia rating scale (CDR) sum of boxes (CDR_SOB). The secondary outcomes were global CDR, memory Z score, executive function Z score and Hamilton depression rating scale (HDRS) score. Results: The clinical characteristics between two groups were well matched, except that the supplement group had better executive function. The supplement effectively lowered serum homocysteine (mean 13.9 ± sd 3.5 mmol at baseline to 9.3 ± 2.4 mmol/L at month 24). At month 24, there was no significant group difference in CDR_SOB or any secondary outcomes (mean changes in CDR_SOB 0.36 versus 0.22 in supplement and placebo groups respectively). At month 12, the supplement group significantly improved in executive function and had lower HDRS score (P ¼ 0.004 and 0.012 respectively). Group difference was significant for HDRS, but borderline significant for executive function. (P ¼ 0.01; 0.06 respectively) These effects were not significant at month 24. Subgroup analysis showed that aspirin use had significant interaction with B supplements in CDR_SOB at month 24 (Beta 0.189, P ¼ 0.005). Conclusions: Vitamin B12 and folic acid supplementation did not reduce cognitive decline in older people with MCI and elevated serum homocysteine, though the cognitive decline over two years in placebo group was small. The supplement led to a significant reduction in depressive symptoms at month 12, though this effect was not sustained. Aspirin use had a negative interaction effect on cognitive functioning with B supplements. Clinical trial registration: Centre for Clinical Research and Biostatistics (CCRB) Clinical Trials Registry: CUHK_CCT00373. © 2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Folic acid Vitamin B12 Mild cognitive impairment (MCI) Elderly Randomized trial
* Corresponding author. Fax: þ852 2637 3852. E-mail addresses: [email protected] (T. Kwok), [email protected] (Y. Wu), [email protected] (J. Lee), [email protected] (R. Lee), [email protected] (C.Y. Yung), [email protected] (G. Choi), [email protected] (V. Lee), [email protected] (J. Harrison), [email protected] (L. Lam), [email protected] (V. Mok). https://doi.org/10.1016/j.clnu.2019.11.005 0261-5614/© 2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
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1. Introduction Preventing cognitive decline in older people is a public health priority. Mild cognitive impairment (MCI) is a transitional clinical state between normal aging related cognitive decline and dementia, a person with MCI is at an increased risk of developing dementia [1,2]. So far, there is no effective drug treatment to prevent cognitive decline in older people with MCI. Elevated plasma concentrations of homocysteine have been shown to be associated with poor cognitive function, cognitive decline and dementia [3,4]. High plasma homocysteine concentration has been associated with silent brain infarcts and atherosclerosis causing cognitive impairment [5,6], and activation of Nmethyl-D-aspartate receptors resulting in neuronal cell damage and hippocampal atrophy [6]. In addition, homocysteine in excess lowers hypomethylation potential, leading to impairment in synthesis of neurotransmitters, myelin, and an increase in amyloid b deposits [6]. Randomized clinical trials (RCTs) and observational studies have shown that B vitamins (folate and vitamin B12) can significantly decrease serum/plasma homocysteine concentration, especially in older people with suboptimal folate and vitamin B12 status [7]. This is because homocysteine is remethylated to methionine by methionine synthase which is a vitamin B12 and folate-dependent enzyme [7]. Randomized trials of B vitamins have shown mixed results in improving cognitive function in older people with or without dementia. A notable VITACOG trial showed that vitamin B12, folic acid and vitamin B6 was effective in slowing down the rate of brain atrophy in MCI patients, especially for subjects with plasma homocysteine 11 mmol/L [8]. There was also a mild improvement in executive function [9]. Interestingly, in subgroup analysis, aspirin use had a negative interaction effect of borderline significance on the protective effect of B vitamins against brain atrophy [8]. However, the VITACOG trial had limited sample size. Additional clinical trials are required to validate its findings. Therefore, we carried out another randomized trial of B vitamins to prevent cognitive decline in older people with MCI. In this trial, we used a lower than usual dose (400 mg) of folic acid, because recent metaanalysis suggested cancer risk may be increased by folic acid use [10]. In addition, we planned to perform subgroup analysis based on aspirin use. 2. Subjects and methods Between April 2013 and July 2016, people aged 65 years or more in the specialist medical outpatient clinics in Prince of Wales Hospital (a publicly funded teaching hospital in Hong Kong) were screened for cognitive impairment by research assistants (RAs) by the Montreal cognitive assessment (MoCA) test (Hong Kong version), after written informed consent. Those who had MoCA score lower than 22, which suggested mild cognitive impairment (MCI) [11], had fasting blood taken for serum homocysteine analysis (see below). Those with serum homocysteine less than 10 mmol/L were excluded because a previous randomized trial showed negative results of B vitamin supplementation in subjects with homocysteine less than 10 mmol/L [12], while another clinical trial (VITACOG) suggested that, B vitamins were effective in slowing down the rate of brain atrophy only in those with plasma homocysteine 11 mmol/L, [8]. Those with elevated serum homocysteine (10 mmol/L) were further clinically assessed in research clinic by a trained geriatrician who took a detailed history of cognitive impairment and coexisting illnesses, and also performed a neurological examination. Clinical diagnosis of MCI was made according to clinical criteria by
Petersen and Morris [13]. Those with dementia or peripheral neuropathy, or those with clinical depression, renal failure, anemia, disabling stroke and those who were receiving centrally acting medications or B vitamins supplementation, as well as those who did not have a reliable caregiver or a family member who was able to regularly inform on cognitive functioning (at least one personal contact each week) were excluded from this study. The trial was approved by the medical ethics committee of Chinese University of Hong Kong and New Territories East Cluster of Hospital Authority of Hong Kong, and was registered at the Clinical Trials Registry of the Centre for Clinical Research and Biostatistics (CCRB) (CUHK_CCT00373). After obtaining another written consent from the eligible subjects, the following tests were administered by a trained RA: 1. Demographic and clinical information including level of education, medical diagnoses and drug history. 2. Chinese version of the Mini-Mental State Examination (MMSE) e this Chinese version has been validated locally. In a previous local study, the optimal cutoff values were 22 or below for people with over 2 years of schooling, 20 for people with 1e2 years of schooling, and 18 for illiterate people [14]. 3. Clinical Dementia Rating (CDR) Scale - It was obtained from the subjects and the informants by a semi-structured interview, covering six domains of functioning: memory, orientation, judgment and problem solving, community affairs, home and hobbies, and personal care. In addition to a patient interview, a caregiver who could have personal contact regularly with the subject was asked to accomplish the interview CDR rating ranges from 0 (refers to normal) to 3 (refers to severe dementia). Its Chinese version has also been validated locally [15]. The global score, determined by an algorithm, ranges from 0 to 3, 0.5 indicating an intermediate state between normal cognitive function and dementia, labeled as ‘Questionable dementia’, 1 or more indicating clinical dementia. The CDR sum of boxes score ranging from 0 to 18 was obtained by summing each of the domain box scores. All RAs administering CDR had accomplished the online training courses for CDR administration. 4. Neurocognitive Test Battery (NTB) - We have compiled a small battery of neurocognitive tests which have been successfully used in drug trials of Alzheimer's Disease (AD), and are more sensitive to changes in early AD [16]. Some of them have been computerized and administered by a touch-screen portable computer. They have been shown to have high reproducibility and sensitivity, and are neutral to practice, education level and cultural differences [17]. The tests included International Shopping List Test (ISLT) [18] and Continuous Paired Associates Learning (CPAL, touch screen computer assisted) for memory, and Controlled Oral Word Association Test (COWAT) and Category Fluency Test (CFT) for executive function. 5. Based on the mean value and standard deviation (SD) of baseline data of all subjects, the z-scores were calculated for each of the individual test scores, with higher scores indicating better performance. For CPAL where higher scores representing poorer performance, the sign was reversed when doing the calculation. The memory and executive domain z-scores were calculated based on the average of the two tests in each domain. For subjects who could not complete a test satisfactorily, the results were regarded as missing. 6. Hamilton Depression Rating Scale (HDRS) - It is widely used to measure severity of depressive symptoms in clinical trials. It has 17 items, rating the severity of symptoms such as low mood, insomnia, agitation, anxiety and weight loss. Its Chinese version has been validated [19].
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
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7. Fasting blood was taken for serum folate, active vitamin B12 (holotranscobalamin), creatinine, complete blood count and thyroid function test. The blood samples were stored in an icebox and transported to the laboratory of the Department of Medicine & Therapeutics, Prince of Wales Hospital (PWH) within 2 h. The serum was then separated and kept under 80 C. The frozen samples were later thawed and analyzed for total L-homocysteine by a chemiluminescent microparticle immunoassay with total CV <7% (ARCHITECT Homocysteine assay) at a local accredited chemical pathology laboratory. The other biochemical analysis was performed at the Chemical Pathology Department of PWH. Active vitamin B12 and folate were measured by immunoassays. Active B12 24 pmol/L detected vitamin B12 deficiency with a specificity of 95%. Reference range of serum folate was 8.8e60.8 nmol/L Creatinine was measured by spectrophotometry. Those with serum homocysteine <10 mmol/L, active vitamin B12 < 28 pmol/L, folate <9.5 nmol/L, creatinine >200 mmol/L and hypothyroidism were excluded from the trial.
outcomes was performed using multiple linear regression models by multiplying the variable aspirin use (or not) with group assignment, with adjustment for age, sex, education level and baseline values. All statistical tests were two-sided and p-value < 0.05 was considered statistically significant for primary outcome. For secondary cognitive or mood outcomes, only p-value 0.01 was considered significant because of multiple comparisons.
The eligible subjects were randomly allocated to supplement and placebo group according to a randomization table compiled by the staff at the Clinical Research Pharmacy, who was blinded to subjects’ identity. The pharmacist did not have any contact or previous knowledge of the enrolled subjects. The pharmacist packaged the trial tablets (B vitamins or placebo) for each month into plain bottles labeled with the name and number of according subject. The pharmacist concealed the allocation of supplement and placebo group from both the investigators and RAs participating in this clinical trial. The subjects, RAs and clinicians were all kept blinded to the group assignment. The supplement group subjects took two active tablets (methylcobalamin 500 mg and folic acid 400 mg respectively) once daily for 24 months, and the placebo group subjects took two similar looking placebo tablets once daily. Active and identical placebo tablets were both placed in medicine bottles. The finished products complied with the microbiological standard in international pharmacopoeia by a testing laboratory. The drug supply was replenished once every three months and a pill count were performed by RA. All subjects were reviewed by a geriatrician in research clinic at twelve monthly intervals for 24 months. At each follow-up visit, adverse events were enquired; medication and new diagnosis were recorded. CDR, NTB and HDRS were repeated by the trained RAs as in baseline. Fasting blood were taken at month 12 and 24 for serum homocysteine, folate and active vitamin B12. All subjects were allowed to take any medication, except vitamin B12, B6 and folic acid. The use of vitamins was specifically enquired at each followup visit. All subjects who dropped out for whatever reasons were invited to continue with the month 12 and 24 follow-up visits. If the subjects were clinically diagnosed to have developed dementia at follow-up, use of AD drugs was allowed. The primary outcome was CDR_SOB. The secondary outcomes were CDR global score, memory Z score, executive function Z score, HDRS score and homocysteine.
Between April 2013 and July 2016, a total of 975 subjects aged 65 years or more were screened for cognitive impairment by the Chinese MoCA test, and 352 subjects (36.1%) with MCI and elevated levels of serum homocysteine were eligible for the randomized trial. Out of these, 279 subjects (79.3%) participated in the trial. The numbers of subjects screened, randomized and followed-up at different time points (month 12, 24) were summarized in Fig. 1. 138 subjects (supplement group) were randomly assigned to take methylcobalamin 500 mg and folic acid 400 mg once daily, and 141 subjects (placebo group) took two placebo tablets once daily. The participation rates at month 12 and month 24 were 88.2%, 85.3% respectively. The reasons for dropout included: withdrew consent (N ¼ 35), death (N ¼ 3) and terminal illness (N ¼ 3). The pill count indicated that the median percentages of trial tablets taken were 97% or more in both trial groups at both follow-up's. The baseline clinical characteristics and cognitive scores of the participants were shown in Table 1. The supplement and placebo group subjects were well matched except that the supplement group had better executive function (average z score 0.13 versus 0.13 in placebo group), and they were slightly younger (76.9 vs. 78.0). There were no significant differences in cognitive function and baseline characteristics between aspirin users and non-users, except that aspirin users had significantly higher serum creatinine (mean 94.6 versus 87.8 mmol/L), lower low density lipoprotein (1.98 vs 2.75 mmol/L), higher prevalence of diabetes mellitus (43.8% vs 27.4%), hypertension (76.6% vs 61.9%), stroke (20.3% vs 2.3%) and statin use (82.8% vs 26.5%). The serum homocysteine, folate and vitamin B12 before and after intervention were shown in Table 2. The supplement lowered serum homocysteine significantly (from 13.83 ± 3.41 at baseline to 9.37 ± 2.21 at month 12, and 9.32 ± 2.39 at month 24 respectively). The changes in cognitive test scores and HDRS at month 12 and 24 were shown in Table 3. Three placebo group subjects and three supplement group subjects took AD drugs for more than three months before either follow-up. Because they were small in number and their exclusion did not alter the results significantly, they were included in the analysis. The mean changes in CDR_SOB over 24 months were 0.36 (95%CI 0.15 to 0.57) versus 0.22 (0.05e0.39) in supplement and placebo groups respectively. There was no significant group difference in CDR_SOB, memory and executive Z scores at month 24. At month 12, the supplement group had a significant increase in executive function Z score and lower HDRS score from baseline (P ¼ 0.004; 0.012 respectively). The group differences were significant in HDRS (P ¼ 0.010), but not in executive function (P ¼ 0.06). These differences were not significant at month 24. The
2.1. Statistical analysis Intention-to-treat analyses were adopted. Paired t-test was used to examine the effects of B vitamin supplement or placebo at follow-up's, except that Wilcoxon signed rank test was used for HDRS as the data was skewed. The group difference in the changes in CDR_SOB and CDR global score was compared by Student t test and Chi-square test respectively. The interaction effects of concomitant use of aspirin on the effect of B vitamins supplement in cognitive functioning and secondary cognitive or mood
2.2. Sample size calculation In a local prospective clinical trial of older people with diabetes mellitus [20], among 50 control group subjects with CDR global score of 0.5 (questionable dementia), the average change in CDR_SOB at 27 months was 1.42 (SD 0.92). Based on this, a group sample size of 105 had 80% power to detect a 25% decrease in CDR SOB. Assuming a dropout rate of 20%, the group sample size required is 126. 3. Results
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
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975 screened for eligibility 696 excluded Reasons: Refusal 133; No carer 13; Memory well (high MMSE score) 30; Vascular cognitive impairment 1; Dementia 19; Depression 12; Parkinson’s disease 1; Creatinine > 150 5; Homocysteine < 10 315; MoCA >= 26 or too low 105; B12 deficiency 13; On vitamin B 30; Hypothyroidism 6; Having CA 1; Join other study 5; Too frail 4; Eye problem 2; Others 1
Randomized (N=279)
Placebo (N=141)
Active (N=138) Dropout: 17 Patient withdraw: 17
12 Months (N=121)
Dropout: 16 Patient withdraw: 13; Died: 2; Terminal illness: 1
12 Months (N=125)
Dropout: 3 Patient withdraw: 1; Died: 1; Terminal illness: 1
24 Months (N=118)
Dropout: 5 Patient withdraw: 4; Terminal illness: 1
24 Months (N=120) Fig. 1. Consort diagram of randomization.
changes in CDR global score were shown in Table 4. There was no significant group difference in the overall changes or an increase in CDR global score at both follow-up's. But supplement group subjects were more likely to have improved CDR global score at month 12 (P ¼ 0.027, Chi-square test). On subgroup analysis, supplement group had significantly higher CDR_SOB at month 12 and 24 among aspirin users after adjustment for confounders and baseline values (Beta ¼ 0.174, 0.292, P ¼ 0.022, 0.013 at month 12 and 24 respectively). The interaction effect of aspirin on CDR_SOB with supplement was significant at month 24 (beta 0.189; P ¼ 0.005). At month 12 only, supplement group had significantly better executive function among aspirin users, (Beta 0.217; P ¼ 0.007) but not in non aspirin users. This interaction effect was borderline significant (P ¼ 0.040). Post hoc subgroup analysis was also performed in those with higher serum homocysteine (15 mmol/L), lowest quartile of serum active vitamin B12 (58.5 pmol/L) and lowest quartile serum folate (21.9 nmol/L) at baseline. No significant interaction effect was observed in any of the cognitive or
mood outcomes at month 12 or 24, except that the supplement group had significantly lower HDRS score at month 24 among those with low baseline serum active vitamin B12 (interaction effect: Beta ¼ 0.191; P ¼ 0.018). 4. Discussion This trial showed that vitamin B12 and folic acid supplement did not significantly attenuate cognitive decline in older people with MCI over two years. But the observed cognitive decline was small. There was significant reduction in depressive symptoms at month 12 but this was not sustained at month 24. The trial groups were well matched except that subjects in the intervention group had better executive function, probably because these subjects tended to be younger. Compared to MCI subjects in previous trials [9,12], our subjects had relatively high vascular burden as reflected by the prevalence of diabetes mellitus (DM), hypertension, and statin and aspirin use. The education level of the subjects was generally low, which is consistent with that of the
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
T. Kwok et al. / Clinical Nutrition xxx (xxxx) xxx Table 1 Clinical characteristics of trial subjects at baseline. Placebo (N ¼ 141)
Supplement (N ¼ 138)
Number (%)
Number (%)
Female DM HBP CVA Aspirin use Statin use CDR ¼ 0 Education < 3 years
62 (43.9%) 43 (30.5%) 88 (62.4%) 9 (6.4%) 33 (23.4%) 58 (41.1%) 20 (14.2%) 38 (27.0%)
51 (36.9%) 44 (31.9%) 94 (68.1%) 9 (6.5%) 31 (22.5%) 54 (39.1%) 19 (13.8%) 26 (18.8%)
Mean (SD)
Mean (SD)
P
Age (yrs) BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) Glucose fasting (mmol/l) Creatinine (umol/L) Triglycerides (g/L) Total cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Non-HDL (mmol/L) MMSE (max 30) HDRS NTBa Memory z-score Executive function z-score
78.0 (5.3) 24.8 (3.2) 139.4 (16.6) 79.0 (8.3) 6.03 (1.63)
76.9 (5.4) 24.6 (3.4) 139.6 (16.0) 79.2 (9.2) 6.05 (1.51)
0.080 0.724 0.908 0.852 0.889
90.0 (24.3) 1.35 (0.73) 4.62 (1.04)
88.6 (20.5) 1.42 (1.30) 4.73 (1.04)
0.615 0.560 0.360
1.51 2.55 3.18 25.7 2.94
1.56 2.60 3.20 26.0 2.92
(0.47) (0.86) (0.93) (3.1) (3.01)
0.412 0.635 0.858 0.354 0.623
0.04 (0.88) 0.13 (0.07)
0.436 0.011
(0.44) (0.86) (0.92) (3.0) (3.03)
0.04 (0.77) 0.13 (0.07)
P
0.233 0.802 0.317 0.962 0.852 0.733 0.588 0.107
Abbreviations: DM - diabetes mellitus; HBP - high blood pressure; CVA e cerebrovascular accident; MMSE - Mini mental state examination; LDL - Low density lipoprotein; HDL - high density lipoprotein; NTB - Neurocognitive tests; CDR Clinical Dementia Rating scale; HDRS - Hamilton depression rating scale. a z-scores as compared with mean of all subjects; higher score indicating better performance.
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current cohort of older population in Hong Kong. Their average baseline serum homocysteine was more above 13 mmol/L because of the exclusion of subjects with low serum homocysteine, and the lack of local folate supplementation of flour. The compliance of the trial tablets was satisfactory, and over 85% of the subjects completed the trial. The supplements were effective in lowering serum homocysteine. The changes in CDR_SOB were however less than expected in the placebo group, thus limiting the power of the trial in detecting an effect from B vitamins supplementation. Nevertheless, we observed a borderline trend of improvement in executive function at month 12. This is consistent with the results of a recent meta-analysis of a small number of randomized trials of B vitamins in older people with MCI [21]. Unfortunately, the improvement was not sustained at month 24. This may be explained by correction of subclinical vitamin B12 or folate deficiency. However, higher serum homocysteine or lower serum vitamin B12 or folate at baseline showed no significant interaction effect on response in cognitive function. The supplement group had significant reduction in depressive symptoms at month 12, even though subjects with clinical depression were excluded at baseline. There is evidence that folate deficiency is associated with depression, and folic acid supplement is effective in treating depression [22,23], though the doses used in previous trials have been suggested to be 2 mg once daily. Few of the trials have been more than one year. This trial suggested that lower doses of folic acid may also be effective in reducing depressive symptoms, though the effect may not last more than one year. The relatively importance of vitamin B12 and folate status in depressive symptoms has been inconclusive [23e26]. It is noteworthy that B supplements had significant effects on depressive symptoms at month 24 only in those with lower baseline serum active vitamin B12. More trials on the potential effect of vitamin B12 with or without folic acid supplement on depression in those with mild vitamin B12 deficiency are warranted.
Table 2 Serum homocysteine and B vitamin concentrations during trial.
Month Number Homocysteine (mmol/L) Folate (nmol/L) Active vit. B12 (pmol/L)
Placebo
Supplement
Mean (SD)
Mean (SD)
0 141 13.8 (3.0) 29.4 (8.6) 85.8 (32.9)
12 125 13.5 (3.6) 28.9 (8.3) 86.8 (31.8)
24 120 14.0 (4.1) 27.8 (9.8) 85.8 (34.0)
0 138 13.8 (3.4) 27.8 (8.0) 85.5 (29.8)
12 121 9.4 (2.2)* 43.3 (4.6)* 125.2 (9.9)*
24 118 9.3 (2.4)* 48.0 (12.6)* 123.6 (13.6)*
*P < 0.0001, paired t test.
Table 3 Mean change from baseline in cognitive test and depressive symptom scores at follow-upb. Month
CDR_SOB Executive Z Memory Z HDRSa
12 24 12 24 12 24 12 24
Placebo
Supplement
Difference due to supplement
N
Mean change (95% CI)
N
Mean change (95% CI)
(95% CI)
126 122 126 122 122 112 126 122
0.05 (0.07, 0.16) 0.22 (0.05, 0.39)y 0.03 (0.08, 0.13) 0.03 (0.08, 0.14) 0.06 (0.05, 0.16) 0.01 (0.15, 0.14) 0.30 (0.23, 0.84) 0.24 (0.30, 0.77)
123 119 123 119 119 110 123 119
0.13 (0.01, 0.26) 0.36 (0.15, 0.57)z 0.18 (0.06, 0.30)z 0.00 (0.12, 0.12) 0.03 (0.09, 0.14) 0.00 (0.13, 0.12) 0.59 (1.01, 0.18)y 0.08 (0.63, 0.46)
0.08 (0.25, 0.10) 0.14 (0.40, 0.13) 0.15 (0.01, 0.31) 0.03 (0.19, 0.13) 0.03 (0.19, 0.13) 0.00 (0.19, 0.19) 0.90 (1.57, 0.22)z 0.32 (1.08, 0.44)
Abbreviations: CDR_SOB e Clinical dementia rating scale sum of boxes; HDRS e Hamilton depression rating scale; Z e Z score. yp < 0.05; zp 0.01. a Wilcoxon signed rank test used to compare baseline and follow-up values. b Follow-up value minus baseline value.
Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005
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T. Kwok et al. / Clinical Nutrition xxx (xxxx) xxx
significant negative interaction effect between aspirin and B vitamins in cognitive functioning.
Table 4 Changes in CDR global score.
DCDR 0.5 0.0 0.5 Total
Placebo
Supplement
Month 12
Month 24
Month 12
Month 24
3 (2.4%) 113 (91.1%) 8 (6.5%) 124
9 (7.9%) 96 (84.2%) 9 (7.9%) 114
11 (8.9%)* 105 (85.4%) 7 (5.7%) 123
17 (14.7%) 88 (75.9%) 11 (9.5%) 116
Conflict of interest The authors declare no conflict of interest. Acknowledgments
DCDR e clinical dementia rating global score at follow-up minus CDR at baseline.
* more likely to improve in CDR global score P ¼ 0.027, Chi-square test.
This study was supported by the General Research Fund of the Research Grants Council of Hong Kong (RGC Reference 466612).
The subgroup analysis of aspirin use was motivated by an incidental finding of a negative interaction effect of aspirin use on the protective effect of B vitamins against brain atrophy among MCI subjects in the VITACOG trial [8]. Our results showed complex interactions between aspirin and B vitamins. On one hand, aspirin had negative interaction with B vitamins in cognitive functioning at month 24. On the other hand, aspirin had borderline positive interaction effect on executive function at month 12. The negative interaction effect of aspirin on CDR_SOB was consistent with the negative interaction effect of aspirin on brain atrophy rate in the VITACOG trial. A possible explanation is that aspirin was interfering with the cerebral effects of folic acid. Folic acid is a synthetic form of folate and requires metabolism into dihydrofolate and then to tetrahydrofolate by dihydrofolate reductase (DHFR). Because DHFR expression in liver is low and variable, there have been concerns about the potential harm of unmetabolized folic acid. Older people in Ireland where there was no folic acid fortification of flour policy have been found to have higher fasting serum concentrations of unmetabolized folic acid with dietary folic acid supplement alone [27]. The concern is the unmetabolized folic acid may impair the active transport of active folates into the brain, though so far the harm of unmetabolized folic acid is yet to be demonstrated. Aspirin has been shown to inhibit the expression of a protein “E2F-1” which regulates DHFR expression [28]. It is therefore possible that aspirin inhibits the upregulation of DHFR which normally occurs with folic acid supplementation [29], resulting in higher serum concentrations of unmetabolized folic acid. A cross sectional study found that individuals with genetically defective DHFR had cognitive impairment when their serum folate concentrations were high [30]. It is intriguing to observe that the use of aspirin had a borderline positive interaction effect on the improvement of executive function at month 12. One of the proposed mechanisms whereby homocysteine contributes to atherosclerosis is the activation of platelets in response to damaged endothelium [31]. It is plausible that homocysteine lowering by B vitamins may work in synergy with aspirin in inhibiting platelet activation in those with vascular diseases. As executive function is primarily impaired in vascular cognitive impairment [32], it is plausible the combined effects of B vitamins and aspirin may enhance executive function in those with cerebrovascular diseases. But why this beneficial effect waned at month 24 remains to be explained. This trial had limitations. The sample size was insufficient to detect a small difference in cognitive function. The high vascular burden and the associated drug use may confound the outcomes of B vitamin supplementation. There was lack of brain imaging to measure brain atrophy rate, which is a more sensitive marker of AD. In conclusion, we did not observe significant effects of vitamin B12 and folic acid supplementation in attenuating cognitive decline in older people with mild cognitive impairment over a two year period, though the trial was underpowered because of limited cognitive decline in placebo group. On the other hand, B vitamins had a short term beneficial effect in mood. Lastly, we found a
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Please cite this article as: Kwok T et al., A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.11.005