Objective Our study aimed to assess the effect of a 12-month vitamin D supplementation on cognitive function and amyloid beta (Aβ)-related biomarkers in subjects with Alzheimer’s disease (AD).
Methods This was a randomised, double-blind, placebo-controlled trial. 210 AD patients were randomly divided into intervention and control groups. Participants received 12-month 800 IU/day of vitamin D or starch granules as placebo. Tests of cognitive performance and Aβ-related biomarkers were measured at baseline, 6 months and 12 months.
Results Repeated-measures analysis of variance showed significant improvements in plasma Aβ42, APP, BACE1, APPmRNA, BACE1mRNA (p<0.001) levels and information, arithmetic, digit span, vocabulary, block design and picture arrange scores (p<0.05) in the intervention group over the control group. According to mixed-model analysis, vitamin D group had significant increase in full scale IQ during follow-up period (p<0.001).
Conclusions Daily oral vitamin D supplementation (800 IU/day) for 12 months may improve cognitive function and decrease Aβ-related biomarkers in elderly patients with AD. Larger scale longer term randomised trials of vitamin D are needed.
Trial registration number ChiCTR-IIR-16009549.
- Alzheimer’s disease
- vitamin D
- cognitive function
- randomised controlled trial
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Dementia includes a series of neurological diseases characterised by memory loss and cognitive dysfunction. And Alzheimer’s disease (AD) is the most common manifestation of dementia, accounting for 50%–70% of cases.1 Nowadays, there is no blockbuster drugs to delay the course of disease, but many researchers have previously explored some factors and biochemical markers associated with AD in search of effective prevention strategies of cognitive impairment and dementia.2 3 And modifying dietary and lifestyle habits may delay the progression of AD, particularly in vitamin supplementation.4 5
Currently, vitamin D is considered a steroid hormone. Vitamin D has been shown to have a wide range of health promoting effects, and it has good clinical application in the prevention and treatment of various diseases.6 7 Vitamin D receptors are present in neurons and glial cells and affect a lot of physiological processes in the hippocampus, hypothalamus and cortex.8–10 Vitamin D status has showed tightly links with the function of the central nervous system, particularly in delays apoptosis,11 suppression synaptic degeneration in hippocampus,12 and inhibition of inflammation.13 So, The neuroprotective effect of vitamin D may delay the progression of AD. Additionally, in recent years, increasing evidence have shown that patients with AD have lower levels of 25-hydroxy vitamin D (25-OH) relative to healthy controls, and vitamin D has been suggested to be related to AD through reducing the accumulation of amyloid beta (Aβ).14 15
As we know, an important hallmark of patients with AD is the deposition of amyloid plaques in the brain.16 Previous studies have shown that abnormal protein aggregation, including the accumulation of Aβ peptide, plays a key role in the course of AD.17 The neurotoxicity of Aβ mainly manifests itself in oxidative stress, mediates inflammatory injury and causes cholinergic nerve damage.18 19 Some studies have reported that vitamin D can promote phagocytic clearance of amyloid plaques.20 21 Therefore, vitamin D may be a potential drug to prevent AD.
This study aimed to determine whether 12-month vitamin D intervention would improve cognitive function, and assess whether cognitive performances are related to vitamin D status and Aβ-related biomarkers at baseline in Chinese elderly with AD.
This is a randomised, double-blind, placebo-controlled trial to evaluated whether vitamin D could effectively delay clinical progression in patients with AD. Participants were enrolled between April 2016 and April 2017 in Huanhu hospital of Tianjin according to the inclusion criteria as follows: (1) conforms to the likely AD diagnostic criteria which is developed by the National Institute on Aging and the Alzheimer’s Association in 201122; (2) perform detailed medical history, neurological examinations, neuropsychological tests, positron emission tomography, MRI and necessary laboratory tests and diagnosed by two neurology specialists; (3) with Chinese as mother tongue, right handedness, clear mind and cooperation; (4) aged 65 years and over. However, there are also some exclusion criteria: (1) neurological examination of hemiplegia, partial sensory disturbances, aphasia and other signs of focal central nervous system disorders, history of cerebrovascular disease (including haemorrhagic and ischaemic), occurrence of intracranial trauma or fracture; (2) has medical conditions such as asthmatic bronchitis, severe hypertension, angina and serious infections; (3) patients with obvious depression, anxiety and other mental disorders or endocrine system diseases (such as hyperthyroidism, hypothyroidism, systemic lupus erythematosus, rheumatoid arthritis, etc); (4) newly discovered or advanced tumours; (5) visual, auditory, or verbal difficulties that obviously affect communication in cognitive function tests; (6) history of alcohol dependence and other psychoactive substance abuse (such as antipsychotics, benzodiazepines, etc), or medications that may affect cognitive function. According to these criteria, 210 eligible patients were enrolled in the study.
In our study, eligible participants were randomly divided into vitamin D group and placebo group. The randomisation sequence was computer generated by the study sponsor.
The study drug was vitamin D3 administered as capsules, and the product was manufactured by Aiweidi soft capsule national medicine Health Industry Co., Shanghai, China. (400 IU/tablet; state medical permit No.: 2 013 012.) The intervention group took a daily oral dose of two tablets consisting of 800 IU vitamin D3 for the entire 12-month period. The placebo was prepared by the identical capsules, using starch granules without vitamin D3, that had same appearance to vitamin D3 capsules. There is no evidence that starch granules at this amount have beneficial or adverse effects on cognitive function. Subjects were instructed to take capsules at the same time for each day and guaranteed a normal diet during the study period. In this trial, all project staff and participants were unaware of groups division. A blinding key linked each patient number to his or her assigned treatment and was protected by an investigator. And the assignment was revealed at the completion of the trial.
On the assessment of cognitive performance, the participants received a standardised neuropsychological assessment conducted by trained physicians at the baseline, 6-month and 12-month during the treatment. In our study, the main outcome was cognitive function during follow-up which was measure by the Chinese version of the Wechsler Adult Intelligence Scale-Revised (WAIS-RC).23 The WAIS-RC includes 11 subtests: information, comprehension, arithmetic, similarities, vocabulary, digit span, digit symbol, block design, object assembly, picture completion and picture arrangement. We calculated IQ and index scores using age-appropriate norms from the Chinese standardisation. At baseline, Activity of Daily Living (ADL) Scale was mainly used to assess the daily life ability of subjects, including dressing, walking, bathing, feeding, shopping, preparing meals, using household appliances and so on. Mini-Mental State Examination (MMSE) was administered as measure of general cognitive function.
Blood sampling were collected by venepuncture at baseline, 6 months and 12 months. In the morning, 5 mL of venous blood is drawn from the patient on an empty stomach. Real-time PCR was used to quantify the gene expression of Aβ protein precursor (APP) mRNA, β-secretase 1 (BACE1) mRNA, presenilin-1 (PS1) mRNA and PS2mRNA. The measurements were performed using the Roche LightCycler 480 sequence detector (Roche, Mannheim, Germany). Protein levels of APP, β-secretase 1, PS1 and PS2 were assessed by western blot assay. The levels of Aβ40 and Aβ42 were detected by ELISA. The concentration of blood 25-D and 1,25-dihydroxy vitamin D (1,25-D) was measured by liquid chromatography tandem mass spectrometry (API4000, AB SCIEX, USA), using high-performance liquid chromatography-mass spectrometry method.
Statistical differences were examined with χ2 tests or Fisher’s exact test for categorical variables and t-test for continuous variables. Repeated-measures analysis of variance was used to evaluate the effects of vitamin D and placebo interventions on cognitive performance and the level Aβ-related biomarkers over 12 months. Data are mean unadjusted scores or levels ±SD, with p value for group (intervention vs control) derived from analysis of covariance adjusted for respective baseline value and baseline characteristics, and the p value included time effect, group effect and interaction effect. A mixed-model repeated-measures ANOVA was used to assess the hypothesis regarding the difference between vitamin D and the control. Within-person variation was modelled by using covariance matrices. The model was developed for the variable of full scale IQ (FSIQ). The critical test of the effectiveness was an effect of the vitamin D supplementation relative to the control over time. All analyses were conducted on the intent-to-treat principle. A two-sided p value of 0.05 or less was considered to have a significant statistical difference. All statistical analyses were performed using SAS V.9.4.
Characteristics of participants
Among the 210 patients meeting the inclusion criteria, 105 were assigned to the vitamin D supplementation group and 105 to the placebo group. One patient in the control group dropped out, resulting in 209 patients who completed the study at last (vitamin D group: n=105; placebo group: n=104).
At baseline, there were no significant differences in the characteristics of the two groups in demography, body mass index, lifestyle and family history. Moreover, intervention group and control group were similar on the concentration of serum 25-D and 1,25-D. Baseline characteristics of the study population are shown in table 1.
Repeated-measures analysis of covariance revealed that, over 12 months, serum 25-D and 1,25-D level both showed substantial percentage increases in intervention group which were significantly different from control group (p<0.001) (table 2). We also could see more clearly the trend of vitamin D levels over time, as shown in figure 1.
Regarding blood biomarkers, the Aβ42 level was significantly lower in the intervention group than in the control group (p<0.001); the intervention group reduced by 11.31%, while control group reduced by 0.27%. The BACE1 level and BACE1mRNA showed significantly lower in the intervention group than in the control group (p<0.001). APP level in intervention group had decreased by 10.70%, while increased by 1.90% in the control group (p<0.001). APPmRNA also showed a greater decrease in vitamin D group (−14.21%) compared with the control group (−2.65%); the difference in the change of APPmRNA was significant (p<0.001). Figure 1 also showed that, BACE1mRNA and APPmRNA levels dropped obviously during the trial in the intervention group which was in marked contrast to control group. PS1mRNA showed a marginally significant difference between the groups (p=0.040). However, there were no significant interaction effect between time and group effect in other biomarkers (p>0.05).
Repeated-measures ANOVA showed few significant interaction effects in the neuropsychological tests except for FSIQ, information, arithmetic, digit span, vocabulary, block design and picture arrange. Over 12 months, the FSIQ was significantly higher in the intervention group than in the control group (p<0.001). Information test showed marked increments in intervention group (+30.73%), while the control group had a decline trend (p<0.001). Arithmetic score showed substantial percentage decreases in both groups (p<0.001), and was lesser in the intervention group (−35.87%) compared with the control group (−42.09%); there was an interaction between time effect and group effect (p=0.027). In addition, the vocabulary, digit span, block design and picture arrangement also showed similar changes in the two groups, and the intervention group showed higher cognitive test score than in the control group. However, comprehension, similarities, digit symbol, picture completion and object assembly only showed substantial percentage decreases in time effects in the two groups (p<0.001) (table 3).
To further indicate that vitamin D supplementation improved cognitive function over time, a mixed-model repeated-measures ANOVA were used for FSIQ score with taking into account baseline biomarker concentrations. Compared with the control group, the vitamin D group had a statistically significant increase in the FSIQ score from baseline to 12 months (p<0.001). In addition to the intervention effects, baseline Aβ42 and APPmRNA concentrations had an important association with cognitive performance over time. Elevated APPmRNA concentrations at baseline were associated with poorer cognitive performance at 12 months for FSIQ score (estimate value=−0.132, p=0.044), and elevated Aβ42 concentrations were associated with better cognitive performance (estimate value=0.031, p=0.001) (table 4).
This study was designed to determine if use of vitamin D supplements in individuals with AD would slow the decline in cognitive function and evaluate cognitive performance in relation to Aβ-related biomarkers at baseline. The results showed that the intervention was successful in reducing some of the Aβ-related biomarkers, including Aβ42, APP, BACE1, APPmRNA and BACE1mRNA. Moreover, in the study population as a whole, there were apparent evidences of improved cognitive function in tests of FSIQ, information, arithmetic, digit span, vocabulary, block design and picture arrange. These findings support the hypothesis that the neuroprotective role of vitamin D may associate with decreasing Aβ-related biomarkers.
The main symptoms of AD are memory loss and decreased ability to think, analyse and judge. In this trial, vitamin D supplementation beneficially affected cognitive functions, especially in information, digit span, vocabulary, block design, picture arrange and arithmetic. The subtests of information, digit span and vocabulary are valid indicator of memory, while block design, picture arrange and arithmetic tests reflect the decline in various behavioural capacities in patients with AD.24 Some studies are similar to our findings. For instance, Ouma’s trial showed that 25-OH is involved in the decrease of MMSE score, and predict mild cognitive impairment, mild AD and moderate AD.25 Vitamin D is a type of steroid hormones with neurosteroid function, and the neuroprotective effect of vitamin D is likely an important contributor to memory development and continued normal cognitive function.26 Moreover, compared with healthy people of the same age, the plasma 25-D level of AD patients seems to be markedly lower.27 According to the above, it suggest that vitamin D may have potential benefits on cognitive function.
Currently, researchers have a variety of perspectives on the biological mechanisms between vitamin D and AD. Some researchers point out the neuroprotective effect of vitamin D, including immunomodulatory, anti-oxidise, neurotrophic effect and maintaining calcium homeostasis in neurons.9 10 28 This neuroprotective effect delays the progression of AD. There are also many researchers indicate that vitamin D could prevent the accumulation of Aβ protein and promote the clearance of Aβ protein through the blood-brain barrier.12 29 In this research, our results showed that Aβ42 level decreased with oral vitamin D supplementation after 12 months, and even after 6 months. We know that accumulation of Aβ protein in the brain is one of the most important causes of AD, and Aβ protein leads to neuronal dysfunction and death, eventually causing dementia.30 31 The mean level of soluble Aβ in the brain is increased threefold in AD and correlates highly with markers of disease severity.32 Moreover, our study showed that Aβ-related biomarkers also had a significant decline trend, such as APP, BACE1, APPmRNA and BACE1mRNA. These results indicate that the level of Aβ protein in the intervention group is decreasing in the brain, and it has a beneficial effect on AD patients. Therefore, vitamin D supplementation may affect cognitive function through its effect on Aβ-related biomarkers. And vitamin D is a promising therapeutic agent for the treatment of AD.
In the research, we observed that APPmRNA level had an inverse association with FSIQ. APP is converted to Aβ by the decomposition of protease. Therefore, the level of APPmRNA can reflect the accumulation of Aβ in the brain to some extent, which conformed to our study demonstrating that increased levels of Aβ-related biomarkers are associated with a higher risk in AD patients. We also found that the level of Aβ42 in the blood had a positive correlation with FSIQ. Nowadays, many studies hold different views on the independent role of Aβ42 concentration in cognitive evaluation. So, explanation of the association between vitamin D and Aβ42 needs to be further explored.
Our study provides several advantages of its rigorous design. First, the random assignment of AD patients resulted in similar characteristics of most background variables in the two groups, so the comparison between the two groups mainly reflected the impact of vitamin D. Second, eligible patients with AD was identified using expert adjudicated diagnosis and rigorous inclusion and exclusion criteria. Third, a standard measure of cognitive status and a broad assessment of different cognitive functions were used for the study.
There are also several limitations have to be noted. First, the optimal dose of vitamin D needed to improve cognitive function is unknown in individuals with AD. Second, this study only measured the concentrations of vitamin D in plasma, which might not reveal in tissues. Third, the potential for plasma Aβ confounders are not measured, such as plasma protein binding, Aβ production from platelets and peripheral organs and hydration. Moreover, many factors which can affect Aβ protein are unknown, and different protective mechanisms may reduce or increase plasma Aβ.15 Therefore, the power of the study could be boosted if confounding factors were controlled better.
In summary, we found obvious evidence that daily oral vitamin D supplementation (800 IU/day) for 12 months can significantly improve cognitive function in patients with Alzheimer’s disease. The beneficial effect of vitamin D supplementation on cognitive function may act by Aβ protein reduction. Larger scale longer term randomised trials of vitamin D are needed.
The authors thank all the subjects for their participation.
Contributors FM designed the research. JH conducted the research. RH and XH provided essential reagents. JJ and YZ performed the statistical analysis. JJ wrote the paper. All authors revised the manuscript and approved the final version to be published.
Funding This study was supported by the National Natural Science Foundation of China (grant number: 81573148; 81874261).
Competing interests None declared.
Patient consent for publication All subjects signed informed consent prior to study initiation.
Ethics approval This study adheres to the principles of the Declaration of Helsinki. The protocol was approved by the Ethics Committees of Tianjin Medical University, China.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement No data are available.
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