Effects of DHA Supplementation on Hippocampal Volume and Cognitive Function in Older Adults with Mild Cognitive Impairment: A 12-Month Randomized,Double-Blind,Placebo-Controlled Trial

Abstract

Docosahexaenoic acid (DHA) is important for brain function, and higher DHA intake is inversely correlated with relative risk of Alzheimer’s disease. The potential benefits of DHA supplementation in people with mild cognitive impairment (MCI) have not been fully examined. Our study aimed to determine the effect of DHA supplementation on cognitive function and hippocampal atrophy in elderly subjects with MCI. This was a randomized, double-blind, placebo-controlled trial in Tianjin, China. 240 individuals with MCI aged 65 years and over were recruited and equalized randomly allocated to the DHA or the placebo group. Participants received 12-month DHA supplementation (2 g/day) or corn oil as placebo. Both global and specific subdomains of cognitive function and hippocampal volume were measured at baseline, 6 months, and 12 months. Both changes were analyzed by repeated-measure analysis of variance (ANOVA). This trial has been registered: ChiCTR-IOR-15006058. A total of 219 participants (DHA: 110, Placebo: 109) completed the trial. The change in mean serum DHA levels was greater in the intervention group (+3.85%) compared to the control group (+1.06%). Repeated-measures analyses of covariance showed that, over 12 months, there was a significant difference in the Full-Scale Intelligence Quotient (ηp² = 0.084; p = 0.039), Information (ηp² = 0.439; p = 0.000), and Digit Span (ηp² = 0.375; p = 0.000) between DHA-treated versus the placebo group. In addition, there were significant differences in volumes of left hippocampus (ηp² = 0.121, p = 0.016), right hippocampus (ηp² = 0.757, p = 0.008), total hippocampus (ηp² = 0.124, p = 0.023), and global cerebrum (ηp² = 0.145, p = 0.032) between the two groups. These findings suggest that DHA supplementation (2 g/day) for 12 months in MCI subjects can significantly improve cognitive function and slow the progression of hippocampal atrophy. Larger, longer-term confirmatory studies are warranted.

Keywords

Alzheimer’s disease cognitive function docosahexaenoic acid hippocampal volume randomized controlled trial

INTRODUCTION

Alzheimer’s disease (AD), the most common form of dementia, is characterized by progressive and profound loss of memory, cognitive function, and ability to carry out daily functional activities of living [1]. Detection and identification of AD at the prodromal stage may allow the delay of disease progression through appropriate treatment intervention [2]. Mild cognitive impairment (MCI) is possibly the earliest stage of detectable dementia [3] and may be the optimal time to intervene with preventive therapies [4].

No pharmacological treatment is currently available to cure AD. Modifying lifestyle habits to delay the onset of AD or MCI has attracted special attention, especially strategies that improve nutrition [5]. Omega-3 polyunsaturated fatty acids (PUFA), due to their central position within the central nervous system, may be good candidates to promote cognitive health during aging. Indeed, omega-3 PUFA, especially docosahexaenoic acid (DHA), are important components of all cell membranes [6] and play a critical role in optimal brain development and function [7]. A number of cross-sectional and prospective studies have highlighted a positive correlation between fish and/or DHA dietary consumption and cognitive performances in healthy elderly [8], as well as with lower accumulation of Aβ peptides [9, 10] and lower brain atrophy [11]. Due to the requirement of DHA to brain functions, new therapeutic strategies to bring it to the central nervous system are promising.

High dietary intake of DHA may confer benefits in cognition [12]. However, no intervention study has examined the impact of DHA supplementation on brain structure (e.g., volume) in elderly subjects with MCI. Hippocampus is a limbic structure involved in cognitive functions such as memory formation [13]. It is well delimited with rather well-defined boundaries, validated, localized, and central to the neurodegenerative pathophysiology. It is central to the pathophysiology of AD as it is one of the earlier and more severely affected regions in AD [14, 15]. Hippocampal atrophy is the most prominent structural hallmark of progression from aMCI to AD. Thus, understanding the effect of DHA on the hippocampus will increase our appreciation of the role that DHA may play in dementia prevention.

The current study was to identify whether 12-month DHA supplementation would improve cognitive function and hippocampal atrophy among elderly subjects with MCI.

METHODS

Sampling methods and ethical considerations

This was a randomized, double-blind, placebo-controlled trial that evaluated effects of 12-month DHA supplementation on cognitive function and hippocampal volume in elderly subjects with MCI. Participants were enrolled between March 2013 and April 2013, on the basis of the following criteria: 1) aged 65 years and over; 2) in generally good health, ambulatory, and with sufficient hearing and vision for compliance with testing procedures; 3) absence of terminal illness or mental disorders (i.e., major depression, schizophrenia, bipolar disorder, etc.); 4) not using any nutritional supplementation known to interfere with nutrition status, fatty acid composition metabolism, or cognitive function in the three months before recruitment; 5) those able to undergo MRI evaluation (i.e., those without a pacemaker or other metal items within the body, and those with MRI brain abnormalities such as multiple microhemorrhages, infarcts, or moderate to severe cortical atrophy); 6) not living in a nursing home or being on a waiting list for a nursing home.

By random cluster sampling, six geographically convenient communities with a high proportion of older residents who were all native Chinese speakers were selected from the Nankai District, Tianjin, China. Of the 4,313 selected individuals, 2,816 (65.3%) agreed to participate, but only 2,555 participants were eligible for the clinical, physical, and neuropsychological examinations. Of these, using previously determined criteria for MCI, 248 elderly subjects with MCI were selected, and 240 met the study criteria and were randomly assigned by means of a computerized randomization schedule to receive either DHA or placebo. The flow of study participants is shown in Fig. 1.

The study was conducted in compliance with the ethical principles of the Declaration of Helsinki and was approved by the medical ethics committee of Tianjin Medical University, China. Each subject provided written informed consent prior to study entry. This trial has been registered with trial number ChiCTR-IOR-15006058 (http://www.chictr.org.cn/showproj.aspx?proj=10530).

Data collection

After giving written consent, all participants completed a basic sociodemographic and medicalhistory questionnaire and reported the list of medications they were taking at baseline. The interview included the following information: age (years), sex, education (years), marital status, occupation, whether they smoked (ever or never), the number of packs smoked per year, number of cardio-cerebrovascular disease(self-reported history of TIA/stroke, myocardial infarction, atrial fibrillation), hypertension (self-reported history, measured blood pressure ≥160 mm Hg systolic or ≥95 mm Hg diastolic, or use of antihypertensive medications), history of stroke (self-report), and diabetes mellitus (self-report or antidiabetic medicationuse).

Assessment of cognitive function

The main outcome in the current study was cognitive function, which was measured using the Chinese version of the Wechsler Adult Intelligence Scale-Revised (WAIS-RC) [16]. The WAIS-RC includes 11 sub-tests: Information, Similarities, Vocabulary, Comprehension, Arithmetic, Digit Span, Block Design, Picture Completion, Digit Symbol-Coding, Object Assembly, and Picture Arrangement. The neuropsychological assessments were administered by trained physicians at baseline, and at the six- and twelve-month time points during treatment. We used age-appropriate norms from the Chinesestandardization to calculate the intelligence quotient (IQ) and index scores [17].

Definition of MCI

MCI was determined in accordance with the modified Petersen’s criteria [17], as follows:

Subjective memory complaint, with a duration of at least 2 weeks;

Objective memory impairment given the individual’s age and education was defined as performing at least 1.5 SD below age- and education-matched controls on the mini-mental state examination memory subtask [18];

Normal general cognitive function impairment was defined as a test performance that was more than 1.5 SD below age- and education-specific norms;

Essentially preserved activities of daily living, as measured by the Activities of Daily Living scale, i.e., a score of <26 [19];

Absence of dementia (Diagnostic and Statistical Manual of Mental Disorders-IV), AD (the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association), psychiatric disorders, cerebral damage, or physical diseases that may account for cognitive impairment.

Study medication, assignments, and masking

After baseline screening, eligible participants were assigned randomly into the DHA group or the placebo group. The randomization sequence was computer generated by the study sponsor.

The study drug was an algal-derived DHA (Martek Biosciences, Columbia, Maryland) administered as capsules, dosed as 2 g/day. Algal DHA contains approximately 45% to 55% of DHA by weight and does not contain eicosapentaenoic acid. The DHA dose was selected based on evidence that plasma levels increase in a dose-dependent manner up to approximately 2 g/d, while at higher doses no further increase in plasma DHA is seen. Placebo capsules (made up of corn oil) were identical in appearance. All capsules were orange-flavored and orange color to protect the study blind. Subjects were instructed to take capsules with food at the same time each day (e.g., 1 capsule/meal), starting at the baseline visit, and to not alter their normal diet during the study. The supplements were packaged into identical pots, each containing 180 capsules, and labeled by staff who were not involved in the study. A blinding key linked each participant number to his or her assigned treatment. This key was kept by an investigator, not involved in any data collection or analyses, in a secure electronic file. The code was revealed at the completion of the trial following analyses of the main study aims.

During the last week of each month, participants were requested to return any remaining tablets in order to measure compliance, together with the replenishment of capsules for the following month. Compliance to the supplementation regimen was defined as the number of capsules actually taken by each subject divided by the number of capsules that should have been taken over the course of the study. Adherence was encouraged and monitored throughout the trial by telephone assessment at 15 time points, and by blood assay at baseline and at the 6-month and 12-month assessments for both groups. All project staff were unaware of group assignments until the completion of the trial and after data analysis.

Gas chromatography

Fasting venous blood samples were collected at baseline and after 6 and 12 months’ intervention. Blood samples, drawn in 2×5 mL serum-separating tubes, were posted overnight to a handling laboratory that prepared and froze sera at –80°C. Total lipid was extracted from plasma with 2:1 (vol./vol.) chloroform and methanol. Fatty acid methyl esters were prepared by incubation of lipid with methanol containing 2% (v/v) sulphuric acid at 50°C for 2 h. Fatty acid methyl esters were separated using a Hewlett Packard 6890 gas chromatograph (Agilent Technologies UK Ltd, Wokingham, Berks., UK) equipped with a 30 m×0.25 mm×0.25 mm BPX-70 fused silica capillary column (SGE, Milton Keynes, UK) and flame ionisation detection on a Schimadzu G-2010 (Schimadzu, Sydney, Australia). The analysis of fatty acid methyl esters is based on areas calculated with Shimadzu Class VP 4.3 software. DHA concentrations were expressed in absolute concentrations (μmol/L) unless otherwise stated. All samples were processed by a technician blinded to treatment.

MRI acquisition and processing

MRI was performed in Wangdingdi community hospital with the same acquisition procedure. Brain imaging was conducted using high resolution Philips 1.5 Tesla scanner (Gyroscan NT, Philips Medical Systems, Best, The Netherlands) at baseline, 6 months, and 12 month. The 3D gradient echo sequence was acquired with the scan parameters TR 8.6 ms, TE 4.0 ms, flip angle 08°. Sagittal slices with a field of view of 240 mm, a slice thickness of 1.2 mm, and an in-slice resolution of 0.94 mm² were reconstructed. The multimodality images were coregistered by standard protocol, extracranial material was removed, and the T1-weighted brain tissue mask was used for segmentation into gray and white matter and cerebrospinal fluid. Anatomical region (i.e., lobes, limbic, basal ganglia, corpus callosum, hippocampus) volumetric measurements were obtained using an automated computer-based template warping method that summed the number of respective voxels [22]. Image-processing steps in all patients are performed by the same research associate, who is blind to clinical information to insure that the volumetric data areunbiased.

Statistical analysis

Baseline characteristics of study population have been checked by means of χ² test or Fisher’s exact test for categorical variables and one-way ANOVA for continuous variables, with post hoc comparison using the Bonferroni test for multiple comparisons. Repeated-measures ANOVA was used to evaluate the effects of DHA and placebo interventions on hippocampus volume and cognitive performance. Data are mean unadjusted scores±standard deviations, with ηp² and p value from repeated-measures ANOVA that included the time-treatment interaction. The MRI volume variables include raw data in cm³ for the right, left, total hippocampus, global cerebral, and ventricular volume at baseline and follow-ups. To account for variations in head size among subjects, prior to analysis, all volumetric data were corrected to adjust for intracranial volume [normalized volumetric volume = (raw volume /total intracranial volume)×1000]. MRI change rates are reported in units of annualized percent change. If V₁ = volume at baseline and V₂ = volume at a later time (either at conversion or month 36), then annualized percent change (APC) = (V₂ – V₁)/V₁. All analyses were performed with the intent-to-treat principle. All statistical testing was two-sided. The significance level was set at a p value of less than 0.05. All analyses were performed using SPSS PASW Statistics for Windows, version 18.0 (Inc. Released 2009, Chicago: SPSS).

RESULTS

Participant flow, characteristics, and follow-up

Of the 248 MCI individuals screened, 240 met the study criteria and were randomized, 120 to DHA and 120 to placebo. At baseline, treatment groups were similar for demographic, health, fish consumption, cognitive function, and MRI volume variables. Baseline characteristics of the study population are shown in Table 1. Dropout rates over the 12 months after randomization were similar between the two groups: 10 (8.33%) participants dropped out from the DHA group and 11 (9.17%) from the placebo group (χ² = 0.276, p = 0.834). Compliance (percentage of capsules taken) was 97% in the DHA group, and 95% in the placebo group. There was no significant difference between the twogroups.

Changes of serum DHA concentration

The mean serum DHA levels (μmol/L) in the DHA-treated group were 286 (95% CI: 268-305) at baseline, 293 (95% CI: 271-311) at 6 months, and 297 (95% CI: 274-323) at 12 months, as compared to 283 (95% CI: 2648-297) at baseline, 284 (95% CI: 266-299) at 6 months, and 287 (95% CI: 263-295) at 12 months in the placebo-treated group. The mean serum DHA levels showed significant but small percentage increases in both groups (p = 0.037, ηp² = 0.092). The change of mean serum DHA levels was greater in the intervention group (+3.85%) compared to the control group (+1.06%).

Cognitive status

Repeated-measures analyses of covariance showed that over 12 months there were significant differences in the Full-Scale Intelligence Quotient (ηp² = 0.084; p = 0.039), Information (ηp² = 0.439; p = 0.000), and Digit Span (ηp² = 0.375; p = 0.000) between DHA-treated versus the placebo group (Table 2). The mean scores of participants in intervention group had a greater increase in Full Scale IQ (+10.31%) than the controls (+0.12%); For Information subtest, the mean scores were greater in the intervention group (+33.33%) than in the control group (+1.22%); For Digit span subtest, it was greater in the intervention group (+48.18%) than the control group(+13.13%).

MRI-based outcome measures

Repeated-measures ANOVA showed that over 12 months there were significant differences in volumes of left hippocampus (ηp² = 0.121, p = 0.016), right hippocampus (ηp² = 0.757, p = 0.008), total hippocampus (ηp² = 0.124, p = 0.023), and global cerebrum (ηp² = 0.145, p = 0.032) between the two groups. Total hippocampus volume in DHA group had increased by 4.00%, while decreased by 0.19% in the placebo group; left hippocampus volume in DHA group increased by 6.13%, decreased by 0.36% in the control group; For right hippocampus, it was greater in the intervention group (+1.89%) compared to the control group (+0.00%); Global cerebral volume showed a greater increase in the intervention group (+0.29%) compared to the control group (+0.12%). However, there were no significant differences in ventricular volume (Table 3).

Correlations between changes observed using MRI and neuropsychological tests

To determine whether the neuropsychological changes observed during the DHA treatment phase were associated with altered rates of change on MRI measures, correlations between MRI APC versus changes in cognitive test performance were calculated for all four MRI measures and cognitive measures (Spearman’s coefficient); the Full Scale IQ, Information, and Digit Span. Each of the 15 possible correlations between change in MRI versus change in cognitive test performance was significant in the expected, biologically plausible, direction (Table 4).

DISCUSSION

The present study aimed to evaluate the effects of DHA supplementation on cognitive function and hippocampal atrophy in elderly people with MCI. Results show that 12-month DHA supplementation significantly improved cognitive function and increase hippocampus volume in MCI subjects. The evidence from randomized controlled trial (RCT) studies in older adults with DHA intervention is conflicted. Recently, Quinn and coworkers [23] showed that 2 g/d DHA supplementation compared with placebo in individuals with mild to moderate AD did not slow the rate of cognitive and functional decline in patients with mild to moderate AD. A meta-analysis agreed that no benefit has been seen for unimpaired elderly people but concluded that cognitively impaired people without dementia may exhibit cognitive benefit [24]. All these studies were in populations with different characteristics differences in baseline cognitive deficit, baseline DHA levels, exposure and testing methods, and dose; none of these studies reported brain volume or brain atrophy data. The results of these studies are therefore difficult to compare with ours.

In this trial, oral DHA supplementation (2 g/d) for 12 months beneficially affected global cognitive function, specifically participants’ performance on the Information and Digit Span tasks. No significant difference in other neuropsychological tests has been observed between treatment groups at endpoint. Potential mechanisms of action are also highlighted: Information test is a valid indicator of long-term memory, while Digit Span test examines attention/short-term memory [25]. The direct role of diet in the modulation of DHA content in specific regions of the brain at key points is likely an important contributor to memory development and continued normal memory function [26].

The hippocampus is a critical brain region for memory formation and plays important roles in the consolidation of information from short-term memory to long-term memory and spatial navigation [27]. It has been hypothesized that the left hippocampus is more involved in episodic or autobiographical memory while the right hippocampus is implicated in spatial memory [28 –30]. Our results suggest that 12-month DHA supplementation significantly increased hippocampus volume. Notably, we observed a 6.13% volume increase in the left hippocampus, a 1.89% increase in the right hippocampus, and a 0.29% increase in total hippocampus. The right hippocampus appears to be involved in standard object-location memory tests, while the right hippocampus appears to be specifically involved in memory tasks requiring allocentric processing of spatial locations. The need for allocentric processes to guide accurate navigation probably accounts for the right hippocampus involvement in accurate large-scale navigation [31]. By contrast, the left hippocampus appears to be involved in episodic/autobiographical memory, although not necessarily through verbal mediation [32]. However, the precise nature of this role remains widely debated.

DHA may increase hippocampal volume by increasing levels of brain derived neurotrophic factor (BDNF), which stimulates neurogenesis and increase the complexity of the dendritic network [33, 34]. Witte and colleagues provided trial evidence that treatment of normal elderly with long-chain omega-3 fatty acids for 26 weeks increased the amount of gray matter in the left hippocampus and also improved several cognitive measures. Cognitive improvements correlated with increases in omega-3-index, BDNF [35]. Recent recognition of the existence of adult neural stem cells and the ability of these cells to support neurogenesis in adulthood supports the possibility that DHA plays a continuous role in hippocampal maintenance that supports memory functionthroughout life [36]. We are very cautious in our interpretation of this result and emphasize that this facet of the study requires further investigation.

In this study, we found that change in MRI mapped onto concurrent change in test performance in a uniformly significant and biologically sensible manner (Table 4). This tends to validate the use of MRI as a measure of disease progression in therapeutic trials for MCI. This result further supports the validity of MRI based rates of brain atrophy as an independent measure of disease progression in therapeutic MCI trials.

In this trial, results show that DHA supplementation (2 g/day) for 12 months in MCI subjects can significantly improve cognitive function and slow the progression of hippocampal atrophy, even though the increase in serum DHA concentration following treatment was very small. In this regard, it is noteworthy that links between ω-3 fatty acids and vitamin B have been suggested. Micronutrients like folic acid, vitamin B₁₂, and DHA are interlinked and any alterations in any of these components may influence other metabolites in the one carbon cycle leading to altered epigenetic changes in proteins involved in angiogenesis, oxidative stress, and growth factors during placental and fetal growth [37 –39]. Results of retrospective exploratory analysis of data from RCT by Jernerén and colleagues have demonstrated that the beneficial effect of ω-3 fatty acids on brain atrophy may be confined to subjects with good B vitamin status [40]. The recent publication by Oulhaj et al. [41] proved that the effects of the interaction between folic acid, vitamin B₁₂, and DHA on brain atrophy and cognition is consistent with the view that they slow down the disease process in MCI. All these findings show that a combination of EPA, DHA, and folic acid could be of significant benefit in dementia and improve cognitive function. There is a significant interaction effect between baseline ω-3 fatty acid status and B vitamin treatment on the secondary cognitive and clinical outcomes of the trial. These results emphasize the importance of identifying subgroups in clinical trials. They may have public health implications.

The strengths of this trial were its randomized placebo controlled design, the relatively large number of carefully selected participants, that few participants were lost to follow-up, that compliance with supplement use was high, and the cognitive performance tests selected assessed a range of cognitive domains, and have good reliability, internal consistency, and validity.

Several limitations to the present analysis have to be noted. First, in this trial, after 12-month follow-up, hippocampal volumes of participants in the control group keep stable, which is inconsistent with other studies on MCI. In Chinese community settings, health education has been carried out extensively. All these subjects have received non-pharmacological interventions for preventing, reducing, or postponing cognitive decline and dietary recommendations based on a booklet (Guides to Enhance Elderly Memory) in late life. All these factors may be beneficial to cognitive function. Second, the use of amyloid PET brain imaging would have also been ideal as a biomarker for inclusion, but this was not available at our research center at the time of the study design. Third, the current study used methods that determined the blood concentrations of DHA, which might not reveal DHA status in tissues. It is still uncertain whether normalization of plasma DHA status reflects DHA status in the cerebrospinal fluid and cells in the central nervous system.

In summary, we found obvious evidence for a beneficial effect of DHA supplementation on cognitive function and slowing the progression of hippocampal atrophy among elderly subjects with MCI. Future studies with larger sample sizes and greater in-depth characterization of MCI subtypes (i.e., single-versus multi-domain MCI) are needed to corroborate our current findings.

Footnotes

ACKNOWLEDGMENTS

This study was also supported by a grant from 2012 Chinese Nutrition Society (CNS) Nutrition Research Foundation— DSM Research Fund (grant number: 2014-003). The authors thank all of the subjects for their participation.

Authors’ disclosures available online ().

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