Designing Newer Omega-3 Supplementation Trials for Cognitive Outcomes: A Systematic Review Guided Analysis

Abstract

Background:

Epidemiology cohorts reveal associations between levels or intake of omega-3 polyunsaturated fatty acids (n-3 PUFA) and a lower risk of Alzheimer’s disease (AD). However, the results of randomized clinical trials have been inconsistent.

Objective:

A systematic review was performed to understand the effects of n-3 PUFA supplementation on cognition in adults. The objective was to present suggestions for new study designs to translate epidemiological findings into effective clinical trials.

Methods:

A database search was conducted on PubMed (MEDLINE) and Web of Science to retrieve articles published between 2000 and 2023 that evaluated the effects of n-3 PUFA supplementation on cognitive function. Subsequently, the search results were filtered to collect randomized controlled trials with 100 or more participants, n-3 PUFA supplementation was one of the interventions, cognition was an outcome of interest, and participants were at least 18 years of age.

Results:

A total of 24 articles met the inclusion criteria. In 5 of the 24 studies reviewed, supplementation with n-3 PUFAs improved cognition. All four trials in persons with AD reported null outcomes. Most of the n-3 PUFA studies in cognitively normal individuals or participants with mild cognitive impairment were null, not powered to detect small effect sizes, or selected participants without dementia risk factors.

Conclusions:

We recommend that newer n-3 PUFA supplement trials targeting AD prevention be personalized. For the general population, the null hypothesis appears to be correct, and future interventions are needed to identify and test dietary patterns that include PUFA-rich food rather than supplements.

Keywords

Alzheimer’s disease cognitive function omega-3 supplementation

INTRODUCTION

Omega-3 (n-3) fatty acids, particularly n-3 long-chain polyunsaturated fatty acids (n-3 PUFAs), have gained significant attention because of their potential role in supporting cognitive function in Alzheimer’s disease (AD), as revealed in several high-quality cohort studies [1]. Consumption of the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increases their concentrations in erythrocyte membranes [2] and cerebrospinal fluid [3]. The concentration of n-3 PUFAs in plasma phospholipids correlates with the size of the entorhinal cortex [4], an area of the brain affected early in AD.

N-3 PUFAs are involved in brain physiology and biological processes such as membrane fluidity, cerebral blood flow, neurotransmitter function [5]. Additionally, n-3 PUFAs play a role in neurogenesis and maintenance of healthy axons and synaptic structures [6]. Although some studies indicate that n-3 PUFAs are essential for early brain development and function [7], the role of n-3 PUFAs in promoting cognition, neuronal preservation, and protection against neurodegeneration is not clear [8]. Most clinical trials have been unable to draw conclusive evidence on the impact of n-3 PUFA intake on cognitive function later in life [9]. Mechanistically, n-3 PUFAs have anti-inflammatory and neuroprotective properties that may improve neural efficiency functions, such as processing speed, reaction time, and certain elements of executive and memory functions [8].

Recent systematic reviews of randomized controlled trials (RCTs) have reported mixed effects of n-3 PUFAs on cognition but have identified subgroups or particular interventions as responsive [10]. We conducted a systematic review of n-3 PUFA supplementation clinical trials for cognitive function in adults older than 18 years, with cognition as one of the major outcomes. The purpose of this review is to identify methodological limitations of past trials that can inform future trial designs and translate them into more conclusive clinical recommendations.

SEARCH METHODOLOGY

A database search was performed on PubMed (MEDLINE) and Web of Science using the initial criteria for articles published in English from 2000 to 2023 with the following search terms: (omega-3s OR omega-3 fatty acids OR ω-3 Fatty Acid OR n-3 fatty acid OR eicosapentaenoic acid OR EPA OR docosahexaenoic acid OR DHA) AND cognitive function AND trials. The search yielded a total of 603 articles, 358 corresponding to Web of Science and 245 to PubMed, in which the human and RCT exclusion criteria were applied.

In this review, we selected RCTs with 100 or more participants, with an n-3 PUFA supplementation arm as one of the interventions, cognition as an outcome of interest, individuals aged 18 years or older, and participants with Alzheimer’s disease (AD), mild cognitive impairment (MCI), or cognitive normal (CN). Nutrition epidemiology indicates that the relationship between n-3 PUFA intake and cognition has a small or medium effect size at best [11] (discussed below). Therefore, we excluded trials with a sample size less than 100, given the lack of power to detect small or medium effect sizes of n-3 PUFAs on cognition.

All 603 articles were revised based on title, and a total of 327 articles were excluded according to the following criteria: duplicates, retracted, involving participants under 18 years old, and participants with other cognitive diseases (e.g., depression, schizophrenia, stroke, etc.) than MCI or AD. Subsequently, 276 articles were screened based on the abstracts or manuscripts based on the inclusion criteria. Figure 1 summarizes the selection flowchart. We excluded two articles due to methodological quality [12, 13]. Finally, 24 studies were selected for analysis.

Fig. 1

Literature search flow diagram.

RESULTS

Of the 24 studies reviewed, only five (21%) reported positive effects of n-3 PUFAs supplementation on predefined cognitive outcomes (Table 1). The remaining 19 studies (79%) did not find any beneficial effects of n-3 PUFA consumption on selected cognitive measures. All four trials involving participants with AD [14 –17] reported null results, suggesting that n-3 PUFAs have no effects once clinical AD develops. Among the 20 trials in non-demented populations, two studies in younger adults (age 25–50) [18, 19] showed cognitive improvements with n-3 PUFA supplementation.

Table 1

Summary of omega-3 supplementation trials on cognition

Study name	Total n and n-3 group	Demographics	Omega-3s supplementation	Length of intervention	Effect on cognition (primary outcome)
Bischoff-Ferrari et al. 2020 [56]	2157/269	CN Age: 70 or older 61.7% women Europe	•1000 mg/day of omega-3s, including 330 mg of EPA plus 660 mg of DHA from marine algae	36 months	None
Lin et al. 2022 [14]**	163/41	MCI and/or AD Age: 65 to 95 34% women Taiwan	•700 mg/day of DHA, •1600 mg/day of EPA •800 mg/day of EPA plus 350 mg/day DHA	24 months	None
Patan et al. 2021 [18]**	337/113	CN Age: 25–49 70% women UK	•DHA-rich oil: 900 mg/day of DHA plus 270 mg/day of EPA •EPA-rich oil: 360 mg/day of DHA plus 900 mg/day of EPA	6 months	Positive
Maltais et al. 2022 [57]	243/120	CN Age: 20 to 80 64.5% female Canada	•1700 mg/day of EPA plus 800 mg/day of DHA in ethyl ester form	6 months	None
Li et al. 2021 [15]**	240/60	MCI Age: 60 or older 35% female China	•800 mg/day of DHA	6 months	None
Kuszewski et al. 2020 [58]**	152/32	CN Age: 50–80 54% female Australia	•2000 mg/day of DHA plus 400 mg/day of EPA	4 months	None
Leckie et al. 2020 [59]	271/134	CN Age: 30–54 56.5% female USA	•1000 mg/day of EPA plus 400 mg/day of DHA	4 months	None
Kang et al. 2022 [47]**	3424/1699	CN Age: 60–91 58.9% female USA	•1000 mg/day marine n-3 fatty acids, including 460 mg/day of EPA plus 380 mg/day DHA	34.5 months	None
Malik et al. 2021 [20]**	285/143	CN Age: 37–80 16.8% female USA	•1860 mg/day of EPA plus 1500 mg/day of DHA	30 months	Positive
Andrieu et al. 2017 [50]**	1525/381	CN Age: 70 or older 64% female France and Monaco	•800 mg/day of DHA plus 225 mg of EPA	36 months	None
Dangour et al. 2010 [60]	867/434	CN Age: 70–79 45% female UK	•200 mg/day of EPA plus 500 mg/day of DHA	24 months	None
Dretsch et al. 2014 [61]	106/44	CN Age: 18–55 8% female USA	•2500 mg/day of EPA plus DHA ethyl esters	2 months	None
Freund-Levi et al. 2006 [16]**	204/103	AD Median age: 74 54% female Sweden	•1720 mg/day of DHA plus 600 mg/day of EPA	12 months	None
Jackson et al. 2012 [62]**	140/46	CN Age: 18–35 67% female UK	•DHA rich = 450 mg/day DHA plus 90 mg/day of EPA •EPA rich = 300 mg/day of EPA plus 200 mg/day of DHA	3 months	None
Quinn et al. 2010 [17]**	402/238	AD Median age: 76 52.2% female USA	•2000 mg/day, including 45% to 55% of DHA algal-derived by weight	18 months	None
Stonehouse et al. 2013 [19]**	228/115	CN Age:18–45 63.5% female New Zealand	•1160 mg/day of DHA plus 170 mg/day of EPA	6 months	Positive
Jackson et al. 2016 [63]	100/33	CN Age: 50–70 62% female UK	•2000 mg/day, including 896 mg of DHA plus 128 mg of EPA	6 months	None
Yurko-Mauro et al. 2010 [21]**	485/242	CN Age: 55 or older 58% female USA	•900 mg/day of algal-derived DHA	6 months	Positive
Pase et al. 2015 [64]	160/41	CN Age: 50–70 53.1% female Australia	•480 mg/day EPA plus 480 mg/day DHA	4 months	None
van de Rest et al. 2008 [43]**	302/96	CN Age: 65 or older 45% female Netherlands	•400 mg/day of EPA and DHA 1800 mg/day of EPA and DHA	6 months	None
Geleijnse et al. 2012 [65]**	4838/1192	CN Age: 60–80 22% female Netherlands	•400 mg/day of EPA–DHA •2000 mg/day of ALA, both EPA–DHA and ALA	40 months	None
Tokuda et al. 2015 [22]**	115/57	CN Age: 55–64 0% female Japan	•1,033 mg/day, including 300 mg of DHA, 100 mg of EPA, and 120 mg of ARA	1 month	Positive
Marriott et al. 2021 [66]	555/276	CN Age: 20–35 1.4% female USA	•2300 mg/day of omega-3 krill oil with an EPA and DHA ratio of approximately 2:1	5 months	None
Danthiir et al. 2018 [67]	403/203	CN Age: 65–90 54% female	•1720 mg DHA and 600 mg EPA	18 months	None

CN, cognitive normal; MCI, mild cognitive impairment; AD, Alzheimer’s disease; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; ALA, A-linolenic acid; ARA, arachidonic acid. Additional comments: : Three Omega-3s supplementation groups: DHA (n = 41), EPA (n = 40) and DHA + EPA (n = 42). [18]: Supplementation with EPA improved global cognitive function and outperformed the oil enriched with DHA. Two Omega-3s supplementation groups: DHA-rich (n = 113) and EPA-rich (n = 112). [15]: DHA+ curcumin and DHA alone slightly improved cognitive function. [58] Improvement in processing speed performance in old and obese male individuals. [47]: Cognition was assessed by phone. [20]: High dose Omega-3s in adults with coronary artery disease. [50]: Individuals with spontaneous memory complaints, limitations in one instrumental activity of daily living, or slow gait speed. [16]: No significant effect on cognition in patients with mild-to-moderate AD. However, patients with mild AD showed positive results. [62]: Two Omega-3s supplementation groups: DHA (n = 46) and EPA (n = 46). [17]: Mild to moderate Alzheimer disease. [19]: Healthy young adults whose habitual diet was low in DHA showed improvement in memory and memory reaction times. [21]: Individuals with subjective memory complaints demonstrate improvements in learning and memory function in age-related cognitive decline. [43]: Two Omega-3s supplementation groups: Low dose (n = 96) and High dose (n = 100). [65]: Coronary heart disease patients. Only 2911 were included in the analysis. Two Omega-3s supplementation groups: EPA + DHA (n = 1192) and EPA + DHA + ALA (n = 1212). [22]: All Japanese men. Cognitive processing speed was assessed using P300 latency.

Interestingly, 80% of the positive trials (4 out of 5) performed a minimum of a 6-month intervention [18 –21], supporting the concept that longer intervention periods are more beneficial in impacting cognitive outcomes, as explained by the long half-life of omega-3s in the brain. Moreover, four out of the five positive studies conducted interventions with n-3 s doses equal to or exceeding 1 g per day [18–20 , 22], which supports that doses higher than 1 g per day may be needed to achieve adequate brain delivery and elicit cognitive changes. The majority of trials accounted for baseline n-3 PUFA consumption. However, the majority of trials were not powered to observe small effect sizes. A review of the major factors that influenced the response to n-3 PUFAs in these trials is presented in Table 2. Overall, the reviewed trials failed to demonstrate the benefits of n-3 PUFA supplementation on cognition in a heterogeneous population, particularly in older individuals with multiple cognitive outcomes, and especially in those with established AD dementia.

Table 2

Factors that help explain the response to n-3 PUFA trials

Study:	Low omega-3 intake	No AD	Omega-3 dose > 1 g per day	Intervention length > 6 month	Age stratified [Age range]	Dementia risk factors	Powered to detect small effect sizes
Bischoff-Ferrari et al. 2020 [56]	X	X	X	X			X
Lin et al. 2022 [14]	X		X	X		X
Patan et al. 2021 [18]	X	X	X	X	X [25–49]
Maltais et al. 2022 [57]	X	X	X	X		X
Li et al. 2021 [15]	X	X		X
Kuszewski et al. 2020 [58]	X	X	X
Leckie et al. 2020 [59]	X	X	X		X [30–54]
Kang et al. 2022 [47]	X	X	X	X			X
Malik et al. 2021 [20]		X	X	X		X
Andrieu et al. 2017 [50]		X	X	X			X
Dangour et al. 2010 [60]	X	X		X	X [70–79]
Dretsch et al. 2014 [61]	X	X	X
Freund-Levi et al. 2006 [16]			X	X		X
Jackson et. al 2012 [62]	X	X			X [18–35]
Quinn et al. 2010 [17]	X		X	X		X
Stonehouse et al. 2013 [19]	X	X	X	X		X
Jackson et.al 2016 [63]	X	X	X	X	X [50–70]
Yurko-Mauro et.al 2010 [21]	X	X		X		X
Pase et al. 2015 [64]	X	X			X [50–70]
van de Rest et al. 2008 [43]	X	X	X	X
Geleijnse et al. 2012 [65]		X	X	X	X [60–80]	X	X
Tokuda et al. 2015 [22]	X	X	X		X [55–64]
Marriott et al. 2021 [66]	X	X	X		X [20–35]
Danthiir et al. 2018 [67]		X	X	X	X [65–90]

X indicates they did meet criteria.

Factors that affect the response to n-3 PUFA supplementation in clinical trials

Given that the majority of clinical trials involving n-3 PUFA supplements and cognition reported null findings, we must consider the possibility that the null hypothesis is correct. That is, there is no true difference between n-3 PUFA supplements and placebo in AD prevention or treatment in the older general population. This is further supported by GWAS studies, in which genetic variation in the enzymes that regulate PUFA levels was not associated with a change in the risk of AD [23]. However, it is plausible that these clinical trial interventions were not long enough to find a true effect, given that the prodromal phase of AD can last 10–20 years. In addition, populations studied in the majority of n-3 PUFA trials were heterogeneous, as AD pathology in dementia cases ranged between 19% and 67% [24]. We propose that carefully selected populations may benefit from n-3 PUFA supplementation and present some of the factors that can help define these individuals for personalized intervention with either an n-3 PUFA supplement or an n-3 PUFA dietary pattern.

Baseline omega-3 status

Many epidemiological studies have reported an association between lower intake or levels of omega-3s fatty acids and worse cognitive outcomes, including greater dementia diagnosis, in populations consuming a Western diet high in saturated fats and sugars. This association between DHA intake and dementia outcomes is more evident when comparing individuals who do not consume fatty fish to those who consume one or two servings [25]. However, the association between baseline n-3 PUFA-enriched dietary intake and dementia differs according to the population characteristics. Some vegetarian and vegan populations that do not consume red meat [26], chicken, or fish do not have an increased risk of dementia despite lower n-3 PUFA levels, suggesting compensation for lower n-3 PUFA intake. It is plausible that such populations have lower vascular risk factors and less vascular dementia; however, further research is needed. We recommend that future trials select participants based on low baseline n-3 PUFA levels in the context of a Western dietary lifestyle.

APOE4 and Alzheimer’s disease stage

Younger persons carrying the APOE4 allele show an increased uptake of plasma-derived DHA, using PET scan imaging studies [27], implying “cognitive stress” with an n-3 PUFA-deficient diet at an early age. The first changes in AD are observed in the locus coeruleus. The earliest microscopic events are fission and fusion of the mitochondrial membranes [28]. The antioxidant effects of n-3 PUFAs protect against early stage oxidative stress-related changes. However, as patients develop MCI or AD, the pathways that metabolize n-3 PUFAs become dysregulated. All 4 trials reviewed with participants with AD dementia reported null outcomes after n-3 PUFA supplementation. Once AD neuropathology advances, n-3 PUFA brain metabolism is altered and any potential benefit from n-3 PUFA intake in the brain is lost. We previously reported alterations in n-3 and n-6 brain PUFA metabolism, indicative of n-6 conversion to eicosanoids, in postmortem brain tissues of patients with AD dementia [29]. These changes are likely driven by the activation of lipid catabolic enzymes such as calcium-dependent phospholipase A2 or cPLA2 [30]. Chronic inflammation in AD depletes anti-inflammatory lipid mediators that result from the metabolism of DHA, EPA, and docosapentaenoic acid, tilting the balance toward AA-derived eicosanoids, which have potent inflammatory effects [29]. N-3 PUFAs are also susceptible to oxidation [31] both in vivo and in vitro. Once catabolic pathways are activated during the disease process, drugs that restore n-3 PUFA metabolism (such as cPLA2 inhibitors) are likely more effective than n-3 PUFAs supplementation [32]. We recommend excluding patients with AD from future n-3 PUFA trials, and targeting high-risk populations at an early age.

Omega-3 dose > 1 gram per day

Overall, n-3 PUFA doses of less than 1 g/day are not likely to significantly increase brain levels within a short period of time of supplementation. In the DHA pilot trial, we observed a 28% increase in CSF DHA levels after 2 g of algal DHA per day over 6 months compared with placebo, despite a > 200% increase in plasma DHA levels. These findings suggest that lower doses (<1 g of DHA per day) are likely to be associated with lower brain DHA delivery. Dementia prevention trials using omega-3 supplementation doses equal to or lower than 1 g/day may have reduced brain effects [33], particularly in APOE4 carriers owing to lower brain uptake or greater brain consumption [3]. Although omega-3 fatty acids have been associated with greater total brain and hippocampal volumes in some studies [6], higher doses of n-3 PUFAs may be necessary to achieve significant increases in brain levels within a shorter time frame. Overall, we recommend supplementation of at least 1 g DHA or EPA (or mixed) per day. The clinical evidence regarding whether DHA or EPA in triglyceride or phospholipid form is superior remains inconclusive.

Intervention length

Given the long half-life of DHA in the brain [34], interventions targeting AD dementia that are less than 6 months in duration are unlikely to directly modify brain structure. N-3 PUFAs, particularly DHA, have a long half-life in the brain of around 2.5 years, and their effects on brain structure may take time to manifest [35]. Therefore, long-term interventions with lower doses of omega-3 fatty acids may not be sufficient to produce significant changes in brain levels or structural changes such as brain volume. Short-term interventions may have other effects, such as anti-inflammatory effects at the gut level [36], which may be associated with greater neuronal efficiency [37], as described in the section below. Overall, a supplementation duration longer than 6 months is recommended.

Population heterogeneity

One of the key factors that can help explain the variability in the response to n-3 PUFA supplementation on cognitive outcomes is the heterogeneity of the study population. Past trials based on clinical assessment tools have misclassified AD from other causes of dementia. The development of AD plasma biomarkers [38] will likely refine the recruited population. Another relevant population included those who carried the APOE4 allele. APOE4 carriers comprise more than 50% of patients with dementia and increase the risk of cerebral amyloidosis. APOE4 carriers activate enzymatic processes that catabolize n-3 and n-6 PUFAs [30], and this population may benefit from n-3 PUFA supplementation when started earlier than the non APOE4 carriers. In an Alzheimer’s Disease Cooperative Study-sponsored clinical trial [17], 2 g of DHA per day over 18 months did not significantly change the primary outcome ADAS-cog in patients with mild AD scores. However, a preplanned analysis revealed that the response differed by APOE genotype: ADAS-cog scores improved following supplementation in APOE4 non-carriers but not in APOE4 carriers. This finding underscores the importance of selecting high-risk populations without evidence of AD dementia or with responsiveness to interventions.

Importance of age stratification for outcome selection

To better define or select the cognitive outcomes that respond to n-3 PUFA supplementation, it is essential to understand the normal and pathological changes in cognition that occur during aging, in AD, and in low n-3 PUFA states in vulnerable groups. Starting in midlife and before the age of 60, a linear decline in processing speed has been observed [39]. After 60 years, there is evidence of an accelerated decline in both executive function and memory. Late-onset AD typically appears after 60 years of age and further increases the rate of memory and executive functioning decline. Therefore, we recommend that trials should be stratified by age group and tailored to the specific cognitive outcomes of the target population [40].

Cardiovascular dementia risk factors

Lifestyle interventions targeting populations with dementia risk factors are more likely to show benefits. One example is the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER trial), in which a combination of exercise and dietary advice (increased seafood consumption) was associated with cognitive benefits in individuals with cardiovascular risk factors [41]. Non-modifiable risk factors include age, family history, and genetic factors such as APOE4. Modifiable risk factors include cardiovascular disease (CVD) risk factors, such as hypertension, midlife obesity and diabetes, loneliness, and lower education. Populations without dementia risk factors will likely show limited clinical disease progression, limiting the ability of the intervention to identify differences between the control and treatment arms when differences exist. We recommend taking advantage of advances in plasma AD biomarkers (such as pTau 217 [42]) to better select participants at risk of AD dementia (cerebral amyloid accumulation) before the onset of clinical dementia.

N-3 Mechanisms of action and matching of clinical outcomes

Neuroinflammation is implicated in several brain diseases, including depression, attention-deficit/hyperactivity disorder, and neurodegenerative diseases, such as AD, and lowers the efficiency of neurotransmission. N-3 PUFAs have anti-inflammatory properties. Increased n-3 PUFA consumption may alter neuronal membrane fluidity and phospholipid composition, which may affect neurotransmission and processing speed [8]. This was evident in some of the trials reviewed in younger populations [19 , 43]. Among n-3 PUFAs, EPA intake appears to be more advantageous than DHA in reducing “brain effort” relative to cognitive performance [37]. Therefore, it is important to consider various factors and appropriate measures to accurately evaluate neuronal efficiency and reaction time. Among the studies selected, neuronal efficiency and reaction time were evaluated using various measures, including latency, speed, and reaction time for tests of working memory and attention. In the context of cognitive processes, latency refers to the time between the initiation of a stimulus and the start of the corresponding response, while speed refers to the rate at which the response is executed. Reaction time, on the other hand, refers to the amount of time it takes to respond to a stimulus. These measures appear to be responsive to n-3 PUFA supplementation in selected populations, such as young APOE4 carriers [19].

Sample sizes sufficient to detect medium and small effect sizes

When considering the cognitive domains affected by AD progression, clinical trials must have sufficient sample sizes to detect small or medium effect sizes. The Three-City (3 C) Study is a longitudinal cohort study of the relationship between vascular diseases and dementia in persons aged 65 years and older. A total of 9,294 participants (3,649 men and 5,645 women) were recruited from three French cities: Bordeaux (2,104), Dijon (4,931), and Montpellier (2,259) [44]. The difference between the bottom and top long-chain omega-3 level quintiles was equivalent to adding 1.4 servings of fish to one’s diet every week (equivalent to approximately 750 mg/day EPA + DHA). Based on these findings, more than 3,000 cognitively healthy older participants would be needed in a 5-year trial comparing EPA + DHA versus placebo (with α= 5% and 80% power) to show an effect size on dementia incidence equivalent to a 48% reduction in risk (95% CI 10–58%) between the top and bottom quintiles of summed EPA and DHA in this cohort [45] (reviewed in [46]). A study with a sufficient sample size is the VITAL clinical trial that randomized 4218 generally healthy older participants followed for 2 to 3 years with marine n-3 PUFA supplementation (1 g/day, including 840 mg of EPA + DHA) versus placebo (olive oil with negligible amount of marine n-3 fats), and the intervention was not associated with a change in cognitive decline between the treatment groups [47]. One of the limitations of VITAL is that it was not designed to measure the progression to dementia, relying on telephone-based cognitive tests. Nevertheless, we must also consider that the association between n-3 PUFA levels and cognitive outcomes in epidemiological cohorts confound n-3 PUFA dietary patterns, as discussed below.

Gut microbiota composition and the response to n-3 PUFAs

Analysis of the gut microbiota and metabolome may guide n-3 PUFA supplementation trials [48]. Western diets promote the formation of inflammatory microbiota taxa [49], and n-3 PUFA supplementation has promising effects in remodeling the gut microbiome to anti-inflammatory taxa [36]. Therefore, it is plausible to identify who might benefit from such interventions based on the ability of n-3 PUFAs to remodel the gut microbiome.

Single versus multidomain interventions

It is likely that multimodal or multi-nutrient interventions are more effective than a single supplementation on cognitive outcomes. Examples include the Multidomain Alzheimer Preventive Trial (MAPT), where the combination of n-3 PUFA supplementation with a multimodal intervention consisting of nutritional counseling, physical exercise, and cognitive stimulation on cognition wasw examined [50]. In MAPT, the intervention was not effective. However, in an analysis of a subgroup of individuals with AD risk (positive blood amyloid levels), the multimodal intervention appeared to have positive effects compared to the n-3 PUFA supplementation alone [51]. Another approach is a multi-nutrient approach, such as the LipiDiDiet, where higher doses of n-3 PUFAs are combined with ridine-5’-monophosphate, choline, phospholipids, selenium, and vitamins B, C, and E [52]. In the LipiDiDiet, the intervention was associated with significant improvements in cognitive outcomes and brain atrophy over a period of 36 months [53]. Among these nutrients, the combination of n-3 PUFAs with vitamin Bs in participants with high homocysteine levels appeared to show the greatest benefit on brain volume [54]. Additional mechanistic studies are needed to better define and select multimodal or multinutrient intervention components.

PreventE4: An example of a personalized intervention

PreventE4 is a randomized clinical trial of 2 g of DHA versus placebo in 368 individuals stratified by APOE4 and enriched for CVD risk factors [35]. PreventE4 clinical trial is testing whether high-dose (2 grams per day) DHA supplementation plays a role in APOE4 carriers when started before clinical dementia. The primary outcome is the change in CSF DHA levels according to the APOE genotype. Secondary outcomes include brain imaging biomarkers, and exploratory outcomes included cognition and gut microbiome, plasma/CSF metabolome. All participants in PreventE4 receive high dose vitamin B supplementation to eliminate any confounding effects of high homocysteine levels on the outcomes. The trial is expected to be reported in 2025. However, the mean age of individuals in PreventE4 is 65. Whether this age is too late for an AD prevention study is yet to be determined. PreventE4 was also not powered to detect small effect sizes on cognition. Future personalized trials may select younger APOE4 carriers with low n-3 index, CVD risk factors, but no evidence of dementia to test longer duration interventions with high-dose n-3 PUFA supplements or alternatively, practical and scalable dietary patterns, or multimodal interventions.

Defining an n-3 PUFA dietary pattern

It is biologically plausible that n-3 PUFAs obtained from seafood work synergistically with other components of the diet (e.g., phospholipids, minerals, and vitamins), lifestyle (exercise, lack of smoking, social connections) and metabolic status (obesity, diabetes, or hypertension) to improve brain function. In such a scenario, defining an n-3 PUFA diet or lifestyle pattern at midlife is useful [55]. This is a promising area and merits active investigation to define the active elements or components of such patterns that drive cognitive benefit. There is an opportunity to design cluster randomized trials in which certain communities with a low n-3 PUFA dietary pattern at baseline are randomized to an n-3 PUFA dietary pattern versus a matched control pattern that does not include active intervention elements. Certain biomarkers (RBC n-3 index) can be utilized to monitor adherence to the patterns and sensitive brain outcomes (imaging or sensitive cognitive tests) used for a population without dementia. These “precision” population studies would better translate epidemiology into clinical recommendations.

DESIGNING FUTURE N-3 PUFA CLINICAL TRIALS

In summary, we propose the following recommendations for designing future n-3 PUFA trials.

Select cognitively normal participants enriched with dementia risk factors: for example, based on APOE4, family history of dementia, and plasma pTau181 or 217 levels.

Select individuals with limited n-3 PUFA consumption or levels in the context of a Western lifestyle dietary pattern.

Utilize a high dose (>1 g) of DHA/EPA per day.

Biomarkers can be used to guide the responses to supplementation. For example, changes in the gut microbiome after supplementation can help identify whether participants are responsive to n-3 PUFA supplementation. Participants with persistent gut dysbiosis after supplementation may not show any clinical benefit.

Combination interventions, such as multi-domain, multi-nutrient, or dietary patterns, are likely more effective than n-3 PUFA supplementation alone. The selection of multi-domain or multi-nutrient combinations should be based on mechanistic experiments.

Select primary outcomes that match the n-3 PUFAs mechanism of action. The reviewed studies with positive outcomes support neural efficiency and executive function makers in cognitively non-demented at-risk groups such as APOE4 carriers.

The study duration needs to match the selected outcomes. Neural efficiency measures, such as processing speed or certain elements of executive function, may respond to short-term (e.g., six months) interventions. For AD-related outcomes, longer durations (≥36 months) are needed to observe meaningful changes in a population with dementia risk factors.

AUTHOR CONTRIBUTIONS

Hussein Yassine (Conceptualization; Funding acquisition; Methodology; Writing – original draft; Writing – review & editing); A. Sofia Carrasco (Methodology, Writing – review & editing); Daniel Badie (Methodology).

Footnotes

ACKNOWLEDGMENTS

The authors have no acknowledgments to report.

FUNDING

HNY holds the Kenneth and Bette Volk Endowed Chair of Neurology at USC. HNY is supported by RF1AG076124, RF1AG078362, R01AG067063, R01AG054434, R01AG055770, R21AG056518, and P30AG066530 from the National Institute on Aging (NIA), GC-201711-2014197 from the Alzheimer’s Drug Discovery Foundation (ADDF), and generous donations from the Vranos and Tiny Foundations and from Ms. Lynne Nauss. USC CTSI UL1TR001855 from the National Center for Advancing Translational Science (NCATS) of the U.S. National Institutes of Health supported recruitment for this study. The microbiome and metabolome sub-study of PreventE4 was supported by the Alzheimer’s Gut Microbiome Project (U19AG063744), funded by NIA.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

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