Introduction
Age-related cognitive decline leading to dementia poses a societal challenge with major medical, social and economic impact. In the absence of curative treatment for dementia, the focus is on prevention by management of risk factors [
1]. Epidemiological studies show that individuals with elevated homocysteine levels are at greater risk of cognitive decline and dementia, identifying homocysteine as a possible modifiable risk factor [
2].
Elevated homocysteine levels may reflect impaired B-vitamin status [
3]. B-vitamin supplementation to lower homocysteine levels and thereby slowing down cognitive decline would seem a straightforward solution, yet proof of clinical benefits is lacking. While clinical trials show that B-vitamin treatment, usually existing of vitamin B12, B6 and/or folic acid, is effective in lowering homocysteine levels, its effect on slowing down cognitive decline remains inconclusive [
4].
It has been hypothesized that the efficacy of B-vitamin supplementation in slowing cognitive decline is dependent on omega-3 fatty acid status, with B-vitamin supplementation being only effective in individuals with higher omega-3 fatty acid plasma levels. Indeed, results from several post-hoc analyses of B-vitamin trials underline this hypothesis [
5,
6]. Surprisingly, opposite results have been demonstrated as well, with only individuals with lower omega-3 fatty acid status benefitting from B-vitamin supplementation [
7]. This merits further research to disentangle the complex interaction between B-vitamins and omega-3 fatty acids in cognitive ageing.
Thus, the current study further investigates the interaction between B-vitamin supplementation and omega-3 fatty acids with respect to cognitive outcomes in healthy older adults without cognitive complaints. To this end, we investigated if the efficacy of B-vitamin supplementation was dependent on baseline omega-3 fatty acid plasma levels in cognitively healthy older adults in the B-proof trial (B-Vitamins for the Prevention of Osteoporotic Fractures). In the main study of the B-proof trial, no effects of B-vitamins on slowing cognitive decline were observed [
8].
Discussion
This post-hoc analysis of the B-proof trial showed that the efficacy of B-vitamin supplementation on global cognition may be related to plasma DHA levels, but not to plasma total omega-3 fatty acid or EPA levels. Individuals with higher DHA plasma levels benefitted from B-vitamin supplementation, while individuals with lower DHA plasma levels did not. With respect to domain-specific cognitive performance, plasma omega-3 fatty acid combined, DHA or EPA levels separately did not modify the treatment effect of B-vitamins on episodic memory, attention & working memory, information processing speed nor executive functioning.
To date, the interaction between B-vitamins and omega-3 fatty acids in relation to cognitive decline have been investigated in three post-hoc analyses and one clinical trial, with mostly similar [
5,
6,
22,
23] but also contrasting [
7] findings. In line with our results, the VITACOG trial, in which older adults (> 70y) with MCI were supplemented with B-vitamins (folic acid, vitamin B6 and B12) versus placebo for 2 years, showed that omega-3 fatty acid status influenced B-vitamin treatment efficacy. Only individuals with higher plasma omega-3 fatty acid levels showed slower rates of cognitive decline [
5] and brain atrophy [
6] following B-vitamin supplementation. Similarly, a post-hoc analysis of the OmegAD randomized controlled trial showed that adequate levels of both omega-3 fatty acids and B-vitamins are needed [
22]. In the OmegAD trial on the effect of 6 month daily supplementation with EPA and DHA versus placebo in AD patients, only subjects with lower homocysteine status benefited from omega-3 fatty acid supplementation. Further proof comes from a recent randomized controlled trial with a factorial design, in which older adults with MCI were supplemented with placebo, 0.8 mg folic acid, 0.8 mg DHA or a combination of the two daily for 6 months. Combined intervention of folic acid and DHA was more effective in improving cognition compared to supplementation with only folic acid or DHA, adding proof for the interaction from a factorial clinical trial [
23].
Contrary to our current results and previous studies, the post-hoc analysis of the FACIT trial [
7], performed by our group, showed that either sufficient availability of omega-3 fatty acids or B-vitamins may be needed. In this randomized controlled trial on the effect of 3-year daily supplementation with folic acid in cognitively healthy middle-aged adults (50–70 years) with elevated plasma homocysteine, folic acid supplementation was only beneficial in improving cognition in individuals with lower omega-3 fatty acid status, while individuals with higher omega-3 fatty acid status did not experience benefits.
The B-proof, VITACOG and OmegAD trials differed from the FACIT trial on various different aspects that could potentially explain the opposite findings. Importantly, B-proof, VITACOG and OmegAD participants were older, with an average age of over 70, versus an average age of 60 in the FACIT trial. In older individuals, needs for omega-3 fatty acids may be higher due to changes in dietary intake, bioavailability and increased membrane synthesis rates, as discussed previously [
7]. Additionally, baseline omega-3 fatty acid status could be different between study populations, yet no direct comparison can be made due to differences in the fatty acid fractions analyzed, analytical methods and expressed measures. However, the omega-3 fatty acid distribution of our study population is similar to that of other study populations from European (non-Scandinavian) countries [
24]. Vitamin B12 status also differed between study populations, as in the FACIT trial individuals with vitamin B12 deficiency were excluded. In our previous publication, we hypothesized that the contrasting findings of the FACIT trial could be attributed to differences in baseline homocysteine status and/or type of B-vitamin intervention. These factors now seem less probable, as homocysteine levels were both elevated in FACIT and B-proof trials and B-vitamin treatment included only folic acid in both the FACIT trial and in the clinical trial of Li and colleagues [
7,
23]. We strongly encourage researchers with access to data on both B-vitamin and omega-3 fatty acid status to perform post-hoc analyses to be able to better define populations that may benefit from a combination of B-vitamins and omega-3 fatty acids. These results can be the basis for the design of future clinical trials with a factorial design (comparing B-vitamin supplementation only, omega-3 fatty acid supplementation only, combined supplementation versus placebo).
A mechanistic explanation for the finding that B-vitamin supplementation was more effective in individuals with higher DHA status, may involve the interaction of B-vitamins with phospholipid metabolism [
25]. Phosphatidylcholine (PC) plays a crucial role in the transport of omega-3 fatty acids, including DHA, to the brain. Interestingly, B-vitamins can influence the formation of PC [
25]. In the one-carbon metabolism, the B-vitamins folic acid, B6 and B12 play an important role in regulating homocysteine levels. Inadequate B-vitamin status results in elevated levels of homocysteine and its precursor, S-adenosyl homocysteine (SAH) [
26]. In turn, the accumulation of SAH slows down the enzyme phosphatidylethanolamine-N-methyltransferase, which converts phosphatidylethanolamine to PC [
25]. In short, adequate B-vitamin status is needed to ensure sufficient PC production, and thus transport of omega-3 fatty acids to the brain. To support this possible mechanistic explanation, for further research it would be interesting to measure the proportion of omega-3 fatty acids bound to PC.
Here we demonstrated that DHA status, but not EPA or total omega-3 fatty acid status, modified efficacy of B-vitamin supplementation. An explanation may again involve the regulatory role of B-vitamins for omega-3 fatty acid transport to the brain. EPA and DHA have different mechanisms to promote brain health. While EPA is particularly known for its anti-inflammatory effects and is only present in the brain in limited amounts, DHA is the most abundant fatty acid in the brain. This omega-3 fatty acid increases membrane fluidity which is critical for synaptic vesicles and transmission of signals, demonstrating the importance of adequate DHA levels in the brain for proper functioning of the neuronal membrane [
27]. Alternatively, the differences in study populations (cognitively healthy versus MCI) and treatment (dose, combination of B-vitamins versus folic acid) between our study and previous studies, may be responsible for the lack of interaction with EPA in the current study.
The current analyses were limited to the interaction between vitamin B12/folic acid and omega-3 fatty acids, yet there are indications that also other nutrients may be involved. Bowman and colleagues [
28] demonstrated a possible role for vitamin D, by showing that adequate vitamin D status further enhances the protective effect of sufficient homocysteine and omega-3 fatty acid levels in cognitive ageing. Additionally, omega-3 fatty acids may interact with antioxidants: a post-hoc analysis of an antioxidant supplementation trial demonstrated that the association of omega-3 fatty acid intake with cognitive functioning was modulated by a multi-nutrient antioxidant supplement containing ascorbic acid, vitamin E, beta-carotene, selenium and zinc [
29], illustrating the importance of a multi-nutrient approach in slowing down cognitive ageing. For the current study, although we did have dietary and blood nutrient assessment data available, unfortunately we were limited by our sample size to further look into the role of other nutrients in the interaction. Further research with larger sample size should consider incorporating vitamin D status and/or antioxidant intake and status.
A major limitation of the current post-hoc analysis is that we performed exploratory analyses not designed and adequately powered to investigate the modifying potential of omega-3 fatty acid status on B-vitamin supplementation efficacy. The small sample size may be responsible for the lack of findings for domain-specific cognitive functioning, and for the lack of significant differences between the low and high DHA tertiles. Additionally, omega-3 fatty acid status was only determined at baseline and in plasma phospholipids rather than red blood cells, which is a better proxy for long-term omega-3 fatty acid status. However, we assume that our measurements do represent longer-term status as dietary patterns (and thus omega-3 intake) in older adults are reasonably stable over time [
30], and other factors that may influence variation (e.g. geographic and genetic reasons) also have remained stable. Though the 2-year duration of the trial is a fairly short period of time to recognize cognitive deteriorations in healthy older individuals, it can still be considered a strength as an intervention period of 2 years is quite long in comparison with other nutrition intervention studies to slow cognitive decline. Another strength of the study is the use of an extensive cognitive test battery with a focus on domain-specific tests, instead of general tests such as the MMSE or Telephone Interview for Cognitive Status.
In conclusion, this post-hoc analysis demonstrated that B-vitamin supplementation effectiveness in cognitive ageing is related to plasma DHA levels, with older adults with higher plasma DHA levels benefitting more from B-vitamin supplementation. The results support earlier observations that positive effects of B-vitamins in cognitive ageing may be subgroup-specific. Further research is needed to optimize defining subgroups that may be susceptible for B-vitamin supplementation, and subsequently to confirm this finding in a clinical trial with a factorial design.