Associations between Serum Omega-3 Fatty Acid Levels and Cognitive Functions among Community-Dwelling Octogenarians in Okinawa,Japan: The KOCOA Study

Abstract

Background:

Epidemiological studies have found frequent consumption of fatty fish is protective against cognitive decline. However, the association between circulating omega-3 polyunsaturated fatty acid (PUFA) levels and cognitive functions among the oldest old is not well known.

Objective:

To examine the association between serum PUFA levels and cognitive function among community-dwelling, non-demented elderly aged over 80 years old.

Methods:

The data came from the Keys to Optimal Cognitive Aging (KOCOA) study; an ongoing cohort of relatively healthy volunteers aged over 80 years old, living in Okinawa, Japan. One hundred eighty five participants (mean age 84.1±3.4 years) assessed in 2011 who were free from frank dementia (defined as Clinical Dementia Rating <1.0) were used for the current cross-sectional study. We examined whether serum omega-3 PUFAs (docosahexaenoic acid [DHA] and eicosapentaenoic acid [EPA]), arachidonic acid (AA), EPA/AA ratio, DHA/AA ratio, and DHA+EPA are associated with (1) age and (2) global cognitive function (Japanese MMSE) and executive function (Verbal Fluency Letter). Data was analyzed univariately by t-test and multivariately by cumulative logistic regression models controlling for age, gender, years of education, obesity, hypertension, diabetes, and dyslipidemia.

Results:

Serum DHA levels decreased with increasing age (p = 0.04). Higher global cognitive function was associated with higher levels of serum EPA (p = 0.03) and DHA + EPA (p = 0.03) after controlling for confounders.

Conclusions:

Higher serum EPA and DHA + EPA levels were independently associated with better scores on global cognitive function among the oldest old, free from dementia. Longitudinal follow-up studies are warranted.

Keywords

Cognitive function DHA EPA KOCOA Okinawa oldest old non-demented subjects PUFA

INTRODUCTION

According to the recent estimate by the Japanese Ministry of Health, Labour and Welfare, the proportion of those aged 65 years and older in Japan was 24% in 2012 and is projected to increase rapidly in the coming decades, up to 39% by 2050 [1, 2]. As populations age, the prevalence of those with cognitive impairment will increase sharply. Identifying therapies that can delay cognitive decline or prevent dementia onset are urgently needed. There is consistent epidemiological evidence showing a decreased risk of dementia among those who consume fish versus those who do not [3 –8]. Studies have also shown higher dietary intake of omega-3 polyunsaturated fatty acids (PUFAs), estimated from a food intake questionnaire as well as circulating omega-3 fatty acid levels, are associated with reduced risk for cognitive decline [9, 10] suggesting a protective effect of dietary omega-3 fatty acid intake on cognitive decline. In animal studies, omega-3 fatty acid administration has been found to improve learning ability [11, 12]. Several studies have also reported that PUFAs have shown promise in reducing cardiovascular disease risk factors including hypertension, hypertriglyceridemia, arrhythmia, inflammation, and coronary calcification [13 –15], although not all studies has found these benefits [16, 17]. There have been several reports indicating that risk factors of late life cognitive decline and dementia could be different than those found in mid-life. For example, the protective effects of lower triglyceride levels, lower blood pressure, and lower BMI in midlife on cognitive decline appear to be less robust or even reversed in the oldest old populations [18 –22]. We previously reported that metabolic syndrome is not associated with cognitive decline among the oldest old, using a sub-group of the same cohort examined in this study [23].

We investigated the association between PUFAs and cognitive function among healthy volunteers aged 80 years and older, free from frank dementia living in Okinawa, Japan, the southern island prefecture (state) of Japan. The island has one of the highest concentrations of centenarians in the world and is known for healthy aging [24]. We examined global cognitive function as well as executive function based on previous study findings [10, 25].

METHODS

Study design and participants

Data came from a cross-sectional study, part of a prospective pilot cohort study of community-dwelling adults aged 80 years and older living in Okinawa, Japan called the Key to Optimal Cognitive Aging Project (KOCOA). A detailed description of the recruitment process has been described elsewhere [26, 27]. Briefly, researchers visited 21 senior centers, explained the study protocol, and asked them to participate in the study. A request to join the study was made at the conclusion of each presentation. We recruited community-dwelling adults aged 80 years and older who needed no or partial assistance for all instrumental activities of daily living. The baseline recruitment occurred in 2007 and participants were assessed annually until 2011. Between November 2011 and April 2012, new participants were recruited using the same methods as before. These participants and those followed from the original cohort were used in the current analyses. Out of 191 participants (60 from the original cohort) who consented to the study and completed a face-to-face interview, four were taking anticholinesterase agents and two had frank dementia defined by Clinical Dementia Rating Scale (CDR) [28]≥1 and thus were excluded from the current study. The remaining 185 participants (CDR = 0 or 0.5) were assessed for their fatty acid levels using serum samples.

This study was approved by the Ethics Committee of the University of the Ryukyus, and the Institutional Review Board at Oregon Health & Science University. Informed consent was obtained from all participants prior to enrollment in the study.

Cognitive measures

Cognitive function was assessed by a trained interviewer using the Japanese version of the Mini-Mental State Examination (J-MMSE) [29, 30]. The J-MMSE is a measure of global cognitive function and ranges from 0 to 30 points, with higher scores representing better cognitive function. It takes approximately 10 minutes to administer and includes questions on orientation to time and place, attention and calculation, recall, language, and visual construction. The Verbal Fluency Letters (VFL) test was used to measure executive function. This test requires the participant to generate as many words as they can beginning with the letter “Ka” (in Japanese) in 60 seconds, and has been validated as an equivalent test to Initial Letter Fluency [31].

Serum fatty acid measurements

Venous blood samples were collected in the morning after an overnight fast. Serum was separated by centrifugation at 3000 r.p.m. for 15 min and stored at –80°C until analysis. The laboratory analyses were performed by SRL Inc. (Tokyo, Japan), using standard laboratory protocols.

PUFA estimates from food intake questionnaire

In our preliminary analysis, we found that serum DHA levels are negatively associated with age. To shed light on whether this is due to age-associated changes in dietary patterns, we examined the association between the amount of DHA and EPA obtained from food using a brief self-administered diet history questionnaire (BDHQ). The measurement is validated and explained in detail elsewhere [32, 33]. Brieflly, the BDHQ is a 4-page fixed-portion questionnaire that asks about the consumption frequency of selected foods to estimate the dietary intake of 58 food and beverage items during the preceding month. The BDHQ consists of five sections: (1) intake frequency of food and nonalcoholic beverage items; (2) daily intake of the Japanese staple rice and miso soup (typical Japanese soup); (3) frequency and amount of alcoholic beverage intake; (4) usual cooking methods, e.g., fried, steamed, or broiled in Japanese or Western Style; and (5) general dietary behavior, e.g., speed of eating and thickness of seasoning. The amount of DHA and EPA consumed from foods is estimated by using the information from these five categories and a validated algorithm.

Potential confounders and covariates

First we assessed the association between age and PUFA levels. Then we examined the association between PUFAs and cognitive functions, controlling for the following confounders: Age, hypertension, HbA1c, LDL Cholesterol, and obesity. Hypertension was defined by systolic blood pressure≥140 mmHg or diastolic blood pressure≥90 mmHg, or currently taking antihypertensive medication. The HbA1c value was estimated as a Japan Diabetes Society (JDS) method equivalent value (%), which was calculated by the formula: HbA1c (%) = HbA1c (%) (National Glycohemoglobin Standardization Program (NGSP) - 0.4% [34]. High HbA1c was defined by HbA1c level≥6.1% (JDS). High LDL-Cholesterol (LDL-C) was defined by LDL-C level≥140 mg/dL. Obesity was defined as Body Mass Index≥25 kg/m² according to guidelines published by the Japan Society for the Study of Obesity [35].

Statistical analysis

Associations between nutrient markers (EPA, DHA, AA, EPA/AA, DHA/AA, DHA + EPA) and age were examined by linear regression models. We then categorized J-MMSE and VFL into two groups and used t-tests to compare levels of nutrient markers by dichotomized J-MMSE (J-MMSE <24 versus J-MMSE≥24) and dichotomized VFL (VFL <5 versus VFL≥5). There is no predefined clinical threshold for VFL. Therefore, we dichotomized below/above the lowest 33% as a cutpoint (score of 5). To address the skewed distributions, we used a log-transformation of nutrient markers for the above univariate analyses except DHA which was normally distributed. Our preliminary results showed no association between nutrient markers and VFL. Therefore, subsequent multivariate analyses were conducted only for J-MMSE. Nutrient markers which were significant on univariate analyses were included in the multivariate models. We divided J-MMSE into three groups (Group 1: Lowest tertile, Group 2: Middle tertile, Group 3: Upper tertile). Those in the lowest tertile of J-MMSE score were compared to participants with higher tertiles using cumulative logit models (i.e., the model compares (a) Group 1 versus 2 and above, and (b) Groups 1 and 2 versus Group 3 in outcomes). This model generates two intercepts and one coefficient, assuming the effects of independent variables on dependent variables are the same for the comparisons of (a) and (b) above. Our preliminary analysis confirmed that this model assumption was met in our data. We divided each nutrient marker into three groups: Lowest 25%, middle 50%, and highest 25%, with the lowest 25% group being the reference group and included each marker separately into models controlling for age, years of education, gender, obesity, hypertension, high HbA1c, and high LDL-C, with the latter five items as categorical variables. All statistical analyses were performed with JMP^® 11 for Windows (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at p <0.05. Additionally, we provided the false discovery rate approximation adjusted p-value in table footnotes as multiple comparion adjusted type I error rates, using equation α(m + 1)/2m, where α is 0.05 and m is the number of tests.

RESULTS

Characteristics of the 185 participants are shown in Table 1. Mean age was 84.1 years (standard deviation (SD) = 3.4), and 77% were women. Mean years of education was 7.6 (SD = 2.5) and 63% were cognitively intact (CDR = 0). Mean J-MMSE score was 24.7 (SD = 3.6). The ranges of J-MMSE were 15–23, 24–27 and 28–30 for the 1st, 2nd, and 3rd tertiles, respectively. Although we excluded those with CDR ≥ 1.0, our cohort is old (aged 80 years and older) with a relatively low education level, thus the lower tertile includes J-MMSE scores of less than 24, the conventional cutpoint for dementia.

Figure 1 graphically depicts the associations between age and serum PUFAs levels. DHA levels decreased as age increased (p = 0.04). Positive univariate associations were found between levels of log EPA (p = 0.01), log (EPA/AA) ratio (p = 0.02), and log (DHA + EPA) (p = 0.03) with dichotomized J-MMSE scores (Table 2). There were no associations between nutrient markers and dichotomized VFL test scores (Table 3).

Table 4 shows the results of cumulative logistic regression analyses with J-MMSE tertile groups as outcomes (lowest J-MMSE group as the reference). After adjustment for cofounders, EPA and DHA + EPA remained significantly associated with J-MMSE scores. The odds of being in a higher J-MMSE tertile group was 2.38 times higher if participants had an EPA value in the highest 25%, compared with those in the lowest 25%. Those with EPA values in the middle 50% had 2.33 times higher odds of being in a higher J-MMSE tertile group compared with those in the lowest EPA 25% tile. Likewise, those with DHA + EPA values in the highest 25%, and those in the middle 50% of DHA + EPA values were 2.31 times and 2.52 times more likely to have higher J-MMSE scores compared with those in the lowest 25%.

To explore the reason for the negative association between DHA and age, we examined whether the association was due to changes in dietary intake patterns. We examined the amount of DHA obtained from food by using the BDHQ. The estimated mean DHA intake from food was 485.1±380.3 mg per day. Mean EPA was 270.0±246.8 mg per day. We found no association between either DHA and age (p = 0.49), or EPA and age (p = 0.68), using linear regressions, suggesting that our finding of a negative association between age and DHA is unlikely due to changes in diet (i.e., not due to lower consumption of DHA from food with older age).

Finally fatty fish, the primary source of omega-3 fatty acids in the Japanese diet, is also a good source of vitamin D. Vitamin D has been shown to be associated with cognitive functions [36]. Therefore, as a post hoc anlaysis, we controlled vitamin D levels estimated from the BDHQ in the cumulative logit models. The mean±SD (μg/day) was estimated as: 11.3±10.2. The levels were positvely but weakly correlated with serum DHA and EPA levels (Pearson’s correlation coefficients between vitamin D and DHA: 0.24 (p <0.01) and log EPA: 0.25 (p <0.01)). Even after adding vitamin D as a control variable, EPA and EPA+DHA maintained its significance in the association with J-MMSE scores. Vitamin D was not associated with J-MMSE in all models, possibly due to a large variability associated with the estimation from the food intake.

DISCUSSION

The current study, where community-dwelling oldest old volunteers free from frank dementia were examined, has two noteworthy findings. First, serum DHA levels decreased with increasing age among those 80 years old and older, while EPA did not. Second, higher levels of serum EPA were associated with better global cognitive function as measured by J-MMSE.

Associations between PUFAs and age

Kuriki et al. found that plasma EPA and DHA increased with age among Japanese female dietitians under 66 years old [37]. Otsuka et al. also reported that omega-3 PUFAs including α-linoleic acid, EPA, DHA, and docosa-pentaenoic acid increased with age, whereas omega-6 fatty acids including linoleic acid and AA decreased with age among those 40 to 88 years [38]. Authors in both studies suggested changes in diet (older adults may eat more fatty fish and less red meat [39]) as a possible reason for this age trend. The participants in the above two studies were younger with a wider age range than those in our study. Using autopsy data from participants between 33 and 92 years old, the level of in vivo omega-3 PUFAs, particularly DHA, was found to decrease with age [40]. In our study, the consumption of EPA and DHA estimated from a food intake questionnaire was stable regardless of age. EPA is transformed to DHA by a converting enzyme. Reduced ability to convert the enzyme or to absorb DHA might be reasons for the age-associated decline in DHA levels seen in our study. Alternatively, age-related defects in antioxidant systems may increase lipid peroxidation and thereby decrease levels of omega-3 PUFAs [41]. Further studies are warranted to confirm the underlying mechanisms of the age-associated decline in DHA among the oldest old.

The levels of DHA, EPA, and EPA/AA ratio found in our cohort were similar to those found in several other Japanese studies; however, our cohort’s AA levels were higher in comparison [42, 43]. For example, in Yanagisawa et al’s study, the mean level of serum DHA was 123.2±27.0 μg/ml, EPA was 81.9±31.1 μg/ml, AA was 119.8±22.7 μg/ml, and EPA/AA ratio was 0.68±0.22 among those aged 65 and older. In Yagi et al’s study, the mean level of serum DHA was 132.5±52.2 μg/ml and EPA was 68.9±40.4 μg/ml. Our mean values were DHA: 129.2±36.3 μg/ml, EPA: 62.5±52.2 μg/ml, AA: 195.1±47.7 μg/ml, and EPA/AA ratio: 0.34±0.34 (Table 1). The reason our cohort has higher AA values may be due to the fact that Okinawa had been under the United States occupancy from the end of World War II until 1972 and therefore western food culture has been more prevalent, leading to higher consumption of red meat. As for comparisons with non-Japanese cohorts, using plasma samples, Bowman et al. [44] reported the mean DHA and EPA values were 68.1±17.8 μg/ml and 16.5±10.5 μg/ml, respectively, among community-dwelling elderly aged 80 and older (mean age of 85.7±10) with normal cognition in Portland (Oregon) in the United States. These values are much lower than those shown in our study. The mean EPA value reported in the study by Bowman et al. is lower than the 25% lowest cutpoint of EPA (33.5 μg/ml) in our study, with which we found significant association with lower cognitive test scores. Sekikawa et al. [45] examined serum PUFAs values among quadragenarians in Japan and the United States. They reported that mean serum percentage of PUFAs was much higher among the Japanese than in the United States (9.08% versus 3.84%, p <0.01), coinciding with our finding among the octogenarians. The implication of large differences in PUFA values across regions or countries on cognitive declines or dementia incidence would be an interesting future research area. As previously suggested, both inter- and intra- cultural examinations are required to give a full perspective on the association between nutrition and health [26].

The differential effect of DHA and EPA on cognition

We found that higher levels of serum EPA, but not DHA, were associated with better cognitive function as measured by J-MMSE. Studies have shown that omega-3 PUFAs, mainly DHA and EPA, have protective effects with reagrds to anti-inflammatory properties [46 –48], cardiovascular disease [45, 49], and cognitive decline. The Framingham Heart Study showed that higher plasma DHA levels were associated with a 47% reduction in the risk of developing all-cause dementia and a 39% reduction in AD over nine years of follow-up. Their mean cohort age at baseline was about 75 years old. The Framingham Study did not examine EPA and its effects on dementia incidence. The Three-City Study from Bordeaux (France) reported that higher plasma EPA levels, but not DHA levels, were associated with a lower incidence of dementia of all types (HR = 0.69; 95% CI: 0.48–0.98) [50]. Martins et al. examined the differential effect of EPA and DHA on depression and cognition through an interventional meta-analysis of 28 randomized controlled trials [51]. They reported that EPA had a larger effect than DHA on depressive status and cognition function. Umegaki et al. found that higher serum triglycerides (TG) were associated with cognitive decline in Japanese non-demented older adults [52], and omega-3 PUFAs, mainly EPA and DHA, were found to reduce serum TG concentrations [53, 54]. Harris et al. reported potential TG-lowering mechanisms of EPA and DHA via inhibition of hepatic very-low-density-lipoprotein cholesterol. In a review of omega-3 PUFA supplementation clinical trials to reduce cardiovascular risk, Itakura et al. reported that an EPA/AA ratio > 0.75 was associated with lower major coronary artery events in a Japanese cohort with mean age of 61 years (SD = 9) [55]. EPA may also have neuroprotective effects [56]; prevention of neuronal apoptosis prevents hippocampal dysfunction [57]. EPA was found to be more effective at reducing inflammation in vivo than DHA [58], although both dietary EPA and DHA incorporate into cell membranes. The excessive production of pro-inflammatory eicosanoids derived from AA are competitively compensated for by the anti-inflammatory products of EPA metabolism [59]. Interestingly, we found that the odds of having higher MMSE scores were similar regardless of being in the highest 25% or the middle 50% of EPA or EPA + DHA levels. No dose response relation beyond the lowest 25% threshold suggests that as long as octogenarians keep their levels higher than the 25% tile in EPA and EPA + DHA, they could lower their risk for worsening cognitive outcomes. The cut off levels for the lowest 25% tile for EPA and EPA + DHA were 33.5 μg/ml and 140.65 μg/ml, respectively.

Vitamin D: A potential confounder

Vitamin D has been found to be associated with cognitive functions. Most recently, Miller et al. reported that those with dementia had a lower level of serum 25-hydroxyvitamin D compared to those with MCI and normal cognition [60]. They also reported that the rates of decline in episodic memory and executive function over time were steeper among vitamin D-deficient participants than those with adequate status [61]. It is possible the effect of EPA on cognition found in our study might be, at least partly, due to vitamin D. Therefore, as a post hoc analysis, we included vitamin D consumption levels estimated from the BDHQ in the multinomial logit models. After controlling for vitamin D level, serum EPA maintained its significant association with J-MMSE scores. Although vitamin D itself was not associated with the J-MMSE scores, its levels estimated from the food intake questionnnarie in our study had a large variability, making inferences difficult. The examination of their protective effects on cognition among the oldest old is further warranted.

The association of DHA and EPA with executive function

Bowman et al. [10, 43] found significant associations between executive function and DHA as well as EPA. We did not find any associations between EPA, DHA, and executive function. Although the test we used to measure executive function in this study (Verbal Fluency starting with “Ka” (Table 3)) taps the executive domain, it also measures phonetic abilities (language-based executive functions), while Bowman et al. used Trail Making Test B. The difference in tests could explain the discrepancy in findings between our study and theirs.

Limitations of our study include its cross-sectional design and thus our inabilityto demonstrate a casual relationship between serum omega-3 PUFAs and cognitive function. Also we could not examine the combined effects of PUFAs with other nutrient markers such as trans fat, saturated fats, or vitamins B, C, D, and E, which could have synergistic effects on cognition. Moreover participants were healthy volunteers living in Okinawa, which may limit the generalizability of the study results.

In conclusion, we found that higher serum EPA and DHA + EPA levels are associated with better cognitive function among community-dwelling non-demented volunteers aged 80 years and older. Higher intake of fatty fish may promote better cognitive function, even in the relatively healthy oldest old population. Further research is needed to understand the association between circulating EPA and DHA and dementia.

Footnotes

ACKNOWLEDGMENTS

We would like to acknowledge the contributions from the study participants and the members of the research team.

This study was supported by a Grant-in-Aid for Scientific Research (C; 23591278) from the Japanese Ministry of Health, Labour and Welfare (Tokyo, Japan) and the National Institute on Aging in the United States (P30 AG008017, K01AG023014.), a Linus Pauling Institute Research Grant, an Oregon Tax Check-off Grant, and a Center for Healthy Aging Research Pilot Grant from Oregon State University.

Authors’ disclosures available online ().

References

Japanese Statistic Bureau, Ministry of Internal Affairs and Communications (2013) Estimate of the Japanese population. http://www.stat.go.jp/data/topics/topi721.htm, Last updated September 15, 2013, Accessed on August 24, 2015.

Japanese Ministry of Health Labour, Welfare (2012) Vital Statistics. http://www.mhlw.go.jp/stf/shingi/...att/2r98520000032f26.pdf, Last updated May 20, 2013, Accessed on August, 25, 2015.

Lopez

, Kritz-Silverstein

, Barrett Connor

(2011) High dietary and plasma levels of the omega-3 fatty acid docosahexaenoic acid are associated with decreased dementia risk: The Rancho Bernardo study. J Nutr Health Aging 15, 25–31.

Kalmijn

, Launer

, Ott

, Witteman

, Hofman

, Breteler

(1997) Dietary fat intake and the risk of incident dementia in the Rotterdam Study. Ann Neurol 42, 776–782.

Albanese

, Dangour

, Uauy

, Acosta

, Guerra

, Huang

, Jacob

, de Rodriguez

, Noriega

, Salas

, Sosa

, Sousa

, Williams

, Ferri

, Prince

(2009) Dietary fish and meat intake and dementia in Latin America, China, and India: A 10/66 Dementia Research Group population-based study. Am J Clin Nutr 90, 392–400.

Barberger-Gateau

, Raffaitin

, Letenneur

, Berr

, Tzourio

, Dartigues

, Alpérovitch

(2007) Dietary patterns and risk of dementia: The Three-City cohort study. Neurology 69, 1921–1930.

Morris

, Evans

, Bienias

, Tangney

, Bennett

, Wilson

, Aggarwal

, Schneider

(2003) Consumption of fish and n-3 fatty acids and risk of incident Alzheimer disease. Arch Neurol 60, 940–946.

van Gelder

, Tijhuis

, Kalmijn

, Kromhout

(2007) Fish consumption, n-3 fatty acids, and subsequent 5-y cognitive decline in elderly men: The Zutphen Elderly Study. Am J Clin Nutr 85, 1142–1147.

Beydoun

, Kaufman

, Satia

, Rosamond

, Folsom

(2007) Plasma n-3 fatty acids and the risk of cognitive decline in older adults: The Atherosclerosis Risk in Communities Study. Am J Clin Nutr 85, 1103–1111.

10.

Bowman

, Silbert

, Howieson

, Dodge

, Traber

, Frei

, Kaye

, Shannon

, Quinn

(2012) Nutrient biomarker patterns, cognitive function, and MRI measures of brain aging. Neurology 78, 241–249.

11.

Luchtman

, Song

(2013) Cognitive enhancement by omega-3 fatty acids from child-hood to old age: Findings from animal and clinical studies. Neuropharmacology 64, 550–565.

12.

Hashimoto

, Tozawa

, Katakura

, Shahdat

, Haque

, Tanabe

, Gamoh

, Shido

(2011) Protective effects of prescription n-3 fatty acids against impairment of spatial cognitive learning ability in amyloid β-infused rats. Food Funct 2, 386–394.

13.

Mozaffarian

, Wu

(2011) Omega-3 fatty acids and cardiovascular disease: Effects on risk factors, molecular pathways, and clinical events. J Am Coll Cardiol 58, 2047–2067.

14.

Yang

, Lu

, Chen

, Meng

, Zheng

, Hui

(2012) Effects of n-3 PUFA supplementation on plasma soluble adhesion molecules: A meta-analysis of randomized controlled trials. Am J Clin Nutr 95, 972–980.

15.

, Micha

, Imamura

, Pan

, Biggs

, Ajaz

, Djousse

, Hu

, Mozaffarian

(2012) Omega-3 fatty acids and incident type 2 diabetes: A systematic review and meta-analysis. Br J Nutr 107(Suppl 2), S214–S227.

16.

Roncaglioni

, Tombesi

, Avanzini

, Barlera

, Caimi

, Longoni

, Marzona

, Milani

, Silletta

, Tognoni

, Marchioli

(2013) n-3 fatty acids in patients with multiple cardiovascular risk factors. N Engl J Med 368, 1800–1808.

17.

Macchia

, Grancelli

, Varini

, Nul

, Laffaye

, Mariani

, Ferrante

, Badra

, Figal

, Ramos

, Tognoni

, Doval

, Investigator GESICA (2013) Omega-3 fatty acids for the prevention of recurrent symptomatic atrial fibrillation: Results of the FORWARD (Randomized Trial to Assess Efficacy of PUFA for the Maintenance of Sinus Rhythm in Persistent Atrial Fibrillation) trial. J Am Coll Cardiol 61, 463–468.

18.

Reynolds

, Gatz

, Prince

, Berg

, Pedersen

(2010) Serum lipid levels and cognitive change in late life. J Am Geriatr Soc 58, 501–509.

19.

Forette

, Seux

, Staessen

, Thijs

, Birkenhäger

, Babarskiene

, Babeanu

, Bossini

, Gil-Extremera

, Girerd

, Laks

, Lilov

, Moisseyev

, Tuomilehto

, Vanhanen

, Webster

, Yodfat

, Fagard

(1998) Prevention of dementia in randomised double-blind placebo-controlled Systolic Hypertension in Europe (Syst-Eur) trial. Lancet 352, 1347–1351.

20.

Peters

, Beckett

, Forette

, Tuomilehto

, Clarke

, Ritchie

, Waldman

, Walton

, Poulter

, Ma

, Comsa

, Burch

, Fletcher

, Bulpitt

, HYVET, investigators (2008) Incident dementia and blood pressure lowering in the Hypertension in the Very Elderly Trial cognitive function assessment (HYVET-COG): A double-blind, placebo controlled trial. Lancet Neurol 7, 683–689.

21.

Ninomiya

, Ohara

, Hirakawa

, Yoshida

, Doi

, Hata

, Kanba

, Iwaki

, Kiyohara

(2011) Midlife and late-life blood pressure and dementia in Japanese elderly: The Hisayama study. Hypertension 58, 22–28.

22.

Łojko

, Pałys

, Czajkowska

, Wieczorowska-Tobis

, Łukasik

, Górna

, Sobieska

, Gajewska

, Suwalska

(2014) Association of cognitive performance with the physical activity and body mass index in middle-aged and older rural inhabitants. Eur Rev Med Pharmacol Sci 18, 3645–3652.

23.

Katsumata

, Todoriki

, Higashiuesato

, Yasura

, Ohya

, Willcox

, Dodge

(2013) Very old adults with better memory function have higher low-density lipoprotein cholesterol levels and lower triglyceride to high-density lipoprotein cholesterol ratios: KOCOA Project. J Alzheimers Dis 34, 273–279.

24.

Willcox

, Willcox

, Wang

, He

, Rosenbaum

, Suzuki

(2008) Life at the extreme limit: Phenotypic characteristics of supercentenarians in Okinawa. J Gerontol A Biol Sci Med Sci 63, 1201–1208.

25.

Tan

, Harris

, Beiser

, Au

, Himali

, Debette

, Pikula

, Decarli

, Wolf

, Vasan

, Robins

, Seshadri

(2012) Red blood cell ω-3 fatty acid levels and markers of accelerated brain aging. Neurology 78, 658–664.

26.

Dodge

, Katsumata

, Todoriki

, Yasura

, Willcox

, Bowman

, Willcox

, Leonard

, Clemons

, Oken

, Kaye

, Traber

(2010) Comparisons of plasma/serum micronutrients between Okinawan and Oregonian elders: A pilot study. J Gerontol A Biol Sci Med Sci 65, 1060–1067.

27.

Katsumata

, Todoriki

, Higashiuesato

, Yasura

, Willcox

, Ohya

, Willcox

, Dodge

(2012) Metabolic syndrome and cognitive decline among the oldest old in Okinawa: In search of a mechanism. The KOCOA Project. J Gerontol A Biol Sci Med Sci 67, 126–134.

28.

Morris

(1993) The Clinical Dementia Rating (CDR): Current version and scoring rules. Neurology 43, 2412–2414.

29.

Dodge

, Meguro

, Ishii

, Yamaguchi

, Saxton

, Ganguli

(2009) Cross-cultural comparisons of the Mini-mental State Examination between Japanese and U.S. cohorts. Int Psychogeriatr 21, 113–122.

30.

Folstein

, Folstein

, McHugh

(1975) “Mini-mental state.” A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 12, 189–198.

31.

Lezak

(1995) Neuropsychological Asessment New York: Oxford University Press.

32.

Kobayashi

, Murakami

, Sasaki

, Okubo

, Hirota

, Notsu

, Fukui

, Date

(2011) Comparison of relative validity of food group intakes estimated by comprehensive and brief-type self-administered diet history questionnaires against 16 d dietary records in Japanese adults. Public Health Nutr 14, 1200–1211.

33.

Kobayashi

, Honda

, Murakami

, Sasaki

, Okubo

, Hirota

, Notsu

, Fukui

, Date

(2012) Both comprehensive and brief self-administered diet history questionnaires satisfactorily rank nutrient intakes in Japanese adults. J Epidemiol 22, 151–159.

34.

Kashiwagi

, Kasuga

, Araki

, Oka

, Hanafusa

, Ito

, Tominaga

, Oikawa

, Noda

, Kawamura

, Sanke

, Namba

, Hashiramoto

, Sasahara

, Nishio

, Kuwa

, Ueki

, Takei

, Umemoto

, Murakami

, Yamakado

, Yatomi

, Ohashi

, Committee on the Standardization of Diabetes Mellitus–Related LaboratoryTesting of Japan Diabetes, Society(2012) International clinical harmonization of glycated hemoglobin in Japan: From Japan Diabetes Society to National Glycohemoglobin Standardization Program values. J Diabetes Investig 3, 39–40.

35.

Examination Committee of Criteria for ‘Obesity Disease’ in, Japan (2002) New criteria for ‘obesity disease’ in Japan. Circ J 66, 987–992.

36.

Toffanello

, Coin

, Perissinotto

, Zambon

, Veronese

, Bolzetta

, Corti

, Crepaldi

, Manzato

, Sergi

(2014) Vitamin D deficiency predicts cognitive decline in older men and women: The Pro. V. A. Study. Neurology 83, 2292–2298.

37.

Kuriki

, Nagaya

, Imaeda

, Tokudome

, Fujiwara

, Sato

, Ikeda

, Maki

, Tokudome

(2002) Discrepancies in dietary intakes and plasma concentrations of fatty acids according to age among Japanese female dietitians. Eur J Clin Nutr 56, 524–531.

38.

Otsuka

, Yuki

, Tomoko

, Fujiko

, Hiroshi

(2013) Serum fatty acid compositions by sex and age group in community-dwelling middle-aged and elderly Japanese. J Jpn Soc Nutr Food Sci 66, 147–153.

39.

Japanese Ministry of Health Labour, Welfare (2013) National Health and Nutrition Survey. http://www.mhlw.go.jp/bunya/kenkou/eiyou/dl/h25-houkoku.pdf#search=’http%3A%2F%2Fwww.mhlw.go.jp%2Fbunya%2Fkenkou%2Feiyou%2Fdl%2Fh25houkoku03.pdf’, Last updated March 2013, Accessed on August 25, 2015.

40.

Söderberg

, Edlund

, Kristensson

, Dallner

(1990) Lipid compositions of different regions of the human brain during aging. J Neurochem 54, 415–423.

41.

Uauy

, Dangour

(2006) Nutrition in brain development and aging: Role of essential fatty acids. Nutr Rev 64, S72–S91; discussion S72–91.

42.

Yanagisawa

, Shimada

, Miyazaki

, Kume

, Kitamura

, Ichikawa

, Kiyanagi

, Hiki

, Fukao

, Sumiyoshi

, Hirose

, Matumori

, Takizawa

, Fujii

, Mokuno

, Daida

(2010) Polyunsaturated fatty acid levels of serum and red blood cells in apparently healthy Japanese subjects living in an urban area. J Atheroscler Thromb 17, 285–294.

43.

Yagi

, Hara

, Aihara

, Fukuda

, Takashima

, Hotchi

, Ise

, Yamaguchi

, Tobiume

, Iwase

, Yamada

, Soeki

, Wakatsuki

, Shimabukuro

, Akaike

, Sata

(2014) Serum concentration of eicosapentaenoic acid is associated with cognitive function in patients with coronary artery disease. Nutr J 13, 112–117.

44.

Bowman

, Dodge

, Mattek

, Barbey

, Silbert

, Shinto

, Howieson

, Kaye

, Quinn

(2013) Plasma omega-3 PUFA and white matter mediated executive decline in older adults. Front Aging Neurosci 5, 92.

45.

Sekikawa

, Miura

, Lee

, Fujiyoshi

, Edmundowicz

, Kadowaki

, Evans

, Kadowaki

, Sutton-Tyrrell

, Okamura

, Bertolet

, Masaki

, Nakamura

, Barinas-Mitchell

, Willcox

, Kadota

, Seto

, Maegawa

, Kuller

, Ueshima

, ERA, Study JUMP, Group (2014) Long chain n-3 polyunsaturated fatty acids and incidence rate of coronary artery calcification in Japanese men in Japan and white men in the USA: Population based prospective cohort study. Heart 100, 569–573.

46.

Krebs

, Browning

, McLean

, Rothwell

, Mishra

, Moore

, Jebb

(2006) Additive benefits of long-chain n-3 polyunsaturated fatty acids and weight-loss in the management of cardiovascular disease risk in overweight hyperinsulinaemic women. Int J Obes (Lond) 30, 1535–1544.

47.

Himmelfarb

, Phinney

, Ikizler

, Kane

, McMonagle

, Miller

(2007) Gamma-tocopherol and docosahexaenoic acid decrease inflammation in dialysis patients. J Ren Nutr 17, 296–304.

48.

Yusof

, Miles

, Calder

(2008) Influence of very long-chain n-3 fatty acids on plasma markers of inflammation in middle-aged men. Prostaglandins Leukot Essent Fatty Acids 78, 219–228.

49.

Djoussé

, Akinkuolie

, Wu

, Ding

, Gaziano

(2012) Fish consumption, omega-3 fatty acids and risk of heart failure: A meta-analysis. Clin Nutr 31, 846–853.

50.

Samieri

, Féart

, Letenneur

, Dartigues

, Pérès

, Auriacombe

, Peuchant

, Delcourt

, Barberger-Gateau

(2008) Low plasma eicosapentaenoic acid and depressive symptomatology are independent predictors of dementia risk. Am J Clin Nutr 88, 714–721.

51.

Martins

(2009) EPA but not DHA appears to be responsible for the efficacy of omega-3 long chain polyunsaturated fatty acid supplementation in depression: Evidence from a meta-analysis of randomized controlled trials. J Am Coll Nutr 28, 525–542.

52.

Umegaki

, Iimuro

, Shinozaki

, Araki

, Sakurai

, Iijima

, Ohashi

, Ito

, Japanese Elderly Diabetes Intervention Trial Study, Group (2012) Risk factors associated with cognitive decline in the elderly with type 2 diabetes: Baseline data analysis of the Japanese Elderly Diabetes Intervention Trial. Geriatr Gerontol Int 12(Suppl 1), 103–109.

53.

Mori

, Burke

, Puddey

, Watts

, O’Neal

, Best

, Beilin

(2000) Purified eicosapentaenoic and docosahexaenoic acids have differential effects on serum lipids and lipoproteins, LDL particle size, glucose, and insulin in mildly hyperlipidemic men. Am J Clin Nutr 71, 1085–1094.

54.

Harris

, Miller

, Tighe

, Davidson

, Schaefer

(2008) Omega-3 fatty acids and coronary heart disease risk: Clinical and mechanistic perspectives. Atherosclerosis 197, 12–24.

55.

Itakura

, Yokoyama

, Matsuzaki

, Saito

, Origasa

, Ishikawa

, Oikawa

, Sasaki

, Hishida

, Kita

, Kitabatake

, Nakaya

, Sakata

, Shimada

, Shirato

, Matsuzawa

, Investigators JELIS (2011) Relationships between plasma fatty acid composition and coronary artery disease. J Atheroscler Thromb 18, 99–107.

56.

Freemantle

, Vandal

, Tremblay-Mercier

, Tremblay

, Blachère

, Bégin

, Brenna

, Windust

, Cunnane

(2006) Omega-3 fatty acids, energy substrates, and brain function during aging. Prostaglandins Leukot Essent Fatty Acids 75, 213–220.

57.

Lonergan

, Martin

, Horrobin

, Lynch

(2004) Neuroprotective actions of eicosapentaenoic acid on lipopolysaccharide-induced dysfunction in rat hippocampus. J Neurochem 91, 20–29.

58.

Sierra

, Lara-Villoslada

, Comalada

, Olivares

, Xaus

(2008) Dietary eicosapentaenoic acid and docosahexaenoic acid equally incorporate as decosahexaenoic acid but differ in inflammatory effects. Nutrition 24, 245–254.

59.

Freeman

, Hibbeln

, Wisner

, Davis

, Mischoulon

, Peet

, Keck

Jr , Marangell

, Richardson

, Lake

, Stoll

(2006) Omega-3 fatty acids: Evidence basis for treatment and future research in psychiatry. J Clin Psychiatry 67, 1954–1967.

60.

Miller

, Haevey

, Beckett

, Green

, Farias

, Reed

, Oilchney

, Mungas

, DeCarli

(2015) Vitamin D status and rates of cognitive decline in a multiethnic cohort of older adults. JAMA Neurol 72, 1295–1303.

61.

, Chen

, Zhang

, Persons

, Kohn

, Berkowitz

, Martinello

, Holick

(2007) An evaluation of the vitamin D3 content in fish: Is the vitamin D content adequate to satisfy the dietary requirement for vitamin D? J Steroid Biochem Mol Biol 103, 642–644.