Omega-3 fatty acids supplementation in Alzheimer s disease: A systematic review

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1 Omega-3 fatty acids supplementation in Alzheimer s disease: A systematic review Scheine Canhada 1, Kamila Castro 2,3, Ingrid Schweigert Perry 3,4, Vivian Cristine Luft 1,3 1 Postgraduate Program in Epidemiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, 2 Postgraduate Program in Child and Adolescent Health, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, 3 Food and Nutrition Research Centre (CESAN), Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, 4 Postgraduate Program in Collective Health, Academic Unit of Health Sciences, Universidade do Extremo Sul Catarinense, Criciúma, Brazil Introduction: Alzheimer s disease (AD) is a neurodegeneration disorder characterized by progressive impairments of memory, language, reasoning, and other cognitive functions. Evidence suggests that omega-3 fatty acids may act as a possible protection factor in AD. Objective: To evaluate the results available in the literature involving omega-3 fatty acids supplementation and its effect on cognitive function in AD patients. Methods: A systematic review of MEDLINE (from PubMed), Excerpta Medica Database, and Cochrane Library databases was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Inclusion criteria consisted in original intervention studies, controlled by placebo, that assessed the impact of supplementation or dietary intake of omega-3 fatty acids on cognitive function, in humans with AD, without limitation for prime date of publication. Results: Initial search resulted in 361 articles. Seven studies fully met the inclusion criteria. Most studies did not find statistically significant results for the omega-3 fatty acids supplementation compared to placebo, and those who show some benefit do it only in a few cognitive assessment scales. However, the effects of omega- 3 fatty acids appear to be most effectively demonstrated in patients with very mild AD. Conclusion: The effects of omega-3 fatty acids supplementation in mild AD corroborate epidemiological observational studies showing that omega-3 fatty acids may be beneficial in disease onset, when there is slight impairment of brain function. Although some studies have shown changes in scales of cognitive function in more severe cases, they are not enough to support omega-3 fatty acids supplementation in the treatment of AD. Keywords: Omega-3 fatty acids, Eicosapentaenoic acid, Docosahexaenoic acid, Alzheimer s disease, Cognition, Neuroinflammation Introduction Alzheimer s disease (AD) is a neurodegeneration disorder, with slow and continuous progression, characterized by progressive impairments of memory, language, reasoning, and other cognitive functions. 1 AD results from nervous cell damage which is associated with the accumulation of insoluble forms of amyloid-β (Aβ) in plaques and aggregation of the microtubule protein tau in neurofibrillary tangles. 1 AD prevalence is estimated to quadruplicate until There is a growing interest in lifestyle and dietary components as possible protection factors for AD, including omega-3 fatty acids. 3 8 Some dietary Correspondence to: Scheine Canhada, Postgraduate Program in Epidemiology, School of Medicine, Federal University of Rio Grande do Sul, Ramiro Barcelos, 2400, Santa Cecília, Porto Alegre, RS, Brazil. scheinelc@gmail.com components may interfere directly in disease s pathologic hallmarks like Aβ s excessive production or deposition and neurodegeneration, although mechanisms still remain uncertain. 9 Evidence suggests that omega-3 fatty acids modulate numerous molecular and cellular processes, which include brain and visual development, inflammatory reactions, thrombosis, and carcinogenesis. 10 Omega-3 fatty acids are considered essential for the human body because we are unable to produce them, so they must be obtained from dietary sources. 11,12 Among them, alpha-linolenic acid is present in flaxseed and canola oils, and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are found in fatty fish such as salmon, trout, and tuna, and in fish-oil supplements. 12 Observational studies have suggested that omega-3 fatty acids and its food 2017InformaUKLimited,tradingasTaylor&FrancisGroup DOI / X Nutritional Neuroscience 2018 VOL. 21 NO

2 sources are associated with lower incidences of AD and dementias in general. 4,6 8 However, omega-3 fatty acids role in AD treatment, when the disease already exists, is still controversial in the literature. The present study systematically reviews the effects of interventions using omega-3 fatty acids supplementation on cognitive function in individuals with AD. Methods This is a systematic review of articles found in MEDLINE (US National Library of Medicine and National Institute of Health) using PubMed, Cochrane Library, and EMBASE (Excerpta Medica Database). Furthermore, the reference lists of the selected relevant papers were screened by hand for potentially additional papers in the subject. The search strategy combined the terms Fatty Acids, Omega-3 [Mesh] AND Alzheimer Disease [Mesh]. Guidelines established from Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) were followed. 13 Inclusion criteria consisted in original intervention studies, controlled by placebo, that assessed omega-3 fatty acids impact on cognitive function markers, in humans with AD, published in any language until March 2017, without limitation for the initial date of publication. Non-original articles, such as reviews, editorials, and letters, were excluded, as well as studies in animals or in vitro. We searched for interventions using omega-3 fatty acids as supplementation (in capsules, or in any other form than in food itself) or as increased dietary intake (throughout its food sources, such as fish or fish oils). We first applied eligibility criteria in titles analysis, followed by abstracts analysis and full texts reading. Search, selection, and information extraction were performed independently by two reviewers. In order to favor reliability, data were collected independently in a table including first author, year, journal, study design, population characteristics, initial and final sample size, type and dosage of supplementation, exposure period, outcome measures, and results. A third investigator solved eventual disagreements. One of the researchers evaluated bias risk for each study included in this review, after data extraction, through the application of a tool available on Cochrane Handbook for Systematic Reviews of Interventions and with the support of the software Review Manager. This information was reported in the discussion section, regarding validity of effects found for each study. Results from the original papers are presented comparing scores of cognitive function derived from questionnaires, between intervention and placebo groups, as in P values and, when statistically significant (P < 0.05), additionally as means and its standard deviation or confidence interval. Results are not described when not available in the original articles. Results The search strategy resulted in a total of 361 articles. After eligibility analyses, a total of seven papers fulfilled all criteria. The selection process is shown in Figure 1. Publication year varied from 2006 to All articles were available in English. The questionnaires that were used in these studies were Alzheimer s Disease Assessment Scale Cognitive section (ADAS-cog), Clinicians Global Impression of Change (CIBIC-plus), Mini-Mental State Examination (MMSE), Hamilton Depression Scale (HDRS), Montgomery Asberg Depression Rating Scale (MADRS), Clinical Dementia Rating Scale (CDR), Alzheimer s Disease Cooperative Study Activities of Daily Living (ADCS-ADL), Alzheimer s Disease Cooperative Study Instrumental Activities of Daily Living (ADCS-IADL), Caregivers Burden Scale (CGB), Disability Assessment for Dementia Scale (DAD), Neuropsychiatric Inventory (NPI), Neuropsychological Test Battery (NTB), and Bristol s Activities of Daily Living Scale (BADLS). The most frequent questionnaires were ADAS-cog and MMSE, both present in four studies The main scales are summarized in Table 1. Sample size varied from 19 to 402 men and women, summing a total of 429 individuals randomized for omega-3 fatty acids intervention and 366 for placebo. Patients were years old and presented mild and moderate AD, according to MMSE questionnaire, used in all seven studies, and additionally CDR questionnaire, used in one study. Information about these studies are shown in Table 2. In six studies, omega-3 fatty acids supplementation combined both DHA and EPA 14 16,24 26 and one study supplemented only DHA. 17 Placebo groups received olive oil capsules, soy oil, corn oil, and/or an isocaloric product. In four studies, both groups, intervention and placebo, received other nutrients additionally, such as tocopherols and hydroquinones. 14,16,24 26 None of the studies supplemented omega-3 fatty acids by increasing intake of its food sources. The intervention period varied from 4 months to 18 months. Concomitant pharmacological treatment for AD was not allowed in some studies. 16,24 In studies that allowed the adjuvant treatment with medicines, 14,15,17,25,26 the stable use for 3 or 4 months prior to the beginning of intervention was required. The first randomized clinical trial controlled by placebo that evaluated omega-3 fatty acids impact in AD was published in Freund-Levi et al. 14 assessed omega-3 fatty acids supplementation in 204 subjects with mild-to-moderate AD (mean age: 74 ± 530 Nutritional Neuroscience 2018 VOL. 21 NO. 8

3 Figure 1 Flow diagram of the systematic review of the impact of omega-3 fatty acids supplementation on cognitive function in AD patients. 9 years). The intervention group received omega-3 fatty acids in the dosages of 1720 mg DHA and 600 mg EPA and the placebo group received 4000 mg of corn oil (containing 2400 mg of linoleic acid) for 6 months, followed by more 6 months of omega-3 fatty acids supplementation for both groups. Medication for AD treatment was allowed. As a result, there was no significant statistical difference at 6 months and 12 months between groups in ADAS-cog (P > 0.05), MMSE (P > 0.05) e CDR (P > 0.05). In a subgroup with very mild AD (MMSE > 27 and CDR 0.5 1), there was a significant difference in MMSE between intervention and placebo in the first 6 months (placebo group had a decline of 2.6 points and intervention group had 0.5 points; P = 0.01). In a second paper published in 2008, Freund-Levi et al. 25, using the exact same sample from 2006, assessed other questionnaires. No significant statistical difference was found at 6 months and 12 months Table 1 Summary of main scales used for cognitive assessment in AD. Main scales ADAS-cog 18 ADCS-ADL; ADCS- IADL 19 CDR 20 CIBIC-plus 21 MMSE 22 NTB 23 Description Is a sensitive and reliable method for the assessment of cognitive function in dementias. It consists of a psychometric scale of 11 items, and scores range from 0 (no impairment) to 70 (very severe impairment). It is considered to have a good sensitivity to evaluate changes over time It measures the functional ability to perform activities of daily life. ADL assess basic living skills such as bathing and eating, whereas IADL measure more complex tasks such as using the telephone or preparing meals. A higher ADL or IADL score indicates a worsening functionality. Scores range from 0 to 27 for ADL and 0 to 14 for IADL It is a global measure that assesses memory, orientation, judgment, and other features. Is based on caregiver interview. Classifies dementia into questionable, mild, moderate, and severe It is a global measure capable of detecting changes in cognition, functionality, and behavior, thus assessing dementia s severity and progression. Requires separate interviews with patients and caregivers. It is a 7-point Likert-type scale, in which 1 represents markedly improved; 4, no change; and 7, markedly worse Evaluates cognitive function in the areas of orientation, memory, attention, calculation, language, and visual construction. It is widely translated and used in clinical practice. Patients score between 0 and 30 points, and cutoffs of 23/24 have typically been used to show significant cognitive impairment This scale assesses changes in cognitive function and is seen as a promising method for mild AD. NTB has shown to be able to detect changes in memory performance Nutritional Neuroscience 2018 VOL. 21 NO

4 532 Nutritional Neuroscience 2018 VOL. 21 NO. 8 Table 2 Clinical trials assessing the impact of omega-3 fatty acid s supplementation on cognitive function in Alzheimer s disease patients. Authors Sample Methods Results Freund-Levi et al. 14 Initial sample: 204 Final sample: 174 patients completed the protocol (85%) Recruitment: patients from a specialist memory clinic Local: Stockholm, Sweden Inclusion criteria: AD (DSM-IV); MMSE- 15 score between 15 and 30 points; patient living in his or her own home; Treatment with a stable dose of acetylcholine esterase inhibitors for at least 3 months; and plan to continue acetylcholine esterase inhibitors for the duration of the study Exclusion criteria: treatment with nonsteroidal antiinflammatory agents (low-dose acetylsalicylic acid was accepted), w3 preparations, or anticoagulants; alcohol abuse; concomitant serious disease; did not have a caregiver Chiu et al. 16 Initial sample: 46 Final sample: 29 completed the protocol (63.04%) Recruitment: newspaper advertisements Local: Tapei, Taiwan Inclusion criteria: AD (DSM-IV), mild-or-moderate severity (MMSE score between 10 and 26, and a CDR score of 1 or 2), or amnesic MCI, age range between 55 and 90 years old Exclusion criteria: inadequate motor or sensory capacity; ischemic lesion on brain computed tomography (CT); a modified Hachinski Ischemic Scale scoren4; a 17-item HDRS (Hamilton, 1960) scoren13; abnormal levels of folic acid, vitamin B12, or thyroid function; severe comorbidity (another neurodegenerative diseases, chronic debilitating neurological illness, brain trauma, tumors, severe pulmonary, renal, liver disease, cardiac disease, or autoimmune disease, or conditions expected to cause death within 1 year, alcoholism, schizophrenia, and bipolar disorder, receiving cholinesterase agents during the screen or taking NSAID on a long-term basis) Design: randomized, double-blind, placebo-controlled study Follow-up: 6 months of placebo-controlled intervention, followed by 6 months of treatment for both groups Group w3/w3: 1720 mg DHA mg EPA, daily (from w3 fatty acid concentrate in triglyceride form) Group placebo/w3: isocaloric placebo oil (4000 mg of corn oil) daily for 6 months, followed by 6 months of treatment with w3 fatty acid supplementation Both groups received 24 mg of vitamin E (tocopherol) added in their capsules Assessments: Baseline, 6 and 12 months: global function (CDR), cognitive function (MMSE), and the modified cognitive portion (ADAS-COG) Design: randomized, double-blind, placebo-controlled study Follow-up: 24 weeks Group w3: 1080 mg EPA mg DHA, daily (the source of the omega-3 fatty acids was menhaden fish body oil concentrate) Group placebo: olive oil esters (dosage not informed) Both groups receive antioxidants in their capsules (1.2 mg of hydroquinone and 12 mg of tocopherols, daily) Assessments: baseline and at weeks 6, 12, 18, and 24: cognitive portion of the ADAS (ADAS-cog), CIBIC-plus scale, and HDRS Baseline, weeks 12, 18, and 24: Erythrocyte membrane fatty acid compositions Baseline and week 24: plasma fatty acids Group w3/w3: 89 individuals, 72.6±9.0 years, 57% women, Group placebo/w3: 85 individuals, 72.9±8.6 years, 46% women Outcomes: MMSE, ADAS-cog, and CDR: There was no statistically significant difference during 6 and 12 months between groups on the ADAS-cog (P > 0.05), MMSE (P > 0.05), or CDR (P > 0.05). In a subgroup analyses (n = 32), with subjects with very mild cognitive dysfunction (MMSE > 27 e CDR 0.5 1), a significant ( 0.5 points vs. 2.6 points; P = 0.01) reduction in decline rate was observed in the omega-3 fatty acids group when compared with placebo between baseline and 6 months. Group 1 (omega-3 fatty acids): 20 individuals, 74.0 years ( ), 8 AD and 12 with MCI, 65% women (at baseline) Group 2 (placebo): 15 individuals, 76.5 ( ), 9 AD and 6 with MCI, 46.7% women (at baseline) Outcomes: ADAS, MMSE, HDRS: there were no differences between groups (P > 0.05, P = 0.87 and, P = 0.76, respectively). In subgroup analyses, the omega-3 fatty acids fatty acids group showed significant improvement in ADAS-cog compared to the placebo group in participants with mild cognitive impairment ( 3.23 ± 3.82 vs ± 1.4, P = 0.03), which was not observed in those with AD. There was a relative improvement in the CIBIC-plus score in omega-3 fatty acids fatty acid group of 0.35 ( 0.61 to 0.09, P = 0.008) compared to placebo group every 6 weeks Continued

5 Table 2 Continued Authors Sample Methods Results Nutritional Neuroscience 2018 VOL. 21 NO Quinn Initial sample: 402 et al. 17 Final sample: 295 completed the protocol (73.4%) Recruitment: Fifty-one US clinical research sites of the Alzheimer s Disease Cooperative Study Local: US Inclusion criteria: probable AD if MMSE score was between 14 and 26, they were medically stable, they consumed on average no more than 200 mg/d of DHA, and they were not taking DHA or omega-3 fatty acids fatty acid supplements Exclusion criteria: taking drugs with central anticholinergic effects or sedatives or receiving any investigational treatment for AD. Stable use (>3 months) of cholinesterase inhibitors or mean time was permitted Scheltens Initial sample: 259 et al. 24 Final sample: 238 completed the protocol (92%) Recruitment: from 27 AD Center Local: The Netherlands, Germany, Belgium, Spain, Italy, and France Inclusion criteria: age 50 years, AD (NINCDS-ADRDA), MMSE score of 20, recent MRI or computed tomography (CT) scan with no evidence of any other potential causes of dementia Exclusion criteria: other neurological diseases; a Geriatric Depression Scale (15-item) score of >6; use of a cholinesterase inhibitor or NMDA-receptor antagonist within 3 months prior to baseline; use of omega-3 fatty acids containing supplements or regular consumption of oily fish (>twice/week) within 2 months prior to baseline; use of atropine, scopolamine, tolterodine, hyoscyamine, biperiden, benztropine, trihexyphenidyl, oxybutynin, antipsychotics, vitamins B, C, and/or E (>200% of the recommended daily intake), consumption of high-energy and/or high-protein nutritional supplements, a change in dose of lipid-lowering medications, antidepressants, antihypertensives, or the use of other investigational products within 1 month prior to baseline; excessive alcohol intake or drug abuse; nursing home institutionalization; or investigator s uncertainty regarding the willingness or ability of the patient to comply with the protocol Design: randomized, double-blind, placebo-controlled trial Follow-up: 18 months Group DHA: 2000 mg of DHA daily (algal-derived) Group placebo: corn or soy oil (dosage not informed) Assessments: baseline, 6 months, 12 months: cognitive subscale (ADAS-cog) and CDR sum of boxes. Brain atrophy [volumetric magnetic resonance imaging (MRI)] in a subsample of participants (n = 102). ADCS-ADL, NPI, and the Quality of Life AD scale. Baseline and 18 months: MMSE Design: randomized, controlled, double-blind, parallel-group, multi-country trial Follow-up: 24 weeks Group active: 1200 mg DHA mg EPA + phospholipids, choline, UMP, vitamin B12, B6, and folate, vitamins C and E, and selenium from Souvenaid Group control: isocaloric control product Both study products were available as a 125 ml drink with an identical taste and appearance, taken once daily Assessments: baseline, 12, and 24 weeks: memory function domain score (NTB); executive function (WMS-r Digit Span, Trail Making Tests parts A and B), category Fluency (Controlled Oral Word Association Test), functional ability (DAD) Nutritional blood parameters (plasma vitamin E, erythrocyte DHA and EPA, and homocysteine), and EEG to assess eyesclosed resting-state oscillatory brain activity and functional brain connectivity in a subset of patients Group DHA: 238 individuals, 76 (9.3) years, 47.1% female sex (at baseline) Group placebo: 164 individuals, 76 (7.8) years, 59.8 female sex (at baseline) Outcomes: There was no statistically significant difference between groups on the ADAS-cog (P = 0.41), CDR (P = 0.68), ADCS-ADL (P = 0.38), NPI (P = 0.11), or MMSE (P = 0.88) on the 18-month follow-up. Brain atrophy decline difference was not statistically significant between groups during 18 months (P = 0.79) Active: 130 individuals, 74.4 (6.9) years, 52.3% male (at baseline) Control: 129 individuals, 73.2 (8.4) years, 49.6% male (at baseline) Outcomes: NTB memory domain: Z-score was significantly increased in the active vs. the control group over the 24-week intervention period (P = 0.023; Cohen s d = 0.21; 95% confidence interval [ 0.06] [0.49]); a trend for an effect was observed on the NTB total composite z- score (P = 0 053); no statistically significant difference between groups was found on the NTB executive function domain (P = 0.686) EEG measures of functional connectivity in the delta band were significantly different between study groups during 24 weeks in favor of the active group (P = 0.011) Continued

6 534 Nutritional Neuroscience 2018 VOL. 21 NO. 8 Table 2 Continued Authors Sample Methods Results Shinto Initial sample: 39 et al. 15 Final sample: 34 completed the protocol (87%) Recruitment: - Local: US Inclusion criteria: age 55 years, AD (NINCDS/ADRDA), MMSE score 15 26, CDR 0.5 to 1.0, not depressed (CESD <4.0), general health status that would not interfere with patient s ability to participate and complete the study, and caregiver/informant able to accompany participant to study visits Exclusion criteria: non-ad dementia, residence in a long-term care facility at screening visit, history of stroke, health conditions such as cancer (prostate cancer gleason grade <3 and non-metastatic cancers were acceptable), liver disease, history of ventricular fibrillation or ventricular tachycardia, major psychiatric disorder, major central nervous system diseases, taking lipid-lowering medication, hyperlipidemic, fish oil or cod liver oil supplementation within 30 days of enrollment, greater than one 6 ounce serving per week of fish or seafood within 30 days of enrollment, lipoic acid supplementation within 30 days of enrollment, taking systemic corticosteroids, neuroleptics, antiparkinsonian agents, and narcotic analgesics. The following medications and supplements were allowed if stable for 4 months prior to study enrollment, acetylcholinesterase inhibitors, memantine, vitamin E, and ginkgo biloba Freud-Levi Initial sample: 204 et al. 25 Final sample: 174 completed the protocol (85%) Recruitment: patients from a specialist memory clinic Local: Stockholm, Sweden Inclusion criteria: AD (DSM-IV), MMSE score inclusive of 15 and 30, living in their own homes; Treatment with a stable dose of acetylcholine esterase inhibitors for at least 3 months before the study start Exclusion criteria: use of NSAID (low-dose acetylsalicylic acid was accepted), ω3 preparations, anticoagulation agents; alcohol abuse; concomitant serious disease; or did not have a caregiver Exclusion criteria: treatment with nonsteroidal antiinflammatory agents (low-dose acetylsalicylic acid was accepted), w3 preparations, or anticoagulants; alcohol abuse; concomitant serious disease; did not have a caregiver Design: 3-arm, parallel group, randomized, double-blind, placebo-controlled pilot clinical trial Follow-up: 12 months Group w3: omega-3 fatty acids fish oil concentrate containing a daily dose of 675 mg DHA and 975 mg EPA Group w3/la: omega-3 fatty acids (fish oil concentrate containing a daily dose of 675 mg DHA and 975 mg EPA) plus LA (600 mg/day) Group placebo: soy oil Assessments: Baseline to 12 months: cognitive subscale (ADAS-cog), cognitive function (MMSE), Activities of Daily Living/Instrumental Activities of Daily Living (ADL/IADL) Design: randomized, double-blind placebo-controlled clinical trial Follow-up: 12 months Group w3: 430 mg DHA and 150 mg EPA in four capsules daily Group placebo: isocaloric placebo oil (containing 1 g of corn oil, including 0.6 g of linoleic acid) for 6 months, followed by 6 months open treatment with omega-3 fatty acids for all patients 4 mg vitamin E (tocopherol) was added to each EPAX1050TG and placebo capsule Assessments: Baseline, 6 and 12 months: Neuropsychiatric symptoms (NPI); Depressive symptoms (MADRS); Burden of the caregiver was evaluated using three items from CGB. Activities of daily living were rated (DAD) Placebo: 13 individuals, 75.2 (10.8) years, 54% female (at baseline) Omega-3 fatty acids: 13 individuals, 75.9 (8.1) years, 39% female (at baseline) Omega-3 fatty acids + LA: 13 individuals, 76.7 (10.6) years, 39% female (at baseline) Outcomes: ADAS-cog: The ω-3 + LA and ω-3 were not significantly different than the placebo group (P = 0.98, P = 0.86) ADL: The ω-3 + LA and ω-3 were not significantly different than the placebo group (P = 0.15, P = 0.82). MMSE: The mean change over 12 months between placebo group and intervention group with ω-3 was not statistically significant ( 4.6 points (±1.4) for the placebo group vs. 4.3 points (±1.3) for the ω-3 group; P = 0.80); but there was a significant difference between placebo and ω-3+la ( 4.6 points (±1.4) for placebo vs. 1.0 points (±0.7) for ω-3+la; P < 0.01), suggesting that the combination therapy decreased the rate of decline in MMSE over 12 months IADL: The mean change over 12 months between placebo group and ω-3 group was statistically significant (4.2 points (±0.9) vs. 0.7 points (±1.0); P < 0.01) and between placebo and ω-3+la (4.2 points (±0.9) vs. 0.9 points (±1.1); P = 0.01) Group w3/w3: 89 individuals, 72.6 ± 9.0 years. 57% women Group placebo/w3: 85 individuals, 46% women, 72.9 ± 8.6 years Outcomes: NPI: no significant difference over 6 and 12 months was observed between the w3/w3 and the placebo/w3 groups (P = 0.52) No significant differences in depressive symptoms occurred in either the v3/v3 or the placebo/v3 groups at 0 6 and 6 12 months, whether assessed using the depressive domain of NPI (P = 0.84) or by MADRS (P = 0.49) No significant differences in DAD score change or in the three items of care givers burden (CGB) between the v3/ v3 and placebo/v3 at 0 and 6 months were found Continued

7 Table 2 Continued Authors Sample Methods Results Group omega-3 fatty acids: Eight individuals, 71.1 (4.8) years, 56.8% female sex (at baseline except for the number of individuals, data refer to 37 CNID and AD patients) Group placebo: Eleven individuals, 71.1 (9.5) years, 53.8% female sex (at baseline except for the number of individuals, data refer to 39 CNID and AD patients) Outcomes: There was no statistically significant difference between groups on the MMSES7 (P = 0.711), MMSEWB (P = 0.576), BADLS (P = 0.595) in the 4 months of followup Design: randomized, double-blind, placebocontrolled study Follow-up: 4 months Group omega-3 fatty acids: 625 mg DHA and 600 mg EPA, daily Group placebo: olive oil (dosage not informed) Both groups also received 20 mg mixed tocopherols, daily, in their capsules Assessments: Baseline, 1 and 4 months: cognitive function (MMSE: MMSES7 and MMSEWB), activities of daily living (BADLS) and others Phillips Initial sample: 19 et al. 26 Final sample: 19 completed the protocol (100%). There were no dropouts but twodid not complete all visits Recruitment: through specialist memory clinics Local: Bristol, UK Inclusion criteria: AD (diagnosis based on NINCDS-ADRDA); have been on a stable dose of a cholinesterase inhibitor for a minimum of 3 months prior to entering the study; MMSE score between 16 and 30 Exclusion criteria: if another dementia was suspected; history of other psychiatric or neurological conditions; current drug or alcohol addiction; disabilities, disorders or special dietary needs that would prevent the study s requirements from being undertaken; already taking omega-3 fatty acids PUFA supplements; did not have a relative or friend that could accompany them to research visits and who lived with them or visited them at least 2 times a week between intervention and placebo in NPI (P = 0.52), MADRS (P = 0.49), DAD (P > 0.05) e CGB (P > 0.05). Chiu et al. 16 studied 46 subjects with mild-to-moderate AD or mild cognitive impairment (mean age: years). During 24 weeks, intervention group received omega-3 fatty acids in the form of 1080 mg of EPA and 720 mg of DHA, while placebo group received olive oil. Medication for AD treatment was not allowed. There was no significant statistical difference in ADAS-cog (P > 0.05), MMSE (P = 0.87), and HDRS (P = 0.76) between the two groups in 24 weeks. A significant improvement was observed in CIBICplus in the intervention group of 0.35 (IC95% 0.61 a 0.09, P = 0.008) every 6 weeks compared to placebo group. In a subgroup analysis, participants with mild cognitive impairment, but not with AD, showed a significant additional delay in ADAS-cog decline compared to placebo group ( 3.23 ± 3.82 vs ± 1.4, P = 0.03). Quinn et al. 17 assessed 402 subjects with mild-tomoderate AD (age: 76 ± 8.7 years). The intervention group received 2000 mg of DHA and the placebo group received corn or soy oil for 18 months. Medication for AD treatment was allowed. A subsample received magnetic resonance imaging (MRI) assessments. There was no significant effect from the intervention in cognitive decline rhythm in ADAScog (P = 0.41), CDR (P = 0.68), ADCS-ADL (P = 0.38), NPI (P = 0.11), or in MMSE (P = 0.88). There was also no change in cerebral volume at 18 months of trial (P = 0.79). Scheltens et al. 24 assessed 259 subjects with mild AD (age of 55 years or higher). The intervention group received 1200 mg of DHA and 300 mg of EPA with the addition of other nutrients in the form of the product Souvenaid, whereas the placebo group received isocaloric control product for 24 weeks. Medication for AD treatment was not allowed. A subsample was assessed through EEG. In the NTB memory domain, a significant increase was found in the intervention group compared to control group at 24 weeks (P = 0.023, additional alteration of 0.21, IC95% ). There was a trend for effect in the NTB total composite in 24 weeks (P = 0.053). No significant effect from the intervention was found in the NTB execution function domain (P = 0.686). EEG measures of functional connectivity were significantly different in one of the bands the delta band in favor of the intervention group (P = 0.011). Shinto et al. 15 studied 39 subjects with more than 55 years with probable AD in a randomized placebo-controlled pilot with three arms. Two groups received omega-3 fatty acids supplementation, one only omega-3 fatty acids (975 mg EPA and 675 mg DHA), and the other with the addition of alpha Nutritional Neuroscience 2018 VOL. 21 NO

8 lipoic acid (975 mg EPA, 675 mg DHA, and 600 mg ALA); placebo group received soy oil. The intervention lasted 12 months and medication for AD was allowed. There were no differences in ADAS-cog and ADL between placebo and omega-3 fatty acids (P = 0.86; P = 0.82) or between placebo and omega-3 fatty acids + ALA (P = 0.98; P = 0.15). In MMSE, the mean alteration in 12 months between placebo group and intervention group with only omega-3 fatty acids was not significant ( 4.6 ± 1.4 vs. 4.3 ± 1.3 points; P = 0.80), whereas the difference between placebo and omega-3 fatty acids + ALA was significant ( 4.6 ± 1.4 points for placebo vs. 1.0 ± 0.7 points for omega-3 fatty acids + ALA; P < 0.01). The mean alteration in IADL in 12 months was significant between placebo group and omega-3 fatty acids group (4.2 ± 0.9 points for placebo group vs. 0.7 ± 1.0 points for omega-3 fatty acids group; P < 0.01) and between placebo and omega-3 fatty acids + ALA (4.2 ± 0.9 points for placebo group vs. 0.9 ± 1.1 points for omega-3 fatty acids + ALA group; P = 0.01). Phillips et al. 26 assessed omega-3 fatty acids supplementation in 19 subjects with AD (mean age: 71.1 ± 4.8 years all data refer to 76 patients, which includes 57 with cognitive impairment and 19 with AD). The intervention group received daily omega-3 fatty acids in the dosages of 625 mg DHA and 600 mg EPA and the placebo group received olive oil for 4 months. Medication for AD treatment was allowed. As a result, there was no significant statistical difference at 4 months between groups in MMSES7 (P = 0.711), MMSEWB (P = 0.576), or BADLS (P = 0.595). Discussion Recent literature reveals weak associations between omega-3 fatty acids supplementation and cognitive outcomes in AD. Most studies do not find statistically significant results when supplementation is compared to placebo. On the other hand, some benefit was found in just a few evaluated scales. 15,16,24 Omega-3 fatty acids supplementation seems more effective in very mild AD. 14 Some studies showed improvements with omega-3 fatty acids supplementation in overall analyses, and not only in subgroups. Chiu et al. 16 found better results for the intervention group, compared to placebo, in the CIBIC-plus scale. Shinto et al. 15 found better results in the MMSE scale with omega- 3 fatty acids in addition to ALA, and in the IADL scale in both groups that received omega-3 fatty acid s supplementation, compared to placebo. Scheltens et al. 24 also found better results with omega-3 fatty acids in the memory domain of NTB scale. The CIBIC-plus 21 is a scale that provides a global measure designed to detect changes not only in cognition, but also in overall function and behavior, thus assessing dementia s severity and progression. Only Chiu et al. 16 used this scale to assess the impact of omega-3 fatty acids supplementation, finding statistically significant improvements with the intervention compared to placebo. However, as this same study did not find benefits in other two scales that specifically measure the cognitive capacity ADAS-cog and MMSE it is possible that the positive results in CIBIC-plus are the reflection of omega-3 fatty acid s action in other systems, such as cardiovascular or immune systems in which the omega-3 fatty acids beneficial effects have already been reported. From the studies that used MMSE scale as an assessment method, 14 17,26 only one demonstrated statistically significant effects in favor of intervention, 15 but only when in combination with ALA. MMSE 22 assess cognitive function in the orientation, memory, attention, calculation, language, and visual construction areas. The mechanism that resulted in the delay of cognitive decline progression remains uncertain, so it is not possible to assert if the effects were the result of the combination or only of ALA, because there was no exclusive intervention with the latter in that study. ADCS-ADL scale 19 was used in two studies. 15,17 This scale measures the subject s functional ability in performing daily living activities. While Quinn et al. 17 used the full scale and did not find statistical significance, Shinto et al. 15 used a modified version to measure ADL and IADL. ADL assessed basic daily functions, such as bathing and eating, whereas IADL assessed more complex skills, such as using the phone and cooking meals. In this study, despite the fact that placebo group has shown an apparent superior decline, no significant difference was found in ADL between placebo and omega-3 fatty acids or between placebo and omega-3 fatty acids + ALA. Regarding IADL, both groups that received omega-3 fatty acids obtained delay in functional decline when compared with placebo. This scale measures more complex daily functions, and maybe for this reason it was more sensitive to Alzheimer s progression than ADL. 15 From the seven studies included in this review, only one used NTB scale for assessing cognitive function 24 to detect changes in early stages of AD. 23 The authors found a significant increase in the NTB memory domain in the intervention group with the product Souvenaid compared to placebo, but not in the executive function and total composite score domains. The effect observed in a subgroup of subjects whose MMSE scores were the highest and cognitive decline was the smaller 14 corroborates findings from 536 Nutritional Neuroscience 2018 VOL. 21 NO. 8

9 epidemiological observational studies. Several cohort studies suggest that fish consumption, omega-3 fatty acids source, would have a role in Alzheimer s prevention, but not in the treatment when the disease is already manifested. 4,6 8 Subgroup analysis of subjects with mild AD also allowed the conclusion that improvements were originated in the memory domain, one of the disease s key symptoms that marks the beginning of the symptomatology. Some limitations in the studies may explain part of their results and, as so, deserve consideration. First, in at least three studies 15,16,26 the sample size was too small, which can limit statistical power. Second, loss during the treatment period was frequent in one of the studies only 63% 16 of the subjects completed the follow-up. Two authors tested if dropout was different between groups, 16,17 not finding statistically significant differences. Reasons for losing participants during the follow-up included lack of adhesion from caregivers, withdrawal of consent, adverse gastrointestinal effects, other diseases, and death. Third, the high heterogeneity of methods used to assess aspects of patient s cognition, along with the heterogeneity in interventions composition and dosages, makes it difficult to establish greater comparative analysis. Other study characteristics can also contribute to the absence of significant results of omega-3 fatty acids in AD in some of these papers. Primarily, in two of the studies 16,26 the placebo included olive oil, which is a source of monounsaturated fatty acids, already shown by other authors as inversely associated with cognitive decline. In this case, the placebo would not be totally inert and, therefore, would limit the differences compared to the intervention group. Second, in five studies 14,15,17,25,26 participants were allowed to receive medications for AD treatment along with omega-3 fatty acids, which could contribute to good outcomes also in the control group. However, in these five studies there was no apparent difference in the use of medications between groups, and in two of them 15,17 tests were applied to detect differences and no statistical significance was found. Third, the trials observed in this review had, as exposure period to treatment, a variation between 4 and 18 months, a time considered appropriate from the ethical point of view in this population, but that can be too short since Alzheimer s is a chronic disease. Fourth, omega-3 fatty acids dosages could have been insufficient to provide significant benefits. Indeed, there is no consensus among AD studies about dosages of EPA e DHA. In Chiu et al. 16, 1080 mg of EPA and 720 mg of DHA was supplemented daily. In this study, there was an increase in the DHA levels and total omega-3 fatty acids in plasma and in membranes of erythrocytes, but not in EPA levels. There was an increase of 1.5-fold in plasma levels after 1.8 g/day of oil fish smaller than in other study, in which increases of 2.4- to 3.6- folds were observed after 2.4 g/day of fish oil. 14,25 In addition, the study of Chiu et al. 16 was performed in Taiwan, a country with a high fish consumption, 27 so perhaps a higher omega-3 fatty acids dosage is necessary to reach a significant contrast compared to control group. Shinto et al. 15 used 975 mg EPA mg DHA, a quite similar dosage to the one used by Chiu et al. 16, but with a DHA:EPA relation much smaller than other studies. 14,17,25 Phillips et al. 26 used practically the same amounts for DHA (625 mg) and EPA (600 mg). Freund-Levi et al. 14,25, on the other hand, supplemented fish oil (DHA 1.7 g/day and EPA 0.6 g/day) in higher doses than studies that revealed benefits in cardiovascular diseases and AD prevention. The authors chose an elevated DHA:EPA relation (2.8-folds more DHA than EPA) based on studies that show DHA deficiency in brains affected by AD and on studies that associate the DHA ingestion with Aβ reduction in hippocampus and parietal cortex in rats in a dose-dependent way. These areas are the first affected by AD in the brain, and they alter the verbal episodic memory. Scheltens et al. 24 also administered a higher DHA quantity throughout the use of the product Souvenaid (1200 mg DHA mg EPA), which contains additional nutrients potentially responsible for part of the observed benefits. Quinn et al. 17, on the other hand, did not observe significant impact with DHA exclusive use (2 g/day). The high heterogeneity in the dosages supplemented made unfeasible to conduct a meta-analysis. To our knowledge, this is the most recent systematic review focusing exclusively in intervention studies, with randomized clinical trials, controlled by placebo, assessing omega-3 fatty acids supplementation in AD, regarding cognitive function outcomes. These findings support other reviews, that previously described omega 3 s supplementation in other populations and other outcomes, as also still inconclusive, as for the treatment of AD. Although few studies have shown alterations in some of the cognitive function scales, it is not enough to support, in a systematic way, omega-3 fatty acids supplementation in AD treatment. The effects found in mild AD corroborate observational epidemiological studies that show omega-3 fatty acids benefits in the early stages of the disease, when there is still a mild cerebral functional impairment. In this sense, more studies are needed, with sufficient sample size and longer intervention period, for different stages of the disease, with the use of standardized cognitive function scales, that also assess the dependency level in daily activities, in which the effect of each of the omega-3 fatty acids is evaluated, in Nutritional Neuroscience 2018 VOL. 21 NO

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