1

APPENDICES

APPENDIX A: Screening questionnaires and laboratory analyses

Geriatric Depression Scale – short form

The GDS-15 [1] is a 15-item, self-reported screening measure for depression, with good

reliability and validity for assessing depressive symptoms in the elderly [2]. Although it cannot

diagnose clinical depression, the GDS-15 indexes the severity of depressive symptoms. A total

score of ≥ 10 (scores of 0 and 1 are given for “yes” and “no” responses, respectively, on each

item) indicates moderate-to-severe depressive symptoms; such participants were excluded from

the study.

Modified telephone interview for cognitive status

The TICS-M is a standard cognitive assessment tool that has been widely used for screening

different aspects of cognition, including recall, attention, comprehension and memory [3]. Total

scores are classified as 29–39, normal; 18–28, possible MCI; and 0–17, possible dementia. Any

participants scoring < 18 was deemed ineligible for the trial.

Establishment of flavonoid rich food intake at screening

We wish to avoid including participants with high habitual flavonoid intake, because such

individuals would be unlikely to be responsive to, or benefit from, the intervention products.

Currently, in the UK and Australia, adults typically consume an average of ~10 servings of

flavonoid-rich foods per day (including tea, coffee, chocolate, fruit, vegetables and other plant-

based products) (National Diet and Nutrition Survey [NDNS], 2011). CANN defines high

flavonoid consumption as 50% more than this average intake (i.e. > 15 servings of flavonoid-rich

foods per day). Intake was indexed by the question “How many portions of the following foods

2

do you consume per day?” separately applied to fruits (including berries), vegetables, fruit juice,

tea, coffee, red wine and cocoa/dark chocolate. Respondents consuming > 15 servings were

asked if they would be willing to reduce their intake for the trial duration, and if yes, were

requested do so at least 1 month prior to V1. Fish oil and oily fish consumption was also noted,

with the requirement that any respondent taking fish oils cease for ≥ 3 months prior to trial

enrolment. Intake of more than one portion of oily fish per week precluded trial entry.

Functional Activities Questionnaire

The FAQ [4] is a 10-item measure of instrumental activities of daily living (IADLs) with items

on paying bills and shopping for groceries, for example. The FAQ can distinguish between

cognitively normal, MCI and dementia populations, with scores ≥ 6 indicating a significant

impairment in IADLs [5], and thus non-suitability for CANN. The highest possible score is 30

with items scored as follows: dependent, 3; requires assistance, 2; has difficulty [with the

activity] but does by self / never did and would have difficulty now, 1; normal / never did [the

activity] but could do now, 0. Of the questionnaires mailed out after the telephone screen, only

the FAQ is non-self-report, completed instead by a relative or friend of the respondent and

preferably someone who they see on at least 3 days per week.

Memory Functioning Questionnaire

The MFQ [6] evaluates perception of everyday memory functioning in adult and elderly

populations and consists of 62 items rated on 7-point scales. The MFQ has four subscales:

Frequency of Forgetting, Seriousness of Forgetting, Retrospective Functioning, and Mnemonics

Usage. Higher scores reflect higher perceived levels of memory functioning, i.e. fewer and less

3

serious forgetting incidents, improvement in current memory ability relative to earlier in life, and

less use of mnemonics. For CANN, no cut-off MFQ score for inclusion/exclusion was set.

NEO Personality Inventory-Five Factor Inventory (NEO-FFI)

The NEO-FFI [7] is the most widely used questionnaire to assess personality traits. Consisting of

60 items, the NEO-FFI is based on the Five Factor Model of personality and assesses participants

according to the facets of Openness, Conscientiousness, Extroversion, Agreeableness and

Neuroticism. As personality traits are considered stable across time, participants only complete

this questionnaire once during CANN. There was no cut-off NEO-FFI score for trial

inclusion/exclusion.

Hospital Anxiety and Depression Scale

The HADS [8] contains 14 items, of which 7 relate to anxiety and 7 to depression. The

respondent rates each item on a 4-point scale ranging from 0 (absence) to 3 (extreme presence).

There was no inclusion/exclusion cut-off set for the HADS, with depression severity instead

being indexed by the GDS-15.

CANN Study General Health and Lifestyle Questionnaire (GHLQ)

The GHLQ captures basic demographic information and includes a brief medical history (disease

status, medication use, vaccination status, etc.).

Fasted blood sample

4

An overnight fasted blood sample was taken to assess APOE genotype, LC n-3 PUFA status, and

for clinical biochemistry. The following indices were measured by an accredited pathology

laboratory:

1. Full blood count analysis: white cell count (lymphocytes, neutrophils, monocytes,

eosinophils, basophils), red cell count, hemoglobin, hematocrit, mean corpuscular volume

(MCV), mean corpuscular hemoglobin (MCH), and platelet count.

2. Kidney function: urea and electrolytes (sodium, potassium, bicarbonate, urea and creatinine)

3. Liver function: bilirubin, total protein, albumin, globulin, alkaline phosphatase (ALP),

alanine aminotransferase (ALT), gamma glutamyl transferase (GGT).

APOE genotyping

For APOE genotyping, the buffy layer was collected from an ethylenediaminetetraacetic acid

(EDTA) tube (BD Biosciences, San Diego, CA, USA) and genomic DNA was extracted using a

DNA extraction kit (Qiagen, Hildenberg, Germany), following the manufacturer’s instructions.

DNA was quantified with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham,

MA, USA) and diluted using MilliQ water (Millipore, Billerica, MA, USA) to achieve a

concentration in the range of 1–10 ng. APOE genotype was determined by two real-time reverse

transcription polymerase chain reaction (RT-PCR) single nucleotide polymorphism (SNP)

genotyping assays, to determine the 112 T/C (rs429358) APOE4 polymorphism and 158 C/T

(rs7412) APOE2 polymorphism per the Applied Biosystems (Foster City, CA, USA) TaqMan

SNP Genotyping Assays protocol (2010) [9]. The analyses were done on a 7500 Fast Real-Time

PCR system (Applied Biosystems).

5

Red blood cell fatty acid status

A high RBC n-3 index (EPA+DHA as > 6% of total FA) was an exclusionary criterion [10]. For

the UEA site, 1 mL of blood was taken from the EDTA tubes and sent to the University of

Stirling (Stirling, UK) for analysis. Separation and quantification of FA was done by gas-liquid

chromatography (GLC; ThermoFisher Trace, Hemel Hempstead, UK) using a 60m × 0.32mm ×

0.25mm film thickness capillary column (ZB Wax; Phenomenex, Macclesfield, UK). Hydrogen

was used as the carrier gas, at a flow rate of 4.0 mL/min. The temperature programme was 50–

1,508°C at 408°C/min, then 1,958°C at 28°C/min, and finally 2,158°C–0.58°C/min. Individual

fatty acid methyl esters (FAMEs) were identified compared to well-characterized in-house

standards as well as commercial FAME mixtures (Supelcoe 37 FAME mix; Sigma-Aldrich Ltd.,

Gillingham, UK). The precision of the whole-blood FA analysis was ensured by measuring

triplicate samples over 4 successive days to provide mean and standard deviation and coefficient

of variation (CV%).

At the SUT site, 6 mL of blood was collected into a lithium heparin tube and sent to a

commercial pathology laboratory for analysis of FA status (Australian Clinical Laboratories,

Australia). The sample of serum was extracted with a mixture of chloroform and methanol. The

chloroform fraction was dried under nitrogen and the lipid fraction trans-esterified to methyl

esters by reconstitution in Meth-Prep II reagent. This methylated extract was analyzed by gas

liquid chromatography with flame ionisation detection (GLC-FID).

6

APPENDIX B: Cognitive assessment instruments

Montreal Cognitive Assessment

The MoCA [11] is a rapid cognitive assessment instrument that assesses the following cognitive

domains: attention and concentration, executive function, memory, language, visuoconstructional

skills, conceptual thinking, calculations, and orientation. The highest possible total score is 30

points; a score ≥ 26 or above is considered to indicate normal cognitive function, while a score <

17 indicates dementia.

Logical Memory I and II

Taken from the WMS-R, Logical Memory [12] is a validated clinical measure frequently used to

assess verbal memory in patients with MCI and AD. Participants are read a short passage, and

after hearing the story are asked to retell it from memory. Recall is measured immediately

(Logical Memory I) and after a 30-minute delay (Logical Memory II).

California Verbal Learning Test-II

The CVLT-II [13] is a reliable and well-validated test of verbal learning and memory.

Participants are read a list of 16 words, belonging to four distinct groups (e.g. animals), and must

recall them across a series of immediate and delayed trials (including an interference trial). The

CVLT-II is sensitive to subtle episodic memory impairments in patients with MCI [14].

Boston Naming Test

The BNT [15] is a confrontation naming task consisting of 60 black line drawings of common

objects, which participants are asked to identify (identification difficulty differs among the

objects). The CANN study uses a 30-item version of this task [16].

7

Figure Copy

Taken from the RBANS, the Figure Copy task [6] assesses visuospatial function. The RBANS is

a well-validated neuropsychological test battery for identifying and characterizing abnormal

cognitive decline in older adults that has been shown to be sensitive to MCI due to AD [17].

Participants copy a modified Rey-Figure comprising 10 elements and receive 2 points (1 for

accuracy and 1 for placement) for correct responses on each of the 10 elements, with a highest

total possible score of 20.

Digit Span task (Forward and Backward)

The Digit Span task, a subtest from WAIS III [18] that assesses attention and short-term

memory, comprises the Digits Forward and Digits Backward subtests. For each Digits Forward

item, a series of digits (e.g. '8, 3, 4') is read out and participants must immediately repeat them

back in order. If they do this successfully, they are given a longer list of digits (e.g. '9, 2, 4, 0').

For Digits Backward, the participant is required to repeat back a series of digits in the reverse

order. The total score on this task is given by the sum score for Digits Forward plus Digits

Backward.

Trail Making Test – A and B

The TMT [19] provides a measure of executive function. Participants are required to connect

numbered circles on a sheet of A4 paper. There are two versions of the test: Version A, in which

targets are all numbers in consecutive order (1,2,3…25), and Version B, in which the participant

alternates between numbers and letters (1, A, 2, B…etc.). The participant must finish the test as

8

quickly as possible, without making errors or removing the pen from the paper, and the time

taken to complete the test indexes performance.

Test of Premorbid Functioning

The ToPF [20] estimates premorbid intelligence. Participants read aloud a list of 70 irregularly

spelled words, such as gnat, lieu and subtle, and the total score is the number of words

pronounced correctly. Unlike many other domains of intelligence and memory abilities, reading

recognition is relatively stable in the presence of the cognitive decline associated with normal

aging or brain injury. The ToPF provides a reliable estimate of intelligence in clinical and

research settings.

Cognitive Drug Research test battery

The CDR Computerized Cognitive Assessment System [21] will be used to measure the effects

of the intervention on cognition. The battery is sensitive to cognitive changes, including in trials

of MCI, dementia and SMI. It has been used in 1,200 clinical trials across 3,000 sites in more

than 60 countries. Tasks are presented via color monitors on laptops; with the exception of the

written Word Recall tasks, performed with pen and paper, with all responses are recorded via a

two-button (YES/NO) response box. The entire battery takes approximately 25–30 minutes to

complete.

For the CANN study, the following tasks are completed, in order: Bond-Lader Visual Analogue

Scale of Mood and Alertness, Leeds Sleep Evaluation Questionnaire, Word Presentation,

Immediate Word Recall, Picture Presentation, Simple Reaction Time, Digit Vigilance, Choice

Reaction Time, Spatial Working Memory, Numeric Working Memory, Delayed Word Recall,

9

Word Recognition and PRT [22]. At V1, the CDR is completed twice in succession so that

participants are well familiarized prior to the clinical visits (V2-4).

iPosition task

The iPosition task [23] assesses relational memory function, which is critically reliant on the

hippocampus. Participants must spatially reconstruct an array of five abstract shapes. In a typical

trial, participants view on a computer screen, and must then memorize, the location of each

shape. Following the stimulus phase, there is a brief delay (4 s), during which the objects

disappear and then reappear in a line at the top of the screen; the participant is then asked to

move them back to their original location. This task has demonstrated high sensitivity to

hippocampal integrity and can detect deficits in relational memory in patients with mild

hippocampal damage [23].

Verbal Fluency Test

The D-KEFS Verbal Fluency Test is composed of three conditions: letter fluency (participant

says as many words beginning with a specific letter as possible); category fluency (participant

says words belonging to a designated semantic category), and category switching (participant

alternates between saying words belonging to two different semantic categories). The category-

switching task can strongly discriminate between healthy controls and MCI and AD patients

[24].

10

APPENDIX C: Schematic of MCI/SMI classification cut-offs

11

12

13

APPENDIX D: Pre-V2 questionnaires for participants who passed the screening visit

EPIC Food Frequency Questionnaire

The EPIC FFQ is a widely used and well-validated instrument [25] assessing habitual diet over

the previous year. It captures information on approximately 130 foods, divided into the

categories of meat and fish, bread and savory biscuits, cereals, potatoes, rice and pasta, dairy

products and fats, sweets and snacks, soups, sauces and spreads, fruits, vegetables, and drinks.

The data is in the form of consumption frequency, based on 9-point scales ranging from never or

less than once per month to 6+ per day. The FFQ also indexes dietary supplement use and

general cooking and food consumption practices. The FFQ data will be mapped to a nutritional

database to derive intakes at the food group, individual food and individual nutrient levels [26].

Additional Food Frequency Questions

Data on the frequency of consumption of additional food items, of particular relevance to CANN

and not included in the EPIC FFQ (i.e. flavonoid or n-3 FA-containing items, such as teas,

coffees, and a wide range fruits, vegetables and oily fish), were additionally obtained, which

included 84 foods. Frequency of consumption was indexed on a 7- or 9-point scales as used in

the EPIC FFQ.

International Physical Activity Questionnaire

The IPAQ is a reliable, well-validated, 27-item measure [27] that assesses physical activity

across five domains: leisure time physical activities; domestic and gardening activities; work-

related physical activity; and transport-related physical activity.

Profile of Mood States

14

The POMS [28] measures six dimensions of mood: tension-anxiety; depression-dejection; anger-

hostility; vigor-activity; fatigue-inertia; and confusion-bewilderment. The POMS comprises 65

adjectives pertaining to emotional states; for each item, the respondent notes on a 5-point, Likert-

type scale, ranging from not at all to extremely, and the extent to which they have experienced

each state during the past week.

15

APPENDIX E: Clinical laboratory analyses and cardiovascular assessment (V2–V4)

Fasted blood sample

At V2–V4, participants provide an overnight fasted blood sample: 35 mL of blood is

collected, as follows (in order): 2 × 6 mL into EDTA tubes; 3 × 5 mL serum

separator tubes (SSTs); and 1 × 8 mL into a cell preparation tube (CPT). The blood

samples will be analyzed for a range of biomarkers of compliance and

cognitive and cardiovascular health, likely to include but not limited to:

1. RBC and plasma FA status

2. Markers of neuronal activity: brain-derived neurotrophic factor (BDNF)

3. Markers of inflammation: select cytokines, C-reactive protein (CRP) and

micro RNAs (miRNAs)

4. Markers of biomechanical stress: cortisol

5. Lipids and apolipoproteins: total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-

C), high-density lipoprotein-cholesterol (HDL-C), triglyceride, ApoE, and ApoA-II

6. Glucose and insulin

Urinary flavonoid analysis

Urine samples are collected at V2–V4 to assess -3- metabolite profiles.

Participants are asked to provide the first morning void (at least 100 mL of

urine into a 200 mL container). Samples will be analyzed by liquid

chromatography-tandem mass spectrometry (LC-MS/MS) using published

methods [29].

Faecal collection

16

Faecal samples are collected by participants within 48 h of V2–V4. Using a

faecal collection kit (EasySampler; Sarstedt, Nümbrecht, Germany)

participants are asked to collect eight pea-sized aliquots of feces, which are

divided into four tubes, two containing 5 mL RNAlater RNA Stabilization

Solution (Applied Biosciences, Carlsbad, CA, USA) and silicone beads, and

two containing no liquid and stored at -80°C in preparation for next-

generation shotgun sequencing [30].

Cardiovascular assessment

Aortic BP is measured non-invasively using the Vicorder (SMT Medical, Würzburg, Germany)

and SphygmoCor XCEL (AtCor Medical, New South Wales, Australia) instruments at the UEA

and SUT sites, respectively, with the patient in the supine position. Following assessment of

brachial BP, the devices automatically and immediately inflate a brachial BP cuff to derive the

aortic pressure waveform, from which indices of cardiovascular health relating to heart rate,

aortic pressure and aortic pressure wave reflection (a risk factor for cardiovascular disease; [31])

are automatically calculated by the bundled software.

Carotid-femoral PWV is the gold standard non-invasive method for measuring aortic stiffness,

which occurs due to aging and arteriosclerosis and is associated with an increased risk of

myocardial infarction and stroke [32]. PWV refers to the speed of pulse wave transit along an

artery, which is directly proportional to the stiffness of the artery. In CANN, PWV is measured

between the carotid and femoral sites because this best captures PWV along the aorta (the

primary site of arterial stiffness). The participant remains in the supine position, cuffs are placed

around the thigh (femoral artery) and neck (carotid artery), and the distance between them is

17

measured and entered into the bundled software. Once adequate signals have been obtained, the

femoral cuff automatically inflates to automatically derive the PWV between the carotid and

femoral artery.

At the UEA site, 24 h ABP is also assessed by an ABP monitor (Spacelabs, Snoqualmie, WA,

USA) in the week prior to V2–V4; three BP measurements are taken per hour between 6 AM and

10 PM, switching to one per hour between 10 PM and 6 AM. Participants record their sleeping

pattern and any notable events likely to have affected their BP during the 24 h recording period.

18

APPENDIX F: Magnetic resonance imaging (V2 and V4)

Approximately half of the participants at each center participate in structural and functional MRI

paradigms at both the initial baseline scan (V2) and the post-treatment follow-up (V4). At SUT,

brain activity is also recorded by magnetoencephalography (MEG). The structural, biochemical

and blood flow measures obtained is evaluated according to the change from an initial baseline

scan, thereby overcoming any heterogeneity introduced into the data due to differences in the

instruments used at the UEA and SUT sites (both are 3 T MRI scanners, the but UEA has a

General Electric Discovery MR750w [Fairfield, CT, USA] and SUT has a Siemens TIM Trio

system [Erlangen, Germany]). Data are collected at both sites using 32-channel head coils. Post-

processing of the MRI measures obtained at both sites will be conducted at the Biomedical

Imaging Centre (University of Illinois) to ensure consistency across study sites. A radiology

consultant will check for incidental findings.

Structural magnetic resonance imaging

To monitor brain structure over the study period, T1-weighted three-dimensional gradient-echo

sequences are conducted. A T2-weighted scan is also acquired. In addition to providing data for

quantification of white and grey matter volume, structural information from these scans is co-

registered with other MRI and MEG methods.

At UEA the T1-weighted image is obtained using a three-dimensional fast spoiled gradient echo

brain volume imaging (FSPGR-BRAVO) sequence in the sagittal orientation, repetition time

(TR)/ echo time (TE)/inversion time (TI) = 7,040/2.612/900 ms, 0.9 mm isotropic resolution,

field of view (FOV) = 230 × 230 mm, number of excitations (NEX) = 0.5. The T2-weighted

structural image is obtained using a three-dimensional CUBE fluid-attenuated inversion recovery

19

(FLAIR) sequence in the sagittal orientation, TR/TE/TI = 6,000/125.8/1,863 ms, 0.9 × 0.9 in-

plane resolution, with a slice thickness of 1 mm and 178 slices, FOV = 230 × 230 mm.

At SUT, the T1-weighted structural image is acquired using a three-dimensional magnetization

prepared rapid gradient echo (MPRAGE) sequence, TR/TE/TI = 1,900/2.32/900 ms, 0.9 mm

isotropic resolution, FOV = 230 × 230 mm, generalized autocalibrating partial parallel

acquisition (GRAPPA), acceleration factor factor of 2. The T2-weighted structural image is

obtained using a three-dimensional sampling perfection with application optimized contrasts

using different flip angle evolution (SPACE) FLAIR sequences in the sagittal orientation,

TR/TE/TI = 6,000/388/2,200 ms, 1.0 mm isotropic resolution, FOV = 256 × 256 mm, GRAPPA

acceleration factor of 2. The SUT site is also collecting a high-resolution in-plane thick-slab T 2-

weighted structural scan in a partial volume centered on the temporal lobes. This T2-weighted

structural image is acquired using an interleaved turbo spin echo sequence in the coronal

orientation perpendicular to the long axis of the hippocampus, TR/TE = 4,230/109 ms, 0.5 × 0.5

in-plane resolution, 30 slices, slice thickness = 2.5 mm, FOV = 224 × 224 mm, bandwidth = 159

Hz/pixel, echo spacing = 13.7 ms, turbo factor = 19. These images are normalized using a pre-

scan as implemented by the scanner manufacturer.

Diffusion tensor imaging (DTI)

To assess the integrity of white matter in the brain, DTI is performed using diffusion-weighted

echo planar imaging scans. AT UEA, scans at b = 1,000 s/mm2 are acquired in 60 directions

using TR/TE = 10,367/131.3, 36 slices, slice thickness = 3 mm, 128 × 128 matrix size, FOV =

240 × 240, 60 bandwidth = 1,953 Hz/Px. At b = 2,000 s/mm2, the scan is acquired with the same

20

parameters, except TR/TE = 11,048/141. The scans are oriented parallel to the anterior

commissure-posterior commissure line (AC-PC).

At SUT, data is acquired at b = 1,000 and 2,000 s/mm2 in 64 directions using TR/TE = 5,000/110

ms, 33 slices, slice thickness = 3 mm, 128 × 128 matrix size, FOV = 240 × 240, bandwidth =

1,698 Hz/Px. The scan orientation is axial with respect to the scanner coordinates.

Magnetic resonance spectroscopy (MRS)

Brain biochemistry information is obtained using single-voxel MRS. At UEA, a spectrum is

obtained using a proton brain exam/single voxel (PROBE-SV) sequence, TR/TE = 2,060/30 ms,

5,000 Hz, 4,096 points, with water suppression. The spectrum is obtained from a voxel that is 2

cm × 2 cm × 2 cm, with saturation bands applied on all eight sides of the voxel. At SUT, a

spectrum is obtained using a single-voxel point resolved spectroscopy (PRESS) sequence,

TR/TE = 2,000/30 ms, 2,000 Hz, 1,024 points, 128 averages, with water suppression. At both

sites a second scan is obtained without water suppression to aid in quantification.

The single voxel is positioned using parallel planes derived from the T1-weighted structural scan.

The voxel is positioned posterior to the corpus callosum, and centered on the midline of the brain

to include mostly medial grey matter, as shown in Fig. S1.

21

Figure S1. Representative placement of a single voxel spectroscopy scan.

Because placement of the voxel is critical for reproducibility and signal quantification, we

performed a T2-weighted overlay scan immediately after acquisition of the spectrum, with the

same orientation and center of the single-voxel scan. By registering this scan to the T1-weighted

structural scan, we are able to characterize the tissue composition of the voxel and produce

images showing voxel placement. These images are generated from pre-intervention data, and

then used by the MRI technologists to place the voxel in the post-intervention scan in the same

location as was scanned before the intervention.

At UEA, the T2-weighted overlay is performed with a rapid acquisition with refocused echoes

(RARE) sequence, TR = 2,500 ms, TE = 102 ms, flip angle = 111°, slice thickness = 2 mm, FOV

= 240 × 240 mm, 256 × 256 matrix size, 10 slices, NEX = 1). At SUT, the T2-weighted overlay

is also obtained using a RARE sequence, TR = 5,000 ms, TE = 84 ms, flip angle = 120°, slice

22

thickness = 2 mm, FOV = 256 × 256 mm, 256 × 256 matrix size, GRAPPA, acceleration factor =

2, 36 slices).

Arterial spin labelling (ASL)

We measure cerebral blood flow (CBF) using ASL sequences.

At UEA, we measure CBF using a pseudo-continuous arterial spin labeling (pCASL) sequence

with a spiral readout, TR/TE = 4852/10.7 ms, postlabeling delay (PLD) = 2,025 ms, labeling

time = 1,450 ms, flip angle = 111°, bandwidth = 976 Hz/pixel, 64 × 64 matrix size, FOV = 240 ×

240 mm, 4 mm thickness, 36 slices, three averages. The scan orientation is axial with respect to

the scanner coordinates.

At SUT, we measure CBF using a pulsed arterial spin labeling (PASL) PICORE Q2TIPS product

sequence with an echo-planar imaging (EPI) readout, TR/TE = 4,000/13 ms, TI1 = 800 ms, TI2

= 2000 ms, TI1s (saturation stop time ) = 1,200 ms, flip angle 90°, bandwidth = 2,232 Hz/Px, 64

× 64 matrix size, FOV = 220 × 220 mm, 5 mm slice thickness with a 25% distance factor, 14

slices, phase partial Fourier 6/8). The labeling plane thickness is 110 mm with a gap of 18.8 mm

and 81 pairs of label/control images are obtained. The scan orientation is axial with respect to the

scanner coordinates.

Resting state fMRI

At SUT, an additional resting state fMRI scan is conducted, during which participants are asked

to rest and look at a centrally located fixation cross. Functional data are obtained with a gradient

echo EPI sequence with multiband acceleration [33], multiband acceleration factor = 6, TR/TE =

870/30 ms, bandwidth = 1,860 Hz/Px, flip angle = 55°, field of view = 192 mm, voxel resolution

23

= 2 × 2 × 2 mm, slice orientation = axial, number of slices = 66. A total of 500 volumes are

acquired (total acquisition time = 7 minutes and 15 seconds).

Magnetoencephalography

MEG scanning is conducted using an Elekta Neuromag TRIUX system (Elekta, Stockholm,

Sweden). Firstly, resting state recordings are obtained. Then, a virtual spatial navigation task is

completed during the MEG scanning; the task is a virtual reality analogue of the Morris Water

Maze, which has been used extensively in rodent research to study spatial learning and memory

[34]. Using MEG recordings in humans, previous research has demonstrated that hippocampal

and parahippocampal theta oscillatory activity is linked with navigation performance on this task

[35].

24

APPENDIX G: ETHICAL AND LEGAL ASPECTS

Finance and Insurance

CANN is funded is funded in part by Abbott Nutrition via a Center for Nutrition, Learning, and

Memory (CNLM) grant to the University of Illinois, US. UEA sponsors the trial and provide

indemnity and insurance.

Confidentiality and data storage

No raw or analyzed participant data, from the tests or questionnaires, will contain information

that could enable identification of individual participants. The principal investigator (PI) at each

site keeps the file containing each participant’s name and file number in a locked cabinet

separate from all other data. This information will not be accessible unless a justified scientific or

ethical reason is provided to the two local PIs, and will be kept confidential and known only to

the study coordinator and other relevant members of the research team. Any electronic

information has restricted access and/or password protection, as appropriate. Data will be

retained for 15 years from the date of publication of the results from the study. In all publications

and findings, participants will be anonymous. Any plasma or serum, collected as a backup

resource and used for repeat data analysis or stored for future assessment/studies of emerging

biomarkers of vascular function and cognition, will be labelled according to study numbers only.

These samples will be stored for up to 10 years and then disposed of according

to UEA and SUT confidential information disposal routes.

Adverse Events

25

Expected Adverse Events (AEs)

Due to the nature of the intervention products, i.e. commercially available food products and

supplements, no AEs are expected. However, if a participant feels in any way adversely affected

by the intervention, or the PI feels an AE necessitates cessation of participation, the participant

will be advised to exit the trial. Clinical staff can terminate the study immediately if there is

cause for concern regarding a high number of adverse events, although this is deemed highly

unlikely.

Adverse Event Monitoring

Participants are encouraged to contact the research team at any time in the event of an AE or

serious AE (SAE), however unlikely that it relates to the intervention. Any non-contraindicatory

medical condition that is present at screening is not reported as an AE. However, if the condition

deteriorates at any point during the study, this is recorded as an AE. All AEs are be graded for

severity and relationship to intervention products (none, remote, possible, probable, highly

probable). Changes in the severity of an AE are documented to allow assessment of the duration

of the event by level of intensity (mild, moderate, severe). If there is any doubt as to whether a

clinical observation is an AE, the event is reported.

If a participant does reports an AE, they are followed-up through regular telephone calls, and

asked about their symptoms and whether medical advice has been sought. Events are described

in detail and documented in the CRF (subject and date; description of event; duration; frequency;

intensity; seriousness; action taken; outcome and sequelae; relationship to intervention products).

For documentation of an SAE, the local ethics committee are notified; such events are reported

immediately by phone, followed by a faxed SAE form within 48 h.

26

APPENDIX H: TRIAL ORGANISATION

Centre Principal Investigators

Neal Cohen, Professor of Psychology, UoI.

Anne-Marie Minihane, Professor of Nutrigenetics, UEA.

Andrew Scholey, Professor of Psychopharmacology, SUT.

Recruitment and Intervention sites

University of East Anglia, Clinical Research Trials Unit, Norwich Medical School, Chancellor’s

Drive, Norwich, NR4 7TJ, UK.

Swinburne University of Technology, Centre for Human Psychopharmacology, Melbourne,

VIC3122, Australia.

27

APPENDIX I: ABBREVIATIONS

α-ACT, alpha-actinin

Aβ, amyloid beta

AA, arachidonic acid

ABP, ambulatory blood pressure

AC-PC, anterior commissure-posterior commissure line

ACN, acetonitrile

AD, Alzheimer’s disease

AE, adverse event

ALA, alpha-linoleic acid

ALT, alanine aminotransferase

ALP, alkaline phosphatase

APOE, apolipoprotein

ASL, arterial spin labelling

BDNF, brain-derived neurotrophic factor

BMI, body mass index

BNT, Boston Naming Test

BP, blood pressure

CANN, Cognitive Ageing, Nutrition and Neurogenesis

CBF, cerebral blood flow

CDR, Cognitive Drug Research

CNLM, Centre for Nutrition, Learning and Memory

CPT, cell preparation tube

CT-proET-1, C-terminal pro-endothelin-1

28

CTSQ, CANN Telephone Screening Questionnaire

CRF, case report form

CRP, C-reactive protein

CV%, coefficient of variation

CVLT-II, California Verbal Learning Test-II

DBP, diastolic blood pressure

D-KEFS, Delis–Kaplan Executive Function System (D-KEFS)

DHA, docosahexaenoic acid

DHA, docosahexaenoic acid

DPA, docosapentaenoic acid

DTI, diffusion tensor imaging

EDTA, ethylenediaminetetraacetic acid

ELISA, enzyme linked immunosorbent assay

EPA, eicosapentaenoic acid

EPI, echo-planar imaging

ET, echo time

ETE, eicosatrienoic acid

FA, fatty acid

FAME, fatty acid methyl ester

FSPGR-BRAVO, fast spoiled gradient echo brain volume imaging

FAQ, Functional Activities Questionnaire

FFQ, Food Frequency Questionnaire

FLAIR, fluid-attenuated inversion recovery

29

FOV, field of view

fMRI, functional magnetic resonance imaging

GLC-FID, gas liquid chromatography with flame ionization detection

GCP, Good Clinical Practice

GDS-15, Geriatric Depression Scale – short form

GGT, gamma glutamyl transferase

GHLQ, CANN Study General Health and Lifestyle Questionnaire

GLC, gas-liquid chromatography

GP, general practitioner

GRAPPA, generalized autocalibrating partial parallel acquisition

H2S, hydrogen sulfide

HADS, Hospital Anxiety and Depression Scale

HDL-C, high-density lipoprotein-cholesterol

HUFA, highly unsaturated fatty acid

HPLC-MS, high-performance liquid chromatography-mass spectrometry

Hs-CRP, high-sensitivity C-reactive protein

IADLS, instrumental activities of daily living

IL-6, interleukin 6

IL-6RC, IL-6 receptor complex

IPAQ, International Physical Activity Questionnaire

LC, long-chain

LC-MS/MS, liquid chromatography-tandem mass spectrometry

LDL-C, low-density lipoprotein-cholesterol

30

MCI, mild cognitive impairment

MCH, mean corpuscular hemoglobin

MCV, mean corpuscular volume

MEG, magnetoencephalography

MFQ, Memory Functioning Questionnaire

miRNA, micro RNA

MPRAGE, magnetization prepared rapid gradient echo

MR-proANP, mid-regional pro-atrial natriuretic peptide

MoCA, Montreal Cognitive Assessment

MRI, magnetic resonance imaging

MR-proADM, mid-regional pro-adrenomedullin

MRS, magnetic resonance spectroscopy

NAA, N-acetylaspartate

NEO-FFI, NEO Personality Inventory-Five Factor Inventory

NEX, number of excitations

NO, nitric oxide

NPD1, neuroprotectin D1

NSAID, non-steroidal anti-inflammatory drug

NVC, neurovascular coupling

PASL, pulsed arterial spin labeling

PI, principal investigator

POMS, Profile of Mood States

PRESS, point resolved spectroscopy

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PROBE-SV, proton brain exam/single voxel

PRT, Picture Recognition Task

PUFA, polyunsaturated fatty acid

PWV, pulse wave velocity

RARE, rapid acquisition with refocused echoes

RBANS, Repeatable Battery for the Assessment of Neuropsychological Status

RBC, red blood cell

RCT, randomised controlled trials

RT-PCR, real-time reverse transcription polymerase chain reaction

SA, stearidonic acid

SAE, serious adverse event

SMI, subjective memory impairment

SBP, systolic blood pressure

SD, standard deviation

SEM, standard error of the mean

SNP, single nucleotide polymorphism

SPACE, sampling perfection with application optimized contrasts using different flip angle

evolution

SST, serum separator tube

SUT, Swinburne University of Technology

TC, total cholesterol

TI, inversion time

TICS-M, Modified Telephone Interview for Cognitive Status

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TMT, Trail Making Test

TNF-a, tumor necrosis factor-alpha

ToPF, Test of Premorbid Functioning

TR, repetition time

UEA, University of East Anglia

WAIS III, Wechsler Adult Intelligence Scale, 3rd edition

WMS-R, Wechsler Memory Scale–Revised

33

1. Sheikh, J.I.a.J.A.Y., Geriatric Depression Scale (GDS): Recent evidence and development of a shorter version. Clinical Gerontologist, 1986. 5(1-2): p. 165-73.

2. Lesher, E.L. and J.S. Berryhill, Validation of the Geriatric Depression Scale - Short-Form among Inpatients. Journal of Clinical Psychology, 1994. 50(2): p. 256-260.

3. Cook, S.E., M. Marsiske, and K.J.M. McCoy, The Use of the Modified Telephone Interview for Cognitive Status (TICS-M) in the Detection of Amnestic Mild Cognitive Impairment. Journal of Geriatric Psychiatry and Neurology, 2009. 22(2): p. 103-109.

4. Pfeffer, R.I., et al., Measurement of Functional Activities in Older Adults in the Community. Journals of Gerontology, 1982. 37(3): p. 323-329.

5. Teng, E., et al., Utility of the Functional Activities Questionnaire for Distinguishing Mild Cognitive Impairment From Very Mild Alzheimer Disease. Alzheimer Disease & Associated Disorders, 2010. 24(4): p. 348-353.

6. Randolph, C., et al., The repeatable battery for the assessment of neuropsychological status (RBANS): Preliminary clinical validity. Journal of Clinical and Experimental Neuropsychology, 1998. 20(3): p. 310-319.

7. Costa, P.T., NEO Inventories Professional Manual for NEO-PI-3, NEO FFI-3 & NEO PIR. Lutz, FL PAR, 1991.

8. Zigmond, A.S. and R.P. Snaith, The Hospital Anxiety and Depression Scale. Acta Psychiatrica Scandinavica, 1983. 67(6): p. 361-370.

9. Biosystems, A. Custom TaqMan® SNP Genotyping Assays Protocol. 2010. DOI: Part Number 4334431 Rev. F.

10. Harris, W.S., The omega-3 index: clinical utility for therapeutic intervention. Curr Cardiol Rep, 2010. 12(6): p. 503-8.

11. Nasreddine, Z.S., et al., The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment. J Am Geriatr Soc, 2005. 53(4): p. 695-9.

12. Wechsler, D., Manual: Wechsler Memory Scale-Revised. San Antonio, Texas: Psychological Corporation., 1987.

13. Delis, D., Kramer, J., Kaplan, E. and Ober, B., California Verbal Learning Test- Second edition. San Antonio, TX: The Psychological Corporation, 2000.

14. Rabin, L.A., et al., Differential Memory Test Sensitivity for Diagnosing Amnestic Mild Cognitive Impairment and Predicting Conversion to Alzheimer's Disease. Aging Neuropsychology and Cognition, 2009. 16(3): p. 357-376.

15. Kaplan, E., Goodglass, H., & Weintraub, S., Boston Naming Test. Philadelphia: Lea & Febriger., 1983.

16. Saxton, J., et al., Normative data on the Boston Naming Test and two equivalent 30-item short forms. Clinical Neuropsychologist, 2000. 14(4): p. 526-534.

34

17. Karantzoulis, S., et al., The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS): Utility in detection and characterization of mild cognitive impairment due to Alzheimer's disease. Arch Clin Neuropsychol, 2013. 28(8): p. 837-44.

18. Wechsler, D., Wechsler Adult Intelligence Scale, third edition. San Antonio, Texas: Psychological Corporation., 1997.

19. Reitan, R.M., The Relation of the Trail Making Test to Organic Brain Damage. Journal of Consulting Psychology, 1955. 19(5): p. 393-394.

20. Wechsler, D., Test of Premorbid Functioning. San Antonio, TX: The Psychological Corporation, 2009.

21. Simpson, P.M., et al., The Cognitive Drug Research Computerized Assessment System for Demented Patients - a Validation-Study. International Journal of Geriatric Psychiatry, 1991. 6(2): p. 95-102.

22. Bond, A.a.M.L., The use of analogue scales in rating subjective feelings. British Journal of Psychology, 1974. 47: p. 211-18.

23. Watson, P.D., et al., Spatial reconstruction by patients with hippocampal damage is dominated by relational memory errors. Hippocampus, 2013. 23(7): p. 570-580.

24. Nutter-Upham, K.E., et al., Verbal fluency performance in amnestic MCI and older adults with cognitive complaints. Archives of Clinical Neuropsychology, 2008. 23(3): p. 229-241.

25. Kroke, A., et al., Validation of a self-administered food-frequency questionnaire administered in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study: comparison of energy, protein, and macronutuient intakes estimated with the doubly labeled water, urinary nitrogen, and repeated 24-h dietary recall methods. American Journal of Clinical Nutrition, 1999. 70(4): p. 439-447.

26. Food Standards Agency, McCance and Widdowson’s The Composition of Foods, Seventh Summary Edition. 2002: Royal Society of Chemistry: Cambridge.

27. Craig, C.L., et al., International physical activity questionnaire: 12-country reliability and validity. Medicine and Science in Sports and Exercise, 2003. 35(8): p. 1381-1395.

28. McNair, D.M., Lorr, M., Droppleman, L.F., Profile of Mood States. Educational and Industrial Testing Service, San Diego, CA, 1992.

29. Czank, C., et al., Human metabolism and elimination of the anthocyanin, cyanidin-3-glucoside: a C-13-tracer study. American Journal of Clinical Nutrition, 2013. 97(5): p. 995-1003.

30. Qin, J., et al., A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature, 2012. 490(7418): p. 55-60.

31. Murakami, T., et al., Aortic pressure wave reflection in children. Hypertension Research, 2010. 33(3): p. 225-228.

32. Sethi, S., et al., Aortic stiffness: pathophysiology, clinical implications, and approach to treatment. Integr Blood Press Control, 2014. 7: p. 29-34.

33. Setsompop, K., et al., Blipped-controlled aliasing in parallel imaging for simultaneous multislice echo planar imaging with reduced g-factor penalty. Magn Reson Med, 2012. 67(5): p. 1210-24.

34. Morris, R.G.M., Spatial localization does not require the presence of local cues. Learning and Motivation, 1981. 12(2): p. 239-60.

35

35. Cornwell, B.R., et al., Human hippocampal and parahippocampal theta during goal-directed spatial navigation predicts performance on a virtual Morris water maze. Journal of Neuroscience, 2008. 28(23): p. 5983-5990.