A clinical trial to investigate how the DailyColors(TM) dietary supplement affects markers of health in older adults

Approved 11/09/2023, University of Exeter Public Health & Sports Science REC (University of Exeter Medical School, Exeter, EX1 2LU, United Kingdom; +44 (0)1392 726621; shs-ethics-admin@exeter.ac.uk), ref: 2929219

Cognition as measured by the FLAME computerised cognitive test system, as described below:
1. Cognition will be assessed at baseline and 60 days on the PROTECT platform within the DailyColorsTM study bespoke study area. Cognition will be assessed using the FLAME cognitive test system which involves eight cognitive tests and takes up to 45 minutes to complete. They can be completed on either a computer with a keyboard and mouse or a touchscreen device. Participants will be encouraged to complete the tests at the same time of day on each test occasion. A video explaining the tests will be shown prior to the completion of the tests. The tests are presented in a set sequence according to the paradigm validated in the published FLAME cognitive composite measure which has proven sensitivity for detecting cognitive status and change over time. The tests are:
2. Picture Recognition Stage One: A series of 20 pictures of everyday scenes and objects is presented on the screen, at the rate of one picture every three seconds, for the participant to remember. The participant is instructed that the pictures will all be reshown later mixed with very similar ones.
3. Self‐Ordered Search (SOS) task assessing spatial working memory: spatial working memory is measured through this widely used test. Participants search a series of on-screen boxes to find a hidden symbol. Once found, participants search for the symbol again, remembering that the symbol will never be hidden in the same box twice. The symbol is hidden in every box once per level. After successfully completing a level, a new level opens with more boxes to search than the previous level. The outcome measure is the average number of boxes in the successfully completed trials. Participants are allowed three errors before the test terminates.
4. Paired Associate Learning (PAL) task assessing visual episodic memory: verbal short-term memory is measured through the paired associates learning test, which is widely used in the assessment of cognitive deterioration in AD. Participants are shown objects, one per “window” in a grid. Then they see the series of objects, one at a time in a random order, and select the correct “window” where the object had previously appeared. This version uses a ratchet-style approach, each successful trial is followed by one with more objects to recall and each unsuccessful trial is followed by the same number of objects as in the unsuccessful attempt. The outcome measure is the average number of correct object-place associations (“paired associates”) in the trials that were successfully completed. Participants are allowed three errors before the test terminates.
5. Digit Span task assessing working memory: numerical working memory is measured through a version of the “digit span” (DS) task, which has been widely cited in the neuropsychological literature and used in many commercially available brain-training devices. A series of numbers is shown to the participant who then enters the numbers in the same sequence as they appeared using a number keypad. The test uses a ratchet-style approach in which each successful trial is followed by a new sequence that is one digit longer than the last and each unsuccessful trial is followed by a new sequence that has the same number of digits as the unsuccessful trial. This allows an accurate estimate of the digit span to be made quickly. The outcome measure is the average number of digits in all successfully completed trials. Participants are allowed three errors before the test terminates.
6. Simple Reaction Time (SRT): the participant is instructed to respond using the right arrow key (keyboard) or touchscreen as quickly as possible every time a stimulus is presented in the centre of the screen. The speed of each response is recorded. The outcome measure is the speed and accuracy of response as a measure of attention. The test terminates after fifty stimuli are presented with an inter-stimulus interval which varies randomly between 1 and 3.5 seconds.
7. Digit Vigilance (DV): a target digit from 0 to 9 is randomly selected and constantly shown on the screen. Digits 0 to 9 are then presented one at a time at the rate of 150 per minute. The participant is required to respond using the right arrow key (keyboard) or touchscreen as quickly as possible every time a digit matches the target digit. The number of correct detections, the speed of the correct detections and the number of responses made in error (False Alarms) are recorded. The outcome measure is speed and accuracy of response as a measure of attention and processing speed. The test terminates after a total of 450 digits is presented, with 15 target digits in each block of 150 digits.
8. Choice Reaction Time (CRT): one of two different stimuli is randomly displayed with equal probability at a frequency between 1 and 3.5 seconds and remains visible until the participant responds. The participant is required to respond using the right/left arrow keys (keyboard) or touchscreen as quickly and accurately as possible using their left or right finger, respectively. The accuracy and speed of each response is recorded, and the outcome measure is attention and processing speed. The test terminates after 50 successive trials.
9. Picture Recognition Stage Two: The original pictures plus 20 very similar distractor pictures are presented one at a time in a counterbalanced order. Half of the original pictures are presented prior to the very similar distractor versions, and half afterwards. For each picture, the participant indicates whether it is the precise picture shown in Picture Recognition Stage One, by clicking or pressing the keyboard or touchscreen respectively using their right finger if it was shown, and their left finger if it was not shown, as quickly and accurately as possible. Each picture remains on the screen until a response is made. The accuracy and speed of each response are recorded, and the outcome measure is attention and processing speed. The test terminates after the participant gives a response for all 40 pictures.
10. Grammatical Reasoning task assessing verbal reasoning: The Baddeley Grammatical Reasoning test correlates with measures of general intelligence and involves determining the accuracy of grammatical statements about a series of pictures. The outcome measure is the total number of trials answered correctly, minus the number answered incorrectly, as a measure of executive function. The test terminates after 90 seconds.

All measured at baseline, 30 and 60 days

Full cohort:
1. Digital outcomes of health and wellbeing: the following validated measures will be completed by the participant on the study website at baseline, 30 and 60 days:
1.1. Self-reported cognition measured by the IQCODE that is clinically validated for the detection of cognitive status
1.2. Quality of life measured by the EQ5D measure with EQ-VAS
1.3. Instrumental Activities of Daily Living measured by a modified self-reported six-item IADL
1.4. Symptoms of mood and depression, measured by the nine-item Patient Health Questionnaire (PHQ-9)
1.5. Symptoms of anxiety, measured by the seven-item Generalised Anxiety Disorder (GAD-7) scale
1.6. Sleep quality measured by the short-form Cognition Online Sleep Monitoring Scale (COSMOS)
1.7. Physical fitness: A series of questionnaires and tasks designed to give a robust measure of current physical fitness. This involves:
1.8. Video-led tasks: Participants will view a video that will lead them through three simple tests of physical fitness which are validated for use in older age groups. This involves:
1.8.1. The Chair Stand Test, in which the participant raises from sitting to standing as many times as they can within 30 seconds, and provides the number of times, how difficult they found it and whether they used their hands to stand up
1.8.2. The Timed Chair-Stand Test, in which the participant reports how long it takes for them to rise from sitting to standing ten times
1.8.3. Two-Minute Step Test, in which the participant reports how many times they can march on the spot in 2 minutes
1.9. Self-Reported Gait Velocity: Participants rank their walking speed using a tick-box selection. This is validated for self-report.
1.10. The Activities-specific-Balance-Confidence (ABC) scale: A widely used 16-item measure for measuring balance in older adults.
2. Epigenetic sampling and processing: Participants will receive two saliva sample kits in the post. One will be sent after the baseline assessment and one prior to the 60-day assessment. Participants will follow the kit instructions to provide a saliva sample at baseline and 60 days. The sample will be returned to Muhdo Inc. for processing and analysis. Kit identification will be enabled through entry of unique kit identifiers and participant date of birth in the online web portal hosted by Muhdo Inc. Samples will be processed by Muhdo Health Inc. to perform DNA analysis using a custom Illumina DNA array to analyse 1000 SNPs and a masked Epigenetic Illumina array to detect 30,000 CpG sites. Raw and interpreted data will be uploaded to the web portal and downloaded by trial coordinators at the University of Exeter for integration into the full trial database.

Sub-group of 45 participants, measured at baseline and 60 days:
1. Blood sampling and processing: Sub-group participants will attend the University of Exeter St Luke’s Campus. Blood samples (up to 30 ml) will be taken by venepuncture by a highly experienced researcher who is a trained phlebotomist. This is a procedure which is routinely carried out by researchers at St Luke’s. This visit will take place on a University of Exeter campus where there is 24-hour access to a first aid trained Estate Patrol Team. Serum separator vacutainers will be centrifuged for 15 min at 4°C and 4000 rpm. Serum will be aliquoted into microcentrifuge tubes (500 µl/tube) and all samples then frozen at -70 °C +/- 10 °C and stored until analysis/end of the sample retention period.
2. Oral microbiome sampling and processing: Sub-group participants attending the University of Exeter St Luke’s Campus will provide a saliva sample for oral microbiome analysis. Participants will be provided with a sample collection tube and 10 ml of mouthwash (Scope, 15 wt% alcohol, Proctor & Gamble) for self-collection of samples. Participants will be instructed to refrain from eating or drinking in the 2 hours prior to providing the sample. They will swish the mouthwash for 30 seconds and expectorate it into a universal tube. Samples will be frozen at -70 °C +/- 10 °C and stored until analysis/end of the sample retention period. For analysis, oral bacteria genomic DNA will be extracted using a Gentra Puregene Buccal Cell Kit (Qiagen, Germantown, MD), according to the manufacturer's instructions. The sample libraries will be prepared for sequencing using the NEXTflex 16S V1-V3 Amplicon-Seq Kit (Bioo Scientific, Austin, USA) and sequenced using a paired-end 300 base pair Illumina MiSeq system as described previously (Vanhatalo et al., 2021).
3. Physiological measurements: Sub-group participants will provide measurements of their height, weight and blood pressure.