BACKGROUND

Alzheimer's Disease and related dementias are debilitating health conditions that can cause individuals to lose their ability to communicate, take care of themselves, and remember people, places, or events.(Alzheimer's Association 2019; Alzheimer's Society of Canada 2010; GBD 2019 Dementia Forecasting Collaborators 2022) By 2031, dementia is expected to affect nearly 1 million people in Canada, with associated healthcare costs in excess of $16.6 billion.(Alzheimer's Society of Canada 2010) Delaying dementia onset by two years could reduce the total economic burden of dementia by 25% over 30 years.(Alzheimer's Society of Canada 2010; GBD 2019 Dementia Forecasting Collaborators 2022) People who report subjective cognitive decline (SCD) have a 3 to 6 times greater risk of progressing to clinical dementia compared to cognitively normal adults.(Cunnane et al. 2020) SCD refers to the subjective experience of worsening cognitive abilities, like attention, flexible thinking, and memory, in the absence of objectively measured cognitive impairment.(Jack et al. 2010; Nation et al. 2019) SCD is considered the first 'clinical' manifestation on the dementia continuum and represents the critical intervention period to prevent or delay dementia progression.(Jessen et al. 2010; Iadecola 2013; McCrimmon et al. 2012)

Impaired cerebral glucose metabolism and cerebral blood flow (CBF) are putative mechanisms underlying dementia pathogenesis that can precede other clinical biomarkers by decades.(Cunnane et al. 2020; Iadecola 2013; McCrimmon et al. 2012) Cerebral hypometabolism and impaired CBF in SCD negatively impact neuronal function, specifically in modelling the Default Mode Network (DMN) from functional magnetic resonance imaging (fMRI) data.(Li et al. 2017) The DMN is activated during wakeful rest, reciprocally inhibited during cognitive engagement, and directly impacts attention, future planning, and memory.(Petrella et al. 2011) Changes in DMN activity can predict eventual conversion to dementia in people with SCD or mild cognitive impairment. Accordingly, mitigation of cerebral hypometabolism and impaired CBF are critical interventional targets to reduce the risk of conversion to dementia from SCD. Despite this knowledge, pharmacological interventions (e.g., anti-amyloid agents)(Mujica-Parodi et al. 2020) are largely ineffective, highlighting the need for innovation to protect brain health in those with SCD.

Emerging evidence suggests that dietary interventions that increase plasma ketone bodies (e.g., ketogenic diets) can improve cerebral metabolism and enhance CBF in SDC.(Krikorian et al. 2012; Roy et al. 2022; Cunnane et al. 2016; Svart et al. 2018) Ketogenic diets have poor adherence given that they are highly restrictive and gastrointestinal complaints are common. Emerging work from our lab shows that ingesting oral ketone monoester (KME)(e.g., beta-hydroxybutyrate (β-OHB)) supplements can mimic the beneficial effects of a ketogenic diet on brain health without dietary restriction or gastrointestinal complaints. KME supplementation has been shown to increase CBF and cognition in healthy adults, those with impaired glucose metabolism (Type 2 Diabetes)(Neudorf et al. 2024;Jensen et al. 2020) and obesity.(Walsh et al. 2021a,b) Although not entirely understood, the improved stability of cerebral energy metabolism provided by elevated KME levels through supplementation may increase CBF.(Walsh et al. 2021a) KME supplementation has also shown improved cerebrovascular function and an increase in neuroprotective proteins (e.g., brain-derived neurotrophic factor (BDNF)) suggesting that β-OHB may signal neuroprotective properties with elevated concentrations.(Walsh et al. 2021b; Shimazu et al. 2013; Han et al. 2018) Promisingly, KME supplementation could be a multi-pronged therapeutic to prevent SCD progression to dementia during this critical intervention window.

Purpose: The purpose and primary outcome of this study is to investigate the effects of a 14-day KME supplementation intervention on indices of CBF and cognitive brain function in older adults with SCD in a fasted, non-supplemented state (AIM 1). The secondary outcome is to investigate if the 14-day intervention alters the acute response to a single KME supplement dose with respect to CBF and cognitive brain function relative to pre-intervention (AIM 2). We hypothesize that compared to pre-intervention and placebo, a 14-day KME supplementation intervention will increase CBF, increase DMN functional connectivity, improve cognitive performance (AIM 1) and produce an elevated response to a single dose (AIM 2).

METHODS

Recruitment: We will obtain permission from potential participants to contact them via email. Participants will be asked to contact the research team to confirm their interest in participating in this study. A phone call or virtual meeting (i.e., Zoom) will be scheduled for participants to confirm their interest in participating and to book Study Visit 1 to obtain written informed consent, perform the cognitive screening tests (MoCA and PRMQ), MRI safety screening, and have body metrics (height, weight, waist circumference) measured.

We will invite SCD participants enrolled in our current Alzheimer's Society funded study to participate. We work closely with the MacSeniors Exercise Program at the Physical Activity Centre of Excellence (PACE), and recruitment posters will be placed within PACE and the Ivor Wynne Centre of McMaster University. We will obtain consent from the staff at PACE before posting any recruitment posters. We will place an ad in "Coffee News", a popular newspaper column local to Hamilton newspapers, as well as various social media websites. We also work closely with the McMaster Institute on Research in Aging (MIRA), geriatricians (e.g., Dr. Papaioannou) at the Geras Centre for Aging Research, and the local Alzheimer's society of Brant, Haldimand, Norfolk, Hamilton, Halton (https://alzda.ca/) to engage with older adults in the Hamilton community. Dr. Mbuagbaw will oversee the random allocation of eligible participants to a group. Recruitment posters will also be placed in local physician offices, adult community centres, and recreation centres throughout the Hamilton region (with permission).

Sample Size Determination: Our previous study of global CBF changes in middle-to-older adults with obesity following a 14-day KME supplementation informed our sample size calculation, which found that global CBF was increased by 12% in adults with obesity as measured by duplex ultrasound of the extracranial arteries;(Walsh et al. 2021a) corresponding to a large effect size. Using G\*Power (v.3.1.9.4), we conservatively estimated a required sample size of 24/group (n=48 total) based on a medium effect size, 90% power, and alpha level of 0.05 (two-tailed t-test of the null hypothesis that there is no difference between groups). Importantly, this sample size also provides adequate power for secondary outcomes.(Mujica-Parodi et al. 2020; Walsh et al. 2021a) To account for 10% drop out and data quality concerns, 60 participants (30/group; 50% female) will be recruited. We expect 4 to 6 eligible participants per month based on the recruitment rate of our current Alzheimer's Society funded KME supplementation study in older adults, allowing a conservative timeline of 20-months for recruitment, screening, and data collection activities. Data processing and statistical analyses will be performed within the following 6-months.

Study Design: In total, the study will involve 3 visits at the Imaging Research Centre located in St. Joseph's Hospital in Hamilton. During Visit 1, all interested individuals will complete an eligibility screening study visit to establish inclusion/exclusion. Participants will also be introduced to the lab, the different tests that we will run, and will be given the chance to practice the tests that will be completed during the experimental visits (Visits 2 and 3). Data will be collected at two time points: 1) Pre-intervention (Visit 2: baseline); and post-intervention (Visit 3: following 14-day intervention). All outcome measures will be collected at the Imaging Research Center at St. Joseph's Healthcare Hamilton (3T GE MRI) with the estimated total time commitment of 5 hours (2.5 hours per visit). Participants will be provided with a standardized dinner and breakfast before both study visits (Energy: \~50% carbohydrate, \~30% fat, \~20% protein)(Harris and Benedict 1918) to control for potential dietary influences on brain function. Meals will be provided via a local meal preparation service (Heart to Home Meals, Hamilton). Participants will be asked to refrain from alcohol and structured exercise and limit physical activity to their activities of daily living on the day before each trial.

Eligible participants will complete two identical study visits for pre/post-intervention comparisons. Written, informed consent will be obtained before data collection at the first study visit. Participants will arrive at the MRI suite in the morning for about 8am or 9am following an 8-hour overnight fast. Both study visits will include outcome measures collection (i.e., brain MRI scanning, cognitive testing, venous blood samples) at a fasted, non-supplemented state and repeated 30-minutes after a single dose of placebo or KME. All participants will then receive a 14-day supply of placebo or KME doses to consume one dose thrice daily. Between Visits 2 and 3, participants will consume a placebo or KME dose 3x daily aligning with breakfast, lunch and dinner. A study Visit 3 will occur 14-days later to repeat the data collection protocol.

Demographic information will also be collected at the beginning of the first study visit to obtain information regarding medication use, medical history, age, years of education, and sex and gender-based variables. This information will be collected using a participant history questionnaire.

Statistical Analysis Plan: Descriptive statistics (means, SD, and frequencies) and Q-Q plots of residuals will be used for normality and skewness testing to assess model assumptions. Linear- mixed effects models with fixed effects of time (pre vs. post), condition (placebo vs. KME), and time by condition interaction and random effects of participants will be used to compare all primary and secondary outcome data. We will recruit equal numbers of males and females, report data disaggregated by sex, and explore potential sex differences for all outcomes in sub-analyses. We will also explore potential influences of gender identity in a sub-analysis. All data will be reported following the 2010 CONSORT guidelines for RCTs.(Moher et al. 2010)