Background
Apart from glucose, ketone bodies (KBs) can cross the blood–brain barrier and are considered to be alternative energy in maintaining brain functions [
1]. 3-β-Hydroxybutyrate (BOHBUT), one of the most abundant KBs, has been reported to enhance cognitive abilities [
2]. BOHBUT plays crucial roles in brain energy supply at the low glucose [
3], neurotransmitter regulation [
4,
5], reduction of oxidative stress [
6], and preventing neuronal cells from pathogenic cellular proteins, including β-amyloid (Aβ) and phosphorylated tau [
7,
8] that can contribute to neurodegenerative diseases.
Many studies have suggested that dietary BOHBUT supplementation may have a positive effect on cognitive function. In small experimental studies, BOHBUT enhanced cognitive memory in patients with type 2 diabetes [
9]. In addition, BOHBUT showed positive outcomes on memory functions in older adults free of dementia [
10], patients with mild cognitive impairment [
11], and those with mild-to-moderate Alzheimer’s disease (AD) [
8,
12]. Small randomized controlled trials (RCTs) also suggested KB treatment in both AD and diabetes patients may provide neuroprotection [
9,
11,
12]. In studies on mice, it was shown that BOHBUT promoted hippocampal brain-derived neurotrophic factor (BDNF) expression (
4,
5), a protein which is positively associated with neuronal survival, neurotransmitter regulation, synaptic plasticity, and memory formation whereas a decrease in BDNF is linked to increased risk of neurodegenerative diseases [
13,
14]. The hypothesized biological pathway connecting KB metabolism and cognitive functions is shown in Additional file
1: Fig. S1. In addition, BOHBUT has been reported to enhance learning and memory in AD transgenic mice via improvement in neuronal mitochondrial energy [
15]. It is unclear whether KBs improve cognitive performance or prevent or delay cognitive impairment in humans as studies verifying the results across disparate lines of evidence are missing, a technique known as triangulation.
In this study, we present a triangulation of evidence using observational studies and Mendelian randomization (MR) studies to assess evidence of the causality of BOHBUT on cognitive performance. First, we used an observational study to investigate the associations between BOHBUT and cognitive functions. Second, we performed MR analyses (a method of using measured variation in associated genes to examine the causal effect of a modifiable exposure on disease in observational studies) to assess the causal relationships of BOHBUT with cognitive performance and AD, a disorder characterized by cognitive impairment. In addition to BOHBUT, we also performed parallel analyses on acetoacetate (ACACE), a precursor to BOHBUT. After fatty acids are broken down in the liver, ACACE is produced before converting to BOHBUT [
16,
17]. ACACE is known as an unstable compound that becomes rapidly decarboxylated and is produced in a small amount; thus, it is a less reliable measurement for KBs compared to BOHBUT [
18].
Discussion
Triangulating evidence from observational and MR studies supports a beneficial causal effect of BOHBUT on cognitive performance. First, an observational study showed that a range of increased levels of BOHBUT was associated with higher general cognitive function scores. Second, using a two-sample MR, BOHBUT showed evidence for causality on cognitive performance with a positive causal effect which was in concordance with the observational study previously observed. Using the MR study, we also observed the nominal protective effect of BOHBUT against AD, which supports the protective role of BOHBUT on cognitive function.
In clinical trials, either ketogenic diet or various types of KB supplement intakes were used to investigate the therapeutic effect on neurodegenerative diseases [
24,
37]. The suggested therapeutic levels of KB for Alzheimer’s and epilepsy were 0.5 mmol/L and 2 mmol/L, respectively [
24]; however, no investigation was shown for an early stage of cognitive decline. In our study, by varying KB cutoffs, we observed that BOHBUT was associated with the general cognitive performance score, when its concentration reached 0.32 mmol/L. Therefore, the lower concentration of KB could give a beneficial effect at an early stage.
In our observational analysis in which KB was categorized into two groups using exploratory therapeutic thresholds, we observed an attenuated estimate when the cutoff was increased. It was previously shown that a very high concentration of KBs (> 3 mmol/L) known as ketoacidosis [
24] leads to delayed brain development [
38] and increased risk of dementia in type 2 diabetes patients [
39]. This may imply a non-linear relationship between KB and cognitive abilities. If this is the case, other possible assumptions on a therapeutic range of KB should be further explored. In our study, the KB range measured in WHII is limited to 0–1.5 mmol/L, with 98% of samples falling within a normal BOHBUT range below 0.5 mmol/L. Consequently, this limitation constrained our exploratory analysis within the framework of an observational study. Further investigation on a non-linear MR should be explored when individual patient data is available.
Clinical examinations using mental status tests, including word memory, verbal fluency, and verbal meaning, are recommended by current diagnostic guidelines to evaluate cognitive impairment. In this present study, we investigated the association of serum BOHBUT with the general cognitive function scores obtained from these mental status tests. These mental performances involve cognitive functions mainly in the prefrontal and left frontal cortices and hippocampus [
40‐
42]. Perhaps further investigations are required to assess the causal role of KB on other parts of the brain, e.g., the occipital and parietal white matter that is myelinated region mediating network messaging and the processing speed [
43,
44]. In addition, although we were less focused on ACACE due to its being an unstable compound [
16], we still observed evidence of the association between increased ACACE and an improvement in general cognitive performance. This provided supportive evidence of what we previously observed in BOHBUT.
We further looked into downstream diseases from the progression of cognitive function decline. Unfortunately, a large GWAS for dementia was not publicly available; therefore, only AD was investigated. The protective causal effect of BOHBUT against AD was observed using IVW, and similar causal effects with less precision were shown using W-median and W-mode. This nominal protective effect gives some supportive evidence for a beneficial effect of BOHBUT on cognitive performance previously found. MR uses genetic variants as a natural experiment which is less prone to bias from confounding factors and reverse causation compared to an observational analysis, and thus, MR infers stronger evidence for causality.
Using genetic variants as an instrument, our work suggests that lifelong naturally elevated KBs have a beneficial effect on cognitive performance and, considering the protective causal role of BOHBUT, on AD. This concept might align with the beneficial effect of consuming a low-carbohydrate diet and perhaps coincide with the concept of adhering to set mealtime schedules, such as intermittent fasting, which was previously suggested to yield cognitive benefits [
45]. It is important to emphasize that findings from our observational and genetic analyses do not inform about the potential immediate benefits of interventions aimed at elevating KB levels on cognitive performance. Further RCTs are needed to investigate these short-term effects. If similar outcomes are replicated in RCTs, short-term therapeutic interventions, like the adoption of a ketogenic diet or the use of ketone supplements, could be considered as a combined therapeutic strategy.
Our study provides a number of strengths that reinforce the validity of the findings. Firstly, KBs were measured using NMR spectroscopy, which is an advanced analytical technique allowing for the identification and precise quantification of metabolites. Secondly, the concordance of the effect direction in triangulating evidence from observational and MR studies strengthens the causal evidence observed between KBs and cognitive functions. In addition, one of the SNPs discovered in our in-house meta-analysis for KB is located near
OXCT1, which is known to be in the pathway for KB catabolism [
46]. Therefore, this partially reassures us that selected instrumental SNPs reflect an underlying mechanism of KB and as a consequence, a good proxy as an instrument variable.
Our study also has limitations that should be taken into consideration. Firstly, due to the challenge to control the fasting period before blood collection, there is a potential for measurement error. Higher levels of KB can be measured if individuals have longer fasting periods. This may affect the identification of the underlying KB SNPs that were used as instruments in MR analyses, as well as the results observed in our observational study. Secondly, our instrumental SNPs provide limited variance explained, i.e., 0.6% for BOHBUT and 0.5% for ACACE. Further discovery of underlying KB SNPs is required in the study with larger sample sizes. Thirdly, as mentioned above, we assumed a linear relationship between KB and cognitive performance. If this is not the case, the linear model would have a limited ability to capture nonlinear complexities in the data and may result in biased estimates. Lastly, our study is based on a sample consisting of Caucasian individuals; therefore, caution should be taken when generalizing the finding to other populations.
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