Alzheimer’s disease (AD) is a neurodegenerative disorder that occurs with high frequency in old age (1) and has significant social and economic consequences (2). For example, Japan is a superaging society, and the dementia population is estimated to grow from 4.4 million in 2018 to over 7 million by 2025, or one in five people aged 65 years or older (3). Caring for patients with cognitive decline requires the support of multiple parties, resulting in a significant social and economic burden (2). The accumulation of amyloid-β (Aβ) in the brain is hypothesized to play a causative, initiating role in AD. Although it is expected that some anti-amyloid agents will be approved as drugs, challenges in terms of coverage, price, and convenience are expected to remain. Even if the development of cognitive impairments could be delayed by pharmacotherapy, it is economically challenging to continue the treatment for such a long time after the preclinical phase.

From this perspective, a lifestyle modification approach is one of the realistic options to address dementia. When considered in relation to body mass index (BMI), low BMI is a clear risk factor for AD in elderly individuals. A recent study of 3,632 participants in the Framingham study reported that a lower BMI is associated with a higher risk of dementia in old age (>50 years) (4). In the Japanese population, it has also been reported that lower BMI is associated with a higher incidence of dementia (5). Inadequate dietary intake triggers low BMI and prefrailty/frailty, and the prefrailty/frailty leads to malnutrition, forming a vicious cycle of deteriorating health outcomes (6).

In this regard, changing dietary habits can be a feasible option. A long-term randomized controlled trial called the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER), a multidomain lifestyle-based intervention that includes dietary intervention to improve vascular and lifestyle-related risk factors, was found to maintain cognitive function and reduce the risk of cognitive decline in older adults at high risk of dementia (7). The World Health Organization (WHO) guidelines for cognitive decline and dementia risk reduction recommends a healthy, balanced diet for all adults (8). In particular, recent findings in nutritional epidemiological studies have shown that protein intake is particularly important for maintaining brain function in the elderly population (9–12). Recently, the quality of protein has also been studied, and a low amino acid score of breakfast has been associated with cognitive impairment (13).

Precision biomarkers linking nutritional status and cognitive decline have been intensively investigated (14) and several candidate markers based on cerebrospinal fluid (CSF) or blood samples were reported in both rodent model and clinical studies (15–18). We previously demonstrated the following as the first report of this clinical study (19). We cross-sectionally compared the concentrations of plasma free amino acid (PFAA) and albumin (Alb) in multicenter clinical study with 219 elderly individuals with MCI and 220 cognitively normal (CN) individuals. We found that the concentrations of essential amino acids (EAAs), such as Lys, His, and Thr, and Alb were lower in the MCI group (19). We are currently following up these individuals with MCI for up to 3 years to assess AD development and are developing a prognostic blood-based biomarker focusing on PFAAs. Here, we report an interim analysis mainly based on the 2-year follow-up status of 155 MCI individuals. Additionally, a stratified analysis based on apolipoprotein E ε4 (APOE ε4) allele possession is reported.

In the current analysis, we investigated the association of AD development within 2 years and the baseline PFAA profile of MCI participants. The AD-converted group showed lower concentrations of EAAs, especially three BCAAs: Val, Leu, and Ile. Additionally, in the stratified analysis, the association of plasma BCAA concentrations and AD development was more pronounced in the APOE ε4-negative group.

Recent findings in nutritional epidemiology have shown that protein intake is important for maintaining brain function in the elderly. For example, the amount of protein intake of dementia patients is significantly lower compared with healthy elderly individuals (9, 10, 26). The protein intake in the elderly is positively associated with memory function (27, 28). Elderly individuals with higher levels of protein intake have a lower risk of MCI (11), and lower levels of Aβ accumulation in the brain (12). These reports provide some insight into the relationship between protein intake and the risk of developing dementia and AD. In a previous study, we reported that MCI individuals showed lower plasma EAA concentrations than CN individuals based on case-control design analysis, demonstrating that among EAAs, Lys, Thr, and Met showed the largest differences (19). In contrast, the current longitudinal study revealed that plasma BCAA concentrations showed the greatest difference between the MCI-stable and AD-converted groups among the MCI population. It is known that some individuals with MCI convert to AD, while others do not, and it is intriguing that these differences are reflected in the differences in plasma BCAA and His concentrations. It is worth noting that in the current study, the differences in serum concentrations of Alb and prealbumin, which are traditionally used indicators of nutritional status, were not statistically significant between the MCI-stable and AD-converted groups (Table 1). Recent position paper by American Society for Parenteral and Enteral Nutrition (ASPEN) pointed out that serum Alb and prealbumin do not serve as valid proxy measures of total body protein or total muscle mass and they must be recognized as inflammatory markers associated with “nutrition risk” (29). Rather, our study indicates that PFAA may be suitable indicator in this context. Interestingly, one study showed that low concentration of serum Val was associated with both brain atrophy and cognitive decline, but not CSF Aβ_1–42 level nor CSF Aβ_1–42/total tau ratio (30). The concentration of BCAAs in the blood might reflect AD pathology other than Aβ or tau accumulation, such as inflammation or oxidative stress in the brain. Compared to Lys and Thr, which are commonly decreased in MCI compared to CN participants, circulating BCAAs and His may be related to pathological conditions and/or nutritional status, and future detailed studies are warranted.

Recently, multiple reports have emerged on the relationship between changes in the PFAA profile and dementia. An NMR-based metabolomics study based on eight prospective cohorts with 22,623 participants showed that lower serum BCAA concentrations were associated with an increased risk of all types of dementia and AD (31). Recently, a study based on circulating plasma metabolomic data of 110,655 participants in the UK Biobank reported similar results regarding the decrease of plasma BCAAs and the development of dementia (32). It should be noted that in our study, a similar association was found even in the East Asian population. Eating habits and body composition vary across countries, which makes it difficult to compare epidemiological findings between different cultural areas. For example, the average meat consumption per person in Japan is less than half of that in the US (33), and the average BMI and obesity rate of Japanese people are the lowest among OECD member countries (34). Additionally, higher milk consumption was associated with a decrease in executive function or global cognition in a US cohort study (35), whereas higher dairy and milk consumption was associated with a lower risk of all-cause dementia or vascular dementia in two independent Japanese cohort studies (36, 37). Nonetheless, a similar association between the PFAA profile and AD development among diverse food cultures suggests that plasma BCAA concentrations have universal importance in this context.

In this study, differences in APOE ε4 possession showed different strengths of association. The Rotterdam Study, which is a previous study on the relationship between lifestyle and the APOE allele, demonstrated that a healthy lifestyle including a healthy diet reduced the risk of dementia in individuals without the APOE ε4 allele (38). This suggests that a healthy lifestyle, including a balanced diet, is an independent protective factor against dementia, and APOE ε4-negative individuals are more sensitive to lifestyle modification, which is in accordance with our findings that APOE ε4-negative MCI individuals had a stronger association between the PFAA profile and AD development. However, the importance of EAAs in APOE ε4-positive individuals needs future investigation because in our stratified analysis, plasma EAA concentrations of the AD-converted group were lower even in the APOE ε4-positive population, but the effect size might be smaller than that in the APOE ε4-negative population.

Precision biomarkers are key to the preventions tailored to an individual’s biological and nutritional status (14). When considering nutritional interventions based on the current results, the molecular mechanism that links circulating EAAs and development of dementia is important. As a lower plasma BCAA concentration is associated with the progression of sarcopenia (39, 40) one reason may be the loss of muscle strength and the associated decrease in the amount of activity. A decline in handgrip strength has been demonstrated to be an important indicator of dementia (41). Additionally, daily exercise has been reported to help prevent dementia (42). A higher non-grain-derived BCAA intake was associated with handgrip strength among Korean older adults (43), and nutritional intake that supports daily exercise might be desirable. Another perspective is that low-protein diets have been reported to decrease essential amino acid influx to the brain (44) and cause inflammation in the brain, which may be one of the contributing factors of dementia (45, 46). In a previous study using tauopathy model mice, a decrease in circulating EAAs triggered inflammation in the brain. EAA ingestion reversed this effect by competitively inhibiting kynurenine, an initiator of a pathway inducing neuroinflammatory gliosis and neurotoxicity in the brain (45). A recent randomized clinical trial also suggested dietary EAA supplementation improved cognitive function in middle-aged and older adults (47). Another group reported that supplementation with a BCAA mixture in senior malnourished patients improved both MMSE scores and mitochondrial function in peripheral blood mononuclear cells (48). In summary, maintaining an adequate concentration of circulating amino acids by a balanced diet may be protective against cognitive decline potentially by (1) maintaining muscle mass, (2) preventing brain inflammation, and (3) improving mitochondrial function. Future intervention studies on these populations are needed.

This study has several limitations. First, our study did not include molecular imaging or CSF biomarkers, so we were unable to stratify participants with MCI or CN based on the underlying molecular pathology. Second, the study design is based on observational research, and there could be reverse causality. Progressing MCI individuals might have poorer appetite and/or more abnormal eating habits compared to stable MCI individuals, which might have reflected to the imbalance of PFAA profile. Third, factors that may affect PFAAs and cognitive function, such as diet, muscle mass, and physical activity were not included in the current analysis. In future studies, it will be particularly important to examine the correlation with protein nutritional status. Follow-up of the participants through 3 years is currently ongoing, and the relationship with the conversion to AD will be analyzed with other indicators that we were unable to analyze this time in future analyses.

This interim analysis of a multicenter longitudinal study revealed a significant relationship with development of AD and the PFAA profile, especially a decrease in circulating BCAAs and His among MCI participants. In the stratified analysis by APOE ε4 allele positive/negative status, the difference in the PFAA profile with or without AD development was more pronounced in the APOE ε4-negative group, suggesting that the PFAA profile is an independent risk indicator of genetic factors. Further studies are necessary to clarify the relationship between the PFAA profile and lifestyle factors, including nutritional intake.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethics Committee on Genetic Analysis of Niigata University and Ethics Committee of Ajinomoto Co., Inc. The patients/participants provided their written informed consent to participate in this study.

TI and KN: conceptualization and funding acquisition. MK: data curation, formal analysis, software, and visualization. FM, STor, CN, KK, TTo, TTa, YKu, NM, STan, KU, KO, SY, TH, SN, RY, NK, CK, HT, AK, YYo, EK, MY, HK, YYa, HJ, and MK: investigation. HK, YYa, MK, YKa, and KN: methodology. TI, HK, and STou: project administration. MK, HK, YKa, and KN: resources. KN: supervision. MK and KN: validation. TI, HK, YYa, MK, and KN: writing—original draft. TI, MK, and KN: writing—review and editing. All authors have read and agreed to the published version of the manuscript.