Increasing evidence suggests the involvement of metabolic alterations in neurological disorders, including Alzheimer's disease (AD), and highlights the significance of the peripheral metabolome, influenced by genetic factors and modifiable environmental exposures, for brain health. In this study, we examined 1,387 metabolites in plasma samples from 1,082 dementia-free middle-aged participants of the population-based Rotterdam Study. We assessed the relation of metabolites with general cognition (G-factor) and magnetic resonance imaging (MRI) markers using linear regression and estimated the variance of these metabolites explained by genes, gut microbiome, lifestyle factors, common clinical comorbidities, and medication using gradient boosting decision tree analysis. Twenty-one metabolites and one metabolite were significantly associated with total brain volume and total white matter lesions, respectively. Fourteen metabolites showed significant associations with G-factor, with ergothioneine exhibiting the largest effect (adjusted mean difference = 0.122, P = 4.65x10-7). Associations for nine of the 14 metabolites were replicated in an independent, older cohort. The metabolite signature of incident AD in the replication cohort resembled that of cognition in the discovery cohort, emphasizing the potential relevance of the identified metabolites to disease pathogenesis. Lifestyle, clinical variables, and medication were most important in determining these metabolites' blood levels, with lifestyle, explaining up to 28.6% of the variance. Smoking was associated with ten metabolites linked to G-factor, while diabetes and antidiabetic medication were associated with 13 metabolites linked to MRI markers, including N-lactoyltyrosine. Antacid medication strongly affected ergothioneine levels. Mediation analysis revealed that lower ergothioneine levels may partially mediate negative effects of antacids on cognition (31.5%). Gut microbial factors were more important for the blood levels of metabolites that were more strongly associated with cognition and incident AD in the older replication cohort (beta-cryptoxanthin, imidazole propionate), suggesting they may be involved later in the disease process. The detailed results on how multiple modifiable factors affect blood levels of cognition- and brain imaging-related metabolites in dementia-free participants may help identify new AD prevention strategies.

R.K.D., G.K., and M.A. are inventors on a series of patents on use of metabolomics for the diagnosis and treatment of CNS diseases and hold equity in Chymia LLC. R.K.D. also holds equity in Metabolon Inc., Chymia LLC and PsyProtix.

The Rotterdam Study is supported by the Erasmus MC University Medical Center and Erasmus University Rotterdam, the Netherlands Organization for Scientific Research (NWO), the Netherlands Organization for Health Research and Development (ZonMW), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry of Health, Welfare and Sport, The European Commission (DGXII), the Netherlands Genomics Initiative (NGI), and the Municipality of Rotterdam. This project was enabled in part by the Alzheimer's Gut Microbiome Project (AGMP), supported by the National Institute on Aging grants: 1U19AG063744 and 3U19AG063744-04S1, awarded to Dr. Kaddurah-Daouk at Duke University in partnership with multiple academic institutions. As such, the investigators within the AGMP not listed in this publication's authors' list, provided analysis-ready data, but did not participate in designing the study, conducting the analyses or writing of this manuscript. A listing of AGMP Investigators can be found at https://alzheimergut.org/meet-the-team/. Metabolomics data used in preparation of this article were generated by the Alzheimer's Disease Metabolomics Consortium (ADMC). As such, the investigators within the ADMC other than named authors provided data but did not participate in analysis or writing of this report. A complete listing of ADMC investigators can be found at: https://sites.duke.edu/adnimetab/team/. The NIA supported the Alzheimer's Disease Metabolomics Consortium which is a part of NIA's national initiatives AMP-AD and M2OVE-AD (R01 AG046171, RF1AG059093, RF1AG058942, RF1 AG051550, 3U01AG061359, 3U01 AG024904-09S4). In addition, this study used data, of which the production was funded by ZonMW Memorabel (project number #733050814).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Medical Ethical Committee of Erasmus Medical Center and the Ministry of Health, Welfare, and Sport of the Netherlands gave ethical approval for this work.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Due to ethical and legal restrictions, individual‐level data from the Rotterdam Study (RS) are only available upon request to the data manager of the Rotterdam Study Frank van Rooij (f.vanrooij@erasmusmc.nl) and subject to local rules and regulations. This includes submitting a proposal to the management team of RS, where upon approval, analysis needs to be done on a local server with protected access, complying with GDPR regulations. All other data produced in the present study are contained in the manuscript or are available upon reasonable request to the authors.