Using a non-invasive biofluid (saliva), we apply a powerful metabolomics workflow for unbiased biomarker discovery in Alzheimer's disease (AD). We profile and differentiate Cognitively Normal (CN), Mild Cognitive Impairment (MCI), and AD groups. The workflow involves differential chemical isotope labeling liquid chromatography mass spectrometry using dansylation derivatization for in-depth profiling of the amine/phenol submetabolome. The total sample (N = 109) was divided in to the Discovery Phase (DP) (n = 82; 35 CN, 25 MCI, 22 AD) and a provisional Validation Phase (VP) (n = 27; 10 CN, 10 MCI, 7 AD). In DP we detected 6,230 metabolites. Pairwise analyses confirmed biomarkers for AD versus CN (63), AD versus MCI (47), and MCI versus CN (2). We then determined the top discriminating biomarkers and diagnostic panels. A 3-metabolite panel distinguished AD from CN and MCI (DP and VP: Area Under the Curve [AUC] = 1.000). The MCI and CN groups were best discriminated with a 2-metabolite panel (DP: AUC = 0.779; VP: AUC = 0.889). In addition, using positively confirmed metabolites, we were able to distinguish AD from CN and MCI with good diagnostic performance (AUC > 0.8). Saliva is a promising biofluid for both unbiased and targeted AD biomarker discovery and mechanism detection. Given its wide availability and convenient accessibility, saliva is a biofluid that can promote diversification of global AD biomarker research.

Background: Among the neurodegenerative diseases of aging, sporadic Alzheimer’s disease (AD) is the most prevalent and perhaps the most feared. With virtually no success at finding pharmaceutical therapeutics for altering progressive AD after diagnosis, research attention is increasingly directed at discovering biological and other markers that detect AD risk in the long asymptomatic phase. Both early detection and precision preclinical intervention require systematic investigation of multiple modalities and combinations of AD-related biomarkers and risk factors. We extend recent unbiased metabolomics research that produced a set of metabolite biomarker panels tailored to the discrimination of cognitively normal (CN), cognitively impaired and AD patients. Specifically, we compare the prediction importance of these panels with five other sets of modifiable and non-modifiable AD risk factors (genetic, lifestyle, cognitive, functional health and bio-demographic) in three clinical groups.Method: The three groups were: CN (n = 35), mild cognitive impairment (MCI; n = 25), and AD (n = 22). In a series of three pairwise comparisons, we used machine learning technology random forest analysis (RFA) to test relative predictive importance of up to 19 risk biomarkers from the six AD risk domains.Results: The three RFA multimodal prediction analyses produced significant discriminating risk factors. First, discriminating AD from CN was the AD metabolite panel and two cognitive markers. Second, discriminating AD from MCI was the AD/MCI metabolite panel and two cognitive markers. Third, discriminating MCI from CN was the MCI metabolite panel and seven markers from four other risk modalities: genetic, lifestyle, cognition and functional health.Conclusions: Salivary metabolomics biomarker panels, supplemented by other risk markers, were robust predictors of: (1) clinical differences in impairment and dementia and even; (2) subtle differences between CN and MCI. For the latter, the metabolite panel was supplemented by biomarkers that were both modifiable (e.g., functional) and non-modifiable (e.g., genetic). Comparing, integrating and identifying important multi-modal predictors may lead to novel combinations of complex risk profiles potentially indicative of neuropathological changes in asymptomatic or preclinical AD.

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