(P11-008-24) Enzyme-Based Identification of Metabolites for Nutritional Metabolomics

Objectives: High-resolution metabolomics (HRM) is extensively used in nutritional epidemiology to test for metabolite and pathway associations with disease. A key limitation for HRM, which includes measures of thousands of metabolites, lies in confident identification without a priori analysis of relevant standards. This project was to test enzyme-based identification (EBI) for metabolite identification in untargeted nutritional metabolomics.

Methods: A 3-step metabolite identification strategy was developed for liquid chromatography-high resolution mass spectrometry data to use 1) metabolome-wide association study (MWAS) and mummichog pathway enrichment analysis to identify pathway associations, 2) xMSannotator to obtain accurate mass matches to predicted metabolite adducts, and 3) precursor-product correlations to test for identification. Untargeted plasma metabolomics data were from a healthy aging study (NCT00336570) of 135 individuals without known disease.

Results: MWAS of plasma glutathione (GSH) followed by pathway analysis showed that pyrimidine metabolism varied in association with GSH levels (p = 0.0073). Two metabolites in this pathway had Level 1 identification and were positively associated with GSH, uridine (p = 0.0024) and uracil (p =0.0021). Other metabolites identified by EBI criteria included methylcytidine (p =0.006), dihydrothymine (p =0.024) and ureidoisobutyrate (p =0.001). xMSannotator provided 44 metabolic features annotated as possible pyrimidine pathway metabolites. These included the above metabolites and others identifiable by EBI criteria as dihydrouracil (p =0.0045), dihydroorotate (p =0.029), ureiodopropionate (p =0.001), cytosine (p =0.004), deoxyuridine (p =0.019), UDP-xylose (p =0.009), CDP-DG (18:1, 22:3) (p =0.012) and CDP-DG (18:0, 22:3) (p =0.036). Use of EBI criteria for two analytical platforms provided additional identification of orotate, thymine, deoxymethylcytidine, UMP, dUMP, dUDP, dTMP and bromouracil.

Conclusions: EBI method offers an orthogonal criterion to complement accurate mass and pathway association, thereby providing three orthogonal criteria for confident metabolite identification in untargeted nutritional metabolomics research.

Funding Sources: NIEHS R01 ES031980 and P30 ES019776, NIA R21 AG080247, and NIDDK, R01 DK125246