Comprehensive Profiling of Human Urinary Mercapturic Acid Conjugates Associated with Exposure to Reactive Chemical...

Glutathione conjugation is a key metabolic detoxification pathway, and its downstream products, mercapturic acid conjugates (MACs), may serve as urinary biomarkers of toxicant exposure. However, MAC profiling is hindered by incomplete filtering coverage and the lack of comprehensive spectral and structural databases. To address these limitations, we developed an analytical workflow integrating novel enzymatic deacetylation with ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS)-based neutral loss filtering. The aminoacylase-1-mediated deacetylation provides an orthogonal approach to detect MACs that lack the characteristic neutral loss of 129.0426 Da. Additionally, an in-house MAC library of 734,170 putative structures, combined with an in silico structure annotation tool, facilitated efficient MAC identification. The strategy was validated using 11 MAC standards spiked into urine and applied to urine samples from 15 participants before and after consuming deep-fried foods. A total of 847 features were mapped to at least one MAC structure, exceeding the ∼100 reported in previous studies, with 581 absent from the Human Metabolome Database (HMDB) or PubChem. Postconsumption, 59 MACs increased and 11 decreased, reflecting exposure-related shifts in reactive aldehydes, lipid oxidation products, acylcarnitines, and acyl-CoA intermediates. These findings demonstrate the utility of the workflow for capturing urinary MAC changes associated with deep-fried food consumption and for prioritizing candidate exposure indicators of reactive chemical species for future validation studies.