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The human body clears both endogenous and exogenous compounds through phase I and phase II biotransformation reactions. These processes rely on enzymes that modify metabolites into more hydrophilic forms, facilitating their excretion. Phase II modifications such as sulfation, glucuronidation and acetylation play a critical role in diverse research fields including toxicology, nutrition and microbiome metabolism. Among these, the two major phase II conjugate classes, glucuronides and sulfates, have been closely linked to the microbiota-host co-metabolism. The microbiome metabolism also converts dietary components and drugs, and has been revealed to impact human health, including the production of potentially toxic compounds.

In this thesis, advanced metabolomics approaches were combined with new analytical and chemical biology tools to selectively investigate the phase II metabolome in multiple biological contexts. In a nutritional study, untargeted LC–MS metabolomics combined with enzymatic hydrolysis enabled the identification of over 150 sulfated and 140 glucuronidated metabolites in urine from a controlled dietary intervention. Several of these metabolites were proposed as dietary biomarkers of (poly)phenol intake, providing tools for objective dietary assessment and highlighting the contribution of gut microbial metabolism. In the next step, the glucuronide profiling was expanded to plasma, feces and cerebrospinal fluid. The results revealed that this conjugation class is also present in these less commonly studied biospecimens, including the novel detection of a drug-derived glucuronide in human CSF, highlighting the translational potential of this approach.

To improve analytical coverage, a novel chemical biology methodology was developed by immobilizing enzymes on magnetic beads. This tool enabled for the first time parallel analysis of glucuronides and sulfates in the same sample, allowing for semi-quantification within global metabolomics workflows. The focus then shifted to acetylation catalyzed by NAT2, where NAT2-dependent cytotoxicity was explored for several clinically relevant anticancer drugs. These findings showcased the importance of genetic variability in NAT2 activity for drug metabolism and potential treatment outcomes. Finally, a pan-cancer plasma metabolomics investigation identified both cancer-specific and shared metabolic alterations, as well as stage-dependent changes across lung, colorectal and ovarian cancers. These results highlight candidate biomarkers with potential applications in diagnosis and disease monitoring.

This thesis demonstrates how advanced analytical strategies can improve the characterization of phase II metabolites and support biomarker discovery across nutrition, pharmacology, and oncology. The results within this thesis provide useful insights for translational metabolomics, with applications in nutrimetabolomics, pharmacometabolomics, and cancer metabolomics.