Defining the Oxysterolome: Analytical Coverage, Metabolic Connectivity, and Biological Validation.

The number of oxygenated derivatives of cholesterol, its precursors, and other biologically relevant sterols that exist in biological systems is unknown and may extend substantially beyond the oxysterols currently characterized. Successive oxidation, reduction, hydrolysis, sulfation, glucuronidation, esterification, and conjugation reactions can generate a large chemical space that we refer to as the oxysterolome. However, chemical possibility does not establish endogenous existence, and endogenous existence does not establish biological function. Defining this metabolic space requires a progression from analytical annotation to structural authentication, metabolic reconstruction, and biological validation. Iterative metabolism broadens physicochemical diversity, making analytical coverage method-dependent and requiring complementary approaches for different metabolite classes. Authenticated metabolites should subsequently be connected through precursor-product relationships, enzyme dependence, isotope tracing, metabolic flux, and spatial organization. Biological relevance requires separate evidence linking endogenous exposure and pathway perturbation to molecular mechanisms and phenotypes. Further metabolism can generate, terminate, or redirect biological activity, but these outcomes cannot be predicted from structural complexity. The oxysterolome should therefore be approached not as an expanding catalogue of oxygenated sterols, but as an experimentally reconstructed metabolic network integrating molecular identity, pathway connectivity, flux, and biological causality.