Identification of Novel Urolithin Metabolites in Human Feces and Urine after the Intake of a Pomegranate Extract

Urolithins are bioactive gut microbiota metabolites of ellagic acid. Here, we have identified four unknown urolithins in human feces after the intake of a pomegranate extract. The new metabolites occurred only in 19% of the subjects. 4,8,9,10-Tetrahydroxy urolithin, (urolithin M6R), was unambiguously identified by H-1 NMR, UV, and HRMS. Three metabolites were tentatively identified by the UV, HRMS, and chromatographic behavior, as 4,8,10-trihydroxy (urolithin M7R), 4,8,9-trihydroxy (urolithin CR), and 4,8-dihydroxy (urolithin AR) urolithins. Phase II conjugates of the novel urolithins were detected in urine and confirmed their absorption, circulation, and urinary excretion. The production of the new urolithins was not specific of any of the known urolithin metabotypes A and B. The new metabolites needed a bacterial 3-dehydroxylase activity for their production, and this is a novel feature as all the previously known urolithins maintained the hydroxyl at 3 position. The ability of production of these R urolithins can be considered an additional metabolic feature for volunteer stratification.