Metabolomic analysis reveals novel ethylated hydroxytyrosol metabolites in colon cancer cells

Plants produce a diverse array of secondary metabolites, with hydroxytyrosol (HT) and its derivatives distinguished by their antioxidant and chemopreventive properties. Upon dietary consumption, native HT undergoes extensive biotransformation, initially facilitated by the gut microbiota. Increasing evidence suggests that several of these downstream products, rather than the parent molecule, are responsible for the most significant biological effects in mammalian cells. The identification of novel metabolites may reveal unknown metabolic pathways, more particularly in distinct pathological contexts such as cancer. The in vitro simulated gastrointestinal digestion (INFOGEST method) of HT, followed by bioavailability in the tumoral metabolically active intestinal Caco-2 cells (50 µM HT for 2, 4, and 6 h), was performed. Subsequently, an untargeted metabolomic analysis on the supernatant revealed novel HT-derived entities compared with water. Among these, ethoxy phenylacetic acid sulfates (4, 5), ethyl hydroxyphenylacetate acid sulfate (6) and ethoxy hydroxyphenylacetic acid or ethyl hydroxyphenylacetate glucuronide were described for the first time in a study and confirmed by MS/MS fragmentation. These accumulated ethylated forms were undetectable in non-tumoral plasma samples from HT-supplemented humans (60 mg day^-1 HT for 28 days) and mice (50 mg kg^-1 day^-1 HT for 2 months), suggesting a tumour-specific or, at the very least, tumour-favoured biotransformation pathway. These findings not only expand the diversity of HT metabolites but also propose ethoxy-phenylacetic acid and its sulfate conjugate as potential biomarkers for cancer detection.