Immobilized Enzymes on Magnetic Beads for Separate Mass Spectrometric Investigation of Human Phase II Metabolite Classes

The human body hasevolved to remove xenobiotics through a multistepclearance process. Non-endogenous metabolites are converted througha series of phase I and different phase II enzymes into compoundswith higher hydrophilicity. These compounds are important for diverseresearch fields such as toxicology, nutrition, biomarker discovery,doping control, and microbiome metabolism. One of the challenges inthese research fields has been the investigation of the two majorphase II modifications, sulfation and glucuronidation, and the correspondingunconjugated aglycon independently. We have now developed a new methodologyutilizing an immobilized arylsulfatase and an immobilized & beta;-glucuronidaseto magnetic beads for treatment of human urine samples. The enzymeactivities remained the same compared to the enzyme in solution. Theseparate mass spectrometric investigation of each metabolite classin a single sample was successfully applied to obtain the dietaryglucuronidation and sulfation profile of 116 compounds. Our new chemicalbiology strategy provides a new tool for the investigation of metabolitesin biological samples with the potential for broad-scale applicationin metabolomics, nutrition, and microbiome studies.