Electrochemical Activation of Metabolic Enzymes in Human Liver S9 Fractions

Abstract

Human liver subcellular fractions such as liver microsomes, liver cytosol fractions, or S9 fractions are widely used in in-vitro assays to predict liver metabolism. The S9 fractions are the supernatants of human liver homogenates consist of both human liver microsomes and the cytosol. Therefore, these fractions contain majority of cytochrome P450 (CYP) enzymes and soluble phase II enzymes like glucuronosyltransferases, N-acetyl transferases, sulfotransferases, and glutathione-S-transferases. Here we report the direct electrochemistry and biocatalytic features of redox active enzymes impregnated in S9 fractions. We investigated the electrochemical properties of S9 films by immobilizing them onto a high purity graphite electrode and performing cyclic voltammetry in anaerobic (Ar saturated) and aerobic (O2 saturated) conditions. The electron transfer between the S9 film and the high purity graphite electrode was observed at the formal potential of -0.48 V, with respect to Ag/AgCl reference electrode, which confirmed the activation of CYP450 reductase enzyme in the film. These films also demonstrated a catalytic oxygen reduction with a maximum current density of ~0.5 mA cm-2 at the scan rate of 0.050V/s. We investigated the CYP activity in the S9 biofilm for phase 1 metabolism using diclofenac hydroxylation as a probe reaction and could able to identify metabolic products using liquid chromatography-mass spectrometry (LC-MS) technique. Eventually, we envision to develop a platform to perform both phase I and II metabolic reactions in a single electrochemical assay.