Daphne Medina, Bhavana Omanakuttan, Ricky Nguyen, Eman Alwarsh, Charuksha Walgama
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引用次数: 0
Abstract
Human liver subcellular fractions, including liver microsomes (HLM), liver cytosol fractions, and S9 fractions, are extensively utilized in in vitro assays to predict liver metabolism. The S9 fractions are supernatants of human liver homogenates that contain both microsomes and cytosol, which include most cytochrome P450 (CYP) enzymes and soluble phase II enzymes such as glucuronosyltransferases and sulfotransferases. This study reports on the direct electrochemistry and biocatalytic features of redox-active enzymes in S9 fractions for the first time. We investigated the electrochemical properties of S9 films by immobilizing them onto a high-purity graphite (HPG) electrode and performing cyclic voltammetry under anaerobic (Ar-saturated) and aerobic (O2-saturated) conditions. The heterogeneous electron transfer rate between the S9 film and the HPG electrode was found to be 14 ± 3 s−1, with a formal potential of −0.451 V vs. Ag/AgCl reference electrode, which confirmed the electrochemical activation of the FAD/FMN cofactor containing CYP450-reductase (CPR) as the electron receiver from the electrode. The S9 films have also demonstrated catalytic oxygen reduction under aerobic conditions, identical to HLM films attached to similar electrodes. Additionally, we investigated CYP activity in the S9 biofilm for phase I metabolism using diclofenac hydroxylation as a probe reaction and identified metabolic products using liquid chromatography–mass spectrometry (LC-MS). Investigating the feasibility of utilizing liver S9 fractions in such electrochemical assays offers significant advantages for pharmacological and toxicological evaluations of new drugs in development while providing valuable insights for the development of efficient biosensor and bioreactor platforms.
MetabolitesBiochemistry, Genetics and Molecular Biology-Molecular Biology
CiteScore
5.70
自引率
7.30%
发文量
1070
审稿时长
17.17 days
期刊介绍:
Metabolites (ISSN 2218-1989) is an international, peer-reviewed open access journal of metabolism and metabolomics. Metabolites publishes original research articles and review articles in all molecular aspects of metabolism relevant to the fields of metabolomics, metabolic biochemistry, computational and systems biology, biotechnology and medicine, with a particular focus on the biological roles of metabolites and small molecule biomarkers. Metabolites encourages scientists to publish their experimental and theoretical results in as much detail as possible. Therefore, there is no restriction on article length. Sufficient experimental details must be provided to enable the results to be accurately reproduced. Electronic material representing additional figures, materials and methods explanation, or supporting results and evidence can be submitted with the main manuscript as supplementary material.