Fully automated electrophoretically mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate approach

Abstract

In this study, a fully automated incapillary system was developed to monitor the activity of CYP1A1 (Cytochrome P450, family 1, subfamily A, polypeptide 1) in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an inline bioreaction in the presence of CYP1A1 supersomes and Nicotinamide adenine dinucleotide phosphate reduced as cofactor, giving rise to hydroxycoumarin, the product that was assayed. The optimization of the experimental conditions was supported by the application of a design of experiment, providing a better understanding of electrophoretic mixing parameters that influence the metabolic reactions. The results obtained in optimal conditions were compared not only to those achieved after offline metabolization but also with liver microsomes. Finally, inhibition studies were conducted showing an important decrease of hydroxycoumarin formation using apigenin as CYP1A1 potent inhibitor. This study demonstrates the usefulness of our inline system for the fully automated in vitro metabolism studies and the screening of new CYP1A1 inhibitors.

Advances in screening enzyme inhibitors by capillary electrophoresis

“…Online enzyme assay integrates together enzyme incubation, reaction, and product detection in a single capillary within one run. Comparing off‐line enzyme assay, it thus greatly simplified enzyme assay process, and has developed two mainly operation modes, at inlet and electrophoretically mediated microanalysis (EMMA) .…”

“…Farcas et al. established an automated capillary system to monitor the activity of CYP1A1 in physiological conditions. In their work, a short‐end injection and separation was selected, which caused a short assay time (<2 min) and achieved the baseline separation of the compounds (7‐EC, 7‐HC, and NADPH).…”

Advances in Capillary Electrophoretically Mediated Microanalysis for On‐line Enzymatic and Derivatization Reactions

“…In a study by Farcaş et al. , a fully automated EMMA system was developed to monitor the activity of CYP1A1. This on‐line study was performed by the partial filling mode and a zero potential amplification step was applied.…”