A versatile EPR toolbox for the simultaneous measurement of oxygen consumption and superoxide production.

Abstract:

:In this paper, we describe an assay to analyze simultaneously the oxygen consumption rate (OCR) and superoxide production in a biological system. The analytical set-up uses electron paramagnetic resonance (EPR) spectroscopy with two different isotopically-labelled sensors: 15N-PDT (4-oxo-2,2,6,6-tetramethylpiperidine-d16-15N-1-oxyl) as oxygen-sensing probe and 14N-CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine, a cyclic hydroxylamine, as sensor of reactive oxygen species (ROS). The superoxide contribution to CMH oxidation is assessed using SOD or PEGSOD as controls. Because the EPR spectra are not superimposable, the variation of EPR linewidth of 15N-PDT (linked to OCR) and the formation of the nitroxide from 14N-CMH (linked to superoxide production) can be recorded simultaneously over time on a single preparation. The EPR toolbox was qualified in biological systems of increasing complexity. First, we used an enzymatic assay based on the hypoxanthine (HX)/xanthine oxidase (XO) which is a well described model of oxygen consumption and superoxide production. Second, we used a cellular model of superoxide production using macrophages exposed to phorbol 12-myristate 13-acetate (PMA) which stimulates the NADPH oxidase (NOX) to consume oxygen and produce superoxide. Finally, we exposed isolated mitochondria to established inhibitors of the electron transport chain (rotenone and metformin) in order to assess their impact on OCR and superoxide production. This EPR toolbox has the potential to screen the effect of intoxicants or drugs targeting the mitochondrial function.

journal_name

Redox Biol

journal_title

Redox biology

authors

Donatienne d'Hose,Danhier P,Northshield H,Isenborghs P,Jordan BF,Gallez B

doi

10.1016/j.redox.2020.101852

subject

Has Abstract

pub_date

2020-12-28 00:00:00

pages

101852

issn

2213-2317

pii

S2213-2317(20)31057-0

journal_volume

40

pub_type

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