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.

在本文中，我们描述了一种可同时分析生物系统中的耗氧率（OCR）和超氧化物产生的分析方法。分析的建立用途电子用两种不同的同位素标记的传感器顺磁共振（EPR）谱：¹⁵的N- PDT（4-氧代-2,2,6,6-四甲基哌d ₁₆ - ¹⁵ N-1氧基）作为氧敏感探针和¹⁴ N-CMH（1-羟基-3-甲氧羰基-2,2,5,5-四甲基吡咯烷，一种环状羟胺，作为活性氧（ROS）的传感器。超氧化物对CMH氧化的贡献是使用SOD或PEGSOD作为对照进行评估。由于EPR光谱不可叠加，因此EPR线宽的变化为¹⁵N-PDT（与OCR相连）和由¹⁴形成的氮氧化物N-CMH（与超氧化物的产生有关）可以在单一制剂上随时间同时记录。EPR工具箱已通过越来越复杂的生物系统认证。首先，我们使用了基于次黄嘌呤（HX）/黄嘌呤氧化酶（XO）的酶促测定方法，该方法是氧气消耗和超氧化物产生的良好模型。其次，我们使用暴露于佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）的巨噬细胞来生产超氧化物的细胞模型，这刺激NADPH氧化酶（NOX）消耗氧气并产生超氧化物。最后，我们将分离的线粒体暴露于已建立的电子传输链抑制剂（鱼藤酮和二甲双胍）中，以评估其对OCR和超氧化物产生的影响。