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Artifacts related to lucigenin chemiluminescence for superoxide detection in a vascular system

There is a growing interest in the pathophysiology of processes involving vascular cell injury and redox signaling, particularly with superoxide generation. Lucigenin chemiluminescence has been extensively used as a method to assess superoxide production and its underlying enzymatic mechanisms in many biological systems, the most studied one being the vascular NAD(P)H oxidase. Recent evidence suggests substantial limitations of this probe because of artifactual superoxide generation. To investigate if lucigenin concentration could affect the detection of vascular NAD(P)H oxidase activity we performed studies with lucigenin chemiluminescence, oxygen consumption and electron paramagnetic resonance (EPR) spectroscopy for superoxide generation detection with vascular homogenates and different concentrations of lucigenin (5, 50 and 250 μM). The NAD(P)H oxidase blocker diphenylene iodonium (DPI, 20 μM), SOD (500 IU/ml), catalase (500 IU/ml) and the electron acceptor NBT were also used to characterize lucigenin behavior in a vascular system.

Our data showed that lucigenin alone, with 5 μM, induced a 2-fold increase in oxygen consumption, while with 250 μM oxygen consumption increased 5-fold. Superoxide generation, assessed by EPR spectroscopy, also increased progressively with 5, 50 and 250 μM lucigenin. These effects were particularly enhanced by addition of NADH, but occurred also with NADPH. Chemiluminescence studies showed that with 5 and 50 μM lucigenin there is greater NADPH induced signal than NADH, while 250 μM lucigenin yields a 1.5-fold greater signal with NADH than with NADPH. Furthermore, all NADPH-driven luminescent signals were inhibited by SOD, DPI, and NBT, as well as NADH-driven luminescence with 5 μM lucigenin. On the other hand, with lucigenin 250 μM, NADH-driven luminescence could not be blocked by SOD or DPI, but was completely inhibited by NBT. Catalase did not show any inhibitory effect on NADPH-induced luminescence, but inhibited 30% of NADH-driven signals.

In conclusion, lucigenin even at low doses undergoes redoxcycling reactions, which are favored by NADH generating artifactual superoxide. Furthermore, it is possible that lucigenin acts as a direct electron acceptor from vascular enzymatic sources other than the superoxide-generating NAD(P)H oxidase, and detects also hydrogen peroxide generated by a vascular NADH oxidase.

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Janiszewski, M., Pedro, M., Souza, H. et al. Artifacts related to lucigenin chemiluminescence for superoxide detection in a vascular system. Crit Care 5 (Suppl 3), P20 (2001).

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