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Updated: Jun 21, 2026
Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals
Published on: May 19, 2023
Flow cytometry-based assay for the activity of NAD(P)H oxidoreductases of the outer mitochondrial membrane
Alexey G Kruglov1, Marina E Solov'eva, Vera V Teplova
1Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia. krugalex@rambler.ru
Abstract:
NAD(P)H oxidoreductases of the outer mitochondrial membrane (OMM) are able to activate various xenobiotics and stimulate the production of reactive oxygen species and the opening of the mitochondrial permeability transition pore. However, the role of these systems in the cell damage by xenobiotics and chemotherapeutic drugs is poorly understood because the methods for the selective assessment of their activity have not been elaborated and specific inhibitors are unknown. Here we propose a method for the semiquantitative assessment of the activity of NAD(P)H oxidoreductases of the OMM in intact and permeabilized cells that is based on the flow cytometry detection of dimethylbiacridene, a fluorescent product of two-electron reduction of lucigenin. The method uses the structural feature of mitochondrial organization: the proximity of the sites of one-electron reduction of lucigenin to cation radical (NAD(P)H oxidoreductases of the OMM) to the sites of its subsequent oxidation (cytochrome c oxidase). The inhibition of cytochrome c oxidase by cyanide selectively activates the dimethylbiacridene formation by oxidoreductases of the OMM but not by other cellular oxidoreductases. The proposed protocol allows one to assess the lucigenin reductase (two-electron) activity of NAD(P)H oxidoreductases of the OMM and to compare it with the activity of other cellular systems that can be used for the analysis of the role of these systems in the cell damage by xenobiotics and antitumor drugs.
Insights
We developed a new flow cytometry method to measure outer mitochondrial membrane NAD(P)H oxidoreductase activity. This technique helps understand how xenobiotics and chemotherapy drugs damage cells.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Outer mitochondrial membrane NAD(P)H oxidoreductases activate xenobiotics, producing reactive oxygen species and opening the mitochondrial permeability transition pore.
- The precise role of these oxidoreductases in xenobiotic and chemotherapy-induced cell damage remains unclear due to a lack of selective assessment methods and specific inhibitors.
Purpose of the Study:
- To develop and validate a novel method for the semiquantitative assessment of outer mitochondrial membrane NAD(P)H oxidoreductase activity in intact and permeabilized cells.
- To enable the analysis of the contribution of these enzymes to cellular damage induced by xenobiotics and anticancer drugs.
Main Methods:
- A flow cytometry-based assay utilizing lucigenin and its fluorescent reduction product, dimethylbiacridene.
- Exploiting the proximity of outer mitochondrial membrane NAD(P)H oxidoreductases and cytochrome c oxidase within the mitochondria.
- Selective activation of dimethylbiacridene formation by inhibiting cytochrome c oxidase with cyanide.
Main Results:
- The proposed method allows for the selective detection of lucigenin reductase (two-electron reduction) activity specifically attributed to outer mitochondrial membrane NAD(P)H oxidoreductases.
- This approach differentiates the activity of outer mitochondrial membrane enzymes from other cellular oxidoreductases.
- The protocol provides a means to compare the activity of outer mitochondrial membrane NAD(P)H oxidoreductases with other cellular redox systems.
Conclusions:
- A novel, selective, and semiquantitative method for assessing outer mitochondrial membrane NAD(P)H oxidoreductase activity has been established.
- This method facilitates research into the role of these mitochondrial enzymes in cellular responses to xenobiotics and chemotherapeutic agents.
- The developed protocol is valuable for investigating drug-induced toxicity and designing more effective therapeutic strategies.
