Gnaiger 2018 AussieMit
|Komlodi T, Sobotka O, Doerrier C, Gnaiger E (2018) Mitochondrial H2O2 production is low under tissue normoxia but high at in-vitro air-level oxygen pressure - comparison of LEAK and OXPHOS states. AussieMit 2018 Melbourne AU.|
Event: AussieMit 2018 Melbourne AU
Hydrogen peroxide (H2O2) production increases at maximum mitochondrial membrane potential in the resting state of LEAK-respiration relative to the ADP-activated OXPHOS state at lower mt-membrane potential. This OXPHOS-LEAK paradigm of mitochondrial ROS production is based on studies which ignore the fact that the normoxic intracellular oxygen level within tissues (tissue normoxia) is far lower than in-vitro air-level normoxia. We show that the OXPHOS-LEAK-ROS paradigm cannot be extended to physiological tissue normoxia.
H2O2 production and oxygen consumption were measured simultaneously in mitochondria isolated from mouse brain and heart, and in intact cells of Saccharomyces cerevisiae, using the O2k-FluoRespirometer (Oroboros Instruments, Austria). Net H2O2 flux was determined in the O2k-chamber with the Amplex UltraRed assay. MiR05-Kit was used upon evaluation of four respiration media (MiR05-Kit; Buffer z; KCl-based medium; Dulbecco‘s phosphate buffer saline).
Oxygen flux remained independent of oxygen concentration from air-level normoxia to tissue normoxia (~200 to 30 µM O2). As expected under typical incubation conditions near air-saturation, H2O2 production was high in the LEAK state (no ADP), and declined sharply in the OXPHOS state (saturating [ADP]). H2O2 production, however, declined linearly from air-level to tissue normoxia in the LEAK state, such that the effect of respiratory state on H2O2 production nearly vanished. Similarly, H2O2 flux declined from air-level to tissue normoxia in intact yeast during ROUTINE respiration.
Our results (1) do not show any transient reductive oxidative stress under hypoxia, (2) suggest that mitochondrial H2O2 production is independent of respiratory activity in the OXPHOS or LEAK state under tissue normoxia, and thus (3) challenge the LEAK-OXPHOS-ROS paradigm, emphasizing the importance of controlling dissolved oxygen in the range of physiological intracellular conditions and avoiding artificially high oxygen levels in studies of ROS production.
Labels: MiParea: Respiration, Comparative MiP;environmental MiP
Stress:Oxidative stress;RONS, Hypoxia Organism: Mouse, Saccharomyces cerevisiae Tissue;cell: Heart, Nervous system Preparation: Intact cells, Isolated mitochondria
Regulation: Redox state Coupling state: LEAK, ROUTINE, OXPHOS Pathway: NS HRR: Oxygraph-2k, O2k-Fluorometer
Komlòdi T(1), Sobotka O(2), Doerrier C()1, Gnaiger E(1,3)
- Oroboros Instruments, Innsbruck, Austria
- Dept Physiology, Fac Medicine Hradec Kralove, Charles Univ Prague, Czech Republic
- D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria