Li Puma 2020 Am J Physiol Regul Integr Comp Physiol
|Li Puma LC, Hedges M, Heckman JM, Mathias AB, Engstrom MR, Brown AB, Chicco AJ (2020) Experimental oxygen concentration influences rates of mitochondrial hydrogen peroxide release from cardiac and skeletal muscle preparations. Am J Physiol Regul Integr Comp Physiol 318:972-80.|
» PMID: 32233925 »
Abstract: Mitochondria utilize the majority of oxygen (O2) consumed by aerobic organisms as the final electron acceptor for oxidative phosphorylation (OXPHOS), but also to generate reactive oxygen species (mtROS) that participate in cell signaling, physiological hormesis and disease pathogenesis. Simultaneous monitoring of mtROS production and oxygen consumption (JO2) from tissue mitochondrial preparations is an attractive investigative approach, but introduces dynamic changes in media O2 concentration ([O2]) that can confound experimental results and interpretation. We utilized high-resolution fluoro-respirometry to evaluate JO2 and hydrogen peroxide release (JH2O2) from isolated mitochondria (Mt), permeabilized fibers (Pf), and tissue homogenates (Hm) prepared from murine heart and skeletal muscle across a range of experimental [O2]s typically encountered during respirometry protocols (400-50 µM). Results demonstrate notable variations in JH2O2 across tissues and sample preparations during non-phosphorylating (LEAK) and OXPHOS-linked respiration states at 250 µM [O2], but a linear decline in JH2O2 of 5-15% per 50 µM decrease in chamber [O2] in all samples. JO2 was generally stable in Mt and Hm across [O2]s above 50 µM, but tended to decline below 250 µM in Pf, leading to wide variations in assayed rates of JH2O2/O2 across chamber [O2]s and sample preparations. Development of chemical background fluorescence from the H2O2 probe (Amplex Red) was also O2-sensitive, emphasizing relevant calibration considerations. These studies highlight the importance of monitoring and reporting the chamber [O2] at which JO2 and JH2O2 are recorded during fluoro-respirometry experiments, and provide a basis for selecting sample preparations for studies addressing the role mtROS in physiology and disease.
- Sobotka et al (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed in yeast cells. MitoFit Preprints 2021.
- Komlodi T et al (2021) Oxygen dependence of hydrogen peroxide production using Amplex UltraRed in yeast cells, isolated mitochondria, and permeabilized cells. MitoFit Preprints 2021 (in prep).
Labels: MiParea: Respiration
Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Heart, Skeletal muscle Preparation: Permeabilized tissue, Homogenate, Isolated mitochondria
Coupling state: LEAK, OXPHOS Pathway: N, NS HRR: Oxygraph-2k, O2k-Fluorometer
2020-04, AmR, O2k-brief, MitoFit 2021 Yeast