Laner 2013 ASMRM
|Laner V, Gradl P, Eigentler A, Fontana-Ayoub M, Fasching M, Gnaiger E (2013) The Oroboros O2k for high-resolution respirometry and O2k-Fluorometry: a new quality in OXPHOS analysis. ASMRM2013.
High-resolution respirometry (HRR) has become established world-wide for the diagnosis of mitochondrial respiratory control and OXPHOS analysis, reflected in >1,000 O2k-publications using the Oroboros Oxygraph-2k. The O2k-Fluorescence LED2-Module is a recent extension of the Oxygraph-2k for combining HRR and fluorometric measurements. Two major applications of O2k-Fluorometry are presented.
- H2O2 production measured with Amplex® Ultrared in mouse heart mitochondria was not only a function of respiratory substrate and coupling state, but depended strongly on experimental oxygen levels below and even above air saturation of the respiration medium.
- Mitochondrial membrane potential was measured with Safranin (2 µM) with high sensitivity particularly in the range of low membrane potentials. However, Safranin specifically inhibited Complex I linked respiration and the phosphorylation system, such that meaningful applications had to be restricted to Complex II linked respiration with succinate and rotenone. Inhibitory side effects must be critically evaluated for any probe of mitochondrial membrane potential.
These examples illustrate the sensitivity and wide range of applicability of O2k-fluorometry and demonstrate the importance of combining measurements of hydrogen peroxide production and mitochondrial membrane potential with simultaneous monitoring of oxygen concentration and oxygen consumption.
• Keywords: OXPHOS analysis, mitochondrial respiration, hydrogen peroxide production, mt-membrane potential, functional mitochondrial diagnosis
Supported by K-Regio project MitoCom Tyrol, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).
Labels: MiParea: Respiration, Instruments;methods
Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Heart Preparation: Homogenate
Regulation: Oxygen kinetics Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS HRR: Oxygraph-2k, O2k-Fluorometer