Gnaiger 2016 Abstract SFRR Australasia 2016
|Facts and artefacts in measurements of H2O2 production under hypoxia.|
Event: SFRR Australasia 2016
The Amplex Red (AmR) assay is one of the most popular fluorescence methods for determination of mitochondrial and cellular net hydrogen peroxide production (H2O2 flux). Quantitative and accurate measurement of ROS production, however, remains a critical issue. Quality control is required to avoid numerous artefacts. Independent of H2O2 flux, increasing fluorescence signals may result from photosensitive auto-oxidation of AmR, which represents a confounding chemical background process . What is fact and artefact in the fluorometric AmR assay?
H2O2 production and oxygen consumption were measured simultaneously in Saccharomyces cerevisiae, using the O2k-Fluorometer  (Oroboros Instruments, Austria). Following storage of baker’s yeast in the freeze-dried state, the cells were resuspended in four different respiration media: Dulbecco‘s phosphate buffer saline (DBPS); KCl-based respiratory medium; respiratory media MiR05 and Buffer z.
Apparent H2O2 production increased significantly during repeated hypoxia/anoxia transitions of yeast suspended in DPBS. The apparent H2O2 flux increased with progressive hypoxia and declined sharply under deep hypoxia and anoxia. Such hypoxic peaks of H2O2 production were not observed in MiR05 and Buffer z. The apparent reductive oxidative stress in DPBS disappeared, however, when AmR was added only after several hypoxia/anoxia transitions. In DPBS but not in MiR05, the chemical background slope (change of the fluorescence signal over time) increased exponentially from normoxia to hypoxia, and the background slope increased as a function of resorufin concentration. Taken together, these background tests explain quantitatively the apparent hypoxic peaks of H2O2 production as an artifact of the AmR assay. The actual H2O2 flux declined monotonuously from normoxia to hypoxia and anoxia, not indicative of any transient reductive oxidative stress under hypoxia.
Labels: MiParea: Respiration, Instruments;methods
Stress:Oxidative stress;RONS Organism: Saccharomyces cerevisiae
Preparation: Intact cells
Regulation: Oxygen kinetics Coupling state: ROUTINE
HRR: Oxygraph-2k, O2k-Fluorometer
1-Dept Physiol, Charles Univ Prague, Fac Med Hradec Kralove, Czech Republic, 2-Oroboros Instruments, Innsbruck, Austria, 3-D Swarowski Research Lab, Dept Visceral Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - firstname.lastname@example.org
References and support
Supported by K-Regio project MitoFit.
- Zhao B Summers FA, Mason RP (2012) Photooxidation of Amplex Red to resorufin: implications of exposing the Amplex Red assay to light. Free Radic Biol Med 53:1080-7.
- Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. »Bioblast link