Facts and artefacts on the oxygen dependence of hydrogen peroxide flux using Amplex UltraRed

Authors

  • Timea Komlodi Oroboros Instruments, Innsbruck, Austria
  • Ondrej Sobotka 3rd Department of Internal Medicine – Metabolic Care and Gerontology, University Hospital Hradec Kralove, Department of Physiology, Faculty of Medicine in Hradec Kralove, Charles University, Czech Republic
  • Erich Gnaiger Oroboros Instruments, Innsbruck, Austria

DOI:

https://doi.org/10.26124/bec:2021-0004

Keywords:

Amplex UltraRed AmR, Amplex UltroxRed xRed, hydrogen peroxide production, hydrogen peroxide flux, respiration media, mitochondrial respiration medium 5 MiR05, yeast, oxygen dependence, reductive stress, anoxia, hypoxia, O2 kinetics, respiration, reoxygenation

Abstract

The fluorometric Amplex™ UltraRed AmR assay is frequently used for quantitative assessment of hydrogen peroxide production. It is specific to H2O2, can be calibrated accurately, and allows continuous real-time measurement. Without correction for the background fluorescence slope, however, H2O2-independent formation of the fluorescent product UltroxRed (or resorufin from Amplex™ Red) leads to artefacts.

We analysed (1) the medium specificity of the background fluorescence slope of the AmR assay, and (2) the oxygen dependence of H2O2 flux in baker´s yeast Saccharomyces cerevisiae. Apparent H2O2 flux, O2 concentration, and O2 flux were measured simultaneously by high-resolution respirometry equipped with the fluorescence module. The apparent H2O2 flux of yeast showed a maximum under hypoxia when incubated in Dulbecco´s Phosphate Buffered Saline DPBS or KCl-medium. This hypoxic peak increased with the sequential number of normoxic-anoxic transitions. Even in the absence of yeast, the fluorescence slope increased at low O2 levels as a function of fluorescence intensity. The hypoxic peak was not observed in mitochondrial respiration medium MiR05. Therefore, the hypoxic peak was a medium-specific background effect unrelated to cell physiology. In MiR05, H2O2 production of yeast decreased linearly from hyperoxia to hypoxia, with a steep decline towards anoxia. Respiration and oxygen dependence expressed as p50 of yeast were higher in MiR05 than DPBS. Respiration was a hyperbolic function of oxygen concentration in the low-oxygen range. The flux-dependence of oxygen affinity explained the higher p50 in MiR05.

Cite:

Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004

Author Biographies

Timea Komlodi, Oroboros Instruments, Innsbruck, Austria

Shared first author

Ondrej Sobotka, 3rd Department of Internal Medicine – Metabolic Care and Gerontology, University Hospital Hradec Kralove, Department of Physiology, Faculty of Medicine in Hradec Kralove, Charles University, Czech Republic

Shared first author

Published

2021-12-21

Issue

Section

Living Communications