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

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) leads to artefacts. 

 We analyzed (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.