Sobotka 2018 MiP2018

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Ondrej Sobotka
Measurement of ROS production under hypoxia and unexpected methodological pitfalls of Amplex UltraRed assay.

Link: MiP2018

Komlodi T, Sobotka O, Doerrier C, Gnaiger E (2018)

Event: MiP2018

COST Action MitoEAGLE

Mitochondrial ROS production under hypoxia is a hot topic in recent literature and there is contradictory evidence about its regulation. Main theories regarding mitochondrial ROS a production involve its increased generation under physiological normoxia (i.e. [O2] < 50µM). In contrary, there are studies which reveal a linear relationship between O2 concentration and H2O2 generation. This discrepancy can be partly explained by the fact that many studies omit important physiological and methodological issues such as mitochondrial respiratory states, methodology of H2O2 assessment and the chemical effect of respiratory medium. Therefore we aimed to shed light on the [O2] dependence of mitochondrial H2O2 production using Amplex UltraRed assay in different respiration media in LEAK (without ADP) and OXPHOS (with saturated amount of ADP).

High-resolution respirometry (Oroboros Instruments, Innsbruck, Austria) was used to simultaneously measure mitochondrial respiration and H2O2 production with Amplex Ultra Red/horseradish peroxidase (AmUR) assay according to [1]. Mouse brain and heart mitochondria were isolated as previously described in [2]. H2O2 production during hypoxia-anoxia-reoxygenation transition was measured using commercially available dry baker´s yeast (Saccharomyces cerevisiae). Different respiratory media such as MiR05-Kit (Oroboros Instruments, Innsbruck, Austria; first described in [3]), KCl-based media [4] and commercially available DPBS (ThermoFisher scientific, Waltham, MA USA) were used in our experiments.

We found different medium-specific sensitivity and fluorescence background of AmUR assay towards H2O2. According to our results the most stable chemical background slope was observed in MiR05-Kit, but in DPBS and KCl-based buffer we measured an increase of chemical fluorescence emitted by AmUR assay over the experimental time or under various [O2].

In MiR05-Kit we reveal that under physiological normoxia (i.e. [O2] < 50µM) the H2O2 flux in LEAK and in OXPHOS overlap with each other both in brain and heart mitochondria. However, there is a linear relationship between H2O2 flux and oxygen concentration in LEAK, but less pronounced in OXPHOS.

Furthermore, we observed an exponential increase of yeast initiated H2O2 production after several hypoxia-anoxia-reoxygenation transitions in DPBS and in KCl-based buffer, but surprisingly not in MiR05-Kit. After series of background experiments we revealed that this phenomenon was the result of an artefact most probably due to AmUR auto-oxidation.

These results let us conclude that under physiological normoxia (< 50 µM), detection of H2O2 production using AmUR assay is only possible in MiR05-Kit to avoid methodological artefacts and to perform corrections of our data.


Bioblast editor: Plangger M O2k-Network Lab: AT Innsbruck Oroboros, CZ Hradec Kralove Cervinkova Z, AT Innsbruck Gnaiger E


Labels: MiParea: Respiration, Instruments;methods 

Stress:Hypoxia  Organism: Mouse, Saccharomyces cerevisiae  Tissue;cell: Heart, Nervous system  Preparation: Isolated mitochondria 


Coupling state: LEAK, OXPHOS 

HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 

Amplex UltraRed, MitoEAGLE, SUIT-013, SUIT-013 AmR ce D023 

Affiliations and support

Komlodi T(1), Sobotka O(3,4), Doerrier C(1), Gnaiger E(1,2)

  1. Oroboros Instruments
  2. Daniel Swarowski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ; Innsbruck, Austria
  3. Dept Physiology
  4. 3rd Dept Internal Medicine, Geriatric Metabolic Care, Univ Hospital; Fac Medicine Hradec Kralove, Charles Univ, Hradec Kralove, Czech Republic
Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.


References

  1. 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
  2. Komlódi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55
  3. Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. Life in the Cold pp:431-42
  4. Hoffman DL, Brookes PS (2009) Oxygen sensitivity of mitochondrial reactive oxygen species generation depends on metabolic conditions. J Biol Chem 284:16236–45