Schmitt 2022 Abstract Bioblast

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2.2. «10+5»
Sabine Schmitt
Schmitt Sabine, Gnaiger E (2022) Using oxygen and hydrogen gas in studies of mitochondrial respiration and hydrogen peroxide production under hyperoxic, normoxic, and hypoxic conditions. Bioblast 2022: BEC Inaugural Conference. In: https://doi.org/10.26124/bec:2022-0001

Link: Bioblast 2022: BEC Inaugural Conference

Schmitt Sabine, Gnaiger Erich (2022)

Event: Bioblast 2022

Oxygen concentration cO2 in tissues is tightly controlled by O2 signaling to ensure sufficient O2 supply for mitochondrial respiration and prevent oxidative stress at excessive O2 levels. Similarly, cO2 in the experimental medium must be controlled in studies with cultured cells and mitochondrial preparations to simulate extracellular and intracellular conditions in the tissues of origin.

Mitochondrial respiration is independent of cO2 down to ~5 % air saturation [1]. In contrast, ROS production is a continuous function of cO2 from hypoxia to hyperoxia [2]. Decreasing the cO2 in the experimental chamber below air saturation is needed to (1) study mitochondrial function under physiologically relevant cO2, and (2) zoom into the low O2 range to study O2 kinetics under hypoxia. Increased cO2 above air saturation is applied in experiments with permeabilized muscle fibers to prevent artificial O2 diffusion limitation of respiration [3] or to induce hyperoxic stress.

Our newly developed Oxia (Oroboros Instruments) generates oxygen and hydrogen gas by electrolysis of H2O [4]. O2 or H2 is injected into the gas phase of the open O2k-chamber which is closed when the desired cO2 is reached. H2 has been described to affect mitochondrial metabolism. Therefore, we evaluated the effect of lowering cO2 on mitochondrial function in permeabilized HEK 293T cells by injecting either H2 or the conventionally used N2 gas. Simultaneous measurements of mitochondrial respiration and H2O2 production were performed in the LEAK and OXPHOS coupling states fueled by the NADH-linked substrates pyruvate & malate. Upon transition of cO2 from ~160 to ~25 µM, O2 and H2O2 flow of the permeabilized cells decreased even above the kinetic cO2 range when cytochrome c oxidase becomes oxygen-limited [1]. However, the observed changes in O2 and H2O2 flow were indistinguishable when either H2 or N2 were used for lowering cO2.

With Oxia H2 and O2 gases can be produced any time in experimentally required volumes directly at the bench besides the O2k. This is a safe, convenient, and economic alternative to the use of high-pressure gas tanks.

  1. Harrison DK, Fasching M, Fontana-Ayoub M, Gnaiger E (2015) Cytochrome redox states and respiratory control in mouse and beef heart mitochondria at steady-state levels of hypoxia. https://doi.org/10.1152/japplphysiol.00146.2015
  2. Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. https://doi.org/10.26124/bec:2021-0004
  3. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. https://doi.org/10.1007/978-1-61779-382-0_3
  4. Schmitt S, Merth A, Walter-Vracevic M, Gnaiger E (2022) Oxia - HyperOxia to HypOxia. https://wiki.oroboros.at/index.php/MiPNet26.14_Oxia

Keywords: tissue normoxia, hypoxia, hyperoxia, High-resolution respirometry, mitochondrial H2O2 production

O2k-Network Lab: AT Innsbruck Oroboros


Affiliation

Oroboros Instruments GmbH, Innsbruck, Austria

List of abbreviations, terms and definitions - MitoPedia

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Labels: MiParea: Respiration 


Tissue;cell: HEK  Preparation: Permeabilized cells 


Coupling state: LEAK, OXPHOS  Pathway:HRR: Oxygraph-2k  Event: A2