Meszaros 2017 MiPschool Obergurgl

(Very) high resolution respirometry – a deep dive into oxygen dependence of mitochondrial respiration.

Link: MITOEAGLE

Meszaros AT, Haider M, Sigh R, Gnaiger E (2017)

Event: MiPschool Obergurgl 2017

During the past three decades, measurement of mitochondrial oxygen (O₂) consumption during respiratory steady-states and at higher-than-physiological O₂ concentrations has become a gold standard not only in mitochondrial biochemistry but in a wide range of health sciences as well [1]. In contrast, kinetic aspects, such as O₂ dependence of mitochondrial respiration are largely neglected nowadays. However, assessment of parameters such as maximal O₂ flux (J_O2,max) and partial pressure of O₂, which limits mitochondrial O₂ consumption rate to half-maximum (p₅₀) has implications in a wide range of biomedical research topics, e.g. in work with biologically active gases (nitric oxide, hydrogen sulfide, carbon monoxide), which directly or indirectly influence cytochrome c oxidase.

To facilitate studies which involve O₂ kinetics, we (1) critically evaluated instrumental limitations of such measurements, (2) developed a new, up-to-date software tool for internal use to evaluate O₂ kinetics in a partly user-independent way, and (3) delineated future directions and objectives.

Working with the latest series of O2k high resolution respirometers (OROBOROS Instruments, Innsbruck, Austria), low instrumental noise and high time resolution (0.2 s – up to five times higher than used previously in such settings) permitted precise determination of p₅₀ even at high volume specific fluxes (J_V,O2 > 500 pmol*s^-1*ml^-1) in mitochondria isolated from mouse liver, brain and kidney. Furthermore, rapid pulse-titrations of H₂O₂ (TIP2k) allowed determination of p₅₀ during repeated anoxic transitions in each respiratory state in the course of SUIT protocols.

Detailed and careful analysis of mitochondrial O₂ kinetics has been assisted by a newly developed software. Although the principles of such analyses were available and used previously [2,3], possibly biased data evaluation resulting from uncertainties of lower time resolution and smoothed O₂ signal was a major concern until now. Although the relation between p_O2 and J_V,O2 is accepted to be monophasic hyperbolic (conf. Michaelis-Menten kinetics) in the O₂-dependent range of isolated mitochondria (< 1.1 kPa or ca. 10 µM O₂), a second, linear or hyperbolic component can be often observed [5]. To overcome the above peculiarities, we used non-smoothed O₂ concentration data with automated algorithms to determine internal zero O₂ levels, correct for drift of zero O₂ signal and choose data points for curve fitting. Both monophasic hyperbolic and biphasic curve fits were used to obtain p₅₀ and J_O2,max values the most accurate way.

As a first future objective, determination of O₂ kinetics is particularly important to get further insight into apparent affinity resting (uncoupled/dyscoupled), coupled and noncoupled respiration of isolated mitochondria and cells. Second, performance of mitochondrial substrate oxidation at low tissue O₂ levels and hypoxia in conjunction with effects of other molecules modulating mitochondrial O₂ utilization is another promising field of research. As a third option, working with small to moderately big cells, our methods might provide valuable data on diffusion limitation and comparability of cells and isolated mitochondria in this respect.

• Bioblast editor: Meszaros A • O2k-Network Lab: AT Innsbruck OROBOROS, HU Szeged Boros M, AT Innsbruck Gnaiger E

Labels: MiParea: Respiration, Instruments;methods 

Organism: Mouse  Tissue;cell: Nervous system, Liver, Kidney  Preparation: Isolated mitochondria 

Regulation: Oxygen kinetics  Coupling state: LEAK, OXPHOS  Pathway: N, S  HRR: Oxygraph-2k  Event: Oral 

Affiliations

Meszaros AT(1,2), Haider M(3), Sigh R(1), Gnaiger E(1,4)

1. OROBOROS INSTRUMENTS, Innsbruck, Austria
2. Inst of Surgical Research, Univ of Szeged, Hungary
3. 3xxxx, Innsbruck, Austria
4. D.Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria

[email protected]

References

1. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopsies of human muscle. Methods Mol Biol 810:25-58.
2. Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96.
3. Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25.
4. Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543:39-55.

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