Meszaros 2017 MiPschool Obergurgl
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 . In contrast, kinetic aspects, such as O2 dependence of mitochondrial respiration remained largely neglected. However, assessment of parameters such as maximal O2 flux (JO2,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, methane), 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 largely eliminating individual bias, and (3) delineated future directions and objectives.
Working with the latest series of Oroboros O2k high-resolution respirometers (Oroboros Instruments, Innsbruck, Austria), low instrumental noise and high samling rate (low sampling interval: 0.2 s compared to 1 or 2 s used previously) permitted accurate determination of p50 even at high volume specific fluxes (JV,O2 > 500 pmol*s-1*ml-1) in mitochondria isolated from mouse liver, brain and kidney. Furthermore, rapid pulse-titrations of H2O2 using the Oroboros 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 the 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. Although the relation between pO2 and JV,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 observed occasionally . To overcome the above peculiarities, we used the data of O2 concentration without smoothing and applied automated algorithms to correct for drift of the zero O2 signal and choose data points for curve fitting. Both monophasic hyperbolic and biphasic curve fits were used to obtain p50 and JO2,max in the most accurate way.
As a first future objective, determination of O2 kinetics is particularly important to get further insight into the apparent O2 affinity of resting, 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 will provide valuable data on oxygen diffusion gradients and comparability of cells and isolated mitochondria.
- Meszaros AT(1,2), Haider M(3), Sigh R(1), Gnaiger E(1,4)
- Oroboros Instruments, Innsbruck, Austria
- Inst of Surgical Research, Univ of Szeged, Hungary
- Steinhauser & Haider Technology Consulting OG, Innsbruck, Austria
- D.Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria
- 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. - »Bioblast link«
- 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. - »Bioblast link«
- Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25. - »Bioblast link«
- Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543:39-55. - »Bioblast link«
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: A1, Oral