Gnaiger IOC62-Introduction

Gnaiger E (2011) The elements of high-resolution respirometry: An introduction to the Oroboros Oxygraph-2k. IOC62.

Link: IOC61

Gnaiger E (2011)

Event: IOC62

High-resolution respirometry (HRR) provides a quantitative approach to bioenergetics and mitochondrial physiology with the Oroboros O2k (Oroboros Instruments) offering several sole-source features.

• O2k-Network Lab: AT Innsbruck Oroboros

Full text

Gnaiger E (2011) The elements of high-resolution respirometry: An introduction to the Oroboros Oxygraph-2k.

High-resolution respirometry (HRR) provides a quantitative approach to bioenergetics and mitochondrial physiology with the Oroboros O2k (Oroboros Instruments) offering several sole-source features.

Hardware and software developments are based on long-term expertise with polarographic oxygen sensors (POS) [1] and continuous evaluation relative to alternative sensors. The POS is superior in the range from zero oxygen to pure oxygen at about 1 mM dissolved O₂, yielding a 500,000-fold dynamic range with a digital resolution of 2 nM in the O2k [2]. Oxygen flux is measured in the closed system as the negative time derivative of oxygen concentration, calculated and displayed on-line with correction for instrumental background oxygen flux, yielding a resolution of 1 pmol O₂.s^-1.ml^-1. Minimization, experimental evaluation, and automatic correction (DatLab) of instrumental background oxygen flux are integral to HRR [2], as emphasized in practical tests and data analysis during the basic O2k-Workshop. Alternatively, oxygen flux can be measured in an open system mode of operation, using the Titration-Injection microPump (TIP2k) for feedback control of oxygen levels by matching oxygen supply to demand, particularly at graded levels of hypoxia in studies of oxygen kinetics [3].

Most applications of HRR take advantage of the high stability and sensitivity of the O2k in coupling-control protocols with intact cells, or substrate-uncoupler-inhibitor titration (SUIT) protocols with isolated mitochondria, permeabilized cells or tissues, in particular permeabilized muscle fibres [4]. Physiological temperatures (electronic Peltier control, within 0.001 °C), optimized incubation media (MiR06), and a rationale for the design of tested SUIT protocols [5] are the hallmark of quantitative and comparative mitochondrial respiratory physiology. Coupling-control ratio and substrate control of mitochondrial respiration are expressed as flux control ratios, resolving some confusion related to the respiratory control ratio (RCR) [6,7]. Applicaton of CI+II substrate combinations in SUIT protocols extends conventional bioenergetic studies to the level of mitochondrial physiology, some principles of which are applied and discussed at the O2k-Workshop, whereas an in-depth introduction is provided at the MiPsummer School.

Compared to the long tradition of applications of polarographic oxygen sensors, replacing the classical manometric (Warburg) apparatus, HRR is a recent development, which now provides a widely applied tool for routine and specific analyses of mitochondrial function/dysfunction where (1) reliability and quality control are important (clinical studies, functional diagnosis), (2) the amount of biological material is limited (<0.5 mill cultured cells, 1-2 mg of fresh tissue from biopsies; <0.05 mg of mitochondrial protein), (3) pathological effects result in reduced respiration, and (4) effects need to be tested at physiological, low intracellular oxygen levels [8].

Finally, the basic O2k-Workshop provides an overview on O2k-MultiSensor System applications, for the simultaneous measurement of respiration and mitochondrial membrane potential (TPP⁺ electrode), acidification (pH electrode), nitric oxide (amperometric), spectrophotometry (cytochrome spectra), and spectrofluorimetry (Amplex red, safranin, etc.). In parallel sessions, a hands-on introduction is provided to the application of the TPP⁺ electrode for advanced users [9]. Taken together, HRR integrates metabolic and physicochemial concepts on fluxes and forces in open and closed systems [10].

1. Gnaiger E, Forstner H, eds (1983) Polarographic Oxygen Sensors. Aquatic and Physiological Applications. Springer, Berlin, Heidelberg, New York:370 pp.
2. Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52.
3. Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97.
4. 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.
5. Gnaiger E ed (2007) Mitochondrial Pathways and Respiratory Control. Oroboros MiPNet Publications, Innsbruck:96 pp. - Electronic 1st ed: http://www.oroboros.at/index.php?mipnet-publications
6. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45.
7. Gnaiger E. MitoPathways: Respiratory states and flux control ratios. Mitochondr Physiol Network 12.15.
8. Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci U S A 97:11080-5.
9. Renner-Sattler K, Fasching M, Gnaiger E. TPP+ and membrane potential. Mitochondr Physiol Network 14.05.
10. Gnaiger E (1993) Nonequilibrium thermodynamics of energy transformations. Pure Appl Chem 65:1983-2002.