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|Gnaiger E (2011) The elements of high-resolution respirometry: An introduction to the Oroboros Oxygraph-2k. IOC62.|
Gnaiger E (2011)
• O2k-Network Lab: AT Innsbruck Oroboros
- Gnaiger E (2011) The elements of high-resolution respirometry: An introduction to the Oroboros Oxygraph-2k.
- Hardware and software developments are based on long-term expertise with polarographic oxygen sensors (POS)  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 . 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 , 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 .
- 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 . Physiological temperatures (electronic Peltier control, within 0.001 °C), optimized incubation media (MiR06), and a rationale for the design of tested SUIT protocols  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 .
- 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 . Taken together, HRR integrates metabolic and physicochemial concepts on fluxes and forces in open and closed systems .
- Gnaiger E, Forstner H, eds (1983) Polarographic Oxygen Sensors. Aquatic and Physiological Applications. Springer, Berlin, Heidelberg, New York:370 pp.
- 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.
- Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97.
- 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.
- 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
- Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45.
- Gnaiger E. MitoPathways: Respiratory states and flux control ratios. Mitochondr Physiol Network 12.15.
- 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.
- Renner-Sattler K, Fasching M, Gnaiger E. TPP+ and membrane potential. Mitochondr Physiol Network 14.05.
- Gnaiger E (1993) Nonequilibrium thermodynamics of energy transformations. Pure Appl Chem 65:1983-2002.