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| '''TPP special interest group during IOC61''' Β | | '''TPP special interest group during IOC61''' Β |
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| '''[[High-resolution respirometry]]''' (HRR) provides a quantitative approach to bioenergetics and mitochondrial physiology with the [[Oxygraph-2k]] (O2k; OROBOROS INSTRUMENTS) offering several sole-source features.
| | During IOC61 OROBOROS Instruments offers a [[TPP special interest group during an International Oxygraph Course]] covering the utilization of the [http://www.oroboros.at/index.php?id=membrane-potential|OROBOROS ISE system] to measure [[mitochondrial membrane potential]]. Some topics of general interest will be covered together with the participants from the "main course". |
| | This special interest groups is intended as a workshop, therefor the exact program will depend on the level of experience and interests of the participants. In an introductory session the principals of measuring mitochondrial membrane potential with a TPP-electrode are covered. The different steps of planning and performing a TPP are discussed. In "hands-on" sessions the participants will follow [[MiPNet15.03 MultiSensor-ISE]] to assembly TPP and reference electrodes, set up an oxygraph-2k with these electrodes, record an instrumental oxygen background in their presence, calibrate the electrodes and determine the effect chemicals used in a biolgicla experiment on the electrodes, see also |
| | In a hands-on experiment this set up will be used to |
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| Hardware and software developments are based on long-term expertise with [[polarographic oxygen sensor]]s (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 O<sub>2</sub>, 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 O<sub>2</sub>.s<sup>-1</sup>.ml<sup>-1</sup>. 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]. Β
| | [[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]] will be intensively discussed and facilitated by the provided [http://www.oroboros.at/index.php?id=protocols_tpp-membranepotential Spreadsheet templates. |
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| Most applications of HRR take advantage of the high stability and sensitivity of the O2k in [[phosphorylation control protocol]]s 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 Β°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]] and [[substrate control]] of mitochondrial respiration are expressed as [[flux control ratio]]s, 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 2011|MiP''summer'' School]].
| | Finally, interested participants can join a special group combining HRR of [[permeabilized fibres]] with measurement of mitochondrial membrane potential. |
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| 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].
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| Finally, the basic O2k-Workshop provides an overview on [[O2k-MultiSensor System]] applications, for the simultaneous measurement of respiration and [[mitochondrial membrane potential]] (TPP<sup>+</sup> 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<sup>+</sup> electrode for advanced users [9]. Taken together, HRR integrates metabolic and physicochemial concepts on fluxes and forces in open and closed systems [10]. | |
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| # [[Gnaiger_1983_POS|Gnaiger E, Forstner H, eds (1983) Polarographic Oxygen Sensors. Aquatic and Physiological Applications. Springer, Berlin, Heidelberg, New York: 370 pp.]]
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| # [[Gnaiger_2008_POS|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: 327-352.]]
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| # [[Gnaiger_2001_RespPhysiol|Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir. Physiol. 128: 277-297.]]
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| # [[Pesta_2010_Protocols|Pesta D, Gnaiger E (2010) High-resolution respirometry. OXPHOS protocols for human cell cultures and permeabilized fibres from small biopisies of human muscle. In: Mitochondrial bioenergetics: methods and protocols (Series Editor: Sir John Walker), edited by Carlos Palmeira and Antonio Moreno. In press.]]
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| # [[Gnaiger_2007_MitoPathways|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
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| # [[Gnaiger_2009_IJBCB|Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int. J. Biochem. Cell Biol. 41: 1837-1845.]]
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| # [[MiPNet12.15_RespiratoryStates|Gnaiger E. MitoPathways: Respiratory states and flux control ratios. Mitochondr. Physiol. Network 12.15.]]
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| # [[Gnaiger_2000_PNAS|Gnaiger E, MΓ©ndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc. Natl. Acad. Sci. USA 97: 11080-11085.]]
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| # [[MiPNet14.05_TPP-MitoMembranePotential|Renner-Sattler K, Fasching M, Gnaiger E. TPP+ and membrane potential. Mitochondr. Physiol. Network 14.05.]]
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| # [[Gnaiger_1993_PAC|Gnaiger E (1993) Nonequilibrium thermodynamics of energy transformations. Pure Appl. Chem. 65: 1983-2002.]]
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| [[Category:IOC61]] | | [[Category:IOC61]] |