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Gnaiger IOC62-Introduction

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Publications in the MiPMap
Gnaiger E (2010) The elements of high-resolution respirometry: An introduction to the OROBOROS Oxygraph-2k. Mitochondr. Physiol. Network 15.10.


Gnaiger E (2010) Mitochondr. Physiol. Network

Abstract: 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.


β€’ O2k-Network Lab: AT Innsbruck OROBOROS


Labels:

Stress:Hypoxia 


Preparation: Intact Cell; Cultured; Primary"Intact Cell; Cultured; Primary" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property., Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property., Permeabilized Cell or Tissue; Homogenate"Permeabilized Cell or Tissue; Homogenate" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

Regulation: Respiration; OXPHOS; ETS Capacity"Respiration; OXPHOS; ETS Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Flux Control; Additivity; Threshold; Excess Capacity"Flux Control; Additivity; Threshold; Excess Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Coupling; Membrane Potential"Coupling; Membrane Potential" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Substrate; Glucose; TCA Cycle"Substrate; Glucose; TCA Cycle" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property. 


HRR: Oxygraph-2k, TIP2k, MultiSensor pH; TPP; NO"MultiSensor pH; TPP; NO" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property., Method"Method" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.  Event: IOC60"IOC60" is not in the list (A1, A2, A3, A4, B1, B2, B3, B4, C1, C2, ...) of allowed values for the "Event" property. 


Full text

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.

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 phosphorylation 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 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 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_1983_POS
  2. Gnaiger_2008_POS
  3. Gnaiger_2001_RespPhysiol
  4. Pesta_2010_Protocols
  5. Gnaiger_2007_MitoPathways
  6. Gnaiger_2009_IJBCB
  7. MiPNet12.15_RespiratoryStates
  8. Gnaiger_2000_PNAS
  9. MiPNet14.05_TPP-MitoMembranePotential
  10. Gnaiger_1993_PAC