Krumschnabel 2013 Abstract MiP2013: Difference between revisions
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{{Abstract | {{Abstract | ||
|title=Gnaiger E (2013) | |title=Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E (2013) Tissue homogenates for OXPHOS analysis in comparative mitochondrial physiology: trout and mouse β heart and liver mitochondria. Mitochondr Physiol Network 18.08. | ||
|authors=Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E | |||
|authors=Gnaiger E | |||
|year=2013 | |year=2013 | ||
|event=MiP2013 | |event=MiP2013 | ||
|abstract= | |abstract=OXPHOS analysis is based on measurement of respiration in various steady-states of substrate supply and coupling of electron transfer to phosphorylation of ADP. To secure full accessibility of various flux control variables, X (substrates, ADP, etc.), to the mitochondria (mt), the plasma membranes have to be either permeabilized or the mitochondria must be mechanically separated from the intact cell in mt-preparations. Permeabilized muscle fibres represent an excellent and gentle type of mt-preparation, but require incubation at artificially high oxygen levels to overcome oxygen diffusion limitations [1]. Owing to large oxygen diffusion gradients and the oxygen dependence of mt-H2O2 production over a wide range of oxygen pressure [2], permeabilized muscle fibres may not represent an adequate model for the combined study of respiration and ROS production. A high-quality preparation of tissue homogenate may represent an optimum compromise for a variety of respirometric and fluorometric studies. These considerations provided the rationale for initiating a study with the PBI-Shredder, an auxiliary HRR-Tool providing a standardized approach to prepare homogenates of various tissues (heart, liver, brain) and species (mouse, rainbow trout). | ||
In the present study with high-resolution respirometry, mitochondrial respiratory control was compared in trout heart and liver tissue homogenate preparations at 15 Β°C [3]. Biochemical coupling efficiency with Complex I (CI)-linked substrates were identical in the two tissues. The ADP-ATP phosphorylation system exerted a higher control over OXPHOS in trout heart than liver. CI-linked substrate control capacity (OXPHOS) was higher whereas CII-linked succinate control capacity was lower in heart than liver. Pyruvate added to glutamate+malate stimulated OXPHOS capacity to a larger extent in heart than liver. For comparison, mouse heart and liver homogenate was measured at 37 Β°C using an identical substrate-uncoupler-inhibitor titration (SUIT) protocol. The cytochrome c test (<5% stimulation in healthy controls) indicated outer mt-membrane integrity in all cases, following an optimization of the PBI-Shredder application with high reproducibility of complete mt-yield and preservation of mitochondrial respiratory control. Β | |||
|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom | |mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom | ||
}} | }} | ||
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|organism=Mouse | |organism=Mouse | ||
|taxonomic group=Fishes | |taxonomic group=Fishes | ||
|tissues=Heart, Liver | |tissues=Heart, Nervous system, Liver | ||
|preparations=Homogenate | |preparations=Homogenate | ||
|topics= | |topics=Coupling efficiency;uncoupling, Cyt c | ||
|couplingstates=LEAK, OXPHOS, ETS | |couplingstates=LEAK, OXPHOS, ETS | ||
|substratestates=CI, CII, CI+II, ROX | |substratestates=CI, CII, CI+II, ROX | ||
|instruments=Oxygraph-2k, | |instruments=Oxygraph-2k, Fluorometry | ||
|additional=MiP2013 | |additional=MiP2013 | ||
}} | }} | ||
__TOC__ | __TOC__ | ||
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Supported by K-Regio project ''[[MitoCom Tyrol]]'' | Supported by K-Regio project ''[[MitoCom Tyrol]]''. | ||
== References == | == References == | ||
# | # Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543: 39-55. | ||
# Boveris A, Chance B (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 134: 707-716. | |||
# | |||
# [[MiPNet17.03 Shredder vs Fibres|Doerrier CV, Draxl A, WiethΓΌchter A, Eigentler A, Gnaiger E (2013) Mitochondrial respiration in permeabilized fibres versus homogenate from fish liver and heart. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03 V3: 1-12.]] | # [[MiPNet17.03 Shredder vs Fibres|Doerrier CV, Draxl A, WiethΓΌchter A, Eigentler A, Gnaiger E (2013) Mitochondrial respiration in permeabilized fibres versus homogenate from fish liver and heart. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03 V3: 1-12.]] | ||
# Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 productionΒ in mouse brain mitochondria. Mitochondr Physiol Network 17.03 V2: 1-4. |
Revision as of 16:54, 22 August 2013
Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E (2013) Tissue homogenates for OXPHOS analysis in comparative mitochondrial physiology: trout and mouse β heart and liver mitochondria. Mitochondr Physiol Network 18.08. |
Link:
Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E (2013)
Event: MiP2013
OXPHOS analysis is based on measurement of respiration in various steady-states of substrate supply and coupling of electron transfer to phosphorylation of ADP. To secure full accessibility of various flux control variables, X (substrates, ADP, etc.), to the mitochondria (mt), the plasma membranes have to be either permeabilized or the mitochondria must be mechanically separated from the intact cell in mt-preparations. Permeabilized muscle fibres represent an excellent and gentle type of mt-preparation, but require incubation at artificially high oxygen levels to overcome oxygen diffusion limitations [1]. Owing to large oxygen diffusion gradients and the oxygen dependence of mt-H2O2 production over a wide range of oxygen pressure [2], permeabilized muscle fibres may not represent an adequate model for the combined study of respiration and ROS production. A high-quality preparation of tissue homogenate may represent an optimum compromise for a variety of respirometric and fluorometric studies. These considerations provided the rationale for initiating a study with the PBI-Shredder, an auxiliary HRR-Tool providing a standardized approach to prepare homogenates of various tissues (heart, liver, brain) and species (mouse, rainbow trout). In the present study with high-resolution respirometry, mitochondrial respiratory control was compared in trout heart and liver tissue homogenate preparations at 15 Β°C [3]. Biochemical coupling efficiency with Complex I (CI)-linked substrates were identical in the two tissues. The ADP-ATP phosphorylation system exerted a higher control over OXPHOS in trout heart than liver. CI-linked substrate control capacity (OXPHOS) was higher whereas CII-linked succinate control capacity was lower in heart than liver. Pyruvate added to glutamate+malate stimulated OXPHOS capacity to a larger extent in heart than liver. For comparison, mouse heart and liver homogenate was measured at 37 Β°C using an identical substrate-uncoupler-inhibitor titration (SUIT) protocol. The cytochrome c test (<5% stimulation in healthy controls) indicated outer mt-membrane integrity in all cases, following an optimization of the PBI-Shredder application with high reproducibility of complete mt-yield and preservation of mitochondrial respiratory control.
β’ O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom
Labels: MiParea: Respiration, Instruments;methods, Comparative MiP;environmental MiP
Organism: Mouse
Tissue;cell: Heart, Nervous system, Liver
Preparation: Homogenate
Regulation: Coupling efficiency;uncoupling, Cyt c Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k, Fluorometry"Fluorometry" 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.
MiP2013
Affiliations, acknowledgements and author contributions
Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck;
OROBOROS INSTRUMENTS, SchΓΆpfstr. 18, Innsbruck, Austria
Email:
[email protected]
Supported by K-Regio project MitoCom Tyrol.
References
- Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543: 39-55.
- Boveris A, Chance B (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 134: 707-716.
- Doerrier CV, Draxl A, WiethΓΌchter A, Eigentler A, Gnaiger E (2013) Mitochondrial respiration in permeabilized fibres versus homogenate from fish liver and heart. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03 V3: 1-12.
- Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production in mouse brain mitochondria. Mitochondr Physiol Network 17.03 V2: 1-4.