Difference between revisions of "MiPNet12.23 FiberRespiration"
| Line 3: | Line 3: | ||
|info=[[Media:MiPNet12.23 FibreRespiration.pdf|'''Open Access-MiPNet12.23.pdf''']]; [http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions] | |info=[[Media:MiPNet12.23 FibreRespiration.pdf|'''Open Access-MiPNet12.23.pdf''']]; [http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions] | ||
|authors=OROBOROS | |authors=OROBOROS | ||
|year= | |year=2014-11 | ||
|journal=Mitochondr Physiol Network | |journal=Mitochondr Physiol Network | ||
|abstract='''Lemieux H, Votion DM, Gnaiger E ( | |abstract='''Lemieux H, Votion DM, Gnaiger E (2014) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(07): 1-8.''' | ||
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS Oxygraph-2k (O2k). The experiment demonstrates [http://bioblast.at/images/e/ef/MiPNet09.12_O2k-Titrations.pdf manual titrations] applied to study respiratory capacity in permeabilized fibres. Application of the [http://www.oroboros.at/?datlab DatLab 4 software] is shown for on-line data analysis [[MiPNet12.09]] The following guideline describes the experimental protocol and includes a short discussion of results. The experiments was carried out by participants of an O2k-Course on HRR in December 2007 [http://www.oroboros.at/index.php?id=ioc44 (IOC44; Schroecken, Austria).] | Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS Oxygraph-2k (O2k). The experiment demonstrates [http://bioblast.at/images/e/ef/MiPNet09.12_O2k-Titrations.pdf manual titrations] applied to study respiratory capacity in permeabilized fibres. Application of the [http://www.oroboros.at/?datlab DatLab 4 software] is shown for on-line data analysis [[MiPNet12.09]] The following guideline describes the experimental protocol and includes a short discussion of results. The experiments was carried out by participants of an O2k-Course on HRR in December 2007 [http://www.oroboros.at/index.php?id=ioc44 (IOC44; Schroecken, Austria).] | ||
| Line 16: | Line 16: | ||
Β Β | Β Β | ||
#<span style="color:red"> '''LEAK state, L:'''</span> Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N). | #<span style="color:red"> '''LEAK state, ''L'':'''</span> Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N). | ||
#<span style="color:green">'''OXPHOS capacity with Complex I substrates, P:'''</span> Respiratory capacity in the active coupled state (with ADP, GMD). | #<span style="color:green">'''OXPHOS capacity with Complex I substrates, ''P'':'''</span> Respiratory capacity in the active coupled state (with ADP, GMD). | ||
#<span style="color:black">'''Cytochrome c test for quality control:'''</span> Further addition of cytochrome c yields a test for integrity of the outer mitochondrial membrane (loss of cytochrome c would be indicated by a stimulation of respiration). | #<span style="color:black">'''Cytochrome ''c'' test for quality control:'''</span> Further addition of cytochrome ''c'' yields a test for integrity of the outer mitochondrial membrane (loss of cytochrome c would be indicated by a stimulation of respiration). | ||
#<span style="color:green">'''OXPHOS capacity with Complex I | #<span style="color:green">'''OXPHOS capacity with Complex I&II substrates, ''P'':'''</span> Respiratory stimulation by convergent electron flow from Complexes I&II at the Q-junction, in the coupled state after further addition of succinate, as an estimate of OXPHOS capacity (Gnaiger 2009). | ||
#<span style="color:blue">'''Electron transfer system (ETS) capacity with CI | #<span style="color:blue">'''Electron transfer system (ETS) capacity with CI&II substrates, ''E'':'''</span> Uncoupling by FCCP titration (avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ETS capacity by the phosphorylation system. | ||
#<span style="color:blue">'''ETS capacity with CII substrate, E:'''</span> ETS capacity with succinate, after blocking Complex I with rotenone. | #<span style="color:blue">'''ETS capacity with CII substrate, ''E'':'''</span> ETS capacity with succinate, after blocking Complex I with rotenone. | ||
#'''Residual oxygen consumption (ROX)''' due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (Inhibitor of Complex III). | #'''Residual oxygen consumption (ROX)''' due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (Inhibitor of Complex III). | ||
:>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]] | :>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]] | ||
| Line 38: | Line 38: | ||
}} | }} | ||
== Further information == | == Further information == | ||
:>> '''[http://www.oroboros.at/ | * Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7: e34890. - [[Votion 2012 PLoS One |Open Access]] | ||
:>> '''[http://www.oroboros.at/?protocols_muscle_horse Excel Templates, DatLab Templates and DatLab-Demo Files]''' | |||
Revision as of 14:25, 7 November 2014
 |
Β» Open Access-MiPNet12.23.pdf; Versions
OROBOROS (2014-11) Mitochondr Physiol Network
Abstract: Lemieux H, Votion DM, Gnaiger E (2014) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(07): 1-8.
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS Oxygraph-2k (O2k). The experiment demonstrates manual titrations applied to study respiratory capacity in permeabilized fibres. Application of the DatLab 4 software is shown for on-line data analysis MiPNet12.09 The following guideline describes the experimental protocol and includes a short discussion of results. The experiments was carried out by participants of an O2k-Course on HRR in December 2007 (IOC44; Schroecken, Austria).
Figure 1. Oxygen concentration ([ΞΌM] blue line) and oxygen flux per mg wet weight of muscle ([pmolβs-1βmg-1] red lines) in O2k chamber B, in permeabilized fibres from horse skeletal muscle with the standard titration protocol.
A multiple substrate-uncoupler-inhibitor titration protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The experiment was aimed at an assessment of a sequence of titrations, inducing stepwise defined respiratory states.
- LEAK state, L: Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N).
- OXPHOS capacity with Complex I substrates, P: Respiratory capacity in the active coupled state (with ADP, GMD).
- Cytochrome c test for quality control: Further addition of cytochrome c yields a test for integrity of the outer mitochondrial membrane (loss of cytochrome c would be indicated by a stimulation of respiration).
- OXPHOS capacity with Complex I&II substrates, P: Respiratory stimulation by convergent electron flow from Complexes I&II at the Q-junction, in the coupled state after further addition of succinate, as an estimate of OXPHOS capacity (Gnaiger 2009).
- Electron transfer system (ETS) capacity with CI&II substrates, E: Uncoupling by FCCP titration (avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ETS capacity by the phosphorylation system.
- ETS capacity with CII substrate, E: ETS capacity with succinate, after blocking Complex I with rotenone.
- Residual oxygen consumption (ROX) due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (Inhibitor of Complex III).
- >> Product: OROBOROS Oxygraph-2k, O2k-Catalogue
β’ O2k-Network Lab: AT_Innsbruck_OROBOROS, BE_Liege_Votion DM
Labels: MiParea: Respiration, Instruments;methods
Organism: Horse
Tissue;cell: Skeletal muscle
Preparation: Permeabilized tissue
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, Protocol"Protocol" 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.
O2k-Demo, O2k-Core
Further information
- Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7: e34890. - Open Access
