Difference between revisions of "MiPNet12.01 Suppl T-issue"

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|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] MitoPathways at the Q-junction: mouse skeletal muscle fibers.
 
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] MitoPathways at the Q-junction: mouse skeletal muscle fibers.
 
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/2/2d/MiPNet12.01_Suppl_T-issue.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.01_Suppl_T-issue.pdf Versions]
 
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/2/2d/MiPNet12.01_Suppl_T-issue.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.01_Suppl_T-issue.pdf Versions]
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|year=2018-11-18
 
|year=2018-11-18
 
|journal=Mitochondr Physiol Network
 
|journal=Mitochondr Physiol Network
|abstract=[[File:MiPNet12.01 SupplementaryT-issue.png|left|500px|Suppl T-issue: MitoPathways at the Q-junction]] '''Oroboros (2018) MitoPathways at the Q-junction: mouse skeletal muscle fibers. Mitochondr Physiol Network 12.01(03): Suppl T-issue.''' » [http://www.bioblast.at/index.php/File:MiPNet12.01 Versions]
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|abstract= Oroboros (2018) MitoPathways at the Q-junction: mouse skeletal muscle fibers. Mitochondr Physiol Network 12.01(03): Suppl T-issue.
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{{MiPNet pdf page linking to MitoPedia}}
  
[[High-resolution respirometry]] with a [[SUIT protocol]]<sup>1</sup> for [[OXPHOS]] analysis<sup>2</sup> is presented as supplementary '''''T-issue''''' ([[Oroboros]] T-shirt).
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|mipnetlab=AT_Innsbruck_Oroboros
  
[[Pyruvate]]&[[glutamate]]&[[malate]] (PGM) were used in combination to induce NADH-linked [[LEAK respiration]] in permeabilized mouse skeletal muscle ([[MiPNet12.14 IOC39  |IOC39]]; Fig. O2).<sup>3,4</sup> Saturating [[ADP]] (D; 2.5 mM final concentration) stimulated respiration to the level of [[OXPHOS capacity]] (''P'' state), with a small effect of 10 µM [[cytochrome c]] (''c''), expressed as the [[Cytochrome c control factor |cytochrome ''c'' control factor]] (''FCF<sub>c</sub>''<0.01; indicating integrity of the outer mt-membrane). Without correction for residual oxygen consumption (ROX), the biochemical coupling efficiency, (''P-L'')/''P'', was 0.68 (RCR=3.1). Addition of [[succinate]] (S) stimulated respiration by convergent e-input through the [[Q-junction]]. The corresponding succinate control factor was (NS-N)/NS=0.47, i.e. succinate increased respiration by 47%. NS-linked OXPHOS capacity was not stimulated further by [[uncoupler]] titration (U). Therefore, the capacity of the [[phosphorylation system]] matched the [[ET-capacity]] (''E'' state). At ''E=P'' the [[Excess E-P capacity |Excess ''E-P'' capacity]] is zero, in striking contrast to human skeletal and cardiac muscle mitochondria.<sup>1,5,6</sup> Inhibition of CI by [[rotenone]] (Rot) inhibited respiration to the level of S-linked ET-capacity. The corresponding CI-control factor is (NS-S)/NS=0.25. S- was higher than N-linked respiratory capacity (''E=P''). NS-linked respiratory capacity was higher than respiration with any single e-input substrate state, indicating an additive effect at the Q-junction. However, since NS < N + S, the additive effect was incomplete, which indicates that any electron channelling through [[Respiratory complexes |supercomplexes]] to CIV was incomplete. Addition of [[azide]] (Azd) inhibited respiration to the level of [[residual oxygen consumption]] (ROX). ROX was 0.18 of NS-linked ET-capacity.
 
|mipnetlab=AT_Innsbruck_Oroboros
 
 
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|additional=MitoPathways, O2k-Demo, O2k-Core
 
|additional=MitoPathways, O2k-Demo, O2k-Core
 
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<ref> Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58. [[Pesta 2012 Methods Mol Biol |»Bioblast link]] </ref>
 
<ref> Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. Oroboros MiPNet Publications, Innsbruck:80 pp. [[Gnaiger 2014 MitoPathways |»Bioblast link]] </ref>
 
<ref> Oroboros IOC39. International course on high-resolution respirometry. Schroecken 13-17 April 2007. Mitochondr Physiol Network 12.14: 1-8. [[MiPNet12.14 IOC39 |»Bioblast link]] - O2k-Demo experiment 2007-04-14 A-03 carried out by [[Lemieux H |Hélène Lemieux]] at [[MiPNet12.14 IOC39  |IOC39]], Schröcken. </ref>
 
<ref> Oroboros (2014) Oxygraph-2k manual titrations: SUIT protocols with mitochondrial preparations. Mitochondr Physiol Network 09.12(11): 1. [[MiPNet09.12 O2k-Titrations |»Bioblast link]] </ref>
 
<ref> Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845. [[Gnaiger 2009 Int J Biochem Cell Biol |»Bioblast link]] </ref>
 
<ref> Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43: 1729–38. [[Lemieux 2011 Int J Biochem Cell Biol |»Bioblast link]] </ref>
 
 
== Limitations of the SUIT protocol ==
 
 
=== Maximum OXPHOS and ET-capacity ===
 
:::: Evaluation of maximum respiratory capacities requires titration of further substrates activating additional [[respiratory complexes]] at the Q-junction ([[Electron-transferring flavoprotein complex |CETF]] and [[glycerophosphate dehydrogenase complex |CGpDH<]]).
 
 
=== Malate concentration ===
 
:::: The [[malate]] concentration was 2 mM, to saturate N-linked respiration. However, at 2 mM malate, the fumarate concentration is increased to a level which inhibits succinate dehydrogenase. Then NS- and S-linked respiratory capacities are underestimated. A malate concentration of 0.5 mM, which saturates N-linked respiration and inhibits S-linked respiration to a lesser extent, represents and improved standard.
 
::::» [[Talk:Malate |Optimum malate concentration in SUIT protocols]]
 
 
=== ROX correction ===
 
:::: The fact that ROX was higher in the NS-substrate state compared to N-linked LEAK respiration indicates that ROX is partially controlled by the substrate state. Therefore, a single measurement of ROX cannot be applied for correction of total oxygen consumption in the different substrate states. Total respiration, therefore, represents apparent coupling states ''L''´, ''P''´ and ''E''´ (Fig. 1). ROX correction is possible in the present experiment only for NS- and S-linked respiration. [[Azide]] inhibits not only CIV but other heme-based oxidases and peroxidases, and therefore may interfere with ROX beyond blocking respiratory electron transfer. Based on this argument, a combination of CII- and CIII-inhibitors (malonic acid, antimycin A, myxothiazol) may yield more consistent results, although any ROS scavenged by cytochrome ''c'' may in the absence of a CIV-inhibitor result in respiratory oxygen consumption through CIV.
 
 
== References ==
 
<references/>
 
::::» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]]
 

Latest revision as of 11:30, 4 December 2019

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MiPNet12.01 Suppl T-issue

Publications in the MiPMap
O2k-Protocols contents
MitoPathways at the Q-junction: mouse skeletal muscle fibers.

» Bioblast pdf »Versions

Oroboros (2018-11-18) Mitochondr Physiol Network

Abstract: Oroboros (2018) MitoPathways at the Q-junction: mouse skeletal muscle fibers. Mitochondr Physiol Network 12.01(03): Suppl T-issue.

O2k-technical support and open innovation
Open the pdf document above.
» Current O2k-series: O2k-FluoRespirometer
» Current software version: DatLab 7


O2k-Network Lab: AT_Innsbruck_Oroboros


Labels: MiParea: Respiration 


Organism: Mouse  Tissue;cell: Skeletal muscle  Preparation: Permeabilized tissue 


Coupling state: LEAK, ROUTINE, ET  Pathway: N, S, NS, ROX  HRR: Oxygraph-2k, O2k-Protocol 

MitoPathways, O2k-Demo, O2k-Core