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Difference between revisions of "SUIT-008 O2 pfi D014"

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{{MitoPedia
{{MitoPedia
|abbr=NS(PGM)
|abbr=
|SUIT number=8_pfi
|description=[[File:1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png|400px]]
|application=O2
|info='''A''' - [[Lemieux 2017 Sci Rep]] - '''[[SUIT-008]]'''
|description=[[File:1PM;2D;3G;4S;5U;6Rot-.png|300px]]
|SUIT number=D014_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd
|info='''A: SUIT 8 is the SUIT protocol used in the MitoEAGLE project (WG2) for testing the skills preparing permeabilized fibers''' [[Lemieux 2017 Sci Rep]]
|application=O2
}}
}}
{{MitoPedia concepts
::: '''[[MitoPedia: SUIT]]''' - O2 pfi D014 protocol was used a a reference protocol in [[MitoEAGLE]] project (WG2) to evaluate the researcher skills preparing permeabilized fibers'''
|mitopedia concept=SUIT protocol, SUIT A
::: '''[[SUIT protocol pattern]]:''' 1PM;2D;3G;4S;5U;6Rot
}}
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(PGM)
::: '''[[SUIT protocol pattern]]:''' diametral.
__TOC__


{{Template:SUIT text D014}}


== 1PM;2D;2c;3G;4S;5U;6Rot- ==
__TOC__
[[File:1PM;2D;2c;3G;4S;5U;6Rot;7Ama.png|300px]] 1PM;2D;2c;3G;4S;5U;6Rot;7Ama
Communicated by [[Huete-Ortega M]], [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019-06-06)
::: '''SUIT states:''' 1-2[[PM]](LPc) 3[[PGM]] 4-5[[PGMS]](PE) 6[[S]] 7[[ROX]]


{| class="wikitable" border="1"
== Representative traces ==
|-
! Step
! Respiratory state
! Pathway control
! Entry through electron transfer Complex
! Comments


|-
[[File:D014 O2 traces.png|600px]]
| 1PM
{{Template:SUIT-008}}
| [[PM]](L)
| N
| CI
|


|-
== Strengths and limitations ==
| 2D
:::+ NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
| [[PM]](P)
:::+ The presence of PGM and S establishes fully operative TCA cycle activity.
| N
:::+ This protocol allows to analyse the convergence of pathways at the Q-junction (N, NS, S).
| CI
:::+ Mitochondrial external membrane can be measured with the addition of cytochrome c. Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of c.
|
:::+ Reasonable duration of the experiment.
:::+ Complex IV activity can be measured.
:::+ GM and PM yield typically identical fluxes in human skeletal muscle fibres. However, PM is the superior alternative to GM: the fraction of the N-pathway is lower and of the S-pathway is higher with GM compared to PM (GMP is inhibited by the CII inhibitor malonic acid to a larger extent than PMP). PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since an impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to SUIT-011 for diagnosis of N-capacity.
:::- Fatty acid oxidation is not analysed.
:::-Β  When evaluating the additive effect of the N- and S-pathway, it has to be considered that NSP- and NSE-capacities can only be compared with NP- and SE-capacities. This is not a problem when NSP = NSE (Gnaiger 2009). In this case, it may be assumed that SP = SE (Votion et al 2012), such that NSP can be compared with NP + SP. SUIT-004 should be chosen for the additive effect in the ET-state.
:::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.


|-
== Compare SUIT protocols ==
| 2D(2c)
::::* [[SUIT-004]] 1PM;2D;3U;4S;5Rot- The SUIT-004 protocols provide a quick assessment of the linear coupling control (''L''- ''P''- ''E'') with NADH linked-substrates (PM) and the control in ET state (N, NS, S)
| [[PM]](c)
::::* [[SUIT-011]] 1GM;2D;3S;4U;5Rot-Β  The SUIT-011 protocols are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control.
| N
| CI
|


|-
== References ==
| 3G
{{#ask:[[Category:Publications]] [[Additional label::SUIT-008 O2 pfi D014]]
| [[PGM]](P)
| ?Was published in year=Year
| N
| ?Has title=Reference
| CI
| ?Mammal and model=Organism
| Β 
| ?Tissue and cell=Tissue;cell
| format=broadtable
| limit=5000
| offset=0
| sort=Was published in year
| order=descending
}}


|-
{{MitoPedia concepts
| 4S
|mitopedia concept=SUIT protocol, SUIT A, Find
| [[PGMS]](P)
}}
| NS
{{MitoPedia methods
| CI&II
|mitopedia method=Respirometry
|
Β 
|-
| 5U
| [[PGMS]](E)
| NS
| CI&II
|
Β 
|-
| 6Rot
| [[S]](E)
| S
| CII
|
Β 
|-
| 7Ama
| ROX
Β 
|}
Β 
Β 
== 1PM;2D;3G;3c;4S;5U;6Rot- ==
[[File:1PM;2D;3G;3c;4S;5U;6Rot;7Ama.png|300px]] 1PM;2D;3G;3c;4S;5U;6Rot;7Ama
::: '''SUIT states:''' 1-2[[PM]](LP) 3[[PGM]](c) 4-5[[PGMS]](PE) 6[[S]] 7[[ROX]]
Β 
{| class="wikitable" border="1"
|-
! Step
! Respiratory state
! Pathway control
! Entry through electron transfer Complex
! Comments
Β 
|-
| 1PM
| [[PM]](L)
| N
| CI
|
Β 
|-
| 2D
| [[PM]](P)
| N
| CI
|
Β 
|-
| 3G
| [[PGM]](P)
| N
| CI
|
Β 
|-
| G(c)
| [[PGM]](c)
| N
| CI
| Limitation: Cytochrome ''c'' should be added after step 2D, such that all OXPHOS states can be compared in the presence of cytochrome ''c''. See 1PM;2D;2c;3G;4S;5U;6Rot-.
Β 
|-
| 4S
| [[PGMS]](P)
| NS
| CI&II
|
Β 
|-
| 5U
| [[PGMS]](E)
| NS
| CI&II
|
Β 
|-
| 6Rot
| [[S]](E)
| S
| CII
|
Β 
|-
| 7Ama
| ROX
Β 
|}
Β 
Β 
== SUIT O2 DLP for SUIT 8 (DatLab 7.3) ==
Β 
''Updating this page (2018-06-13) for DatLab 7.3 DLP files''
Β 
Β 
== References ==
{{#ask:[[Category:Publications]] [[Additional label::1PM;2D;3G;4S;5U;6Rot-]]
|?Was published in year=Year
|?Has title=Reference
|?Mammal and model=Organism
|?Tissue and cell=Tissue;cell
|format=broadtable
|limit=5000
|offset=0
|sort=Was published in year
|order=descending
}}
}}
{{MitoPedia O2k and high-resolution respirometry}}
{{MitoPedia topics}}

Revision as of 14:56, 26 September 2019


high-resolution terminology - matching measurements at high-resolution


SUIT-008 O2 pfi D014

Description

1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png


Reference: A - Lemieux 2017 Sci Rep - SUIT-008

SUIT number: D014_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd

O2k-Application: O2

MitoPedia: SUIT - O2 pfi D014 protocol was used a a reference protocol in MitoEAGLE project (WG2) to evaluate the researcher skills preparing permeabilized fibers
SUIT protocol pattern: 1PM;2D;3G;4S;5U;6Rot

The SUIT-008 O2 pfi D014 protocol is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity in permeabilized fibers. It also serves as a diagnostic tool for the activity of the glutamate dehydrogenase and its linked pathways, which could be relevant in some pathologies. SUIT-008 O2 pfi D014 can be easily extended with the CIV assay module.

Communicated by Huete-Ortega M, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-06)

Representative traces

D014 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

SUIT-008

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1PM PML(n) N CI 1PM
2D PMP N CI 1PM;2D
2c PMcP N CI 1PM;2D;2c
3G PGMP N CI 1PM;2D;2c;3G
4S PGMSP NS CI&II 1PM;2D;2c;3G;4S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5U PGMSE NS CI&II 1PM;2D;2c;3G;4S;5U
6Rot SE S CII 1PM;2D;2c;3G;4S;5U;6Rot
7Ama ROX 1PM;2D;2c;3G;4S;5U;6Rot;7Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).
Step Respiratory state Pathway control ET-Complex Comment
## AsTm AsTmE CIV CIV
## Azd CHB


Questions.jpg


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Strengths and limitations

+ NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
+ The presence of PGM and S establishes fully operative TCA cycle activity.
+ This protocol allows to analyse the convergence of pathways at the Q-junction (N, NS, S).
+ Mitochondrial external membrane can be measured with the addition of cytochrome c. Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of c.
+ Reasonable duration of the experiment.
+ Complex IV activity can be measured.
+ GM and PM yield typically identical fluxes in human skeletal muscle fibres. However, PM is the superior alternative to GM: the fraction of the N-pathway is lower and of the S-pathway is higher with GM compared to PM (GMP is inhibited by the CII inhibitor malonic acid to a larger extent than PMP). PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since an impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to SUIT-011 for diagnosis of N-capacity.
- Fatty acid oxidation is not analysed.
- When evaluating the additive effect of the N- and S-pathway, it has to be considered that NSP- and NSE-capacities can only be compared with NP- and SE-capacities. This is not a problem when NSP = NSE (Gnaiger 2009). In this case, it may be assumed that SP = SE (Votion et al 2012), such that NSP can be compared with NP + SP. SUIT-004 should be chosen for the additive effect in the ET-state.
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

  • SUIT-004 1PM;2D;3U;4S;5Rot- The SUIT-004 protocols provide a quick assessment of the linear coupling control (L- P- E) with NADH linked-substrates (PM) and the control in ET state (N, NS, S)
  • SUIT-011 1GM;2D;3S;4U;5Rot- The SUIT-011 protocols are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control.

References

 YearReferenceOrganismTissue;cell
Votion 2012 PLoS One2012Votion 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.HorseSkeletal muscle


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry