Difference between revisions of "SUIT-004 O2 pfi D010"

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high-resolution terminology - matching measurements at high-resolution

SUIT-004 O2 pfi D010

Description

Abbreviation: RP1-short pfi

Reference: SUIT-004 - Short protocol linked to SUIT-001 O2 pfi D002 SUIT RP1 (human skeletal muscle)

SUIT number: D010_1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd

O2k-Application: O2

The SUIT-004 O2 pfi D010 protocol provides information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS and S) can be evaluated by using this SUIT protocol. The SUIT-004 O2 pfi D010 can be extended with the CIV assay module. Permeabilized muscle fibers are sensitive to oxygen supply due to limited diffusion of oxygen to the fiber bundle core. To counteract this limitation, hyperoxic conditions (400-250 µM O₂) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.

Communicated by Doerrier C, Gnaiger E (last update 2019-06-05)

Representative traces

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PM	PM_L(n)	N	CI	1PM NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	PM_P	N	CI	1PM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PMc_P	N	CI	1PM;2D;2c NADH-linked substrates (type N-pathway to Q). Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3U	PM_E	N	CI	1PM;2D;3U NADH-linked substrates (type N-pathway to Q). Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency. Noncoupled electron transfer state, ET state, with ET capacity E.
4S	PMS_E	NS	CI&II	1PM;2D;3U;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. Noncoupled electron transfer state, ET state, with ET capacity E.
5Rot	S_E	S	CII	1PM;2D;3U;4S;5Rot Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. Noncoupled electron transfer state, ET state, with ET capacity E.
6Ama	ROX			1PM;2D;3U;4S;5Rot;6Ama 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	AsTm_E	CIV	CIV
## Azd	CHB

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

A succinate concentration of >10 mM may be required for saturating S_E capacity.

CIV assessment is skipped if a prolonged protocol is not practical due to time limitation.

RP1-6Ama may be skipped since correction of the initial states by ROX measured in RP1-6 Ama may represent an over-correction, and inhibition takes a very long time in several cases (Pesta 2012 Methods Mol Biol).

+ Linear coupling control (L- P- E) with NADPH-linked substrate substrates (PM).

+ Pathway control in ET state (N, NS, S).

+ This protocol can be extended with the Complex IV module.

+ Short experimental time.

- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-004 in favor of reducing the time consumed during the experiment.

Compare SUIT protocols

SUIT-001 O2 pfi D002: A comparable but more comprehensive protocol comprising additional substrate states.
Harmonized with SUIT-005 O2 pfi D011.

Specific cases

imt, ROX: NS(PM)01_imt,1PM,2D(c),3U,4S,5Rot,6Ama
pfi, high O₂, no ROX: NS(PM)01_pfiO2,1PM,2D(c),3U,4S,5Rot

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1PM	Pyruvate (P) and Malate (M)
2D	ADP (D)
2c	Cytochrome c (c)
3U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
4S	Succinate (S)
5Rot	Rotenone (Rot)
6Ama	Antimycin A (Ama)

Step	Chemical(s) and link(s)	Comments
## AsTm	Ascorbate (As) and TMPD (Tm)
## Azd	Azide (Azd)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
+|abbr=RP1-short pfi
+|description=[[File:1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd.png|450px]]
+|info='''[[SUIT-004]]''' - Short protocol linked to [[SUIT-001 O2 pfi D002]] '''SUIT RP1''' (human skeletal muscle)
 |application=O2
-|abbr=NS(PM)
+|SUIT number=D010_1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd
-|description=[[File:1PM;2D;3U;4S;5Rot-.png|300px]]
-|SUIT number=D10_1PM;2D;3U;4S;5Rot
-|info='''A''': '''SUIT 4''' is a short SUIT protocol  linked to [[SUIT-1 O2 pfi D02]] - '''SUIT RP1''' (human skeletal muscle)
 }}
-{{MitoPedia concepts
+{{Template:SUIT text D010}} {{Template:Setting oxygen pfi}}
-|mitopedia concept=SUIT protocol, SUIT A
-}}
-::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(PM)
-::: '''[[SUIT protocol pattern]]:''' orthogonal
+__TOC__
+ Communicated by [[Doerrier C]], [[Gnaiger E]] (last update 2019-06-05)
+== Representative traces ==
-::::* Linked to [[SUIT-1 O2 pfi D02]] - SUIT RP1, specifically for human skeletal muscle mitochondria.
+[[File:SUIT 004.png|600px]]
-::::* Harmonized with [[SUIT-5 O2 pfi D11]].
+{{Template:SUIT-004}}
-__TOC__
+== Strengths and limitations ==
+:::* A succinate concentration of >10 mM may be required for saturating S<sub>''E''</sub> capacity.
+::::* CIV assessment is skipped if a prolonged protocol is not practical due to time limitation.
+::::# '''RP1-6Ama''' may be skipped since correction of the initial states by ROX measured in RP1-6 Ama may represent an over-correction, and inhibition takes a very long time in several cases ([[Pesta 2012 Methods Mol Biol]]).
+:::+ Linear coupling control ([[LEAK respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[ET capacity| ''E'']]) with NADPH-linked substrate substrates ([[PM-pathway control state|PM]]).
+:::+ Pathway control in ET state ([[NADH_Electron_transfer-pathway_state|N]], [[NS-pathway control state| NS]], [[Succinate pathway control state| S]]).
+:::+ This protocol can be extended with the Complex IV module.
+:::+ Short experimental time.
+:::- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-004 in favor of reducing the time consumed during the experiment.
-== 1PM;2D;2c;3U;4S;5Rot-==
+== Compare SUIT protocols ==
-[[File:1PM;2D;2c;3U;4S;5Rot.png|300px]] 1PM;2D;2c;3U;4S;5Rot-
+:::* [[SUIT-001 O2 pfi D002]]: A comparable but more comprehensive protocol comprising additional substrate states.
+:::* Harmonized with [[SUIT-005 O2 pfi D011]].
 :::'''Specific cases'''
 ::::* [[Isolated mitochondria |imt]], ROX: NS(PM)01_imt,1PM,2D(c),3U,4S,5Rot,6Ama
-::::* [[Permeabilized muscle fibres |pfi]], high O<sub>2</sub>, no ROX: NS(PM)01_pfiO2,1PM,2D(c),3U,4S,5Rot
+::::* [[Permeabilized muscle fibers |pfi]], high O<sub>2</sub>, no ROX: NS(PM)01_pfiO2,1PM,2D(c),3U,4S,5Rot
+== Chemicals and syringes ==
+{{Template:Chemicals SUIT-004}}
+{{Template:Chemicals CIV}}
+::: Suggested stock concentrations are shown in the specific DL-Protocol.
-== Link to RP1 ==
+== References ==
-:::: The '''1PM;2D;3U;4S;5Rot-''' protocol is linked to [[1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp-]] for healthy human skeletal muscle mitochondria on the basis of the following rationale.
+{{#ask:[[Category:Publications]] [[Additional label::SUIT-004 O2 pfi D010]]
+| ?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
+}}
-[[File:1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp-.png|right|300px|file=SUIT_FNSGp(PGM)01 |RP1]]
-::::# The first coupling control steps (1-4) are identical.
+{{MitoPedia concepts
-::::# '''RP1-5G''' is skipped, since [[PM]] and [[PGM]] yield nearly identical OXPHOS capacity. However, [[PM]]<[[PGM]] is a potential diagnostic for a defect in the PM-linked pathway upstream of CI.
+|mitopedia concept=SUIT protocol, SUIT A, Find
-::::# '''RP1-7Oct''' is skipped, since octanoylcarnitine exerts practically no stimulatory effect on OXPHOS capacity in state [[PMS]] or [[PGMS]], but in conjunction with prolonged exposure may even yield a slight decline of respiration.
+}}
-::::# '''RP1-9Gp''' is skipped, since glycerolphosphate exerts only a minor stimulatory effect on OXPHOS capacity in state [[SRot]].
+{{MitoPedia methods
-::::# Omission of the three steps described above shortens the SUIT protocol, thus reducing the risk of diminishing respiratory capacity over time, reducing the number of necessary reoxygenations, and providing more time for detailed evaluation of steady-states. The loss of diagnostic information may be minor relative to the benefits of simplification of the protocol, particularly in studies of exercise in healthy humans.
+|mitopedia method=Respirometry
-::::# '''RP1-10Ama''' may be skipped, since correction of the initial states by ROX measured in RP1-10 Ama may represent an over-correction, and inhibition takes a very long time in several cases ([[Pesta 2012 Methods Mol Biol]]).
+}}
-::::# '''RP1-11Tm''' and '''RP1-12Azd''' are skipped, if a prolonged protocol is not practical due to limination of time.