SUIT-008 O2 pfi D014

high-resolution terminology - matching measurements at high-resolution

SUIT-008 O2 pfi D014

Description

Reference: A - Lemieux 2017 Sci Rep - SUIT-008- O2 pfi D014 protocol was used a a reference protocol in MitoEAGLE project (WG2) to evaluate the researcher skills preparing permeabilized fibers

SUIT number: D014_pfi;1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd

O2k-Application: O2

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. 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 Huete-Ortega M, Gnaiger E (last update 2024-09-05)

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.
3G	PGM_P	N	CI	1PM;2D;2c;3G NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	PGMS_P	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	PGMS_E	NS	CI&II	1PM;2D;2c;3G;4S;5U Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. 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.
6Rot	S_E	S	CII	1PM;2D;2c;3G;4S;5U;6Rot Succinate, S ( type S-pathway to Q). 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.
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	AsTm_E	CIV	CIV
## Azd	CHB

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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

+ 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 analyze 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 (GM_P is inhibited by the CII inhibitor malonic acid to a larger extent than PM_P). 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 analyzed.

- When evaluating the additive effect of the N- and S-pathway, it has to be considered that NS_P- and NS_E-capacities can only be compared with N_P- and S_E-capacities. This is not a problem when NS_P = NS_E (Gnaiger 2009). In this case, it may be assumed that S_P = S_E (Votion et al 2012), such that NS_P can be compared with N_P + S_P. 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-014: similar version starting with GM, and then adding P. Used in combination with SUIT-008 in Lemieux 2017 for pfi.
SUIT-004: 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: The SUIT-011 protocols are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1PM	Pyruvate (P) and Malate (M)
2D	ADP (D)
2c	Cytochrome c (c)
3G	Glutamate (G)
4S	Succinate (S)
5U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
6Rot	Rotenone (Rot)
7Ama	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

	Year	Reference	Organism	Tissue;cell
Votion 2012 PLoS One	2012	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. https://doi.org/10.1371/journal.pone.0034890	Horse	Skeletal muscle

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry