SUIT-005 O2 pfi D011

high-resolution terminology - matching measurements at high-resolution

SUIT-005 O2 pfi D011

Description

Abbreviation: RP2-short pfi

Reference: A: - SUIT-005 short protocol linked to SUIT-002 O2 pfi D006 - SUIT RP2 (human skeletal muscle)

SUIT number: D011_pfi;1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd

O2k-Application: O2

SUIT-005 O2 pfi D011 protocol provides information on the F-pathway, the combined FN pathway, and the convergence FNS pathways in the OXPHOS state. FNS comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum OXPHOS- and ET capacity. SUIT-005 can be extended with the CIV assay module. This protocol is linked to SUIT-002 O2 pfi D006 - SUIT RP2, specifically for human skeletal muscle mitochondria. SUIT-005 O2 pfi D011 is harmonized with SUIT-004 O2 pfi D010. The F-pathway has to be tested previously. 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 2024-08-14)

Representative traces

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1OctM	OctML(n)	F(N)	FAO	1OctM Respiratory stimulation of the FAO-pathway, F, by fatty acid FA in the presence of malate M. Malate is a type N substrate (N), required for the F-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	OctMP	F(N)	FAO	1OCtM;2D Respiratory stimulation of the FAO-pathway, F, by fatty acid FA in the presence of malate M. Malate is a type N substrate (N), required for the F-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	OctMcP	F(N)	FAO	1OCtM;2D;2c Respiratory stimulation of the FAO-pathway, F, by fatty acid FA in the presence of malate M. Malate is a type N substrate (N), required for the F-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. 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.
3P	OctPMP	FN	F&CI	1OctM;2D;2c;3P Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	OctPMSP	FNS	F&CI&II	1OctM;2D;2c;3P;4S Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5U	OctPMSE	FNS	F&CI&II	1OctM;2D;2c;3P;4S;5U Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F. 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	SE	S	CII	1OctM;2D;3P;4S;5U;6Rot 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			1OctM;2D;3P;4S;5U;6Rot;7Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTmE	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

+ The protocol provides information on FAO capacity in the absence of other potentially interfering pathways, both in the LEAK state and in OXPHOS state.

+ FNS OXPHOS capacity comprises the most important pathways in many cell types and, thus, provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.

+ FNS ET capacity is a good estimate of overall ET capacity in many cell types.

+ The presence of PM & S establishes a fully operative TCA cycle activity (PMS) if the 2-oxoglutarate carrier does not outcompete the sources of 2-oxoglutarate

+ 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 (1.5 to 2 h); 2-3 h when the CIV module is added.

- F- OXPHOS capacity may be underestimated. In human heart muscle, addition of Oct to palmitoylcarnitine (Pal) + malate increased OXPHOS by 26% (Lemieux et al 2011).

- SRot_E may be underestimated if S is not saturating.

Compare SUIT protocols

• SUIT-002: A comparable but more comprehensive protocol comprising additional substrate states.
• SUIT-002 O2 pfi D006: A comparable but more comprehensive pfi-specific protocol comprising additional substrate states and considering the existence of anaplerotic pathways.
• SUIT-025: to measure F-OXPHOS capacity when malate anaplerotic activity is present
• SUIT-027: to check for malate-linked anaplerotic activity
• SUIT-017: to measure N-pathway with a combination of GM substrates instead of PM substrates

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1OctM	Octanoylcarnitine (Oct) and Malate (M)
2D	ADP (D)
2c	Cytochrome c (c)
3P	Pyruvate (P)
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

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry