SUIT-001 O2 pfi D002

high-resolution terminology - matching measurements at high-resolution

SUIT-001 O2 pfi D002

Description

Abbreviation: RP1 pfi

Reference: SUIT-001 - SUIT reference protocol Reference protocol RP1 for permeabilized fibers

SUIT number: D002_pfi;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd

O2k-Application: O2

The SUIT-001 O2 pfi D002 protocol in combination with SUIT-002 O2 pfi D006 is specially designed to provide a common reference for comparison of respiratory control of permeabilized fibers in a wide variety of species and tissues. SUIT-001 O2 pfi D002 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 pfi D002 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 and Gnaiger E (last update 2024-04-09)

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). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state. 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]).
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.
4G	PGM_E	N	CI	1PM;2D;3U;4G NADH-linked substrates (type N-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
5S	PGMS_E	NS	CI&II	1PM;2D;3U;4G;5S 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.
6Oct	OctPGMS_E	FNS	FAO&CI&II	1PM;2D;3U;4G;5S;6Oct 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. Noncoupled electron transfer state, ET state, with ET capacity E.
7Rot	S_E	S	CII	1PM;2D;3U;4G;5S;6Oct;7Rot 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.
8Gp	SGp_E	SGp	CII&GpDH	1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp Respiratory stimulation by action of succinate and glycerophosphate, Gp, with convergent electron flow in the SGp-pathway (CII&GpDH-linked pathway to the Q-junction). Noncoupled electron transfer state, ET state, with ET capacity E.
9Ama	ROX			1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp;9Ama 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

SUIT-001 in combination with SUIT-002 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:

1. to obtain reference values.

2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.

3. to screen specific defects.

SUIT-001 and SUIT-002 are used in the MitoFit Proficiency test for inter-individual and inter-laboratory reproducibility quality control.
A succinate concentration of >10 mM may be required for saturating S_E capacity.

+ Linear coupling control (L-P-E) with N substrates (PM).

+ Pathway control in ET state (N, NS, FNS, S and SGp pathways).

+ Harmonization with SUIT-002 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.

+ In mtprep, SUIT-001 and SUIT-002 harmonize in: SGp_E (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV.

+ Harmonization with many SUIT protocols (up to step 5S).

+ Oct is added in ET state to avoid uncoupler effect with fatty acids.

+ If NS- and FNS-pathways are similar (NS_E ~ FNS_E), the additivity index of N- and S-pathways in ET state can be evaluated.

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

- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring S_E.

- Lengthy duration of the experiment.

Compare SUIT protocols

SUIT-002_O2_pfi_D006 (RP2): Harmonized SUIT protocol for permeabilized fibers.

SUIT-004: Short version of RP1; without G, F and Gp.
SUIT-006: Very short initial coupling control module.

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
4G	Glutamate (G)
5S	Succinate (S)
6Oct	Octanoylcarnitine (Oct)
7Rot	Rotenone (Rot)
8Gp	Glycerophosphate (Gp)
9Ama	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
MiPNet21.06 SUIT RP	2018-06-25	SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.
Doerrier 2018 Methods Mol Biol	2018	Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3	Human Mouse Rat Saccharomyces cerevisiae	Heart Skeletal muscle Endothelial;epithelial;mesothelial cell Blood cells HEK Platelet

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry