SUIT-001 O2 mt D001

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high-resolution terminology - matching measurements at high-resolution

SUIT-001 O2 mt D001

Description

Abbreviation: RP1 mt

Reference: SUIT-001 - SUIT reference protocol Reference protocol RP1 for isolated mitochondria, tissue homogenate and permeabilized cells

SUIT number: D001_mt;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd

O2k-Application: O2

The SUIT-001 O2 mt D001 protocol in combination with SUIT-002 O2 mt D005 is specially designed to provide a common reference for comparison of respiratory control of mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species and tissues. SUIT-001 O2 mt D001 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 mt D001 can be extended with the CIV assay module.

Communicated by Doerrier C, Gnaiger E (last update 2024-03-05)

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). 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.

- We do not generally recommended the addition of permeabilized cells (already permeabilized), however we recommend to perform the permeabilization of the cell plasma membrane into the chambers (see SUIT-001 O2 ce-pce D003).

Compare SUIT protocols

SUIT-001 O2 ce-pce D003: SUIT-001 for non-permeabilized cells. Permeabilization of the cell plasma membrane occurs into the chambers through digitonin addition. Therefore, SUIT-001 O2 ce-pce D003 protocol provides information of non-permeabilized cell respiration (ce) and permeabilized cell respiration (pce).

SUIT-002 O2 mt D005 (RP2): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized).

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