SUIT-014 O2 pfi D042

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SUIT-014 O2 pfi D042



Abbreviation: NS(PGM)

Reference: A SUIT-014

SUIT number: D042_1GM;2D;3P;4S;5U;6Rot;7Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT-category: NS(PGM)
SUIT protocol pattern: diametral 1GM;2D;3P;4S;5U;6Rot-

The SUIT-014 O2 pfi D042 protocol is designed for tissue fibers that display a preference for glutamate & malate (GM) over pyruvate & malate (PM) to support NADH-linked respiration. Adding pyruvate in the OXPHOS state enables to check if NADH-linked respiration can be further stimulated to determine N-pathway P capacity. SUIT-014 protocols provide an assessment of the coupling control L- P with NADH linked-substrates (GM) and P- E with N- and S-pathways combined (PGMS). The pathway control in OXPHOS state (N, NS) and ET state (NS, S) is also evaluated, covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types. 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.

Representative traces

D042 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
2c GMcP N CI 1GM;2D;2c
  • NADH-linked substrates (type N-pathway to Q).
  • Addition of cytochrome c yields a test for integrity of the mtOM. 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 (P, OXPHOS capacity with saturating [ADP]).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
3P PGMP N CI 1GM;2D;2c;3P
  • 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 PGMSE NS CI&II 1GM;2D;2c;3P;4S;5U
6Rot SE S CII 1GM;2D;2c;3P;4S;5U;6Rot
7Ama ROX 1GM;2D;2c;3P;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 entry into Q-junction Comment
## Azd ROX


Click to expand or collaps

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 analyse the convergence of pathways at the Q-junction (N, NS, S).
+ Mitochondrial oter membrane integrity 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.
- It was observed that addition of P (5 mM) after GM inhibited respiration in horse skeletal muscle fibres (Votion et al 2012), which was not the case when P was titrated in steps of 1 mM.
- When evaluating the additive effect of the N- and S-pathway, it has to be considered that NSP- and NSE-capacities can only be compared with NP- and SE-capacities. This is not a problem when NSP = NSE (Gnaiger 2009). Otherwise, it may be assumed that SP = SE (Votion et al 2012), such that NSP can be compared with NP + SP. SUIT-004 should be chosen for the additive effect in the ET-state.
- Rox may be lower in substrate states earlier in the SUIT protocol. Therefore, this Rox measurement is frequently taken as a methodological control rather than as the final basis of Rox correction of mitochondrial respiration (mt).
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
+ This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min.

Compare SUIT protocols

  • SUIT-008: 1PM;2D;2c;3G;4S;5U-; similar version starting with PM, and then adding G. Used in combination with SUIT-014 in Lemieux 2017 for pfi.
  • SUIT-011: 1GM;2D;2c;3S;4U;5Rot-; similar version omitting P.
  • SUIT-012: 1PM;2D;2c;3G;4U-; shorter version with PM preceding PGM and omitting S.


MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry