SUIT-011 O2 pfi D024

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SUIT-011 O2 pfi D024

Description

1GM;2D;2c;3S;4U;5Rot;6Ama.png

Abbreviation: NS physiological maximum capapcity in fibres

Reference: A pfi: permeabilized fibers - SUIT-011

SUIT number: D024_1GM;2D;2c;3S;4U;5Rot;6Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT protocol pattern:1GM;2D;2c;3S;4U;5Rot-

The SUIT-011 O2 pfi D024 protocol is designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control states. SUIT-011 gives information of the linear coupling control (L- P) with NADH linked-substrates (GM). GM and PM yield practically identical fluxes in human skeletal muscle fibers. However, PM is the superior alternative to GM, since the fractions of the N-pathway is lower and of the S-pathway is higher with GM compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-linked pathway (compare SUIT-001 and SUIT-004). Moreover, SUIT-011 allows the evaluation of the coupling control state (P- E) with NADH and succinate linked-substrates (NS) and the pathway control in OXPHOS (NS) and ET state (NS and S). SUIT-011 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2019-03-06)

Representative traces

D024 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

SUIT-011

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1GM GML(n) N CI 1GM
2D GMP N CI 1GM;2D
2c GMcP N CI 1GM;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. 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]).
3S GMSP NS CI&II 1GM;2D;2c;3S
4U GMSE NS CI&II 1GM;2D;2c;3S;4U
5Rot SE S CII 1GM;2D;2c;3S;4U;5Rot
6Ama ROX 1GM;2D;2c;3S;4U;5Rot;6Ama
  • 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
## AsTm AsTmE CIV CIV
## Azd ROX


Questions.jpg


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Strengths and limitations

  • Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI (Lemieux et al. 2017; Votion et al. 2012).
  • A succinate concentration of >10 mM may be required for saturating SE capacity.
  • Rox might be inhibited slightly further by inhibition of CIV by cyanide (KCN; 1 μM). But cyanide inhibits not only CIV, but also catalase and other oxygenases involved in ROX.
+ NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
+ Glutamate is easier to prepare compared to pyruvate.
+ 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.
- 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 (GMP is inhibited by the CII inhibitor malonic acid to a larger extent than PMP). 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 a disadvantage compared to SUIT-004 and SUIT-008 for diagnosis of N-capacity.
- To detect an additive effect of P after GMP, pyruvate would have to be added as step 3 (before S). However, inhibition of respiration was observed after titration of P (5 mM) 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.
- CIV activity is not measured, to save experimental time.


Compare SUIT protocols

  • GM and PM yield typically identical fluxes in human skeletal muscle fibres.
  • SUIT-004 1PM;2D;3U;4S;5Rot-: SUIT-004 allows the evaluation of the linear coupling control (L- P) with PM (instead of GM) as NADH linked-substrates. Moreover, in SUIT-004 the linear coupling control from P to E (with PM) and the ET-pathway state in NS- and S-pathways can be assessed.
  • SUIT-008 1PM;2D;3G;4S;5U;6Rot-: SUIT-008 protocols are 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.
  • SUIT-001 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp-: SUIT reference protocol 1 (RP1)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.
  • 1PGM;2D;3S;4U;5Rot-: PGM as NADH linked-substrates.

References

 YearReferenceOrganismTissue;cell
Votion 2012 PLoS One2012Votion 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.HorseSkeletal muscle


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry