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. 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 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.
  • SUIT-028: 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