Keppner 2017 MiPschool Obergurgl
Event: MiPschool Obergurgl 2017
Substrate-uncoupler-inhibitor-titration (SUIT) protocols are a combination of different substrates, inhibitors and uncouplers to assess the OXPHOS and ET-capacity. LEAK respiration is defined as the oxygen consumption compensating for the proton leak which is measured in the presence of substrates and in the absence of ADP or inhibition of the ATP-synthase. In a standardized SUIT protocol of permeabilized cells LEAK respiration is determined after applying digitonin, when the respiration has settled down to LEAK. Another way to determine leak respiration is the inhibition of the ATP-synthase by oligomycin.
In this outline two different SUIT protocols are described analyzing the difference in LEAK respiration measured with digitonin and oligomycin respectively and subsequently the effect of oligomycin on the uncoupled respiration by FCCP.
The comparison of different SUIT protocols was conducted on freshly isolated peripheral blood mononuclear cells (PBMC). In the first protocol LEAK respiration was determined in the presence of oligomycin and the OXPHOS capacity was measured directly after permeabilizing the cells by digitonin. The second protocol determined LEAK respiration directly after cells were permeabilized by digitonin, afterwards the OXPHOS capacity was measured (Figure 1). Additionally another measurement was conducted to analyze the effect of oligomycin on the uncoupled respiration under FCCP (Figure 2 B).
Determining LEAK respiration after permeabilizing cells by digitonin with a prolonged waiting time led to a decreased OXPHOS-capacity in PBMCs compared to a SUIT protocol where the OXPHOS capacity was determined directly after permeabilizing the cells. An additional measurement showed a reduced ET-capacity by FCCP in the presence of oligomycin.
LEAK respiration in the presence of digitonin led to a reduced OXPHOS capacity whereas the application of oligomycin showed an inhibitory effect on the uncoupled respiration under FCCP. Based on the given situation it should be carefully considered how to determine the different respiratory states.
Labels: MiParea: Respiration
Tissue;cell: Blood cells Preparation: Permeabilized cells
Coupling state: LEAK, OXPHOS, ET Pathway: N, NS HRR: Oxygraph-2k Event: C1, Oral PBMCs
- Keppner G(1,2), Hauner H(2,3), Skurk T(2,3), Klingenspor M(1,2)
- Chair Molecular Nutritional Medicine, Else Kröner-Fresenius Zentrum Ernährungsmedizin
- ZIEl – Inst Food Health
- Chair Clinical Nutritional Medicine, Else Kröner-Fresenius Zentrum Ernährungsmedizin
- Technische Univ München. [email protected]
Figure 1. (A) Structure of the SUIT protocol 2. (B) Representative trace from SUIT protocol 2, using digitonin and oligomycin to determine LEAK respiration
Figure 2. (A) Comparison of two different SUIT protocols. Protocol 1 used oligomycin to determine LEAK respiration. Protocol 2 determined LEAK respiration after digitonin and oligomycin. The OXPHOS capacity is analyzed between these two protocols. (B) Inhibitory effect of oligomycin on the uncoupled respiration by FCCP.
- Recepient of a MitoEAGLE scholarship.