Doerrier 2016 Abstract MitoFit Science Camp 2016
|Development of a SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.|
Link: MiPNet21.06 SUIT RP
We developed a substrate-uncoupler-inhibitor titration (SUIT) protocol with the aim to provide a common reference for comparison of respiratory control in mt-preparations obtained from a large variety of species, tissues and cell types. A SUIT reference protocol (SUIT-RP) is required for establishing a database on comparative mitochondrial physiology. The SUIT-RP is applied in the MitoFit proficiency test with HEK 293T cells. It includes a large number of chemicals used in various specific SUIT protocols, subjecting these to quality control in the MitoFit proficiency test. The SUIT-RP consists of two harmonized SUIT protocols. SUIT RP1 starts with linear coupling control, L – P - E with the type N substrates pyruvate and malate (CI-linked), thus separating coupling control from the subsequent linear sequence of pathway control in the ET-pathway state (Figure 1). The SUIT RP2 has a focus on OXPHOS capacity of fatty acid oxidation (FAOP) compared to OXPHOS capacity with combined NF-type substrates (CI&FAO). RP2 adds a sequence of pathway control steps to measure maximum OXPHOS and ET capacity with a NFSGp substrate combination to activate pathways converging at the Q-junction through Complex I (CI), electron-transferring flavoprotein complex (CETF), Complex II (CII), and glycerophosphate dehydrogenase complex (GpDH) (PGMSOctGpE; Figure 2). Finally, RP1 and RP2 provide information on the activity of the single enzyme step of Complex IV (CIV) downstream of Q. These SUIT-RP are harmonized (Figure 3) such that they can be statistically evaluated as repeat measurements of cross-linked respiratory states, while additional information is obtained when the two protocols are conducted in parallel. Therefore, RP1 and RP2 are complementary with their focus on specific respiratory coupling and pathway control aspects, extending previous strategies for respirometric OXPHOS analysis.
Labels: MiParea: Respiration
Organism: Human, Mouse Tissue;cell: Heart, Blood cells, HEK, Platelet Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria
Coupling state: LEAK, ROUTINE, OXPHOS, ET Pathway: F, N, S, Gp, CIV, NS, ROX HRR: Oxygraph-2k, O2k-Protocol Event: A1 MitoFit Science Camp 2016
1-Oroboros Instruments, Innsbruck; 2-D. Swarovski Research Lab, Dept Visceral, Transplant and Thoracic Surgery, Innsbruck Med Univ, Austria. - [email protected]
Figure 1. RP1.
Figure 2. RP2.
Figure. 3:Harmonization of RP1&2.
The project was supported by K-Regio project MitoFit, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).