Doerrier 2016 Abstract MitoFit Science Camp 2016
SUIT reference assay for OXPHOS analysis by high-resolution respirometry. |
Link:
Doerrier C, Sumbalova Z, Lamberti G, Krumschnabel G, Gnaiger E (2016)
Event: MitoFit Science Camp 2016 Kuehtai AT
A substrate-uncoupler-inhibitor titration (SUIT) reference assay (SUIT-RA) has been developed to provide a common baseline for comparison of mitochondrial respiratory control in a large variety of species, tissues and cell types, mt-preparations and laboratories, for establishing a database on comparative mitochondrial physiology. The SUIT-RA 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 chemicals to quality control in the MitoFit proficiency test. The SUIT-RA consists of two coordinated SUIT reference protocols (SUIT-RP). The SUIT-RP1 (RP1) allows to evaluate CI-linked linear coupling control: L β P - E, thus separating coupling control (CI-linked) and substrate control (in the ETS state) (Figure 1). The SUIT-RP2 (RP2) provides information about fatty acid oxidation (FAO) FAO_P compared to CI&FAO_P. Moreover, RP2 permits to measure maximum ETS capacity through CI, from FAO oxidation through electron-transferring flavoprotein complex (CETF), from succinate (S) through CII, from glycerophosphate (Gp) through glycerophosphate dehydrogenase (GpDH) on the pathway level of converging into CoQ: PGMSOctGpE (Figure 2). RP1 and RP2 also both supply information on electron fluxes downstream of CoQ, giving an evaluation of CIV activity. These SUIT-RP are harmonized (Figure 3) such that they can be statistically evaluated as replicate measurements for carefully selected respiratory states, while additional information is obtained when the two protocols are conducted in parallel. Therefore, the two SUIT-RP are complementary with their focus on specific respiratory coupling and substrate control aspects, extending previous strategies for respirometric OXPHOS analysis.
β’ O2k-Network Lab: AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E
Labels: MiParea: Respiration
Organism: Human, Mouse
Tissue;cell: Heart, Blood cells
Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria
Coupling state: LEAK, ROUTINE, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k, O2k-Protocol Event: A1 MitoFit Science Camp 2016
Affiliations
1-OROBOROS INSTRUMENTS, Innsbruck; 2-D. Swarovski Research Lab, Dept Visceral, Transplant and Thoracic Surgery, Innsbruck Med Univ, Austria. - [email protected]
Figures
Figure 1. RP1.
Figure 2. RP2.
Figure. 3:Harmonization of RP1&2.
Support
The project was supported by K-Regio project MitoFit, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).