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Difference between revisions of "Doerrier 2016 Abstract MitoFit Science Camp 2016"

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{{Abstract
{{Abstract
|title=SUIT reference assay for OXPHOS analysis by high-resolution respirometry.
|title=Development of a SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.
|info=[[MiPNet21.06 SUIT RP]]
|authors=Doerrier C, Sumbalova Z, Lamberti G, Krumschnabel G, Gnaiger E
|authors=Doerrier C, Sumbalova Z, Lamberti G, Krumschnabel G, Gnaiger E
|year=2016
|year=2016
|event=MitoFit Science Camp 2016 Kuehtai AT
|event=MitoFit Science Camp 2016 Kuehtai AT
|abstract=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 phyisology. 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 under 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 – 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) FAOP 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 glycoreophosphate (Gp) through glycerophosphate dehydrogenase (GpDH) on the pathway level of converging into CoQ: PGMSOctGpE (Figure 2). RP1 and RP2, both supply information downstream CoQ, giving an evaluation of CIV activity. These SUIT-RP are harmonized (Figure 3) such that they can be statistically evaluated as replicate measurement 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 respirometrc OXPHOS analysis.
|abstract=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 [[Development MitoFit proficiency test|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-001|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_FNSGp(PGM)02 |SUIT RP2]] has a focus on OXPHOS capacity of fatty acid oxidation (FAO<sub>''P''</sub>) compared to OXPHOS capacity with combined NF-type substrates (CI<small>&</small>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) (PGMSOctGp<sub>''E''</sub>; 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.
|mipnetlab=AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E
|mipnetlab=AT Innsbruck Oroboros
}}
}}
== Affiliations ==
1-Oroboros Instruments, Innsbruck; 2-D. Swarovski Research Lab, Dept Visceral, Transplant and Thoracic Surgery, Innsbruck Med Univ, Austria. - [email protected]
== Figures ==
[[Image:MitoFit Training Camp 2016_Doerrier_Figure1.jpg|left|500px]] Figure 1. RP1.
<br /> <br /> <br /> <br /> <br /> <br /> <br />
[[Image:MitoFit Training Camp 2016_Doerrier_Figure2.jpg|left|500px]] Figure 2. RP2.
<br /> <br /> <br /> <br /> <br /> <br /> <br />
[[Image:MitoFit Training Camp 2016_Doerrier_Figure3.jpg|left|500px]] Figure. 3:Harmonization of RP1&2.
<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br />
== Support ==
The project was supported by K-Regio project MitoFit, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).
{{Labeling
{{Labeling
|area=Respiration
|area=Respiration
|organism=Human
|organism=Human, Mouse
|tissues=Kidney
|tissues=Heart, Blood cells, HEK, Platelet
|model cell lines=HEK
|preparations=Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria
|couplingstates=LEAK, OXPHOS, ETS
|couplingstates=LEAK, ROUTINE, OXPHOS, ET
|substratestates=CI, CII, FAO
|pathways=F, N, S, Gp, CIV, NS, ROX
|instruments=Oxygraph-2k
|instruments=Oxygraph-2k, O2k-Protocol
|event=A1
|event=A1
|additional=MitoFit Science Camp 2016
|additional=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]
== Figure ==
[[Image:MitoFit Training Camp 2016_Doerrier_Figure1.jpg|left|500px]]
[[Image:MitoFit Training Camp 2016_Doerrier_Figure2.jpg|left|500px]]
[[Image:MitoFit Training Camp 2016_Doerrier_Figure3.jpg|left|500px]]
== Support ==
The project was supported by K-Regio project MitoFit, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).

Latest revision as of 17:40, 10 January 2022

Development of a SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.

Link: MiPNet21.06 SUIT RP

Doerrier C, Sumbalova Z, Lamberti G, Krumschnabel G, Gnaiger E (2016)

Event: MitoFit Science Camp 2016 Kuehtai AT

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.


β€’ O2k-Network Lab: AT Innsbruck Oroboros


Affiliations

1-Oroboros Instruments, Innsbruck; 2-D. Swarovski Research Lab, Dept Visceral, Transplant and Thoracic Surgery, Innsbruck Med Univ, Austria. - [email protected]

Figures

MitoFit Training Camp 2016 Doerrier Figure1.jpg

Figure 1. RP1.








MitoFit Training Camp 2016 Doerrier Figure2.jpg

Figure 2. RP2.









MitoFit Training Camp 2016 Doerrier Figure3.jpg

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).


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