Sumbalova 2012 Abstract Bioblast: Difference between revisions
No edit summary |
No edit summary Β |
||
(17 intermediate revisions by 6 users not shown) | |||
Line 1: | Line 1: | ||
{{Abstract | {{Abstract | ||
|title=Sumbalova Z (2012) | |title=Sumbalova Z, Fasching M, Gnaiger E (2012) Evaluation of mitochondrial respiration and membrane potential in mouse brain homogenate. Mitochondr Physiol Network 17.12:61. | ||
|info=[[MiPNet17.12 Bioblast | |info=[[MiPNet17.12 Bioblast 2012|MiPNet17.12 Bioblast 2012 - Open Access]] | ||
|authors=Sumbalova Z | |authors=Sumbalova Z, Fasching M, Gnaiger E | ||
|year=2012 | |year=2012 | ||
|event=[[Bioblast | |event=[[Bioblast 2012]] | ||
|abstract=[[File:ZS.jpg|right|250px|Zuzana Sumbalova]] | |||
Experimental and diagnostic studies require precise evaluation of mitochondrial function in small samples. Using tissue homogenates instead of isolated mitochondria would require minimal amount of tissue and time for preparation and therefore could be widely used for diagnostic purposes. | |||
The aim of our study was to compare mitochondrial function in 3 preparations from mouse brain: crude homogenate (Hmt), supernatant after low-speed centrifugation (Smt), and isolated mitochondria (Imt). | |||
Mitochondrial respiration, JO<sub>2</sub>, and membrane potential, ΞΞ¨ were measured simultaneously at 37Β°C in Oxygraph-2k MultiSensor system with DatLab software equipped with an ion selective electrode system (Oroboros Instruments, Innsbruck, Austria), in MiRO6 and tetraphenylphosphonium (TPP<sup>+</sup>) concentration 1 and 1.5 Β΅M. Coupling and substrate control states [1] were established in 6 different [[SUIT]] protocols. In calculation of ΞΞ¨, mitochondrial protein was normalized to citrate synthase (CS) activity. The value of 11 Β΅l/mg for Kβout and Kβin constants describing external and internal binding of TPP<sup>+</sup>, and 1 Β΅l/mg for mitochondrial volume, were applied for all preparations. The signal of the TPP<sup>+</sup> electrode was corrected for side effects of titrated chemicals [2]. | |||
Mitochondria in all preparations were well coupled, without significant damage of outer membrane, respiration and ΞΞ¨ were stable for 4-5 hours. JO<sub>2</sub>/CS were similar in all preparations, only [[State 4]] respiration was slightly lower in Imt (-21 % vs Hmt).The ratio of respiration stimulated by 0.25 mM and 2.25 mM ADP in Hmt reached 88% of the value in other preparations, [[RCR]] (CI) was significantly higher (+50%) in Smt in comparison with Hmt and Imt. Absolute values of calculated ΞΞ¨ were lowest in Imt. The differences in ΞΞ¨ between respiratory states, ΞΞΞ¨, were systematicaly highest in Imt and lowest in Hmt. Variation of Kβout would reduce differences in ΞΞ¨ and ΞΞΞ¨ between preparations, however, exact determination of Kβout is problematic. | |||
High sensitivity and stability of Oroboros TPP<sup>+</sup> electrode enables determination of mitochondrial respiration and membrane potential simultaneously in Oxygraph-2k MultiSensor system. Homogenates could be a good alternative to Imt for determination of mitochondrial function in small samples. | |||
Β | |||
# [[Gnaiger 2009 Int J Biochem Cell Biol|Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845.]] | |||
# [[MiPNet14.05 TPP-MitoMembranePotential|Renner-Sattler K, Fasching M, Gnaiger E. TPP+ and membrane potential. Mitochondr Physiol Network 14.05.]] | |||
|keywords=ΞΞ¨, Mitochondrial preparations, [[SUIT]] protocols | |||
|mipnetlab=SK Bratislava Sumbalova Z, AT Innsbruck Oroboros | |||
|journal=Mitochondr Physiol Network | |journal=Mitochondr Physiol Network | ||
|articletype=Abstract | |articletype=Abstract | ||
}} | }} | ||
Line 16: | Line 24: | ||
== Affiliations and author contributions == | == Affiliations and author contributions == | ||
Zuzana SumbalovΓ‘ (1), Mario Fasching (2), Erich Gnaiger (2,3) | |||
(1) Pharmacobiochemical Laboratory, Faculty of Medicine, Comenius University, Bratislava, Slovakia; Email: [email protected] | |||
(2) OROBOROS INSTRUMENTS, Innsbruck | |||
== | (3) D. Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Austria | ||
Β | |||
* [[MitoPedia | Β | ||
Supported by FEMtech (NMVIT, Austria) | |||
Β | |||
== MitoPedia == | |||
* [[MitoPedia: Terms and abbreviations]] | |||
Β | |||
{{Labeling | |||
|organism=Mouse | |||
|tissues=Nervous system | |||
|preparations=Homogenate, Isolated mitochondria | |||
|topics=mt-Membrane potential | |||
|pathways=N | |||
|instruments=Oxygraph-2k, TPP | |||
|journal=Mitochondr Physiol Network | |||
|articletype=Abstract | |||
}} |
Latest revision as of 18:26, 10 January 2022
Sumbalova Z, Fasching M, Gnaiger E (2012) Evaluation of mitochondrial respiration and membrane potential in mouse brain homogenate. Mitochondr Physiol Network 17.12:61. |
Link: MiPNet17.12 Bioblast 2012 - Open Access
Sumbalova Z, Fasching M, Gnaiger E (2012)
Event: Bioblast 2012
Experimental and diagnostic studies require precise evaluation of mitochondrial function in small samples. Using tissue homogenates instead of isolated mitochondria would require minimal amount of tissue and time for preparation and therefore could be widely used for diagnostic purposes. The aim of our study was to compare mitochondrial function in 3 preparations from mouse brain: crude homogenate (Hmt), supernatant after low-speed centrifugation (Smt), and isolated mitochondria (Imt). Mitochondrial respiration, JO2, and membrane potential, ΞΞ¨ were measured simultaneously at 37Β°C in Oxygraph-2k MultiSensor system with DatLab software equipped with an ion selective electrode system (Oroboros Instruments, Innsbruck, Austria), in MiRO6 and tetraphenylphosphonium (TPP+) concentration 1 and 1.5 Β΅M. Coupling and substrate control states [1] were established in 6 different SUIT protocols. In calculation of ΞΞ¨, mitochondrial protein was normalized to citrate synthase (CS) activity. The value of 11 Β΅l/mg for Kβout and Kβin constants describing external and internal binding of TPP+, and 1 Β΅l/mg for mitochondrial volume, were applied for all preparations. The signal of the TPP+ electrode was corrected for side effects of titrated chemicals [2]. Mitochondria in all preparations were well coupled, without significant damage of outer membrane, respiration and ΞΞ¨ were stable for 4-5 hours. JO2/CS were similar in all preparations, only State 4 respiration was slightly lower in Imt (-21 % vs Hmt).The ratio of respiration stimulated by 0.25 mM and 2.25 mM ADP in Hmt reached 88% of the value in other preparations, RCR (CI) was significantly higher (+50%) in Smt in comparison with Hmt and Imt. Absolute values of calculated ΞΞ¨ were lowest in Imt. The differences in ΞΞ¨ between respiratory states, ΞΞΞ¨, were systematicaly highest in Imt and lowest in Hmt. Variation of Kβout would reduce differences in ΞΞ¨ and ΞΞΞ¨ between preparations, however, exact determination of Kβout is problematic. High sensitivity and stability of Oroboros TPP+ electrode enables determination of mitochondrial respiration and membrane potential simultaneously in Oxygraph-2k MultiSensor system. Homogenates could be a good alternative to Imt for determination of mitochondrial function in small samples.
- Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845.
- Renner-Sattler K, Fasching M, Gnaiger E. TPP+ and membrane potential. Mitochondr Physiol Network 14.05.
β’ Keywords: ΞΞ¨, Mitochondrial preparations, SUIT protocols
β’ O2k-Network Lab: SK Bratislava Sumbalova Z, AT Innsbruck Oroboros
Affiliations and author contributions
Zuzana SumbalovΓ‘ (1), Mario Fasching (2), Erich Gnaiger (2,3)
(1) Pharmacobiochemical Laboratory, Faculty of Medicine, Comenius University, Bratislava, Slovakia; Email: [email protected]
(2) OROBOROS INSTRUMENTS, Innsbruck
(3) D. Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Austria
Supported by FEMtech (NMVIT, Austria)
MitoPedia
Labels:
Organism: Mouse
Tissue;cell: Nervous system
Preparation: Homogenate, Isolated mitochondria
Regulation: mt-Membrane potential
Pathway: N HRR: Oxygraph-2k, TPP