Sumbalova 2012 Abstract Bioblast
|Sumbalova Z, Fasching M, Erich Gnaiger E (2012) Evaluation of mitochondrial respiration and membrane potential in mouse brain homogenate. Mitochondr Physiol Network 17.12:61.|
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, ΔΨ were measured simultaneously at 37°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 µM. Coupling and substrate control states  were established in 6 different SUIT protocols. In calculation of ΔΨ, mitochondrial protein was normalized to citrate synthase (CS) activity. The value of 11 µl/mg for K’out and K’in constants describing external and internal binding of TPP+, and 1 µl/mg for mitochondrial volume, were applied for all preparations. The signal of the TPP+ electrode was corrected for side effects of titrated chemicals . Mitochondria in all preparations were well coupled, without significant damage of outer membrane, respiration and ΔΨ 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 ΔΨ were lowest in Imt. The differences in ΔΨ between respiratory states, ΔΔΨ, were systematicaly highest in Imt and lowest in Hmt. Variation of K’out would reduce differences in ΔΨ and ΔΔΨ between preparations, however, exact determination of K’out 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.
Labels: Mammal;model: Mouse Tissue;cell: Nervous system Preparation: Homogenate, Isolated mitochondria Regulation: mt-Membrane potential Substrate state: CI HRR: Oxygraph-2k, TPP
Zuzana Sumbalová (1), Mario Fasching (2), Erich Gnaiger (2,3)
(1) Pharmacobiochemical Laboratory, Faculty of Medicine, Comenius University, Bratislava, Slovakia; Email: firstname.lastname@example.org
(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)