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Difference between revisions of "EBEC2014"

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== EBEC Abstracts ==
== EBEC Abstracts ==


{{Abstract
|title=The relationship between cytochrome redox state and oxygen consumption in isolated mouse and beef heart mitochondria during hypoxia.
|authors=Harrison DK, Fasching M, Fontana-Ayoub M, Gnaiger E
|year=2014
|event=EBEC2014
|abstract=This study describes a low-noise, rapid spectrophotometric system using visible light (440-605 nm) for the measurement of cytochrome redox state combined with high-resolution respirometry. The system was tested in an investigation using beef and mouse heart isolated mitochondria (BHImt, MHImt) in order to determine the relationship between respiratory rate and cytochrome redox state at steady-state levels of hypoxia. Monophasic hyperbolic relations were observed between respiratory rate,  ''j'' (with glutamate+malate and saturating ADP concentrations), and oxygen partial pressure, ''p''<sub>O2</sub>, in the range <1.1 kPa for both BHImt and MHImt with ''p''<sub>50,''j''</sub> (''p''<sub>O2</sub> at ''j'' = 0.5 ''J''<sub>max</sub>) of  0.015 and 0.021 kPa respectively. The oxidation fractions of cytochromes ''aa''3 and ''c'' were biphasic hyperbolic functions of ''p''<sub>O2</sub>. The relationships between cytochrome oxidation states and ''j'' were more complex with an initial steep decrease in the oxidation fraction of cytochrome ''c'' to a value of ''j'' of approximately 0.7 followed by a plateaux and a further steep decrease at ''j'' < 0.2. This relationship was less apparent with cytochrome ''b'' redox state. Using these functions, it was possible to create a model that successfully described the measured relationship between cytochrome oxidation state and oxygen consumption.


=== The relationship between cytochrome redox state and oxygen consumption in isolated mouse and beef heart mitochondria during hypoxia ===
|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom
[[Harrison David K | David K Harrison]]<sup>1,2</sup>, [[Fasching Mario | Mario Fasching]]<sup>1</sup>, [[Fontana-Ayoub Mona | Mona Fontana-Ayoub]]<sup>1</sup> and [[Gnaiger Erich | Erich Gnaiger]]<sup>1,3</sup>
}}
{{Labeling
|area=Respiration
|organism=Mouse, Bovines
|tissues=Heart
|preparations=Isolated Mitochondria
|injuries=Hypoxia
|topics=O2
|couplingstates=OXPHOS
|substratestates=CI
|instruments=Oxygraph-2k, TIP2k, Spectrophotometry
}}
    
    
<sup>1</sup>Oroboros Instruments, Innsbruck, Austria; <sup>2</sup>Microvascular Measurements, St Lorenzen, Italy; <sup>3</sup>D Swarowski Research Laboratory, Department of Visceral Transplant and Thoracic Surgery, Medical University of Innsbruck, Austria
 
This study describes a low-noise, rapid spectrophotometric system using visible light (440-605 nm) for the measurement of cytochrome redox state combined with high-resolution respirometry. The system was tested in an investigation using beef and mouse heart isolated mitochondria (BHImt, MHImt) in order to determine the relationship between respiratory rate and cytochrome redox state at steady-state levels of hypoxia. Monophasic hyperbolic relations were observed between respiratory rate,  ''j'' (with glutamate+malate and saturating ADP concentrations), and oxygen partial pressure, ''p''<sub>O2</sub>, in the range <1.1 kPa for both BHImt and MHImt with ''p''<sub>50,''j''</sub> (''p''<sub>O2</sub> at ''j'' = 0.5 ''J''<sub>max</sub>) of  0.015 and 0.021 kPa respectively. The oxidation fractions of cytochromes ''aa''3 and ''c'' were biphasic hyperbolic functions of ''p''<sub>O2</sub>. The relationships between cytochrome oxidation states and ''j'' were more complex with an initial steep decrease in the oxidation fraction of cytochrome ''c'' to a value of ''j'' of approximately 0.7 followed by a plateaux and a further steep decrease at ''j'' < 0.2. This relationship was less apparent with cytochrome ''b'' redox state. Using these functions, it was possible to create a model that successfully described the measured relationship between cytochrome oxidation state and oxygen consumption.


=== Combined high-resolution respirometry and fluorometry. Validation of safranin for determination of mitochondrial membrane potential ===
=== Combined high-resolution respirometry and fluorometry. Validation of safranin for determination of mitochondrial membrane potential ===

Revision as of 09:18, 15 July 2014

Publications in the MiPMap
EBEC2014.png
Lisbon PT, 2014 Jul 12-17. 18th European Bioenergetics Conference - EBEC2014

» EBEC2014

EBEC (2014-07-12) MitoGlobal

Abstract: 18th European Bioenergetics Conference - EBEC2014.


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ORO, 2014, MitoCom, Next 

Erich Gnaiger Gnaiger Erich, CEO, OROBOROS INSTRUMENTS Fleischmann Sandra Fleischmann Sandra; Administrative assistant

EBEC Abstracts

The relationship between cytochrome redox state and oxygen consumption in isolated mouse and beef heart mitochondria during hypoxia.

Link:

Harrison DK, Fasching M, Fontana-Ayoub M, Gnaiger E (2014)

Event: EBEC2014

This study describes a low-noise, rapid spectrophotometric system using visible light (440-605 nm) for the measurement of cytochrome redox state combined with high-resolution respirometry. The system was tested in an investigation using beef and mouse heart isolated mitochondria (BHImt, MHImt) in order to determine the relationship between respiratory rate and cytochrome redox state at steady-state levels of hypoxia. Monophasic hyperbolic relations were observed between respiratory rate, j (with glutamate+malate and saturating ADP concentrations), and oxygen partial pressure, pO2, in the range <1.1 kPa for both BHImt and MHImt with p50,j (pO2 at j = 0.5 Jmax) of 0.015 and 0.021 kPa respectively. The oxidation fractions of cytochromes aa3 and c were biphasic hyperbolic functions of pO2. The relationships between cytochrome oxidation states and j were more complex with an initial steep decrease in the oxidation fraction of cytochrome c to a value of j of approximately 0.7 followed by a plateaux and a further steep decrease at j < 0.2. This relationship was less apparent with cytochrome b redox state. Using these functions, it was possible to create a model that successfully described the measured relationship between cytochrome oxidation state and oxygen consumption.


O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom


Labels: MiParea: Respiration 

Stress:Hypoxia  Organism: Mouse, Bovines  Tissue;cell: Heart  Preparation: Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

Regulation: O2"O2" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.  Coupling state: OXPHOS 

HRR: Oxygraph-2k, TIP2k, Spectrophotometry"Spectrophotometry" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 



Combined high-resolution respirometry and fluorometry. Validation of safranin for determination of mitochondrial membrane potential

Gerhard Krumschnabel1, Andrea Eigentler2, Mario Fasching1 and Erich Gnaiger1,2

1OROBOROS INSTRUMENTS, Innsbruck, Austria; 2D Swarowski Research Laboratory, Department of Visceral Transplant and Thoracic Surgery, Medical University of Innsbruck, Austria

Mitochondrial membrane potential (mtMP) is closely intertwined with oxidative phosphorylation (OXPHOS). The exact nature of the interactions of respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. We introduce the combination of high-resolution respirometry and fluorometry with the OROBOROS Oxygraph-2k, using the widely applied mtMP indicator safranin. OXPHOS analysis with mouse brain homogenate revealed that safranin inhibits Complex I linked OXPHOS capacity at commonly applied concentrations and targets primarily the phosphorylation system, without effect on LEAK respiration. Complex II linked OXPHOS capacity was inhibited by <20% at 2 µM safranin sufficient for mtMP monitoring. mtMP was higher in the LEAK state without adenylates (LN) than at identical LEAK respiration after ADP stimulation and inhibition by oligomycin (LOmy). Maximum ETS capacity was reached in uncoupler titrations before mtMP was fully collapsed, whereas respiration was inhibited at increasing uncoupler concentrations and further reduction of mtMP. Examining a pharmacologically induced state of Complex II dysfunction, mtMP was rather insensitive to 50% inhibition of OXPHOS, but responded strongly to addition of inhibitors when respiration was minimized by substrate depletion. The optimum uncoupler concentration supporting maximum ETS capacity varied as a function of pharmacological intervention. Taken together, combined measurement of respiration and mtMP greatly enhances the diagnostic potential of OXPHOS analysis. Respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore applied for probing mtMP, in any respiratory state, type of tissue and pathophysiological condition.


Supported by K-Regio project MitoCom.


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