Difference between revisions of "Krumschnabel 2013 Abstract MiP2013"

Revision as of 16:54, 22 August 2013

Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E (2013) Tissue homogenates for OXPHOS analysis in comparative mitochondrial physiology: trout and mouse – heart and liver mitochondria. Mitochondr Physiol Network 18.08.

Link:

Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E (2013)

Event: MiP2013

OXPHOS analysis is based on measurement of respiration in various steady-states of substrate supply and coupling of electron transfer to phosphorylation of ADP. To secure full accessibility of various flux control variables, X (substrates, ADP, etc.), to the mitochondria (mt), the plasma membranes have to be either permeabilized or the mitochondria must be mechanically separated from the intact cell in mt-preparations. Permeabilized muscle fibres represent an excellent and gentle type of mt-preparation, but require incubation at artificially high oxygen levels to overcome oxygen diffusion limitations [1]. Owing to large oxygen diffusion gradients and the oxygen dependence of mt-H2O2 production over a wide range of oxygen pressure [2], permeabilized muscle fibres may not represent an adequate model for the combined study of respiration and ROS production. A high-quality preparation of tissue homogenate may represent an optimum compromise for a variety of respirometric and fluorometric studies. These considerations provided the rationale for initiating a study with the PBI-Shredder, an auxiliary HRR-Tool providing a standardized approach to prepare homogenates of various tissues (heart, liver, brain) and species (mouse, rainbow trout). In the present study with high-resolution respirometry, mitochondrial respiratory control was compared in trout heart and liver tissue homogenate preparations at 15 °C [3]. Biochemical coupling efficiency with Complex I (CI)-linked substrates were identical in the two tissues. The ADP-ATP phosphorylation system exerted a higher control over OXPHOS in trout heart than liver. CI-linked substrate control capacity (OXPHOS) was higher whereas CII-linked succinate control capacity was lower in heart than liver. Pyruvate added to glutamate+malate stimulated OXPHOS capacity to a larger extent in heart than liver. For comparison, mouse heart and liver homogenate was measured at 37 °C using an identical substrate-uncoupler-inhibitor titration (SUIT) protocol. The cytochrome c test (<5% stimulation in healthy controls) indicated outer mt-membrane integrity in all cases, following an optimization of the PBI-Shredder application with high reproducibility of complete mt-yield and preservation of mitochondrial respiratory control.

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

Labels: MiParea: Respiration, Instruments;methods, Comparative MiP;environmental MiP

Organism: Mouse Tissue;cell: Heart, Nervous system, Liver Preparation: Homogenate

Regulation: Coupling efficiency;uncoupling, Cyt c Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.

HRR: Oxygraph-2k, Fluorometry"Fluorometry" 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.

MiP2013

Affiliations, acknowledgements and author contributions

Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck;

OROBOROS INSTRUMENTS, Schöpfstr. 18, Innsbruck, Austria

Email: erich.gnaiger@oroboros.at

Supported by K-Regio project MitoCom Tyrol.

References

Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543: 39-55.
Boveris A, Chance B (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 134: 707-716.
Doerrier CV, Draxl A, Wiethüchter A, Eigentler A, Gnaiger E (2013) Mitochondrial respiration in permeabilized fibres versus homogenate from fish liver and heart. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03 V3: 1-12.
Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production in mouse brain mitochondria. Mitochondr Physiol Network 17.03 V2: 1-4.

@@ Line 1: / Line 1: @@
 {{Abstract
-|title=Gnaiger E (2013) Normalization of respiration for OXPHOS analysis in comparative mitochondrial physiology: A study of trout heart and liver mitochondria. Mitochondr Physiol Network 18.08.
+|title=Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E (2013) Tissue homogenates for OXPHOS analysis in comparative mitochondrial physiology: trout and mouse – heart and liver mitochondria. Mitochondr Physiol Network 18.08.
-|info=[[Flux control capacity]]
+|authors=Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E
-|authors=Gnaiger E
 |year=2013
 |event=MiP2013
-|abstract=[[File:Gnaiger Erich.jpg|120px|right|Erich Gnaiger]]
+|abstract=OXPHOS analysis is based on measurement of respiration in various steady-states of substrate supply and coupling of electron transfer to phosphorylation of ADP. To secure full accessibility of various flux control variables, X (substrates, ADP, etc.), to the mitochondria (mt), the plasma membranes have to be either permeabilized or the mitochondria must be mechanically separated from the intact cell in mt-preparations. Permeabilized muscle fibres represent an excellent and gentle type of mt-preparation, but require incubation at artificially high oxygen levels to overcome oxygen diffusion limitations [1]. Owing to large oxygen diffusion gradients and the oxygen dependence of mt-H2O2 production over a wide range of oxygen pressure [2], permeabilized muscle fibres may not represent an adequate model for the combined study of respiration and ROS production. A high-quality preparation of tissue homogenate may represent an optimum compromise for a variety of respirometric and fluorometric studies. These considerations provided the rationale for initiating a study with the PBI-Shredder, an auxiliary HRR-Tool providing a standardized approach to prepare homogenates of various tissues (heart, liver, brain) and species (mouse, rainbow trout).
-Analysis of oxidative phosphorylation (OXPHOS) is a component of mitochondrial phenotyping. OXPHOS analysis is based on measurement of respiration in various steady-states of substrate supply and coupling of electron transfer to phosphorylation of ADP [1]. Mitochondrial respiration is conventionally normalized for mitochondrial (mt) markers such as mt-protein or citrate synthase (CS). No rationale is available to suggest a universal best mitochondrial marker. A general normalization of mt-respiration is presented here.
+	In the present study with high-resolution respirometry, mitochondrial respiratory control was compared in trout heart and liver tissue homogenate preparations at 15 °C [3]. Biochemical coupling efficiency with Complex I (CI)-linked substrates were identical in the two tissues. The ADP-ATP phosphorylation system exerted a higher control over OXPHOS in trout heart than liver. CI-linked substrate control capacity (OXPHOS) was higher whereas CII-linked succinate control capacity was lower in heart than liver. Pyruvate added to glutamate+malate stimulated OXPHOS capacity to a larger extent in heart than liver. For comparison, mouse heart and liver homogenate was measured at 37 °C using an identical substrate-uncoupler-inhibitor titration (SUIT) protocol. The cytochrome c test (<5% stimulation in healthy controls) indicated outer mt-membrane integrity in all cases, following an optimization of the PBI-Shredder application with high reproducibility of complete mt-yield and preservation of mitochondrial respiratory control.
-'''[[Flux control ratio]]s''', ''j'', are oxygen fluxes normalized relative to a common respiratory reference state [2]. For a given protocol or set of respiratory protocols, flux control ratios provide a fingerprint of coupling and substrate control independent of (i) mt-content of cells or tissues, (ii) purification in preparations of isolated mitochondria, and (iii) assay conditions for determination of tissue mass or mt-markers external to a respiratory protocol (CS, protein, stereology, etc.).
-Complementary to the concept of flux control ratios and analogous to elasticities of metabolic control analysis [3], '''[[Flux control capacity|flux control capacities]]''' express the control of respiration by a specific metabolic variable, ''X'', as a dimensionless (normalized) fractional change of flux, Δ''j''. ''Z'' is the reference sate with high (stimulated or un-inhibited) flux; ''Y'' is the background state at low flux, upon which ''X'' acts (''j<sub>Y</sub>''=''Y/Z''); ''X'' is either added (stimulation, activation) or removed (reversal of inhibition) to yield a flux ''Z'' from background ''Y''. Note that ''X'', ''Y'' and ''Z'' denote both, the metabolic control variable (''X'') or respiratory state (''Y, Z'') and the corresponding respiratory flux, ''X''=''Z-Y''. Experimentally, inhibitors are added rather than removed; then ''Z'' is the reference state and ''Y'' the background state in the presence of the inhibitor. The flux control capacity of ''X'' upon background ''Y'' is expressed as the change of flux from ''Y'' to ''Z'', normalized for the reference state ''Z'':
-:: Δ''j<sub>Z-Y</sub>'' = (''Z-Y'')/''Z'' = 1-''j<sub>Y</sub>''
-Substrate control capacities express the relative change of oxygen flux in response to a transition of substrate availability in a defined coupling state. Coupling and phosphorylation control capacities are determined in an [[Substrate control state|ETS-competent substrate state]].
-In the present study with high-resolution respirometry, mitochondrial respiratory control was compared in trout heart and liver tissue homogenate preparations at 15 °C [4]. Phosphorylation control capacities, i.e. [[LEAK]] (''Y''=''L'') to [[OXPHOS]] state (''Z''=''P''), with Complex I (CI)-linked substrates were identical in the two tissues. The ADP-ATP phosphorylation system exerted a higher control over OXPHOS in heart than liver. CI-linked substrate control capacity (OXPHOS) was higher whereas CII-linked succinate control capacity was lower in heart than liver. Pyruvate added to glutamate+malate stimulated OXPHOS capacity to a larger extent in heart than liver. Normalization of respiration in terms of flux control capacities is generally applicable to mitochondrial preparations and intact cells, eliminating any errors in separate measurements of mitochondrial markers.
 |mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom
 }}
@@ Line 28: / Line 13: @@
 |organism=Mouse
 |taxonomic group=Fishes
-|tissues=Heart, Liver
+|tissues=Heart, Nervous system, Liver
 |preparations=Homogenate
-|topics=ADP, Coupling efficiency;uncoupling, Cyt c, Flux control, Threshold;excess capacity
+|topics=Coupling efficiency;uncoupling, Cyt c
 |couplingstates=LEAK, OXPHOS, ETS
 |substratestates=CI, CII, CI+II, ROX
-|instruments=Oxygraph-2k, Theory
+|instruments=Oxygraph-2k, Fluorometry
-|additional=MiP2013, S02
+|additional=MiP2013
 }}
 __TOC__
@@ Line 51: / Line 36: @@
-Supported by K-Regio project ''[[MitoCom Tyrol]]''. I thank Anna Draxl for excellent experimental assistance.
+Supported by K-Regio project ''[[MitoCom Tyrol]]''.
 == References ==
-# [[Gnaiger 2012 MitoPathways|Gnaiger E (2012) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 3rd ed. Mitochondr Physiol Network 17.18. OROBOROS MiPNet Publications, Innsbruck: 64 pp.]]
+# Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543: 39-55.
-# [[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.]]
+# Boveris A, Chance B (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 134: 707-716.
-# Fell D (1997) Understanding the control of metabolism. Portland Press, London.
 # [[MiPNet17.03 Shredder vs Fibres|Doerrier CV, Draxl A, Wiethüchter A, Eigentler A, Gnaiger E (2013) Mitochondrial respiration in permeabilized fibres versus homogenate from fish liver and heart. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03 V3: 1-12.]]
+# Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production  in mouse brain mitochondria. Mitochondr Physiol Network 17.03 V2: 1-4.