Krumschnabel 2013 Abstract MiP2013

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.

Link: Flux control capacity

Gnaiger E (2013)

Event: MiP2013

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.

Flux control ratios, 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 capacities express the control of respiration by a specific metabolic variable, X, as a dimensionless (normalized) fractional change of flux, Δj. 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_Y=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:

Δj_Z-Y = (Z-Y)/Z = 1-j_Y

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 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.

• 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, Liver Preparation: Homogenate

Regulation: ADP, Coupling efficiency;uncoupling, Cyt c, Flux control, Threshold;excess capacity 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, Theory

MiP2013, S02

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. I thank Anna Draxl for excellent experimental assistance.

References