Gnaiger 2014 Abstract MiP2014

Cell ergometry: OXPHOS and ETS coupling efficiency.

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Mitochondr Physiol Network 19.13 - MiP2014

Gnaiger E (2014)

Event: MiP2014

Biochemical cell ergometry aims at measurement of J_O2,max (compare V_O2,max in exercise ergometry of humans and animals) of cell respiration linked to phosphorylation of ADP to ATP. The corresponding OXPHOS capacity is based on saturating concentrations of ADP, [ADP]*, and inorganic phosphate, [Pi]*, available to the mitochondria. This is metabolically opposite to uncoupling respiration, which yields ETS capacity. The OXPHOS state can be established experimentally by selective permeabilization of cell membranes with maintenance of intact mitochondria, titrations of ADP and P_i to evaluate kinetically saturating conditions, and establishing fuel substrate combinations which reconstitute physiological TCA cycle function.

Labels: MiParea: Respiration 

Coupling state: OXPHOS 

HRR: Oxygraph-2k  Event: A4, Oral  MiP2014 

Affiliation

1-Daniel Swarovski Research Lab, Mitochondrial Physiol, Dep Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck; 2-OROBOROS INSTRUMENTS, Innsbruck, Austria. - [email protected]

Abstract

Analogous to ergometric measurement of V_O2max or V_O2peak on a cycle or treadmill, cell ergometry is based on OXPHOS analysis to determine OXPHOS capacity, J_O2P=P [pmol O2·s^-1·mg^-1], at the cellular and mitochondrial level. V_O2peak and J_O2P provide reference values for a subject’s or a cell’s aerobic or mitochondrial competence. Aerobic catabolic flux (1 ml O2·min^-1·kg^-1 = 0.744 µmol·s^-1·kg^-1) is multiplied by the corresponding Gibbs force (Δ_kG_O2=∂G/∂_kξ_O2; typically -470 kJ/mol or -0.48 J/µmol O₂) to obtain the mass-specific aerobic input power [W·kg^-1]. The corresponding mechanical output power, P_peak [W·kg^-1], in cycle ergometry results in ergodynamic efficiencies [1] of about 0.25,

εpeak  =  Ppeak/-(JO2peak·ΔkGO2)  =  (Ppeak/JO2peak) / -ΔkGO2                 (1)

In OXPHOS analysis the output power is mitochondrial ATP production, J_~P=~P, times the Gibbs force of phosphorylation (Δ_pG~P=∂G/∂_pξ_~P), which is typically 48 to 62 kJ/mol ~P [1]. Ergodynamic efficiency is a power ratio, partitioned into a flux ratio (the famous ~P/O₂ ratio; ATP yield per oxygen consumed, Y_~P/O2 = J_~P/J_O2P = ~P/P) and force ratio,

ε_P = (J_~P·Δ_pG_~P)/-(J_O2P·Δ_kG_O2) = ~P/P ∙ Δ_pG_~P/-Δ_kG_O2 = j_≈P ∙ f_≈P (2)

The upper limit of ~P/P is the mechanistic ~P:O₂ ratio or stoichiometric number, ν_~P/O2. The free respiratory OXPHOS capacity, ≈P=P-L, is potentially available to drive phosphorylation, ~P (Figure 1). Quantitatively justified in cases [3] but better adjusted to the protonmotive force, Δp_mt, the dissipative LEAK component, L, in the OXPHOS state P can be assessed by respiration, L, measured in the LEAK state,

ν~P/O2 = ~Plimit/P = ~P/(P-L) = ~P/≈P                              (3)

~P/P divided by ~P/≈P defines the OXPHOS coupling efficiency, j_≈P, as a normalized flux ratio, which is a hyperbolic function of RCR (Figure 2),

j≈P = ≈P/P = (L-P)/P = 1-L/P = 1-RCR-1                            (4)

References and acknowledgements

Supported by K-Regio project MitoCom Tyrol.

1. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.

Figure 1: Phosphorylation control protocol in the intact cell

>> Cell ergometry File:OROBOROS Poster HRR.pdf

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