Gnaiger 2014 Abstract MiP2014
Cell ergometry: OXPHOS and ET-pathway coupling efficiency. |
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, [P_{i}]*, available to the mitochondria. This is metabolically opposite to uncoupling respiration, which yields ET-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. - erich.gnaiger@oroboros.at
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_{k}G_{O2}=∂G/∂_{k}ξ_{O2}; typically -470 kJ/mol or -0.47 J/µmol O_{2}) 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,
ε_{peak} = P_{peak}/-(J_{O2peak}·Δ_{k}G_{O2}) = (P_{peak}/J_{O2peak}) / -Δ_{k}G_{O2} (1)
In OXPHOS analysis the output power is mitochondrial ATP production, J_{~P}=~P, times the Gibbs force of phosphorylation (Δ_{p}G~P=∂G/∂_{p}ξ_{~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/O_{2} ratio; ATP yield per oxygen consumed, Y_{~P/O2} = J_{~P}/J_{O2P} = ~P/P) and force ratio,
ε_{P} = (J_{~P}·Δ_{p}G_{~P})/-(J_{O2P}·Δ_{k}G_{O2}) = ~P/P ∙ Δ_{p}G_{~P}/-Δ_{k}G_{O2} = j_{≈P} ∙ f_{≈P} (2)
The upper limit of ~P/P is the mechanistic ~P:O_{2} 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} = ~P_{limit}/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) [5],
j_{≈P} = ≈P/P = (P-L)/P = 1-L/P = 1-RCR^{-1} (4)
OXPHOS coupling efficiency in Equation (4) is determined by respirometric OXPHOS analysis. At the limit of maximum j_{≈P}=1.0 the dissipative LEAK processes, L, are zero. Ergodynamic efficiency, ε, not only depends on mechanistic coupling but also on the force ratio or force efficiency. At ergodynamic equilibrium, ε=1.0, fluxes vanish to zero when j_{≈P} = f_{≈P} = 1 (Equation 2).
The OXPHOS state can be established experimentally in cells or tissues by selective permeabilization of plasma membranes, with ADP and P_{i} at kinetic saturation and fuel substrate combinations which reconstitute physiological TCA cycle function (Figure 1). The free OXPHOS capacity may be kinetically limited by the phosphorylation system to utilize Δp_{mt}. Then ET-capacity is in excess of OXPHOS capacity by the factor j_{ExP}=(E-P)/E. Such kinetic limitation diminishes the effective j_{≈P} independent of coupling control. Therefore, the ET-pathway coupling efficiency is defined as j_{≈E}=(E-L)/E (compare Eq. 4) and related to j_{≈P} by taking into account the apparent ET-pathway excess capacity (Figure 1),
j_{≈E} = j_{≈P}∙(1-j_{ExP}) + j_{ExP} (5)
Flux control factors and coupling efficiencies were derived from principles of thermodynamics rather than arbitrarily introduced as jargon of a specialized discipline.
Figures 1 and 2
Figure 1. Capacities for ET-pathway, OXPHOS and LEAK respiration (E, P, L). Free devided by total OXPHOS capacity, ≈P/P, is the OXPHOS coupling efficiency; free divided by total ET-capacity, ≈E/E, is the ET coupling efficiency [2]. The ET-pathway excess capacity, ExP, is available for coupled processes other than phosphorylation.
Figure 2. Respiratory acceptor control ratio as a function of OXPHOS coupling efficiency, j_{≈P}. RCR is the State 3/State 4 flux ratio [4], equal to P/L if State 3 is at saturating [ADP] and [Pi]. RCR from 1.0 to infinity is highly non-linear in the typical experimental range of RCR 3 to 10: when j_{≈P} increases from 0.8 to 0.9, RCR doubles from 5 to 10. RCR increases to infinity at the limit of j_{≈P}=1.0. Statistical analyses of RCR±SD require linearization by transformation to j_{≈P}.
References and acknowledgements
Supported by K-Regio project MitoCom Tyrol.
- Gnaiger E (1993) Efficiency and power strategies under hypoxia. Is low efficiency at high glycolytic ATP production a paradox? In: Surviving hypoxia: mechanisms of control and adaptation. Hochachka PW, Lutz PL, Sick T, Rosenthal M, Van den Thillart G (eds) CRC Press: 77-109. »Bioblast Access
- Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. Oroboros MiPNet Publications, Innsbruck. »Open Access
- Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128: 277-97. »Bioblast Access
- Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation: III. The steady state. J Biol Chem 217: 409-27. »Open Access
- Correction of printed version: In Eq.(4), L-P was written instead of the corrected P-L.
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