MitoPedia: Respirometry

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MitoPedia: Respirometry

MitoPedia - high-resolution terminology - matching measurements at high-resolution.
The MitoPedia terminology is developed continuously in the spirit of Gentle Science.

Additive effect of convergent electron flowAα&βAdditivity describes the princple of substrate control of mitochondrial respiration with convergent electron flow. The additive effect of convergent electron flow is a consequence of electron flow converging at the Q-junction from respiratory Complexes I and II (NS or CI&II e-input). Further additivity may be observed by convergent electron flow through glycerophosphate dehydrogenase and electron-transferring flavoprotein complex. Convergent electron flow corresponds to the operation of the TCA cycle and mitochondrial substrate supply in vivo. Physiological substrate combinations supporting convergent NS e-input are required for reconstitution of intracellular TCA cycle function. Convergent electron flow simultaneously through Complexes I and II into the Q-junction supports higher OXPHOS-capacity and ET-capacity than separate electron flow through either CI or CII. The convergent NS effect may be completely or partially additive, suggesting that conventional bioenergetic protocols with mt-preparations have underestimated cellular OXPHOS-capacities, due to the gating effect through a single branch. Complete additivity is defined as the condition when the sum of separatly measured respiratory capacities, N + S, is identical to the capacity measured in the state with combined substrates, NS (CI&II). This condition of complete additivity, NS=N+S, would be obtained if electron channeling through supercomplex CI, CIII and CIV does not interact with the pool of redox intermediates in the pathway from CII to CIII and CIV, and if the capacity of the phosphorylation system (≈P) does not limit OXPHOS-capacity (excess E-P capacity factor is zero). In most cases, however, additivity is incomplete, NS < N+S.
Advancement per volumedtrY [MU∙L-1]Advancement per volume or volume-specific advancement, dtrY, is related to advancement of a transformation, dtrY = dtrξV-1 [MU∙L-1]. Compare dtrY with the amount of substance j per volume, cj (concentration), related to amount, cj = njV-1 [mol∙V-1]. Advancement per volume is particularly introduced for chemical reactions, drY, and has the dimension of concentration (amount per volume [mol∙L-1]). In an open system at steady-state, however, the concentration does not change as the reaction advances. Only in closed systems and isolated systems, specific advancement equals the change in concentration divided by the stoichiometric number,
drY = dcj/νj (closed system) 
drY = drcj/νj (general) 

With a focus on internal transformations (i; specifically: chemical reactions, r), dcj is replaced by the partial change of concentration, drcj (a transformation variable or process variable). drcj contributes to the total change of concentration, dcj (a system variable or variable of state). In open systems at steady-state, drcj is compensated by external processes, decj = -drcj, exerting an effect on the total concentration change of substance j,

dcj = drcj + decj = 0 (steady state)
dcj = drcj + decj (general)
Air calibrationR1Air calibration of an oxygen sensor (polarographic oxygen sensor) is performed routinely on any day before starting a respirometric experiment. The volume fraction of oxygen in dry air is constant. An aqueous solution in equilibrium with air has the same partial pressure as that in water vapour saturated air. The water vapour is a function of temperature only. The partial oxygen pressure in aqueous solution in equilibrium with air is, therefore, a function of total barometric pressure and temperature. Bubbling an aqueous solution with air generates deviations from barometric pressure within small gas bubbles and is, therefore, not recommended. To equilibrate an aqueous solution ata known partial pressure of oxygen [kPa], the aqueous solution is stirred rigorously in a chamber enclosing air at constant temperature. The concentration of oxygen, cO2 [µM], is obtained at any partial pressure by multiplying the partial pressure by the oxygen solubility, SO2 [µM/kPa]. SO2 is a function of temperature and composition of the salt solution, and is thus a function of the experimental medium. The solubility factor of the medium, FM, expresses the oxygen solubility relative to pure water at any experimental temperature. FM is 0.89 in serum (37 °C) and 0.92 in MiR06 or MiR05 (30 °C and 37 °C).
Barometric pressurepb [Pa]Barometric pressure, pb, is an important variable measured for calibration of oxygen sensors in solutions equilibrated with air. The atm-standard pressure (1 atm = 760 mmHg = 101.325 kPa) has been replaced by the SI standard pressure of 100 kPa. The partial pressure of oxygen, pO2, in air is a function of barometric pressure, which changes with altitude and locally with weather conditions. The partial oxygen pressure declines by 12% to 14% per 1,000 m up to 6,000 m altitude, and by 15% to 17% per 1,000 m between 6,000 and 9,000 m altitude. The O2k-Barometric Pressure Transducer is built into the Oroboros O2k as a basis for accurate air calibrations in high-resolution respirometry. For highest-level accuracy of calculation of oxygen pressure, it is recommended to compare at regular intervals the barometric pressure recording provided by the O2k with a calibrated barometric pressure recording at an identical time point and identical altitude. The concept of gas pressure or barometric pressure can be related to the generalized concept of isomorphic pressure.
Basal respirationBMRBasal respiration or basal metabolic rate (BMR) is the minimal rate of metabolism required to support basic body functions, essential for maintenance only. BMR (in humans) is measured at rest 12 to 14 hours after eating in a physically and mentally relaxed state at thermally neutral room temperature. Maintenance energy requirements include mainly the metabolic costs of protein turnover and ion homeostasis. In many aerobic organisms, and particularly well studied in mammals, BMR is fully aerobic, i.e. direct calorimetry (measurement of heat dissipation) and indirect calorimetry (measurement of oxygen consumption multiplied by the oxycaloric equivalent) agree within errors of measurement (Blaxter KL 1962. The energy metabolism of ruminants. Hutchinson, London: 332 pp [1]). In many cultured mammalian cells, aerobic glycolysis contributes to total ATP turnover (Gnaiger and Kemp 1990 [2]), and under these conditions, 'respiration' is not equivalent to 'metabolic rate'. Basal respiration in humans and skeletal muscle mitochondrial function (oxygen kinetics) are correlated (Larsen et al 2011 [3]). » MiPNet article
Biochemical threshold effectDue to threshold effects, even a large defect diminishing the velocity of an individual enzyme results in only minor changes of pathway flux.
Calorespirometric ratioCR ratio [kJ/mol]The calorimetric/respirometric or calorespirometric ratio (CR ratio) is the ratio of calorimetrically and respirometrically measured heat and oxygen flux, determinded by calorespirometry. The experimental CR ratio is compared with the theoretically derived oxycaloric equivalent, and agreement in the range of -450 to -480 kJ/mol O2 indicates a balanced aerobic energy budget (Gnaiger and Staudigl 1987). In the transition from aerobic to anaerobic metabolism, there is a limiting pO2, plim, below which CR ratios become more exothermic since anaerobic energy flux is switched on.
CalorespirometryCRCalorespirometry is the method of measuring simultaneously metabolic heat flux (calorimetry) and oxygen flux (respirometry). The calorespirometric ratio (CR ratio; heat/oxygen flux ratio) is thus experimentally determined and can be compared with the theoretical oxycaloric equivalent, as a test of the aerobic energy balance.
Cell ergometryBiochemical cell ergometry aims at measurement of JO2max (compare VO2max or VO2peak 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 ET-capacity. The OXPHOS state can be established experimentally by selective permeabilization of cell membranes with maintenance of intact mitochondria, titrations of ADP and Pi to evaluate kinetically saturating conditions, and establishing fuel substrate combinations which reconstitute physiological TCA cycle function. Uncoupler titrations are applied to determine the apparent ET-pathway excess over OXPHOS capacity and to calculate OXPHOS- and ET-coupling efficiency , j≈P and j≈E. These normalized flux ratios are the basis to calculate the ergometric or ergodynamic efficiency, ε = j · f, where f is the normalized force ratio. » MiPNet article
Cell respirationCell respiration channels metabolic fuels into the chemiosmotic coupling (bioenergetic) machinery of oxidative phosphorylation, being regulated by and regulating oxygen consumption (or consumption of an alternative final electron acceptor) and molecular redox states, ion gradients, mitochondrial (or microbial) membrane potential, the phosphorylation state of the ATP system, and heat dissipation in response to intrinsic and extrinsic energy demands. See also respirometry. In internal or cell respiration in contrast to fermentation, redox balance is maintained by the use of external electron acceptors, transported into the cell from the environment. The chemical potential from electron donors to electron acceptors is converted in the Electron transfer-pathway to generate a chemiosmotic potential that in turn drives ATP synthesis.
Chemical background correction of oxygen fluxChemical background correction of oxygen flux is the correction of oxygen flux for the side reaction of autooxidation, as a function of oxygen concentration.
Closed systemA closed system is a system with boundaries that allow external exchange of energy (heat and work), but do not allow exchange of matter. A limiting case is light and electrons which cross the system boundary when work is exchanged in the form of light or electric energy. If the surroundings are maintained at constant temperature, and heat exchange is rapid to prevent the generation of thermal gradients, then the closed system is isothermal. A frequently considered case are closed isothermal systems at constant pressure (and constant volume with aqueous solutions). Changes of closed systems can be partitioned according to internal and external sources. Closed systems may be homogenous (well mixed and isothermal), continuous with gradients, or discontinuous with compartments (heterogenous).
Convergent electron flown.a.
Convergent electron flow
Convergent electron flow is built into the metabolic design of the Electron transfer-pathway. The glycolytic pathways are characterized by important divergent branchpoints: phosphoenolpyruvate (PEPCK) branchpoint to pyruvate or oxaloactetate; pyruvate branchpoint to (aerobic) acetyl-CoA or (anaerobic) lactate or alanine. The mitochondrial Electron transfer-pathway, in contrast, is characterized by convergent junctions: (1) the N-junction and F-junction in the mitochondrial matrix at ET-pathway level 4, with dehydrogenases (including the TCA cycle) and ß-oxidation generating NADH and FADH2 as substrates for Complex I and electron-transferring flavoprotein complex, respectively, and (2) the Q-junction with inner mt-membrane respiratory complexes at ET-pathway level 3, reducing the oxidized ubiquinone and partially reduced semiquinone to the fully reduced ubiquinol, feeding electrons into Complex III.
Coupled respirationCoupled respiration drives oxidative phosphorylation of the diphosphate ADP to the triphosphate ATP, mediated by proton pumps across the inner mitochondrial membrane. Intrinsically uncoupled respiration, in contrast, does not lead to phosphorylation of ADP, despite of protons being pumped across the inner mt-membrane. Coupled respiration, therefore, is the coupled part of respiratory oxygen flux that pumps the fraction of protons across the inner mt-membrane which is utilized by the phosphorylation system to produce ATP from ADP and Pi. In the OXPHOS state, mitochondria are in a partially coupled state, and the corresponding coupled respiration is the free OXPHOS capacity. In the state of ROUTINE respiration, coupled respiration is the free ROUTINE activity.
Coupling control factorCCFCoupling control factors, CCF, are flux control factors, FCF, at a constant ET-pathway competent state.
Coupling control protocolCCPA coupling control protocol, CCP, induces different coupling control states at a constant electron transfer-pathway state. Residual oxygen consumption (Rox) is finally evaluated for Rox correction of flux. The CCP may be extended, when further respiratory states (e.g. cell viability test; CIV assay) are added to the coupling control module consisting of three coupling control states. The term phosphorylation control protocol, PCP, has been introduced synonymous for CCP. » MiPNet article
Coupling control ratioCCRCoupling control ratios, CCR, are flux control ratios, FCR, at a constant mitochondrial pathway control state. In mitochondrial preparations, there are three well-defined coupling states of respiration, L, P, E (LEAK, OXPHOS, ET-pathway). In intact cells, state P cannot be induced, but a ROUTINE state of respiration, R, can be measured. The reference state, Jref, is defined by taking Jref as the maximum flux, i.e. flux in the ET state, E, such that the lower and upper limits of the CCR are defined as 0.0 and 1.0. Then there are two mitochondrial CCR, L/E and P/E, and two CCR for intact cells, L/E and R/E.
Coupling control stateCCSCoupling control states are defined in mitochondrial preparations (isolated mitochondria, permeabilized cells, permeabilized tissues, homogenates) as LEAK, OXPHOS, and ET-pathway states, with corresponding respiration rates (L, P, E) in any electron transfer-pathway state which is competent for electron transfer. These coupling states are induced by application of specific inhibitors of the phosphorylation system, titration of ADP and uncouplers. In living cells, the coupling control states are LEAK, ROUTINE, and ET-pathway states of respiration (L, R, E), using membrane-permeable inhibitors of the phosphorylation system (e.g. oligomycin) and uncouplers (e.g. CCCP). Coupling control protocols induce these coupling control states sequentially at a constant electron transfer-pathway state.
Crabtree effectThe Crabtree effect describes the observation that respiration is frequently inhibited when high concentrations of glucose or fructose are added to the culture medium - a phenomenon observed in numerous cell types, particularly in proliferating cells, not only in tumor cells, in bacteria, and yeast. The Pasteur effect (suppression of glycolysis by oxygen) is the converse of the Crabtree effect (aerobic glycolysis to lactate or ethanol).
Cytochrome c control factorFCFcThe cytochrome c control factor expresses the control of respiration by externally added cytochrome c, c, as a fractional change of flux from substrate state CHO to CHOc. In this flux control factor (FCFc), CHOc is the reference state with stimulated flux; CHO is the background state with CHO substrates, upon which c is added,
» MiPNet article
Dilution effectDilution of the concentration of a compound or sample in the experimental chamber by a titration of another solution into the chamber.
DithioniteDitZero oxygen solution powder, Na2S2O4, used for calibration of oxygen sensors at zero oxygen, or for stepwise reduction of oxygen concentration in instrumental O2 background tests.
Dyscoupled respirationDyscoupled respiration is LEAK respiration distinguished from intrinsically (physiologically) uncoupled and from extrinsic experimentally uncoupled respiration as an indication of extrinsic uncoupling (pathological, toxicological, pharmacological by agents that are not specifically applied to induce uncoupling, but are tested for their potential dyscoupling effect). Dyscoupling indicates a mitochondrial dysfunction. In addition to intrinsic uncoupling, dyscoupling occurs under pathological and toxicological conditions. Thus a distinction is made between physiological uncoupling and pathologically defective dyscoupling in mitochondrial respiration.
ET-capacityEE.jpg ET-capacity is the respiratory electron transfer-pathway capacity, E, of mitochondria measured as oxygen consumption in the noncoupled state at optimum uncoupler concentration. This optimum concentration is obtained by stepwise titration of an established protonophore to induce maximum oxygen flux as the determinant of ET-capacity. The experimentally induced noncoupled state at optimum uncoupler concentration is thus distinguished from (i) a wide range of uncoupled states at any experimental uncoupler concentration, (ii) physiological uncoupled states controlled by intrinsic uncoupling (e.g. UCP1 in brown fat), and (iii) pathological dyscoupled states indicative of mitochondrial injuries or toxic effects of pharmacological or environmental substances. ET-capacity in mitochondrial preparations requires the addition of defined fuel substrates to establish an ET-pathway competent state. » MiPNet article
ET-coupling efficiencyj≈EET-coupling efficiency The ET-coupling efficiency (E-L coupling control factor) is a normalized flux ratio, j≈E = ≈E/E = (E-L)/E = 1-L/E. j≈E is 0.0 at zero coupling (L=E) and 1.0 at the limit of a fully coupled system (L=0). The background state is the LEAK state which is stimulated to ET-pathway reference state by uncoupler titration. LEAK states LN or LT may be stimulated first by saturating ADP (State P) with subsequent uncoupler titration to State E. The ET-coupling efficiency is based on measurement of a coupling control ratio (LEAK control ratio, L/E), whereas the thermodynamic or ergodynamic efficiency of coupling between ATP production (DT phosphorylation) and oxygen consumption is based on measurement of the output/input flux ratio (~P/O2 ratio) and output/input force ratio (Gibbs force of phosphorylation/Gibbs force of oxidation). Biochemical coupling efficiency is either expressed as the ET-coupling efficiency, j≈E, or OXPHOS coupling efficiency, j≈P, obtained in a coupling control protocol (phosphorylation control protocol). » MiPNet article
Electron flowIeElectron flow through the mitochondrial Electron transfer-pathway (ET-pahway) is the scalar component of chemical reactions in oxidative phosphorylation (OXPHOS). Electron flow is most conveniently measured as oxygen consumption (oxygraphic measurement of oxygen flow), with four electrons being taken up when oxygen(02) is reduced to water.
Electron leakElectrons that escape the electron transfer system without completing the reduction of oxygen to water at cytochrome c oxidase, causing the production of ROS. The rate of electron leak depends on the topology of the complex, the redox state of the moiety responsible of electron leakiness and usually on the protonmotive force (Δp). In some cases, the Δp dependance relies more on the ∆pH component than in the ∆Ψ.
Electron transfer-pathwayET-pathwayIn the mitochondrial electron transfer-pathway (ET-pathway) electrons are transferred from externally supplied reduced fuel substrates to oxygen. Based on this experimentally oriented definition (see ET-capacity), the ET-pathway consists of (1) the membrane-bound ET-pathway with respiratory complexes located in the inner mt-membrane, (2) TCA cycle and other mt-matrix dehydrogenases generating NADH and succinate, and (3) the carriers involved in metabolite transport across the mt-membranes. » MiPNet article
Electron transfer-pathway stateET-pathway state
SUIT-catg FNSGpCIV.jpg

Electron transfer-pathway states are obtained in mitochondrial preparations (isolated mitochondria, permeabilized cells, permeabilized tissues, tissue homogenate) by depletion of endogenous substrates and addition to the mitochondrial respiration medium of fuel substrates (CHNO) activating specific mitochondrial pathways, and possibly inhibitors of specific pathways. Mitochondrial electron transfer-pathway states have to be defined complementary to mitochondrial coupling control states. Coupling control states require ET-pathway competent states, including oxygen supply. Categories of SUIT protocols are defined according to mitochondrial ET-pathway states.

» MiPNet article
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Ergodynamic efficiencyεThe ergodynamic efficiency, ε (compare thermodynamic efficiency), is a power ratio between the output power and the (negative) input power of an energetically coupled process. Since power [W] is the product of a flow and the conjugated thermodynamic force, the ergodynamic efficiency is the product of an output/input flow ratio and the corresponding force ratio. The efficiency is 0.0 in a fully uncoupled system (zero output flow) or at level flow (zero output force). The maximum efficiency of 1.0 can be reached only in a fully (mechanistically) coupled system at the limit of zero flow at ergodynamic equilibrium. The ergodynamic efficiency of coupling between ATP production (DT phosphorylation) and oxygen consumption is the flux ratio of DT phosphorylation flux and oxygen flux (P»/O2 ratio) multiplied by the corresponding force ratio. Compare with the OXPHOS coupling efficiency.
External flowIe [MU·s-1]External flows across the system boundaries are formally reversible. Their irreversible facet is accounted for internally as transformations in a heterogenous system (internal flows, Ii).
The F-junction is a junction for convergent electron flow in the electron transfer-pathway (ET-pathway) from fatty acids through fatty acyl CoA dehydrogenase (reduced form FADH2) to electron transferring flavoprotein (CETF), and further transfer through the Q-junction to Complex III (CIII). The concept of the F-junction and N-junction provides a basis for defining categories of SUIT protocols. Fatty acid oxidation, in the F-pathway control state, not only depends on electron transfer through the F-junction (which is typically rate-limiting) but simultaneously generates NADH and thus depends on N-junction throughput. Hence FAO can be inhibited completely by inhibition of Complex I (CI). In addition and independent of this source of NADH, the N-junction substrate malate is required as a co-substrate for FAO in mt-preparations, since accumulation of AcetylCoA inhibits FAO in the absence of malate. Malate is oxidized in a reaction catalyzed by malate dehydrogenase to oxaloacetate (yielding NADH), which then stimulates the entry of AcetylCoA into the TCA cycle catalyzed by citrate synthase.
Fatty acid oxidationFAOFatty acid oxidation (β-oxidation) is a multi-step process by which fatty acids are broken down to generate acetyl-CoA, NADH and FADH2 for further energy production. Fatty acids (short chain with 4–8, medium-chain with 6–12, long chain with 14-22 carbon atoms) are activated by fatty acyl-CoA synthases (thiokinases) in the cytosol. The outer mt-membrane enzyme carnitine palmitoyltransferase I (CPT 1) generates an acyl-carnitine intermediate for transport into the mt-matrix. Octanoate, but not palmitate, (eight- and 16-carbon saturated fatty acids) may pass the mt-membranes, but both are frequently supplied to mt-preparations in the activated form of octanoylcarnitine or palmitoylcarnitine. Electron-transferring flavoprotein complex (CETF) is located on the matrix face of the inner mt-membrane, and supplies electrons from fatty acid β-oxidation (FAO) to CoQ. FAO cannot proceed without a substrate combination of fatty acids & malate, and inhibition of CI blocks FAO completely. Fatty acids are split stepwise into two carbon fragments forming acetyl-CoA, which enters the TCA cycle by condensation with oxaloacetate (CS reaction). Therefore, FAO implies simultaneous electron transfer into the Q-junction through CETF and CI.
FluxJFlux, J, is a specific quantity. Flux is flow, I [MU·s-1 per system] (an extensive quantity), divided by system size. Flux (e.g., Oxygen flux) may be volume-specific (flow per volume [MU·s-1·L-1]), mass-specific (flow per mass [MU·s-1·kg-1]), or marker-specific (e.g. flow per mtEU).
Flux / SlopeJFlux / Slope is the pull-down menu in DatLab for (1) normalization of flux (chamber volume-specific flux, sample-specific flux or flow, or flux control ratios), (2) flux baseline correction, (3) Instrumental background oxygen flux, and (4) flux smoothing, selection of the scaling factor, and stoichiometric normalization using a stoichiometric coefficient. A Savitzky-Golay smoothing filter is used in DatLab as a basis of calculating the time derivative (Flux / Slope) of the signal (oxygen, fluorescence, ..). For each signal channel, the signal for the measured substance X is typically calibrated as an amount of substance concentration, cX [µM = nmol/mL]. The signal of the potentiometric channel, however, is primarily expressed logarithmically as pX=-log(cX/c°) and then transformed to cX. The slope is calculated as the change of concentration over time, dcX/dt [nmol/(s · mL)]. In a chemical reaction, the change of substance X is stoichiometrically related to the changes of all other substrates and products involved in the reaction. If the stoichiometry of the reaction is normalized for substance X, then its stoichiometric coefficient is unity and νX equals 1 if the substance is a product formed in the reaction, but νX equals -1 if the substance is a substrate consumed in the reaction. Oxygen is formed in photosynthesis and νX=1 when expressing photosynthesis as oxygen flux. Oxygen is consumed in aerobic respiration and νX=-1 when expressing respiration as oxygen flux.
Flux baseline correctionbcFlux baseline correction provides the option to display the plot and all values of the flux (or flow, or flux control ratio) as the total flux, J, minus a baseline flux, J0.
JV(bc) = JV - JV0
JV = (dc/dt) · ν-1 · SF - V
For the oxygen channel, JV is O2 flux per volume [pmol/(s·ml)] (or volume-specific O2 flux), c is the oxygen conentration [nmol/ml = µmol/l = µM], dc/dt is the (positive) slope of oxygen concentration over time [nmol/(s · ml)], ν-1 = -1 is the stoichiometric coefficient for the reaction of oxygen consumption (oxygen is removed in the chemical reaction, thus the stoichiometric coefficient is negative, expressing oxygen flux as the negative slope), SF=1,000 is the scaling factor (converting units for the amount of oxygen from nmol to pmol), and V is the volume-specific background oxygen flux (Instrumental background oxygen flux). Further details: Flux / Slope.
Flux control factorFCFFlux control factors express the control of respiration by a metabolic control variable, X, as a fractional change of flux from YX to ZX, normalized for ZX. ZX is the reference state with high (stimulated or un-inhibited) flux; YX is the background state at low flux, upon which X acts.
ΔjX = (ZX-YX)/ZX = 1-YX/ZX

Complementary to the concept of flux control ratios and analogous to elasticities of metabolic control analysis, the flux control factor of X upon background YX is expressed as the change of flux from YX to ZX normalized for the reference state ZX.

» MiPNet article
Flux control ratioFCRFlux control ratios (FCR), are ratios of oxygen flux in different respiratory control states, normalized for maximum flux in a common reference state, to obtain theoretical lower and upper limits of 0.0 and 1.0 (0% and 100%). 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 in 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.). FCR obtained from a single respirometric incubation with sequential titrations (sequential protocol; SUIT protocol) provide an internal normalization, expressing respiratory control independent of mitochondrial content and thus independent of a marker for mitochondrial amount. FCR obtained from separate (parallel) protocols depend on equal distribution of subsamples obtained from a homogenous mt-preparation or determination of a common mitochondrial marker.
High-resolution respirometryHRR

High-resolution respirometry, HRR, is the state-of-the-art approach in mitochondria and cell research to measure respiration in various types of mitochondrial preparations and living cells combined with MultiSensor modules. Mitochondrial function and dysfunction have gained an increasing interest over the past years, reflecting growing awareness of the fact that mitochondria play a pivotal role in human health and disease. Combining instrumental accuracy and reliability with versatility of applicable protocols - allowing practically unlimited addition and combination of substrates, inhibitors and uncouplers - mitochondrial respiratory pathways may be analyzed in detail to evaluate even minor alterations in respiratory pathway control and/or capacity. The most advanced way to analyze mitochondrial function is by means of high-resolution respirometry with the Oroboros O2k. The O2k is a sole source apparatus, with no other available instrument meeting its specifications for high-resolution respirometry. Substrate-uncoupler-inhibitor titration (SUIT) protocols allow the diagnosis of numerous mitochondrial pathway and coupling defects in a single respirometric assay. Technologically, HRR is based on the Oroboros O2k, combining optimized chamber design, application of oxygen-tight materials, electrochemical sensors, Peltier-temperature control, and specially developed software features (DatLab) to obtain the unique sensitive and quantitative resolution of oxygen concentration and oxygen flux, with both, a closed-chamber or open-chamber mode of operation (TIP2k). Standardized calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the experimental basis for high accuracy of quantitative results and quality control in HRR.

HRR can be extended for MultiSensor analysis by using the O2k-FluoRespirometer. Smart Fluo-Sensors are integrated into the O2k to measure simultaneously fluorometric signals using specific fluorophores. Potentiometric modules are available with ion selective electrodes (pH, TPP+). The NextGen-O2k is the all-in-one device including the Q-redox sensor and a PhotoBiology (PB) module.
International oxygraph courseIOCInternational Oxygraph Course (IOC), see O2k-Workshops.
L/P coupling control ratioL/PL/P coupling control ratio The L/P coupling control ratio or LEAK/OXPHOS coupling control ratio combines the effects of coupling (L/E) and limitation by the phosphorylation system (P/E); L/P = (L/E) / (P/E) = 1/RCR.
LEAK control ratioL/ELEAK control ratio The LEAK control ratio, or L/E coupling control ratio [1,2], is the flux ratio of LEAK respiration over ET-capacity, as determined by measurement of oxygen consumption in sequentially induced states L and E of respiration. The ET-pathway control ratio is an index of uncoupling or dyscoupling at constant ET-capacity. L/E increases with uncoupling from a theoretical minimum of 0.0 for a fully coupled system, to 1.0 for a fully uncoupled system [3].
LEAK-respirationLL.jpg EAK-respiration or LEAK oxygen flux, L, compensating for proton leak, proton slip, cation cycling and electron leak, is a dissipative component of respiration which is not available for performing biochemical work and thus related to heat production. LEAK-respiration is measured in the LEAK state, in the presence of reducing substrate(s), but absence of ADP - abbreviated as L(n) (theoretically, absence of inorganic phosphate presents an alternative), or after enzymatic inhibition of the phosphorylation system, which can be reached with the use of oligomycin - abbreviated as L(Omy). The LEAK state is the non-phosphorylating resting state of intrinsic uncoupled or dyscoupled respiration when oxygen flux is maintained mainly to compensate for the proton leak at a high chemiosmotic potential, when ATP synthase is not active. In this non-phosphorylating resting state, the electrochemical proton gradient is increased to a maximum, exerting feedback control by depressing oxygen flux to a level determined mainly by the proton leak and the H+/O2 ratio. In this state of maximum protonmotive force, LEAK-respiration, L, is higher than the LEAK component of OXPHOS capacity, P. The conditions for measurement and expression of respiration vary (oxygen flux in the LEAK state, JO2L, or oxygen flow, IO2L). If these conditions are defined and remain consistent within a given context, then the simple symbol L for respiratory rate can be used as a substitute for the more explicit expression for respiratory activity. » MiPNet article
Limiting pO2plimIn the transition from aerobic to anaerobic metabolism, there is a limiting pO2, plim, below which anaerobic energy flux is switched on and CR ratios become more exothermic than the oxycaloric equivalent. plim may be significanlty below the critical pO2.
LinearityLinearity is the ability of the method to produce test results that are proportional, either directly or by a well-defined mathematical transformation, to the concentration of the analyte in samples within a given range. This property is inherent in the Beer-Lambert law for absorbance alone, but deviations occur in scattering media. It is also a property of fluorescence, but a fluorophore may not exhibit linearity, particularly over a large range of concentrations.
Living cellsvceCell viability in living cells should be >95% for various experimental investigations, including cell respirometry. Viable cells (vce) are characterized by an intact plasma membrane. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce). In contrast, the cell membrane of cells can be permeabilized selectively by mild detergents (digitonin), to obtain the mt-preparation of permeabilized cells used for cell ergometry. Living cells are frequently labelled as intact cells in the sense of the total cell count, but intact may suggest the alternative meaning of viable.
Malate-aspartate shuttleThe malate-aspartate shuttle involves the glutamate-aspartate carrier and the 2-oxoglutarate carrier exchanging malate2- for 2-oxoglutarate2-. Cytosolic and mitochondrial malate dehydrogenase and transaminase complete the shuttle for the transport of cytosolic NADH into the mitochondrial matrix. It is most important in heart, liver and kidney.
MicroplatesMicroplate readers allow large numbers of sample reactions to be assayed in well format microtitre plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well (8 by 12 matrix) with a typical reaction volume between 100 and 200 µL per well. a wide range of applications involve the use of fluorescence measurements , although they can also be used in conjunction with absorbance measurements.
Mitochondrial markermt-markerMitochondrial markers are structural or functional properties that are specific for mitochondria. A structural mt-marker is the area of the inner mt-membrane or mt-volume determined stereologically, which has its limitations due to different states of swelling. If mt-area is determined by electron microscopy, the statistical challenge has to be met to convert area into a volume. When fluorescent dyes are used as mt-marker, distinction is necessary between mt-membrane potential dependent and independent dyes. mtDNA or cardiolipin content may be considered as a mt-marker. Mitochondrial marker enzymes may be determined as molecular (amount of protein) or functional properties (enzyme activities). Respiratory capacity in a defined respiratory state of a mt-preparation can be considered as a functional mt-marker, in which case respiration in other respiratory states is expressed as flux control ratios. » MiPNet article
Mitochondrial membrane potentialmtMP, Δψ [V]The mitochondrial membrane potential, mtMP, is the electric part of the protonmotive force, ΔpH+.

Δψ = ΔpH+ - ΔµH+ / F

mtMP or Δψ is the potential difference across the inner mitochondrial (mt) membrane, expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ electron or per electron moved across the transmembrane potential difference, with the number of ‘motive’ electrons expressed in the unit coulomb [C].

The chemical part of the protonmotive force, µH+ / F stems from the difference of pH across the mt-membrane. It contains a factor that bridges the gap between the electric force [J/C] and the chemical force [J/mol]. This factor is the Faraday constant, F, for conversion between electric force expressed in joules per coulomb or Volt [V=J/C] and chemical force with the unit joules per mole or Jol [Jol=J/mol],

F = 96.4853 kJol/V = 96,485.3 C/mol
Mitochondrial respirationIntegrative measure of the dynamics of complex coupled metabolic pathways, including metabolite transport across the mt-membranes, TCA cycle function with electron transfer through dehydrogenases in the mt-matrix, membrane-bound electron transfer mET-pathway, the transmembrane proton circuit, and the phosphorylation system.
The N-junction is a junction for convergent electron flow in the electron transfer-pathway (ET-pathway) from type N substrates (further details »N-pathway control state) through the mt-NADH pool to Complex I (CI), and further transfer through the Q-junction to Complex III (CIII). Representative type N substrates are pyruvate (P), glutamate (G) and malate (M). The corresponding dehydrogenases (PDH, GDH, MDH) and some additional TCA cycle dehydrogenases (isocitrate dehydrogenase, oxoglutarate dehydrogenase generate NADH, the substrate of Complex I (CI). The concept of the N-junction and F-junction provides a basis for defining categories of SUIT protocols based on Electron transfer-pathway states.
NADH Electron transfer-pathway stateN
The NADH electron transfer-pathway state (N) is obtained by addition of NADH-linked substrates (CI-linked), feeding electrons into the N-junction catalyzed by various mt-dehydrogenases. N-supported flux is induced in mt-preparations by addition of NADH-generating substrate combinations of pyruvate (P), glutamate (G), malate (M), oxaloacetate (Oa), oxoglutarate (Og), citrate, hydroxybutyrate. These N-junction substrates are (indirectly) linked to Complex I by the corresponding dehydrogenase-catalyzed reactions reducing NAD+ to NADH+H+. The most commonly applied N-junction substrate combinations are: PM, GM, PGM. The malate anaplerotic pathway control state (M alone) is a special case related to malic enzyme (mtME). The glutamate anaplerotic pathway control state (G alone) supports respiration through glutamate dehydrogenase (mtGDH). Oxidation of tetrahydrofolate is a NAD(P)H linked pathwaynwith formation of formate. In mt-preparations, succinate dehydrogenase (SDH; CII) is largely substrate-limited in N-linked respiration, due to metabolite depletion into the incubation medium. The residual involvement of S-linked respiration in the N-pathway control state can be further suppressed by the CII-inhibitor malonic acid). In the N-pathway control state ET pathway level 4 is active.
NS-S control factorjNS-SThe NS-S control factor (CI&II-CII substrate control factor) expresses the relative stimulation of succinate supported respiration (S) by NADH-linked substrates (N), with the S-pathway control state as the background state and the NS-pathway control state as the reference state. In typical SUIT protocols with type N and S substrates, flux in the NS-pathway control state, NS, is inhibited by Rotenone to measure flux in the S-pathway control state, SRot or S. Then the NS-S control factor is
jNS-S = (NS-S)/NS
The NS-S control factor expresses the fractional change of flux in a defined coupling control state when inhibition by rotenone is removed from flux under S-pathway control in the presence of a type N substrate combination. Experimentally rotenone (Rot) is added to the NS-state. The reversed protocol, adding N-substrates to a S-pathway control background does not provide a valid estimation of S-respiration with succinate in the absence of Rot, since oxaloacetate accumulates as a potent inhibitor of succinate dehydrogenase (CII).
NigericinNigericin is a H+/K+ antiporter, which allows the electroneutral transport of these two ions in opposite directions across the mitochondrial inner membrane following the K+ concentration gradient. In the presence of K+, nigericin decreases pH in the mitchondrial matrix, thus, almost fully collapses the transmembrane pH gradient, which leads to the compensatory increase of mt-membrane potential. Therefore, it is ideal to use to dissect the two components of the protonmotive force, delta pH and mt-membrane potential. It is recommended to use the lowest possible concentration of nigericin, which creates a maximal mitochondrial hyperpolarization. In the study of Komlodi 2018 J Bioenerg Biomembr, 20 nM was applied on brain mitochondria isolated from guinea-pigs using 5 mM succinate in the LEAK state which caused maximum hyperpolarisation, but did not fully dissipate transmembrane pH gradient. Selivanov and his co-workers [1] and Lambert [2], however, used 100 nM nigericin, which in their hands fully collapsed transmembrane pH gradient using succinate as a respiratory substrate on isolated rat brain and skeletal muscle in the LEAK state.
NoiseIn fluorometry and spectrophotometry, noise can be attributed to the statistical nature of the photon emission from a light source and the inherent noise in the instrument’s electronics. The former causes problems in measurements involving samples of analytes with a low extinction coefficient and present only in low concentrations. The latter becomes problematic with high absorbance samples where the light intensity emerging from the sample is very small.
Noncoupled respirationEE.jpg Noncoupled respiration is distinguished from general (pharmacological or mechanical) uncoupled respiration, to give a label to an effort to reach the state of maximum uncoupler-activated respiration without inhibiting respiration. Noncoupled respiration, therefore, yields an estimate of ET-capacity. Experimentally uncoupled respiration may fail to yield an estimate of ET-capacity, due to inhibition of respiration above optimum uncoupler concentrations or insufficient stimulation by sub-optimal uncoupler concentrations. Optimum uncoupler concentrations for evaluation of (noncoupled) ET-capacity require inhibitor titrations (Steinlechner-Maran 1996 Am J Physiol Cell Physiol; Huetter 2004 Biochem J; Gnaiger 2008 POS). Noncoupled respiration is maximum electron flow in an open-transmembrane proton circuit mode of operation (see ET-capacity).
Nuclear respiratory factor 1NRF-1Nuclear respiratory factor 1 is a transcription factor downstream of PGC-1alpha involved in coordinated expression of nDNA and mtDNA.
O2kO2kO2k - Oroboros O2k: the modular system for high-resolution respirometry.
O2k-FluoRespirometerThe Oroboros O2k-FluoRespirometer (O2k-Series H) - the experimental system complete for high-resolution respirometry (HRR), including fluorometry, the TIP2k and the O2k-sV-Module allowing simultaneous monitoring of oxygen consumption together with either ROS production (AmR), mt-membrane potential (TMRM, Safranin and Rhodamine 123), Ca2+ (CaG) or ATP production (MgG).

The O2k-FluoRespirometer supports all add-on O2k-Modules: O2k-TPP+ ISE-Module, O2k-pH ISE-Module, O2k-NO Amp-Module, enabling measurement of mt-membrane potential with ion sensitive electrodes (ISE for TPP+ or TPMP+ ) or pH.


  • oxygen consumption
  • ROS production
  • ATP production
  • Ca2+
  • pH
  • mitochondrial membrane potential

Features of O2k-Series H:

  • Fluo-Control Unit integrated into O2k-Main Unit.
  • Smart Fluo-Sensors with pre-calibrated light intensities for direct input into DatLab 7
  • Added: spare OroboPOS
  • DL-Protocols as real-time guides for titrations in DatLab 7, context-sensitive help. Further details » MitoPedia: DatLab.
  • MiR05-Kit
  • MitoKit-CII

The O2k is a sole source apparatus with no other instruments meeting its specifications.
OXPHOS control ratioP/EOXPHOS control ratio The OXPHOS control ratio or P/E coupling control ratio (OXPHOS/ET-pathway; phosphorylation system control ratio) is an expression of the limitation of OXPHOS capacity by the phosphorylation system. The relative limitation of OXPHOS capacity by the capacity of the phosphorylation system is better expressed by the excess E-P capacity factor, jExP = 1-P/E. The P/E ratio increases with increasing capacity of the phosphorylation system up to a maximum of 1.0 when it matches or is in excess of ET-capacity. P/E also increases with uncoupling. P/E increases from the lower boundary set by L/E (zero capacity of the phosphorylation system), to the upper limit of 1.0, when there is no limitation of P by the phosphorylation system or the proton backpressure (capacity of the phosphorylation system fully matches the ET-capacity; or if the system is fully uncoupled). It is important to separate the kinetic effect of ADP limitation from limitation by enzymatic capacity at saturating ADP concentration. » MiPNet article
OXPHOS coupling efficiencyj≈POXPHOS coupling efficiency The OXPHOS coupling efficiency (P-L or ≈P control factor), j≈P = ≈P/P = (P-L)/P = 1-L/P. OXPHOS capacity corrected for LEAK respiration is the free OXPHOS capacity, ≈P = P-L. The OXPHOS coupling efficiency is the ratio of free to total OXPHOS capacity. j≈P = 1.0 for a fully coupled system (when RCR approaches infinity); j≈P = 0.0 (RCR=1) for a system with zero respiratory phosphorylation capacity (≈P=0) or zero ET-coupling efficiency (E-L=0 when L=P=E). If State 3 is measured at saturating ADP and Pi concentrations (State 3 = P), then the respiratory acceptor control ratio, RCR, is P/L. Under these conditions, the RCR and OXPHOS coupling efficiency are related by a hyperbolic function, j≈P = 1-RCR-1. » MiPNet article
OXPHOS-capacityPP.jpg OXPHOS-capacity (P) is the respiratory capacity of mitochondria in the ADP-activated state of oxidative phosphorylation, at saturating concentrations of ADP (possibly in contrast to State 3), inorganic phosphate, oxygen, and defined reduced substrates. » MiPNet article
OctanoateOcaOctanoate (octanoic acid). C8H16O2 Common name: Caprylic acid.
Open systemAn open system is a system with boundaries that allow external exchange of energy and matter; the surroundings are merely considered as a source or sink for quantities transferred across the system boundaries (external flows, Iext).
Oroboros Instruments Corp
Oroboros Instruments distributes the gold standard O2k-technology for high-resolution respirometry - HRR - world-wide. The Oroboros Company is a scientifically oriented organization, with emphasis on continuous innovation. The extension of the Oroboros O2k to the O2k-FluoRespirometer sets a new standard. Its modular design provides the flexibility for add-on O2k-Modules (see Oroboros O2k-Catalogue). The O2k is established internationally, with »3360 O2k-Publications in the scientific literature covering areas ranging from fundamental bioenergetics to the analysis of mitochondrial and metabolic diseases, advancing the rapidly growing field of preventive mitochondrial medicine. The Oroboros science team actively participates in science and research (see: publications). Moreover, the Oroboros MitoFit Laboratory and the MitoFit laboratory at the Medical University of Innsbruck (two labs - one team) frequently host international researchers (visiting scientists). Oroboros Instruments organizes international O2k-Workshops on a regular basis. The O2k-Network includes and connects 626 reference laboratories worldwide. The NextGen-O2k will extend HRR to include a Q-redox sensor and PhotoBiology module.
Oxidative phosphorylationOXPHOSP.jpg Oxidative phosphorylation (OXPHOS) is the oxidation of reduced fuel substrates by electron transfer to oxygen, chemiosmotically coupled to the phosphorylation of ADP to ATP (P») and accompanied by an intrinsically uncoupled component of respiration. The OXPHOS state of respiration provides a measure of OXPHOS-capacity (P), which is frequently corrected for residual oxygen consumption (ROX).
Oxycaloric equivalentDeltakHO2The oxycaloric equivalent is the theoretically derived enthalpy change of the oxidative catabolic reactions per amount of oxygen respired, DeltakHO2, ranging from -430 to -480 kJ/mol O2. The oxycaloric equivalent is used in indirect calorimetry to calculate the theoretically expected metabolic heat flux from the respirometrically measured metabolic oxygen flux. Calorimetric/respirometric ratios (CR ratios; heat/oxygen flux ratios) are experimentally determined by calorespirometry. A CR ratio more exothermic than the oxycaloric equivalent of -480 kJ/mol indicates the simultaneous involvement of aerobic and anaerobic mechanisms of energy metabolism.
Oxygen calibration - DatLabO2 calibration is the calibration in DatLab of the oxygen sensor. It is a prerequisite for obtaining accurate measurements of respiration. Accurate calibration of the oxygen sensor depends on (1) equilibration of the incubation medium with air oxygen partial pressure at the temperature defined by the experimenter; (2) zero oxygen calibration; (3) high stability of the POS signal tested for sufficiently long periods of time; (4) linearity of signal output with oxygen pressure in the range between oxygen saturation and zero oxygen pressure; and (5) accurate oxygen solubility for aqueous solutions for the conversion of partial oxygen pressure into oxygen concentration. The standard oxygen calibration procedure is described below for high-resolution respirometry with the automatic calibration routine by DatLab.^
Oxygen flowIO2 [mol·s-1]Respiratory oxygen flow is the oxygen consumption per total system, which is an extensive quantity. Flow is advancement of a transformation in a system per time. Oxygen flow or respiration of a cell is distinguished from oxygen flux (e.g. per mg protein or wet weight).
Oxygen fluxJO2Oxygen flux, JO2, is a specific quantity. Oxygen flux is oxygen flow, IO2 [mol·s-1 per system] (an extensive quantity), divided by system size. Flux may be volume-specific (flow per volume [pmol·s-1·mL-1]), mass-specific (flow per mass [pmol·s-1·mg-1]), or marker-specific (flow per mtEU). Oxygen flux (e.g. per body mass, or per cell mass) is distinguished from oxygen flow (per subject, or per cell).
Oxygen flux - instrumental backgroundJ°O2Instrumental background oxygen flux, J°O2, in a respirometer is due to oxygen consumption by the POS, and oxygen diffusion into or out of the aqueous medium in the O2k-Chamber. It is a property of the instrumental system, measured in the range of experimental oxygen levels by a standardized instrumental background test. The oxygen regime from air saturation towards zero oxygen is applied generally in experiments with isolated mitochondria and living or permeabilized cells. To overcome oxygen diffusion limitation in permeabilized fibres and homogenates, an elevated oxygen regime is applied, requiring instrumental background test in the same range of elevated oxygen.

Instrumental background correction eliminates errors by systemic flux compensation, automatically performed by DatLab. If no experimental background test has been performed, the system default values are used, which are a°=-2.0 pmol/(s·ml) for the intercept at zero oxygen concentration, and b°=0.025 for the slope of background flux as a function of oxygen concentration.

Automatic correction for the instrumental background oxygen flux is an essential standard in high resolution respirometry. At the same time an instrumental background experiment is the ultimate test for instrumental performance, evaluating chamber performance after completion of all elements of the Oxygen sensor test. The instrumental background oxygen flux measured at air saturation should reflect the theoretically predicted volume-specific oxygen consumption by the oxygen sensor. The actual agreement using experimental respiration medium provides at the same time a test that excludes microbial contamination of the medium or serves to evaluate any autoxidation processes in newly tested experimental media.
Oxygen kineticsOxygen kinetics describes the dependence of respiration of isolated mitochondria or cells on oxygen partial pressure. Frequently, a strictly hyperbolic kinetics is observed, with two parameters, the oxygen pressure at half-maximum flux, p50, and maximum flux, Jmax. The p50 is in the range of 0.2 to 0.8 kPa for cytochrome c oxidase, isolated mitochondria and small cells, strongly dependent on Jmax and coupling state.

Oxygen dependence of respiration

In isolated mitochondria and many types of small cells (such as endothelial cells, fibroblasts etc), zero-order kinetics with respect to oxygen pressure (i.e. independence of flux with declining oxygen concentration) applies over a wide range down to very low oxygen levels, where then a hyperbolic oxygen dependence is observed. With a p50 (c50, apparent Km) in the order of <1 µM and a hyperbolic oxygen dependence of flux, 99% saturation of flux and hence zero-order kinetics is observed at an oxygen concentration >19 µM (i.e. >2 kPa, >2% oxygen saturation or >10% air saturation). For reviews see Gnaiger et al 1995, Scandurra and Gnaiger 2010.

Compared to the difficulties with classical chart recorder tracings, our 'modern' approaches over the past 20 years allowed us to drop the linearity assumption and statistically test for it, frequently rejecting this assumption to describe a non-linear oxygen dependence beyond the low-oxygen range governed by cytochrome c oxidase. For review see Gnaiger 2003.

In permeabilized muscle fibers (30 to 37 °C), oxygen kinetics is shifted 100-fold due to artifially high oxygen gradients, forcing us to apply elevated oxygen levels for obtaining near-zero-order kinetics.

For details, see Pesta and Gnaiger 2012, Gnaiger 2003, Bezuidenhout 2016a and Oxygen kinetics of permeabilized fibers.
Oxygen pressurepO2 [kPa]Oxygen pressure or partial pressure of oxygen [kPa], related to oxygen concentration in solution by the oxygen solubility, SO2 [µM/kPa].
Oxygen pressure, intracellularpO2,iPhysiological, intracellular oxygen pressure is significantly lower than air saturation under normoxia, hence respiratory measurements carried out at air saturation are effectively hyperoxic for cultured cells and isolated mitochondria.
Oxygen signalThe oxygen signal of the Oroboros O2k is transmitted from the electrochemical polarographic oxygen sensor (OroboPOS) for each of the two O2k chambers to DatLab. The primary signal is a current [mAmp] which is converted into a voltage [V] (raw signal), and calibrated in SI units for amount of substrance concentration [µmol·L-1 or µM].
Oxygen solubilitySO2 [µM/kPa]The oxygen solubility, SO2 [µM/kPa], expresses the oxygen concentration in solution in equilibrium with the oxygen pressure in a gas phase, as a function of temperature and composition of the solution. SO2 is 10.56 µM/kPa in pure water at 37 °C. At standard barometric pressure (100 kPa), the oxygen concentration at air saturation is 207.3 µM at 37 °C (19.6 kPa partial oxygen pressure). In MiR06 and serum, the corresponding saturation concentrations are 191 and 184 µM. The oxygen solubility depends on temperatue and the concentrations of solutes in solution. See also: Oxygen solubility factor
Oxygen solubility factorFMThe oxygen solubility factor of the incubation medium, FM, expresses the effect of the salt concentration on oxygen solubility relative to pure water. In mitochondrial respiration medium MiR06, FM is 0.92 determined at 30 and 37 °C, and FM is 0.89 in serum at 37 °C. FM for other media may be estimated using Table 4 in MiPNet06.03. For this purpose KCl based media can be described as "seawater" of varying salinity.
P50p50p50 is the oxygen partial pressure at which (a) respiratory flux is 50% of maximum oxygen flux, Jmax, at saturating oxygen levels. The oxygen affinity is indirectly proportional to the p50. The p50 depends on metabolic state and rate. (b) p50 is the oxygen partial pressure at which oxygen binding (on myoglobin, haemoglobin) is 50%, or desaturation is 50%.
PH calibration bufferspH calibration buffers are prepared to obtain two or more defined pH values for calibration of pH electrodes and pH indicator dyes.
POS calibration - dynamicCalibration of the sensor response time. See also POS calibration - static.
POS calibration - staticF5Two-point calibration of the polarographic oxygen sensor, comprising Air calibration and Zero calibration. See also POS calibration - dynamic.
Pathway control ratioSCRSubstrate control ratios, SCR, are flux control ratios, FCR, at a constant mitochondrial coupling control state. Whereas there are only three well-defined coupling control states of mitochondrial respiration, L, P, E (LEAK, OXPHOS, ET-pathway), numerous Electron transfer-pathway states are possible. Careful selection of the reference state, Jref, is required, for which some guidelines may be provided without the possibility to formulate general rules. FCR are best defined by taking Jref as the maximum flux (e.g. NSE), such that flux in various other respiratory states, Ji, is smaller or equal to Jref. However, this is not generally possible with SCR. For instance, the N/S substrate control ratio (at constant coupling control state) may be larger or smaller than 1.0, depending on the mitochondrial source and various mitochondrial injuries. The S-pathway control state may be selected preferentially as Jref, if mitochondria with variable N-linked injuries are studied. In contrast, the reference state, Z, is strictly defined for flux control factors.
Physiological pathway control stateSee Electron transfer-pathway state.
Polyether ether ketonePEEKPolyether ether ketone (PEEK) is a semicrystalline organic polymer thermoplastic, which is chemically very resistant, with excellent mechanical properties. PEEK is compatible with ultra-high vacuum applications, and its resistance against oxygen diffusion make it an ideal material for high-resolution respirometry (POS insulation; coating of stirrer bars; stoppers for closing the O2k-Chamber).
Polyvinylidene fluoridePVDFPolyvinylidene fluoride (PVDF) is a pure thermoplastic fluoropolymer, which is chemically very resistant, with excellent mechanical properties. It is used generally in applications requiring the highest purity, strength, and resistance to solvents, acids, bases and heat (Wikipedia). PVDF is resistant against oxygen diffusion which makes it an ideal material for high-resolution respirometry (coating of stirrer bars; stoppers for closing the O2k-Chamber).
Power O2k-FluoRespirometerPower O2k-FluoRespirometer - optional configuration as additional system for increasing output combined with the O2k-FluoRespirometer (O2k-Series H). The Power O2k-FluoRespirometer includes the TIP2k and the O2k-sV-Module, and supports all add-on O2k-Modules of the Oroboros O2k. It can be added to an existing Oroboros O2k of any O2k-Series. This application does not require an additional ISS-Integrated Suction System and O2k-Titration Set. Furthermore, the OroboPOS-Mounting Tool of the OroboPOS Service Tools can be used from the available O2k and is not included.
Proton fluxJH+Volume-specific proton flux is measured in a closed system as the time derivative of proton concentration, expressed in units [pmol·s-1·mL-1]. Proton flux can be measured in an open system at steady state, when any acidification of the medium is compensated by external supply of an equivalent amount of base. The extracellular acidification rate (ECAR) is the change of pH in the incubation medium over time, which is zero at steady state. Volume-specific proton flux is comparable to volume-specific oxygen flux [pmol·s-1·mL-1], which is the (negative) time derivative of oxygen concentration measured in a closed system, corrected for instrumental and chemical background. pH is the negative logarithm of proton activity. Therefore, ECAR is of interest in relation to acidification issues in the incubation buffer or culture medium. The physiologically relevant metabolic proton flux, however, must not be confused with ECAR.
Proton leakFlux of protons driven by the protonmotive force across the inner mt-membrane, bypassing the ATP synthase and thus contributing to LEAK respiration. Proton leak-flux depends non-linearly (non-ohmic) on the protonmotive force. Compare: Proton slip.
Proton pumpMitochondrial proton pumps are large enzyme complexes (CI, CII, CIV, CV) spanning the inner mt-membrane, partially encoded by mtDNA. CI, CII and CIV are proton pumps that drive protons against the electrochemical protonmotive force, driven by electron transfer from reduced substrates to oxygen. In contrast, CV is a proton pump that utilizes the energy of proton flow along the protonmotive force to drive phosphorylation of ADP to ATP.
Proton slipProton slip is a property of the proton pumps (Complexes CI, CIII, and CIV) when the proton slips back to the matrix side within the proton pumping process. Slip is different from the proton leak, which depends on Δp and is a property of the inner mt-membrane (including the boundaries between membrane-spanning proteins and the lipid phase). Slip is an uncoupling process that depends mainly on flux and contributes to a reduction in the biochemical coupling efficiency of ATP production and oxygen consumption. Together with proton leak and cation cycling, proton slip is compensated for by LEAK respiration or LEAK oxygen flux, L. Compare: Proton leak.
The Q-junction is a junction for convergent electron flow in the Electron transfer-pathway (ET-pathway) from type N substrates and mt-matix dehydrogenases through Complex I (CI), from type F substrates and FA oxidation through electron-transferring flavoprotein complex (CETF), from succinate (S) through Complex II (CII), from glycerophosphate (Gp) through glycerophosphate dehydrogenase complex (CGpDH), from choline through choline dehydrogenase, from dihydro-orotate through dihydro-orotate dehydrogenase, and other enzyme complexes into the Q-cycle (ubiquinol/ubiquinone), and further downstream to Complex III (CIII) and Complex IV (CIV). The concept of the Q-junction, with the N-junction and F-junction upstream, provides the rationale for defining Electron transfer-pathway states and categories of SUIT protocols.
ROUTINE-respirationRR.jpg In the living cell, ROUTINE-respiration (R) or ROUTINE-activity in the physiological coupling state is controlled by cellular energy demand, energy turnover and the degree of coupling to phosphorylation (intrinsic uncoupling and pathological dyscoupling). The conditions for measurement and expression of respiration vary (oxygen flux in state R, JO2R or oxygen flow in state R, IO2R). If these conditions are defined and remain consistent within a given context, then the simple symbol R for respiratory state can be used to substitute the more explicit expression for respiratory activity. R and growth of cells is supported by exogenous substrates in culture media. In media without energy substrates, R depends on endogenous substrates. R cannot be measured in permeabilized cells or isolated mitochondria. R is corrected for residual oxygen consumption (ROX), whereas R´ is the uncorrected apparent ROUTINE-respiration or total cellular oxygen consumption of cells including ROX.
Respiratory acceptor control ratioRCRThe respiratory acceptor control ratio (RCR) is defined as State 3/State 4 [1]. If State 3 is measured at saturating [ADP], RCR is the inverse of the OXPHOS control ratio, L/P (when State 3 is equivalent to the OXPHOS state, P). RCR is directly but non-linearly related to the OXPHOS coupling efficiency, j≈P = 1-L/P. Whereas the normalized flux ratio j≈P has boundaries from 0.0 to 1.0, RCR ranges from 1.0 to infinity, which needs to be considered when performing statistical analyses. In intact cells, the term RCR has been used for the ratio State 3u/State 4o, i.e. for the inverse L/E ratio [2,3]. Then for conceptual and statistical reasons, RCR should be replaced by the ET-coupling efficiency, j≈E= 1-L/E [4].
Respiratory chainRCThe mitochondrial respiratory chain (RC) consists of enzyme complexes arranged to form a metabolic system of convergent pathways for oxidative phosphorylation. In a general sense, the RC includes (1) the electron transfer-pathway (ET-pathway), with transporters for the exchange of reduced substrates across the inner mitochondrial membrane, enzymes in the matrix space (particularly dehydrogenases of the tricarboxylic acid cycle), inner membrane-bound electron transfer complexes, and (2) the inner membrane-bound enzymes of the phosphorylation system.
Respiratory complexesCiRespiratory complexes are membrane-bound enzymes consisting of several subunits which are involved in energy transduction of the respiratory system. » MiPNet article
Respiratory stateRespiratory states of mitochondrial preparations and intact cells are defined in the current literature in many ways and with a diversity of terms. Mitochondrial respiratory states must be defined in terms of both, the coupling control state and the electron transfer-pathway state.
RespirometryRespirometry is the quantitative measurement of respiration. Respiration is therefore a combustion, a very slow one to be precise (Lavoisier and Laplace 1783). Thus the basic idea of using calorimetry to explore the sources and dynamics of heat changes was present in the origins of bioenergetics (Gnaiger 1983). Respirometry provides an indirect calorimetric approach to the measurement of metabolic heat changes, by measuring oxygen uptake (and carbon dioxide production and nitrogen excretion in the form of ammonia, urea or uric acid) and converting the oxygen consumed into an enthalpy change, using the oxycaloric equivalent. Liebig (1842) showed that the substrate of oxidative respiration was protein, carbohydrates, and fat. The sum of these chemical changes of materials under the influence of living cells is known as metabolism (Lusk 1928). The amount (volume STP) of carbon dioxide expired to the amount (volume STP) of oxygen inspired simultaneously is the respiratory quotient, which is 1.0 for the combustion of carbohydrate, but less for lipid and protein. Voit (1901) summarized early respirometric studies carried out by the Munich school on patients and healthy controls, concluding that the metabolism in the body was not proportional to the combustibility of the substances outside the body, but that protein, which burns with difficulty outside, metabolizes with the greatest ease, then carbohydrates, while fats, which readily burns outside, is the most difficultly combustible in the organism. Extending these conclusions on the sources of metabolic heat changes, the corresponding dynamics or respiratory control was summarized (Lusk 1928): The absorption of oxygen does not cause metabolism, but rather the amount of the metabolism determines the amount of oxygen to be absorbed. .. metabolism regulates the respiration.
SUITSUITSUIT is the abbreviation for Substrate-Uncoupler-Inhibitor Titration. SUIT protocols are used with mt-preparations to study respiratory control in a sequence of coupling and pathway control states induced by multiple titrations within a single experimental assay.
SUIT-001 O2 ce-pce D003RP1 ce-pceSUIT-RP1
SUIT-001 O2 ce-pce D004RP1 ce-pce bloodSUIT-RP1 for PBMCs and PLTs
SUIT-001 O2 mt D001RP1 mtSUIT-RP1
SUIT-001 O2 pfi D002RP1 pfiSUIT-RP1
SUIT-002 O2 ce-pce D007RP2 ce-pceSUIT-RP2
SUIT-002 O2 ce-pce D007aRP2 ce-pce bloodSUIT-RP2 for PBMCs and PLTs
SUIT-002 O2 mt D005RP2 mt1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png
SUIT-002 O2 pfi D006RP2 pfiSUIT-RP2
SUIT-003CCP-ceCe1;ce2(Omy);ce3U-.png Ce5S;1Dig;1c-.png
SUIT-003 AmR ce D058AmR effect on ceCe1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama.png
SUIT-003 AmR ce D059Amr effect on ce - controlCe1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama.png
SUIT-003 Ce1;ce1P;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4AzdcePMGlc,SCe1;ce1P;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4Azd.png
SUIT-003 Ce1;ce2U-ceCe1;ce2U-.png
SUIT-003 Ce1;ce3U-ce1ce;3ceU-.jpg
SUIT-003 O2 ce D009CCP-ce shortCe1;ce2Omy;ce3U-.png
SUIT-003 O2 ce D012CCP-ce(P)Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama.png
SUIT-003 O2 ce D028CCP-ce S permeability testCe1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama.png
SUIT-003 O2 ce D037CCP-ce Crabtree_RCe1;ce1Glc;ce2(Omy);ce3U;ce4Ama.png
SUIT-003 O2 ce D038CCP-ce Crabtree_ECe1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.png
SUIT-003 O2 ce D039CCP-ce microalgaeCe1;(ce2Omy);ce3U;ce4Rot;ce5Ama.jpg
SUIT-003 O2 ce D050CCP-ce SnvCe1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama.png
SUIT-003 O2 ce D060CCP-ce Snv,MnanvCe1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama.png
SUIT-003 O2 ce D061CCP-ce Snv,Mnanv - controlCe1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama.png
SUIT-003 O2 ce D062CCP-ce Snv - controlCe1;ce1DMSO;(ce2Omy);ce3U;ce4Rot;ce5Ama.png
SUIT-003 O2 ce-pce D013CCVP-Glc,MCe1;ce1P;ce2Omy;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4Azd.png
SUIT-003 O2 ce-pce D018CCVP-GlcCe1;ce1P;ce2Omy;ce3U;ce4Glc;ce5Rot;ce6S;1Dig;1U;1c;2Ama;3AsTm;4Azd.png
SUIT-003 O2 ce-pce D020CCVPCe1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd.png
SUIT-004 O2 pfi D010RP1-short pfi1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd.png
SUIT-005 O2 pfi D011RP2-short pfi1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd.png
SUIT-006 O2 ce-pce D029CCP ce-pce PMCe1;1Dig;1PM;2D;2c;3Omy;4U;5Ama.png
SUIT-006 O2 mt D022CCP mt S(Rot)1SRot;2D;2c;3(Omy);4U;5Ama.png
SUIT-006 O2 mt D047CCP mt PM1PM;2D;2c;3Omy;4U;5Ama.png
SUIT-007Glutamate anaplerosis1G;2D;3M;4U-.png
SUIT-007 O2 ce-pce D030Glutamate anaplerotic pathwayCe1;1Dig;1G;2D;2c;3M;4U;5Ama.png
SUIT-008 O2 ce-pce D025Q-junction ce-pceCe1;1Dig;1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png
SUIT-008 O2 mt D026Q-junction mtprep1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png
SUIT-008 O2 pce D25NS(PGM)1PM;2D;3G;4S;5U;6Rot-.png
SUIT-008 O2 pfi D0141PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png
SUIT-009 O2 ce-pce D016SUIT-009
SUIT-009 O2 mt D015SUIT9
SUIT-010Digitonin testRespirometric test of optimum digitonin concentration
SUIT-010 O2 ce-pce D008Dig titration-pceRespirometric test of optimum digitonin concentration
SUIT-011 O2 pfi D024NS physiological maximum capapcity in fibres1GM;2D;2c;3S;4U;5Rot;6Ama.png
SUIT-012 O2 ce-pce D052N(PGM)Ce1;1Dig;1PM;2D;2c;3G;4U;5Ama.png
SUIT-012 O2 mt D027N CCP mtprep1PM;2D;2c;3G;4U;5Ama.png
SUIT-013 AmR ce D023O2 dependence of H2O2 production ce</td>SUIT013 AmR ce D023.png</td></tr>
SUIT-014 O2 pfi D042</td>NS(PGM)</td>1GM;2D;2c;3P;4S;5U;6Rot;7Ama.png</td></tr>
SUIT-015 O2 pti D043</td>FNS(Oct,PGM)</td>1OctM;2D;3G;4P;5S;6U;7Rot;8Ama.png</td></tr>
SUIT-016 O2 pfi D044</td>FNS(Oct,GM)</td>1OctM;2D;3G;4S;5Rot;6Omy;7U;7c;8Ama.png</td></tr>
SUIT-017 O2 mt D046</td>FNS(Oct,GM)</td>1OctM;2D;2c;3G;4S;5U;6Rot;7Ama.png</td></tr>
SUIT-017 O2 pfi D049</td>FNS(Oct,GM)</td>1OctM;2D;2c;3G;4S;5U;6Rot;7Ama.png</td></tr>
SUIT-018 O2 mt D054</td></td>1GMS;2D;2c;3Ama.png</td></tr>
SUIT-019 O2 pfi D045</td>FNS(PalOct,PGM)</td>1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot;9Ama.png</td></tr>
SUIT-020 O2 mt D032</td>Q-junction additivity and respiratory control for membrane potential</td>1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama.png</td></tr>
SUIT-021</td>OXPHOS (GM+S+Rot+Omy)</td>1GM;2D;3S;4Rot;5Omy;6U-.png</td></tr>
SUIT-021 O2 mt D035</td>NS(GM)</td>1GM;2D;2c;3S;4Rot;5Omy;6U;7Ama.png</td></tr>
SUIT-022</td>AOX (ce CN+SHAM)</td>Ce1;ce2KCN;ce3SHAM.v2.png</td></tr>
SUIT-022 O2 ce D051</td>AOX-ce CN+SHAM</td>Ce1;ce2KCN;ce3SHAM.v2.png Ce1;ce2KCN;ce3SHAM.png</td></tr>
SUIT-023</td>AOX-ce SHAM+CN</td>Ce1;ce2SHAM;ce3KCN.png</td></tr>
SUIT-023 O2 ce D053</td>AOX-ce SHAM+CN</td>Ce1;ce2SHAM;ce3KCN.png</td></tr>
SUIT-024</td>ATPase (PM)</td>Ce1;1Dig;1PM;2T;2D;3Omy-.png</td></tr>
SUIT-024 O2 ce-pce D056</td>N(PM)</td>Ce1;1Dig;1PM;2T;2D;3Omy;4Ama.png</td></tr>
SUIT-025</td>OXPHOS (F+M+P+G+S+Rot)</td>1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Rot-.png</td></tr>
SUIT-025 O2 mt D057</td>FNS(Oct,PGM)</td>1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Rot;9Ama.png</td></tr>
SUIT-027</td>Malate anaplerosis</td>1M;2D;3M;4P;5G-.png</td></tr>
SUITbrowser</td></td>Use the SUITbrowser to find the best substrate-uncoupler-inhibitor-titration (SUIT) protocol for your research questions.
Open the SUITbrowser:
Each SUIT protocol receives a score from 0 to 3 in different categories that correspond to a research question (e.g. information on coupling control, information on fatty acid oxidation, among others). This score is set depending on the information which one can get from each SUIT protocol (3: this SUIT protocol is ideal, highly informative for the specific question; 2: information provided; 1: information provided, but is not a SUIT protocol mainly focused for the study of this question; 0: the specific information/question is not provided by the SUIT protocol). It is possible to choose multiple research questions at a time, to find the best protocols that address this combination of problems.</td></tr>
Selectivity</td></td>Selectivity is the ability of a sensor or method to quantify accurately and specifically the analyte or analytes in the presence of other compounds.</td></tr>
Sensitivity</td></td>Sensitivity refers to the response obtained for a given amount of analyte and is often denoted by two factors: the limit of detection and the limit of quantification.</td></tr>
Smoothing</td></td>Various methods of smoothing can be applied to improve the signal-to-noise ratio. For instance, data points recorded over time [s] or over a range of wavelengths [nm] can be smoothed by averaging n data points per interval. Then the average of the n points per smoothing interval can be taken for each successively recorded data point across the time range or range of the spectrum to give a n-point moving average smoothing. This method decreases the noise of the signal, but clearly reduces the time or wavelength resolution. More advanced methods of smoothing are applied to retain a higher time resolution or wavelength resolution.</td></tr>
Stability</td></td>Stability determines the accuracy of intensity and absorbance measurements as a function of time. Instability (see drift introduces systematic errors in the accuracy of fluorescence and absorbance measurements.</td></tr>
Substrate control state</td></td>See Electron transfer-pathway state</td></tr>
Substrate-uncoupler-inhibitor titration</td>SUIT</td>Mitochondrial Substrate-uncoupler-inhibitor titration (SUIT) protocols are used with mitochondrial preparations to study respiratory control in a sequence of coupling and substrates states induced by multiple titrations within a single experimental assay.</td></tr>
TPP+ inhibitory effect</td></td>A major task in establishing a procedure for measurement of mitochondrial membrane potential using probe molecules is the evaluation of inhibitory concentrations of the probe molecule on the activity of respiration. The TPP+ inhibitory effect (this also applies to TPMP+ and other indicator molecules) is frequently ignored. Accurate knowledge of a threshold concentration is required to evaluate the necessary limit of detection of TPP+, and for restriction of experimental TPP+ concentrations below the inhibitory range.</td></tr>
Tetraphenylphosphonium</td>TPP+</td>Tetraphenylphosphonium (TPP+). A lipophilic molecular probe in conjunction with an ion selective electrode (ISE) for measuring the mitochondrial membrane potential.</td></tr>
Time resolution</td></td>Time resolution in respirometric measurements is influenced by three parameters: the response time of the POS, the data sampling interval and the number of points used for flux calculation.</td></tr>
Uncoupling control ratio</td>UCR</td>The uncoupling control ratio, UCR, is the ratio of ET-pathway/ROUTINE respiration (E/R) in intact cells, evaluated by careful uncoupler titrations (Steinlechner et al 1996). Compare ROUTINE control ratio (R/E) (Gnaiger 2008).</td></tr>
Unspecific binding of TPP+</td></td>Unspecific binding of the probe molecule TPP+ in the matrix phase of mitochondria is taken into account as a correction for measurement of the mitochondrial membrane potential. External unspecific binding is the binding outside of the inner mt-membrane or on the outer side of the inner mt-membrane, in contrast to internal unspecific binding.</td></tr>
Warburg effect</td></td>Requires definition</td></tr>
Zero calibration</td>R0</td>Zero calibration is together with air calibration one of the two steps of the OroboPOS calibration. It is performed in the closed chamber after all the oxygen has been removed by the addition of dithionite or by respiration of mitochondria or cells. Any incubation medium can be used for zero calibration with dithionite or sample. Unlike air calibration it is not necessary to perform a zero calibration each day.</td></tr></table>
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