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Glossary: Respirometry
High-resolution terminology - matching measurements at high-resolution >>> Respiratory states, Fluorometry, Spectrophotometry (n.a. = no abbreviation)
| Term | Abbreviation | Description |
|---|---|---|
| ADP | D | Adenosine 5’diphosphate (D), C10H15N5O10P2K, potassium salt; substrate of ANT and ATP synthase. |
| Accuracy | n.a. | The accuracy of a method is the degree of agreement between an individual test result generated by the method and the true value. |
| Additive effect of convergent CI+II electron flow | Aα+β | Additivity describes the princple of substrate control of mitochondrial respiration, where the additive effect of convertent CI+II electron flow is a consequence of electron flow converging at the Q-junction from respiratory Complexes I and II (CI+II e-input). Further additivity may be observed by convergent electron flow through glycerophosphate dehydrogenase and electron-transferring flavoprotein. Convergent electron flow corresponds to the operation of the TCA cycle and mitochondrial substrate supply in vivo. Convergent electron flow simultaneously through CI+II into the Q-junction supports higher OXPHOS capacity and ETS capacity than separate electron flow through either CI or CII. Physiological substrate combinations supporting convergent CI+II e-input are required for reconstitution of intracellular TCA cycle function. The convergent CI+II 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, corresponding to additivity. |
| Basal respiration | BMR | Basal respiration or basal metabolic rate (BMR) is the minimal rate of metabolism required to support basic body functions, required for maintenance only. BMR (in humans) is measured at rest 12 to 14 hours after eating. Maintenance energy requirements include particularly the metabolic costs of ion homeostasis and protein turnover. 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 very closely (Blaxter KL 1962. The energy metabolism of ruminants. Hutchinson, London: 332 pp). In many cultured mammalian cells, aerobic glycolysis contributes to total ATP turnover (Gnaiger and Kemp 1990), and under these conditions, basal 'respiration' is not equivalent to basal 'metabolic rate'. |
| Biochemical threshold effect | n.a. | Due to threshold effects, even a large defect diminishing the velocity of an individual enzyme results in only minor changes of pathway flux. |
| CI+II e-input | CI+II e-input is electron input though Complexes CI plus CII simultaneously into the Q-junction corresponding to TCA cycle function in vivo, with convergent electron flow through the ETS. In mt-preparations, CI+II e-input requires addition not only of CI substrate (pyruvate+malate or glutamate+malate), but of succinate simultaneously, since metabolite depletion in the absence of succinate prevents a significant stimulation of CII. | |
| Calorespirometric ratio | CR ratio | 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. |
| Calorespirometry | Calorespirometry 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 culture media | Cell culture media, like RPMI or DMEM, used for HRR of intact cells. | |
| Cell respiration | Cell respiration channels metabolic fuels into the bioenergetic machinery of oxidative phosphorylation, regulating oxygen consumption and being regulated by molecular redox states, ion gradients, mitochondrial membrane potential, the phosphorylation state of the ATP system, and heat dissipation in response to intrinsic and extrinsic energy demands. | |
| Cellular substrates | Ce; Cm | 1) Cellular substrates in vivo, endogenous; Ce. 2) Cellular substrates in vivo, with exogenous substrate supply from culture medium or serum; Cm. |
| Chemical background correction of oxygen flux | Chemical background correction of oxygen flux is the correction of oxygen flux for the side reaction of autooxidation, as a function of oxygen concentration. | |
| Closed system | A 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. Changes of closed systems can be partitioned according to internal and external sources. | |
| Coupled respiration | Coupled respiration drives oxidative phosphorylation of ADP to ATP mediated by proton pumps across the inner mitochondrial membrane. 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. | |
| Coupling control ratio | CCR | Coupling control ratios, CCR, are flux control ratios, FCR, at a constant mitochondrial substrate state. In mitochondrial preparations, there are three well-defined coupling states of respiration, L, P, E (LEAK, OXPHOS, ETS). 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 ETS state, E, such that the lower and upper limits of 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 state | n.a. | Coupling control states are defined in mitochondrial preparations (isolated mitochondria, permeabilized cells, permeabilized tissues, homogenates) as LEAK, OXPHOS, and ETS states of respiration (L, P, E). These coupling states are induced by application of specific inhibitors of the phosphorylation system, titration of ADP and uncouplers. In intact cells, the coupling control states are LEAK, ROUTINE, and ETS states of respiration (L, R, E). Coupling control protocols induce these coupling control states sequentially at a constant substrate control state. |
| Crabtree effect | n.a. | The 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 oygen) is the converse of the Crabtree effect (aerobic glycolysis to lactate or etanol). |
| Dyscoupled respiration | n.a. | Dyscoupled respiration is 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. |
| ETF+CI(L) - ETF+CI(P) - CI(P) - CI+CII(P) - CII(P) - CII(L) - CII(E) | ||
| ETS capacity | E | Respiratory electron transfer system capacity, E, of mitochondria in the experimentally induced non-coupled (fully uncoupled) state, in mitochondrial preparations with defined substrates, or in intact cells, by titration of an established uncoupler to optimum concentration at maximum flux. Non-coupled respiration yields an estimate of ETS capacity. In this state E, the mt-membrane potential is collapsed, which provides a reference state for flux control ratios and measurement of mt-membrane potential. In intact mitochondria, the ETS capacity depends not only on the inner membrane-bound ETS (mETS, with respiratory Complexes I to IV, ETF, and glycerophosphate dehydrogenase) but also integrates transporters across the inner mt-membrane, the TCA cycle and other matrix dehydrogenases. |
| Electron flow | Electron flow through the mitochondrial electron transfer system (ETS) 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 transfer system | ETS | The mitochondrial electron transfer system (ETS) transfers electrons at steady state from externally supplied reduced substrates to oxygen. It consists of the membrane-bound ETS (mETS) with enzyme complexes located in the inner mt-membrane, mt-matrix dehydrogenases generating NADH, and the transport systems involved in metabolite exchange across the mt-membranes (see ETS capacity). |
| Extensive quantity | Extensive quantities pertain to a total system, e.g. oxygen flow. | |
| External flow | External flows across the system boundaries are formally reversible. Their irreversible facet is accounted for internally as part of a heterogenous system. | |
| Flux control ratio | FCR | Flux 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%). FCR obtained from a single respirometric incubation (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 determination of a common mitochondrial marker. |
| High-resolution respirometry | HRR | High-resolution respirometry (HRR) is based on the OROBOROS Oxygraph-2k, combining chamber design, application of oxygen-tight materials, electrochemical sensors and electronics, Peltier-temperature control and software features (DatLab) to obtain a unique level of quantitative resolution of oxygen concentration and oxygen flux, with a closed-chamber or open-chamber mode of operation (TIP2k). Standardized two-point 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. |
| Instrumental background oxygen flux | J°O2 | Instrumental 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 measured in the range of experimental oxygen levels by a standardized instrumental background test, and is a property of the instrumental system. Instrumental background correction eliminates errors by systemic flux compensation, automatically performed by DatLab. See also Chemical background correction of oxygen flux. |
| Internal flow | Within the system boundaries, irreversible internal flows of heat and matter along gradients contribute to the internal entropy production, diS. | |
| International oxygraph course | IOC | International Oxygraph Course, O2k-workshop. |
| Jmax | Jmax | Jmax is the maximum pathway flux (e.g. oxygen flux) obtained at saturating substrate concentration. Jmax is a function of metabolic state. In hyperbolic oxygen kinetics, Jmax is calculated by extrapolation of the hyperbolic function, with good agreement between the calculated and directly measured fluxes, when substrate levels are >20 times the p50. |
| LEAK control ratio | L/E | The LEAK control ratio, L/E, is the flux ratio of LEAK respiration over ETS capacity, as determined by measurement of oxygen consumption in sequentially induced states L and E of respiration. The L/E ratio is an index of uncoupling or dyscoupling at constant ETS 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 (non-coupled) system. |
| LEAK respiration | L | LEAK respiration or LEAK oxygen flux, compensating for proton leak, slip and cation cycling, is measured as mitochondrial respiration in state L, in the presence of reducing substrate(s), but absence of inorganic phosphate or ADP, or after inhibition of the phosphorylation system. 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 by the proton leak and the H+/O ratio. In this state of maximum protonmotive force, LEAK respiration is higher than the LEAK component in state P (OXPHOS capacity). |
| LEAK state | L | The LEAK state, L, is the non-phosphorylating resting state of intrinsic uncoupled or dyscoupled respiration when oxygen flux is maintained mainly to compensate for the proton leak when ATP synthase is not active. |
| LEAK state with ATP | LT | LEAK state with ATP or State 4 where - in mt-preparations without ATPase activity - LT is obtained after ADP is fully phosphorylated to ATP (Chance and Williams 1955) or after addition of high ATP in the absence of ADP (Gnaiger et al. 2000). |
| LEAK state with oligomycin | LOmy | The LEAK state with Omy is a LEAK state induced by inhibition of ATP synthase by oligomycin (LOmy; State 4o). |
| LEAK state without adenylates | LN | In the LEAK state without adenylates, LN (N for no adenylates), mitochondrial respiration is measured after addition of substrates, which decreases slowly to the LEAK state after oxidation of endogenous substrates with no adenylates. |
| Level flow | E | Level flow is a steady state of a system with an input process coupled to an output process (coupled system), in which the output force is zero. Clearly, energy must be expended to maintain level flow, even though output is zero (Caplan and Essig 1983; referring to zero output force, while output flow may be maximum). |
| Limiting pO2 | plim | In 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. |
| Membrane-bound ETS | mETS | The membrane-bound electron transport system (mETS) consists in mitochondria mainly of respiratory complexes CI, CII, electron transferring flavoprotein (ETF), glycerophosphate dehydrogenase (GpDH), and choline dehydrogenase, with convergent electron flow at the Q-junction (Coenzyme Q), and the two downstream respiratory complexes connected by cytochrome c, CIII and CIV, with oxygen as the final electron acceptor. |
| MiR05 | MiR05 | Mitochondrial respiration medium, MiR05, developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 + catalase. MiR07 = MiR06 + creatine. |
| MiR06 | MiR06 | Mitochondrial respiration medium, MiR06, developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. |
| MiR07 | MiR07 | Mitochondrial respiration medium, MiR07, developed for oxygraph incubations of mitochondrial preparations - permeabilized muscle fibres. MiR07 = MiR06 + 20 mM creatine. |
| MitoOx2 | MitoOx2 | Mitochondrial respiration medium, MitoOx2, developed for oxygraph incubations of mitochondrial preparations to measure the H2O2 production. MitoOx2 yields a higher optical sensitivity and lower "drift" (oxidation of the fluorophore precurcor without H2O2 present) for Amplex UltraRed(R) than e.g. MiR05. |
| Mitochondrial competence | mt-competence; MitoCom | Mitochondrial metabolic competence is the organelle's capacity to provide adequate amounts of ATP in due time, by adjusting the mt-membrane potential, mt-redox states and the ATP/ADP ratio according to the metabolic requirements of the cell.
The term mitochondrial competence is also known in a genetic context: Mammalian mitochondria possess a natural competence for DNA import.
|
| Mitochondrial preparations | mt-preparations | mt-preparations are isolated mitochondria, submitochondrial particles, tissue homogenate, mechanically or chemically permeabilized fibres and permeabilized cells. In these preparations the cell membranes are either removed (isolated mitochondria and submitochondrial particles) or mechanically (homogenate) and chemically permeabilized. |
| Mitochondrial respiration | Integrative 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 mETS, the transmembrane proton circuit, and the phosphorylation system. | |
| Mitophagy | Mitophagy | |
| Na-P buffer | Na-PB | Sodium phosphate buffer, Na-PB, for HRR with freeze-dried baker´s yeast. |
| NetROUTINE control ratio | (R-L)/E | The netROUTINE control ratio, (R-L)/E, expresses phosphorylation-related respiration (corrected for LEAK respiration) as a fraction of ETS capacity. (R-L)/E remains constant, if dyscoupling is fully compensated by an increase of ROUTINE respiration and a constant rate of oxidative phosphorylation (OXPHOS) is maintained. |
| Non-coupled respiration | E | Non-coupled respiration is distinguished from general (pharmacological or mechanical) uncoupled respiration, to give a label to an effort to reach the fully uncoupled (non-coupled) state without inhibiting respiration. Non-coupled respiration, therefore, yields an estimate of ETS capacity. Experimentally uncoupled respiration may fail to yield an estimate of ETS capacity, due to inhibition of respiration above optimum uncoupler concentrations or insufficient stimulation by sub-optimal uncoupler concentrations. Optimum uncoupler concentrations for evaluation of (non-coupled) ETS capacity require inhibitor titrations (Steinlechner-Maran_1996_AJP; Huetter_2004_BJ; Gnaiger_2008_POS). Non-coupled respiration is maximum electron flow in an open-transmembrane proton circuit mode of operation (see ETS capacity). |
| O2k | O2k | O2k - OROBOROS Oxygraph-2k: the modular system for high-resolution respirometry. |
| O2k versus multiwell respirometer | O2k stands for Oxygraph-2k and high-resolution respirometry, meeting powerful quality criteria thus securing high output. 'High throughput' stands for multiwell systems. In respirometry, this is not equivalent to high output. | |
| OXPHOS capacity | P | OXPHOS capacity (P; compare State 3) is the respiratory capacity of mitochondria in the ADP-activated state of oxidative phosphorylation, at saturating concentrations of ADP, inorganic phosphate, oxygen, and defined reduced substrates. Since OXPHOS is partially coupled, intrinsic uncoupling and dyscoupling contribute to the control of flux in the OXPHOS state (state P). OXPHOS capacity is expressed (i) per mt-marker (O2 flux per mt-protein, CS, etc); if ETS capacity, E, is used as a functional mitochondrial marker, then OXPHOS capacity is expressed as the P/E ratio (flux control ratio). (ii) OXPHOS capacity is expressed per tissue or cell mass, integrating mt-quantity (density) and mt-quality (O2 flux). (iii) OXPHOS capacity is expressed per cell (O2 flow), which then is a function of mt-density, mt-quality, and cell size. |
| Open system | An 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 boundary. | |
| Oxidative phosphorylation | OXPHOS | Oxidative phosphorylation (OXPHOS) is the oxidation of reduced substrates with electron transfer to oxygen, incompletely coupled to the phosphorylation of ADP to ATP. The OXPHOS state (P) of respiration provides a measure of OXPHOS capacity. |
| Oxycaloric equivalent | DeltakHO2 | The 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 flow | IO2 | Respiratory oxygen flow is the oxygen consumption per total system, extensive quantity. Flow is advancement of a transformation in a system per time. Oxygen flow of a cell, or respiration per million cells, is distinguished from oxygen flux (e.g. per mg protein or wet weight). |
| Oxygen flux | JO2 | Oxygen flux, J, is a specific quantity: oxygen flow, I, divided by system size. Flux may be volume-specific (flow per volume), mass-specific (flow per mass), or marker-specific (e.g. flow per mtDNA). Oxygen flux (e.g. per body mass, or per cell mass) is distinguished from oxygen flow (per subject, or per million cells). |
| Oxygen kinetics | Oxygen 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 pressure | pO2 | Oxygen pressure or partial pressure of oxygen [kPa], related to oxygen concentration in solution by the oxygen solubility, SO2 [µM/kPa]. |
| Oxygen pressure, intracellular | pO2,i | Physiological, 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 solubility | SO2 | The oxygen solubility [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. See also: Oxygen solubility factor |
| Oxygen solubility factor | FM | The 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. |
| P50 | p50 | p50 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%. |
| PEEK | PEEK | Polyether 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). |
| POS calibration - dynamic | Calibration of the sensor response time. See also POS calibration - static. | |
| POS calibration - static | Two-point calibration of the polarographic oxygen sensor. See also POS calibration - dynamic. | |
| PVDF | PVDF | Polyvinylidene 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). |
| Permeabilized muscle fibres | Pfi | Permeabilized muscle fibres (Pfi) are used as a mitochondrial preparation in respirometry to access mitochondrial function comparable to isolated mitochondria. Pfi are obtained by selectively permeabilizing the plasma membrane mechanically and chemically, for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the mt-membranes. Add MitoPedia topic: Mitochondrial preparations |
| Permeabilized tissue or cells | Ptic | Permeabilized tissue (see permeabilized muscle fibres) or cells are mitochondrial preparations obtained by selectively permeabilizing the plasma membrane mechanically or chemically, for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the mt-membranes. Permeabilized cells are, therefore, not any longer intact cells or viable cells, since the intactness of cells implies the intactness of the plasma membrane, and any typical quantiative cell viability test (trypan blue etc) evaluating the intactness of the plasma membrane, yields a 100% negative result on fully permeabilized cells. |
| Phosphorylation control protocol | PCP | A phosphorylation control protocol induces different coupling control states at constant substrate supply. In intact cells, the PCP can be applied by using membrane-permeable inhibitors of the phosphorylation system (e.g. oligomycin) and uncouplers (e.g. FCCP). Coupling control states in intact cells include R, L, E; LEAK, ROUTINE, and ETS. Coupling control states in isolated mitochondria, permeabilized cells or homogenates include L, P, E; LEAK, OXPHOS, and ETS. |
| Phosphorylation system | PS | The phosphorylation system is a functional unit consisting of adenylate nucleotide translocase, phosphate carrier, and ATP synthase. Mitochondrial adenylate kinase, mt-creatine kinase and mt-hexokinase constitute extended components of the phosphorylation system, controlling local AMP and ADP concentrations and forming metabolic channels. |
| Phosphorylation system control ratio | P/E | The phosphorylation system control ratio or OXPHOS/ETS (P/E) ratio is an expression of the limitation of OXPHOS capacity by the phosphorylation system. 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 ETS 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 ETS 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. |
| Physiological substrate control state | n.a. | Physiological substrate control states are substrate control states obtained in intact cells respiring on endogenous substrates or in media with physiological exogenous substrates, or designed for reconstitution of TCA cycle function in isolated mitochondria, permeabilized cells or permeabilized tissues. In all cases, electron flow converges at the Q-junction with multiple entry sites of electron transfer throgh CI+II, CI+II+ETF, CI+II+GPDH. |
| Proton leak | Flux of protons along the electrochemical proton gradient across the inner mt-membrane, contributing to LEAK respiration. | |
| Proton pump | Mitochondrial 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 proton motive 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 proton motive force to drive phosphorylation of ADP to ATP. | |
| Q-junction | Q | The Q-junction is a junction for convergent electron flow in the ETS from CI, CII, electron-transferring flavoprotein, glycerophosphate dehydrogenase, choline dehydrogenase and other enzymes into the Q-cycle (ubiquinol/ubiquinone), and further to CIII. |
| ROUTINE control ratio | R/E | The ROUTINE control ratio (R/E) is the ratio of (partially coupled) ROUTINE respiration and (non-coupled) ETS capacity. The R/E control ratio is an expression of how close ROUTINE respiration operates to ETS capacity. |
| ROUTINE respiration | R | In the intact cell, ROUTINE respiration in the physiological coupling state R, is controlled by cellular energy demand, energy turnover and the degree of coupling to phosphorylation (intrinsic uncoupling and pathological dyscoupling). 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. |
| Residual oxygen consumption | ROX | Residual oxygen consumption, ROX, is the respiration due to oxidative side reactions remaining after application of ETS inhibitors to mitochondrial preparations or cells, or in mt-preparations incubated without substrates (in the presence of ADP: State 2). ROX may be related to, but is different from ROS production. |
| Respiratory Complex-specific substrate control state | n.a. | Respiratory Complex-specific substrate control states are substrate control states for selective entry of electron transfer through one particular respiratory Complex; for instance through CI (PM; GM; PMG with or without malonic acid: MiPNet11.04 MitoPathways-CI), CII (Suc(Rot): MiPNet11.09_MitoPathways-CII), CIV (As+Tm: MiPNet06.06_ChemicalBackground). |
| Respiratory chain | RC | The 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 system (ETS), 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 control ratio | RCR | The respiratory control ratio (RCR) is defined as State 3/State 4 (Chance and Williams 1955). Considering an index of uncoupling, RCR should be replaced by the L/E ratio, if P/E<1.0 (Gnaiger 2009). In intact cells, RCR has been used for the ratio State 3u/State 4o, i.e. for the inverse L/E ratio (Huetter et al., 2004). |
| Respiratory state | n.a. | Respiratory 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 substrate control state. |
| Respirometry | n.a. | Respirometry 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 conclusion 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 meabolism determines the amount of oxygen to be absorbed. .. metabolism regulates the respiration. |
| State 1 | n.a. | State 1 is the first respiratory state in an oxygraphic protocol described by Chance and Williams (1955), when isolated mitochondria are added to mitochondrial resipration medium containing oxygen and inorganic phosphate, but no ADP and no reduced respiratory substrates. In State 1, LEAK respiration may be supported to some extent by undefined endogenous substrates, which are oxidized and slowly exhausted. After oxidation of endogenous substrates, only residual oxygen consumption remains (ROX). |
| State 2 | ROXD | Substrate limited state of residual oxygen consumption, after addition of ADP to isolated mitochondria suspended in mitochondrial respiration medium in the absence of reduced substrates (ROXD). Residual endogenous substrates are oxidized during a transient stimulation of oxygen flux by ADP. The peak – supported by endogenous substrates – is, therefore, a pre-steady state phenomenon preceding State 2. Subsequently oxygen flux declines to a low level (or zero) at the steady State 2 (Chance and Williams 1955). ADP concentration (D) remains high during ROXD. |
| State 3 | P | State 3 respiration is the ADP stimulated respiration of isolated coupled mitochondria in the presence of high ADP and Pi concentrations, supported by a defined substrate or substrate combination at saturating oxygen levels (Chance and Williams, 1955). State 3 respiration can also be induced in permeabilized cells, including permeabilized tissue preparations and tissue homogenates. ADP concentrations applied in State 3 are not necessarily saturating, whereas OXPHOS capacity is measured at saturating concentrations of ADP and Pi (state P). For instance, non-saturating ADP concentrations are applied in State 3 in pulse titrations to determine the P/O ratio in State 3→4 (D→T) transitions, when saturating ADP concentrations would deplete the oxygen concentration in the closed oxygraph chamber before State 4 is obtained (Gnaiger et al 2000; Puchowicz et al 2004). Respiration in the OXPHOS state or in State 3 is partially coupled, and partially uncoupled (physiological) or partially dyscoupled (pathological). A high mt-membrane potential provides the driving force for oxidative phosphorylation, to phosphorylate ADP to ATP and to transport ADP and ATP across the inner mt-membrane through the adenine nucleotide translocase (ANT). The mt-membrane potential is reduced, however, in comparison to the LEAK state of respiration, whereas the cytochromes are in a more oxidized redox state. |
| State 3u | E | Non-coupled state of ETS capacity. State 3u (u for uncoupled) has been used frequently in bioenergetics, without sufficient emphasis (e.g. Villani et al 1998) on the fundamental difference between OXPHOS capacity (P, coupled with an uncoupled contribution; State 3) and non-coupled ETS capacity (E; State 3u) (Gnaiger 2009; Rasmussen and Rasmussen 2000). |
| State 4 | LT | State 4 is the respiratory state obtained in isolated mitochondria after State 3, when added ADP is phosphorylated completely to ATP driven by electron transfer from defined respiratory substrates to O2 (Chance and Williams, 1955). State 4 represents LEAK respiration, LT (L for LEAK; T for ATP), or an overestimation of LEAK respiration if ATPase activity prevents final accumulation of ATP and maintains a continuous stimulation of respiration by recycled ADP. This can be tested, by inhibition of ATP synthase by oligomycin (State 4o; LOmy). In the LEAK state (state of non-phosphorylating resting respiration; static head), oxygen flux is decreased to a minimum (correctd for ROX), and the mt-membrane potential is increased to a maximum for a specific substrate or substrate combination. |
| State 5 | n.a. | State 5 is the respiratory state obtained in a protocol with isolated mitochondria after a sequence of State 1 to State 4, when the concentration of O2 is depleted in the closed oxygraph chamber and zero oxygen (the anaerobic state) is reached (Chance and Williams, 1955; Table I). State 5 is defined in the original publication in two ways: State 5 may be obtained by antimycin A treatment or by anaerobiosis (Chance and Williams, 1955; page 414). Antimycin A treatment yields a State 5 equivalent to a state for measurement of residual oxygen consumption, ROX (which may also be induced by rotenone+myxothiazol; Gnaiger 2009). Setting State 5 equivalent to ROX or anoxia (Chance and Williams 1955) can be rationalized only in the context of measurement of cytochrome redox states, whereas in the context of respiration State 5 is usually referred to as 'zero oxygen'. |
| Static head | L | Static head is a steady state of a system with an input process coupled to an output process (coupled system), in which the output force is maximized at constant input or driving force up to a level at which the conjugated output flow is reduced to zero. In an incompletely coupled system, energy must be expended to maintain static head, even though the output is zero (Caplan and Essig 1983; referring to output flow at maximum output force). LEAK respiration is a measure of input flow at static head, when the output flow of phosphorylation (ADP->ATP) is zero at maximum phosphorylation potential (Gibbs force of phosphorylation; Gnaiger 1993a). In a completely coupled system, not only the output flux but also the input flux are zero at static head, which then is a state of ergodynamic equilibrium (Gnaiger 1993b). Whereas the output force is maximum at ergodynamic equilibrium compensating for any given input force, all forces are zero at thermodynamic equilibrium. Flows are zero at both types of equilibria, hence entropy production or power (power = flow x force) are zero in both cases, i.e. at thermodynamic equilibrium in general, and at ergodynamic equilibrium of a completely coupled system at static head. |
| Substrate control | n.a. | Substrate control with electron entry separately through Complex I (pyruvate+malate or glutamate+malate) or Complex II (succinate+rotenone) restricts ETS capacity and artificially enhances flux control upstream of the Q-cycle, providing diagnostic information on specific branches of the ETS. Physiological combinations of Complex I+II substrates support maximum ETS and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. |
| Substrate control ratio | SCR | Substrate control ratios, SCR, are flux control ratios, FCR, at a constant mitochondrial coupling state. Whereas there are only three well-defined coupling states of mitochondrial respiration, L, P, E (LEAK, OXPHOS, ETS), numerous substrate 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. CI+IIE), such that flux in various other respiratory states, Ji, smaller or equal to Jref. However, this is not generally possible with SCR. For instance, the CI/CII substrate control ratio (at constant coupling state) may be larger or smaller than 1.0, depending on the mitochondrial source and various mitochondrial injuries. The CII-respiration may be selected preferentially as Jref, is mitochondria with variable CI-related injuries are studied. |
| Substrate control state | n.a. | Substrate control states are defined in mitochondrial preparations (isolated mitochondria, permeabilized cells, permeabilized tissues, tissue homogenate) by depletion of endogenous substrates and addition of defined substrates to the mitochondrial respiration medium. Mitochondrial substrate control states have to be defined complementary to mitochondrial coupling control states. |
| Substrate-uncoupler-inhibitor titration | SUIT | Mitochondrial SUIT protocols are used with mt-preparations to study respiratory control in a sequence of coupling and substrates states induced by multiple titrations. |
| Substrates as electron donors | Mitochondrial respiration depends on a continuous flow of electron-supplying substrates across the mitochondrial membranes into the matrix space. Many substrates are strong anions that cannot permeate lipid membranes and hence require carriers. | |
| Uncoupler | u | An uncoupler is a protonophore (FCCP, CCCP, DNP) which cycles across the inner mt-membrane with transport of protons and dissipation of the electrochemical proton gradient. Mild uncoupling may be induced at low uncoupler concentrations, the non-coupled state of ETS capacity is obtained at optimum uncoupler concentration for maximum flux, whereas at higher concentrations an uncoupler-induced inhibition is observed. See also Non-coupled respiration. |
| Uncoupling control ratio | UCR | The uncoupling control ratio, UCR, is the ratio of ETS/ROUTINE respiration (E/R) in intact cells (Steinlechner et al 1996). See also Uncoupler, ROUTINE control ratio (R/E) (Gnaiger 2008). |
| Viton | n.a. | Viton® is a fluoroelastomer with excellent resistance to aggressive fuels and chemicals. Viton is resistant against oxygen diffusion which makes it an ideal material for high-resolution respirometry (Viton O-rings). |
| … further results | ||
