|News and Events||About COST Action MitoEAGLE||Working Groups||MitoEAGLE Summit 2020 Obergurgl AT||MitoEAGLE Preprint States and rates||Short-Term Scientific Missions||Inclusiveness Target Countries||Management Committee||Members|
- COST Action CA15203 MitoEAGLE
Evolution-Age-Gender-Lifestyle-Environment: mitochondrial fitness mapping
- COST Action CA15203 MitoEAGLE
- Gnaiger E, Aasander Frostner E, Abdul Karim N, Abumrad NA, Acuna-Castroviejo D, Adiele RC et al (2019) Mitochondrial respiratory states and rates. MitoFit Preprint Arch doi:10.26124/mitofit:190001. - »Bioblast link«
Towards a Preprint publication: Mitochondrial respiratory states and rates
- Concept of joint publications on nomenclature and terminology in mitochondrial physiology
- Working Group 1 (WG1) took first steps at the meeting MitoEAGLE Verona 2016 towards a consensus paper, starting with the topic of respiratory states.
- Table 1: Participants selected terms for discussion and further clarification. A list of recommended terms will be prepared until the meeting MitoEAGLE Barcelona 2017, by (i) elaborating the terms recommended in Table 1, and (ii) addition of missing terms for discussion and recommendation.
- Table 2: Synonyms, historically used and controversial terms.
- At the WG1 workshop in Barcelona, a series of two MitoEAGLE publications will be further considered in detail: (i) a conceptual review paper on Mitochondrial respiratory control states by authors joining the Task Group and actively contributing to writing this manuscript, where controversies and recommendations are discussed, (ii) a consensus paper co-authored by an as-large-as-possible number of scientists. (iii) The scope of the nomenclature publication should be defined, possible extending beyond 'respiratory states' to include respiratory control ratios.
- Concept of joint publications on nomenclature and terminology in mitochondrial physiology
- The MitoEAGLE network is open as a COST Action to all scientists interested to collaborate in this project.
- We invite contributions and critical comments.
- Communications will be posted on the MitoEAGLE website under the contributor's name as the author.
- Entries on the MitoEAGLE website are considered as pre-publication, inviting critical feedback and comments from a large-scale audience. All feedback will be carefully documented and evaluated in terms of justification of co-authorship. The final manuscript(s) will be published Open Access in a peer-reviewed journal as original article(s), with reference to the pre-publication history including reviewer's reports.
- Please contact:
- The MitoEAGLE network is open as a COST Action to all scientists interested to collaborate in this project.
- See also
Respiratory states - Table 1: recommended terms
|Background state||Y||The background state, Y, is the non-activated or inhibited respiratory state at background flux, which is low in relation to the higher flux in the reference state, Z. The transition from the background state to the reference state is a step change. A metabolic control variable, X (substrate, activator), is added to the background state to stimulate flux to the level of the reference state. Alternatively, the metabolic control variable, X, is an inhibitor, which is present in the background state, Y, but absent in the reference state, Z. The background state is the baseline of a single step in the definition of the flux control factor. In a sequence of step changes, the common baseline state is the state of lowest flux in relation to all steps, which can be used as a baseline correction.|
|Basal respiration||BMR||Basal 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 ). In many cultured mammalian cells, aerobic glycolysis contributes to total ATP turnover (Gnaiger and Kemp 1990 ), 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 ). » MiPNet article|
|Baseline state||The baseline state in a sequence of step changes is the state of lowest flux in relation to all steps, which can be used as a baseline correction. Correction for residual oxygen consumption, ROX, is an example where ROX is the baseline state. In a single step, the baseline state is equivalent to the background state.|
|Coupling control state||CCS||Coupling 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.|
|Dyscoupled respiration||Dyscoupled 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-capacity||E||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|
|Electron transfer-pathway state||ET-pathway state|
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
|Excess E-P capacity||ExP||The excess E-P capacity, ExP, is the difference of the ET-capacity and OXPHOS-capacity, ExP = E-P. At ExP > 0, the capacity of the phosphorylation system exerts a limiting effect on OXPHOS capacity. In addition, ExP depends on coupling efficiency, since P aproaches E at increasing uncoupling.|
|Excess E-R capacity||ExR||The Excess E-R capacity, ExR, is the difference of ET-capacity and ROUTINE respiration, ExR = E-R. For further information, see Cell ergometry.|
|FN||FN||NADH-generating substrates (N-pathway control state, or CI-linked pathway control) in combination with one or several fatty acids, which are supplied to feed electrons into the F-junction through fatty acyl CoA dehydrogenase (reduced form FADH2), to electron transferring flavoprotein (CETF), and further through the Q-junction to Complex III (CIII). FAO not only depends on electron transfer through the F-junction (which is typically rate-limiting), but simultaneously generates FADH2 and NADH and thus depends on N-junction throughput. Hence FAO can be inhibited completely by inhibition of Complex I (CI). This physiological substrate combination is required for partial reconstitution of TCA cycle function and convergent electron-input into the Q-junction, to compensate for metabolite depletion into the incubation medium. FS in combination exerts an additive effect of convergent electron flow in most types of mitochondria.|
|FNS||FNS||NADH-generating substrates (N-pathway control state, or CI-linked pathway control) in combination with succinate (S-pathway control state; S- or CII-linked) and one or several fatty acids, which are supplied to feed electrons into the F-junction through fatty acyl CoA dehydrogenase (reduced form FADH2), to electron transferring flavoprotein (CETF), and further through the Q-junction to Complex III (CIII). FAO not only depends on electron transfer through the F-junction (which is typically rate-limiting), but simultaneously generates FADH2 and NADH and thus depends on N-junction throughput. Hence FAO can be inhibited completely by inhibition of Complex I (CI). This physiological substrate combination is required for partial reconstitution of TCA cycle function and convergent electron-input into the Q-junction, to compensate for metabolite depletion into the incubation medium. FNS in combination exerts an additive effect of convergent electron flow in most types of mitochondria.|
|Fatty acid oxidation pathway control state||F, FAO||F-junction through fatty acyl CoA dehydrogenase (reduced form FADH2), to electron transferring flavoprotein (CETF), and further through the Q-junction to Complex III (CIII). FAO not only depends on electron transfer through the F-junction (which is typically rate-limiting), but simultaneously generates FADH2 and 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 type N substrate malate is required as a co-substrate for FAO in mt-preparations, since accumulation of Acetyl-CoA 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 Acetyl-CoA into the TCA cycle catalyzed by citrate synthase. Peroxysomal β-oxidation carries out few β-oxidation cycles, thus shortening very-long-chain fatty acids (>C20) for entry into mitochondrial β-oxidation. Oxygen consumption by peroxisomal acyl-CoA oxidase is considered as residual oxygen consumption rather than cell respiration.|
|Free ET-capacity||≈E||The Free ET-capacity, ≈E, is the ET-capacity corrected for LEAK respiration, ≈E = E-L. ≈E is the respiratory capacity potentially available for ion transport and phosphorylation of ADP to ATP. Oxygen consumption in the ET-pathway state, therefore, is partitioned into the free ET-capacity, ≈E, and LEAK respiration, LP, compensating for proton leaks, slip and cation cycling: E = ≈E+LP (see free OXPHOS capacity).|
|Free OXPHOS-capacity||≈P||The free OXPHOS-capacity, ≈P, is the OXPHOS-capacity corrected for LEAK respiration, ≈P = P-L. ≈P is the scope for ADP stimulation, the respiratory capacity potentially available for phosphorylation of ADP to ATP. Oxygen consumption in the OXPHOS state, therefore, is partitioned into the free OXPHOS-capacity, ≈P, strictly coupled to phosphorylation, ~P, and nonphosphorylating LEAK respiration, LP, compensating for proton leaks, slip and cation cycling: P = ≈P+LP. It is frequently assumed that LEAK respiration, L, as measured in the LEAK state, overestimates the LEAK component of respiration, LP, as measured in the OXPHOS state, particularly if the protonmotive force is not adjusted to equivalent levels in L and LP. However, if the LEAK component increases with enzyme turnover during P, the low enzyme turnover during L may counteract the effect of the higher Δpmt.|
|Free ROUTINE activity||≈R||The free ROUTINE activity, ≈R, is ROUTINE respiration corrected for LEAK respiration, ≈R = R-L. ≈R is the respiratory activity available for phosphorylation of ADP to ATP. Oxygen consumption in the ROUTINE state of respiration measured in intact cells, therefore, is partitioned into the free ROUTINE activity, ≈R, strictly coupled to phosphorylation, ~P, and nonphosphorylating LEAK respiration, LR, compensating for proton leaks, slip and cation cycling: R = ≈R+LR. It is frequently assumed that LEAK respiration, L, as measured in the LEAK state, overestimates the LEAK component of respiration, LR, as measured in the ROUTINE state, particularly if the protonmotive force is not adjusted to equivalent levels in L and LR. However, if the LEAK component increases with enzyme turnover during R, the low enzyme turnover during L may counteract the effect of the higher Δpmt.|
|Glycerophosphate pathway control state||Gp||ET-pathway level 3 control state, supported by the fuel substrate glycerophosphate and electron transfer through glycerophosphate dehydrogenase complex into the Q-junction. The glycerolphosphate shuttle represents an important pathway, particularly in liver and blood cells, of making cytoplasmic NADH available for mitochondrial oxidative phosphorylation. Cytoplasmic NADH reacts with dihydroxyacetone phosphate catalyzed by cytoplasmic glycerophos-phate dehydrogenase. On the outer face of the inner mitochondrial membrane, mitochondrial glycerophosphate dehydrogenase oxidises glycerophosphate back to dihydroxyacetone phosphate, a reaction not generating NADH but reducing a flavin prosthesic group. The reduced flavoprotein donates its reducing equivalents to the electron transfer-pathway at the level of CoQ.|
|LEAK state with ATP||L(T)||The LEAK state with ATP is obtained in mt-preparations without ATPase activity after ADP is maximally phosphorylated to ATP (State 4; Chance and Williams 1955) or after addition of high ATP in the absence of ADP (Gnaiger et al 2000). Respiration in the LEAK state with ATO, L(T), is distinguished from L(n) and L(Omy).|
|LEAK state with oligomycin||L(Omy)||The LEAK state with Omy is a LEAK state induced by inhibition of ATP synthase by oligomycin. ADP and ATP may or may not be present. LEAK respiration with oligomycin, L(Omy), is distinguished from L(n) and L(T).|
|LEAK state without adenylates||L(n)||In the LEAK state without adenylates mitochondrial LEAK respiration, L(n) (n for no adenylates), is measured after addition of substrates, which decreases slowly to the LEAK state after oxidation of endogenous substrates with no adenylates. L(n) is distinguished from L(T) and L(Omy).|
|LEAK-respiration||L||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|
|Mitochondrial membrane potential||mtMP, Δψ [V]||The mitochondrial membrane potential, mtMP, is the electric part of the protonmotive force, ΔpH+.
Δψ = ΔpH+ - ΔµH+ / FmtMP 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].
|NADH Electron transfer-pathway state||N||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-pathway control state||NS, CI&II||NADH-generating substrates (N-pathway control state in combination with succinate (Succinate-pathway; S). Whereas NS expresses substrate control in terms of substrate types (N and S), CI&II defines the same concept in terms of convergent electron transfer to the Q-junction (pathway control). NS is the abbreviation for the combination of N- or NADH-linked substrates and S- or succinate-linked substrates. This physiological substrate combination is required for partial reconstitution of TCA cycle function and convergent electron-input into the Q-junction, to compensate for metabolite depletion into the incubation medium. NS in combination exerts an additive effect of convergent electron flow in most types of mitochondria.|
|Noncoupled respiration||E||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).|
|OXPHOS-capacity||P||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|
|Oxidative phosphorylation||OXPHOS||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).|
|ROUTINE-respiration||R||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.|
|Reference state||Z||The reference state, Z, is the respiratory state with high flux in relation to the background state, Y. The transition between the background state and the reference state is a step brought about by a metabolic control variable, X. If X stimulates flux (ADP, fuel substrate), it is present in the reference state but absent in the background state. If X is an inhibitor of flux, it is absent in the reference state but present in the background state. The reference state is specific for a single step to define the flux control factor. In contrast, in a sequence of multiple steps, the common reference state is frequently taken as the state with the highest flux in the entire sequence, as used in the definition of the flux control ratio.|
|Residual oxygen consumption||ROX||Residual oxygen consumption, ROX, is respiration due to oxidative side reactions remaining after inhibition of the Electron transfer-pathway (ET-pathway) in mitochondrial preparations or cells, or in mt-preparations incubated without addition of fuel substrates (in the presence of ADP following a stimulation of the consumption of endogenous fuel substrates: State 2). Different conditions designated as ROX states (different combinations of inhibitors of CI, CII, CIII and CIV; or respiration of mt-preparations without addition of fuel substrates) may result in consistent or significantly different levels of oxygen consumption. Hence the best quantitative estimate of ROX has to be carefully evaluated. Mitochondrial respiration is frequently corrected for ROX as the baseline state. Then total ROUTINE, LEAK, OXPHOS or ET-pathway (R, L, P and E) respiration is distinguished from the corresponding ROX-corrected, mitochondrial (ET-pathway linked) fluxes: R(mt), L(mt), P(mt) and E(mt). When expressing ROX as a fraction of ET-capacity (flux control ratio), total flux, E (not corrected for ROX), should be taken as the reference. ROX may be related to, but is of course different from ROS production. » MiPNet article|
|Respiratory state||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 electron transfer-pathway state.|
|Resting metabolic rate||RMR||Resting respiration or resting metabolic rate (RMR) is measured under standard conditions of an 8–12-h fast and a 12-h abstinence from exercise. In an exemplary study (Haugen 2003 Am J Clin Nutr), "subjects rested quietly in the supine position in an isolated room with the temperature controlled to 21–24° C. RMR was measured for 15–20 min. Criteria for a valid RMR was a minimum of 15 min of steady state, determined as a <10% fluctuation in oxygen consumption and <5% fluctuation in respiratory quotient". The main difference between RMR and BMR (basal metabolic rate) is the position of the subject during measurement. Resting metabolic rate is the largest component of the daily energy budget in most human societies and increases with physical training state (Speakman 2003 Proc Nutr Soc).|
|SGp-pathway control state||SGp||SGp: Succinate & Glycerophosphate.
MitoPathway control state: SGp; obtained with OctPGMSGp(Rot)SUIT protocol: SUIT-001 and ((SUIT-002
The Succinate-pathway (S-pathway; S) supports succinate-linked respiration (succinate-induced respiratory state; previously used nomenclature: CII-linked respiration; SRot; see Gnaiger 2009 Int J Biochem Cell Biol). Succinate is the single substrate, at ET-pathway level 3. S supports electron flux through Complex II (CII; see succinate dehydrogenase, SDH) to CII-bound flavin adenine dinucleotide (FADH2) to the Q-junction. Inhibition of Complex I (CI) by rotenone (Rot; or amytal, piericidine) prevents accumulation of oxaloacetate which is a potent inhibitor of SDH. After inhibition of CI by rotenone, the NADH-linked dehydrogenases become inhibited by the redox shift from NAD+ to NADH. SDH is activated by succinate and ATP, which explains in part the time-dependent increase of respiration in isolated mitochondria after addition of rotenone (first), succinate and ADP.The S-pathway control state is induced in mt-preparations by addition of succinate&rotenone. In this case, only Complex III and Complex IV are involved in pumping protons from the matrix (positive phase, P-phase) to the negative phase (N-phase) with a P»/O2 of 3.5 (P»/O ratio = 1.75).
Respiratory states - Table 2: find synonyms, historically used and controversial terms
|Complex I&II-linked substrate state||NS||See NS-pathway control state (previous: CI&II-linked)|
|Complex I-linked substrate state||N||See N-pathway control state (previous: CI-linked)|
|Complex II-linked substrate state||SRot, S||See S-pathway control state (previous: CII-linked)|
|ET-pathway competent state||ET-pathway competent state, see Electron transfer-pathway state.|
|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).|
|State 1||State 1 is the first respiratory state in an oxygraphic protocol described by Chance and Williams (1955), when isolated mitochondria are added to mitochondrial respiration 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 (OXPHOS state). 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 well 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 mitochondrial inner membrane (mtIM) 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||Noncoupled state of ET-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 noncoupled ET-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 maximally 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; LOmy). In the LEAK state (state of non-phosphorylating resting respiration; static head), oxygen flux is decreased to a minimum (corrected for ROX), and the mt-membrane potential is increased to a maximum for a specific substrate or substrate combination.|
|State 5||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 anoxic.|
|Substrate control state||See Electron transfer-pathway state|