MitoPedia: Respiratory states

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MitoPedia

MitoPedia: Respiratory states

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

Coupling states
OXPHOS ROUTINE ETS LEAK - ROX
Free and excess capacities of respiration
Free OXPHOS capacity Free ROUTINE activity Free ETS capacity Excess E-P capacity Excess E-R capacity
See also
» MitoPedia: Respiratory control ratios
» Respirometry
» Cell ergometry
Term Abbreviation Description
Aerobic ox The aerobic state of metabolism is defined by the presence of oxygen (air) and therefore the potential for oxidative reactions (ox) to proceed, particularly in oxidative phosphorylation (OXPHOS). Aerobic metabolism (with involvement of oxygen) is contrasted with anaerobic metabolism (without involvement of oxygen): Whereas anaerobic metabolism may proceed in the absence or presence of oxygen (anoxic or oxic conditions), aerobic metabolism is restricted to oxic conditions. Below the critical oxygen pressure, aerobic ATP production decreases.
Anaerobic Anaerobic metabolism takes place without the use of molecular oxygen, in contrast to aerobic metabolism. The capacity for energy assimilation and growth under anoxic conditions is the ultimate criterion for facultative anaerobiosis. Anaerobic metabolism may proceed not only under anoxic conditions or states, but also under hyperoxic and normoxic conditions (aerobic glycolysis), and under hypoxic and microxic conditions below the limiting oxygen pressure.
Anoxic anox Ideally the term anoxic (anox, without oxygen) should be restricted to conditions where molecular oxygen is strictly absent. Practically, effective anoxia is obtained when a further decrease of experimental oxygen levels does not elicit any physiological or biochemical response. The practical definition, therefore, depends on (i) the techiques applied for oxygen removal and minimizing oxygen diffusion into the experimental system, (ii) the sensitivity and limit of detection of analytical methods of measuring oxygen (O2 concentration in the nM range), and (iii) the types of diagnostic tests applied to evaluate effects of trace amounts of oxygen on physiological and biochemical processes. The difficulties involved in defining an absolute limit between anoxic and microxic conditions are best illustrated by a logarithmic scale of oxygen pressure or oxygen concentration. In the anoxic state (State 5), any aerobic type of metabolism cannot take place, whereas anaerobic metabolism may proceed under oxic or anoxic conditions.
Background state Y The background state, Y, is the non-activated or inhibited respiratory state at low flux in relation to the reference state, Z. A metabolic control variable, X, acts on Y (substrate, activator) or is removed from Y (inhibitor) to yield Z.
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 [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
Cell ergometry Biochemical 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 ETS 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 ETS excess over OXPHOS capacity and to calculate OXPHOS- and ETS coupling efficiencies, 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
Complex I&II-linked substrate state CI&II, NS See NS-substrate state
Complex I-linked substrate state CI-linked, N See N-junction; NADHComplex I
Complex II-linked substrate state CII, CII-linked, SRot, S CII-linked; SRot: Succinate (S) supports electron flux through Complex II via flavin adenine dinucleotide (FADH2) into the membrane-bound electron transfer system. Inhibition of Complex I by rotenone (Rot; or amytal, piericidine) prevents accumulation of oxaloacetate which is a potent inhibitor of succinate dehydrogenase. After inhibition of CI by rotenone, the NADH-linked dehydrogenases become inhibited by the redox shift from NAD+ to NADH. Succinate dehydrogenase 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 Complex II-linked substrate state is induced in mt-preparations by addition of succinate&rotenone (Complex I inhibitor). Succinate is the direct substrate of Complex II (succinate dehydrogenase). In CII-linked respiration, only Complex III and Complex IV are involved in pumping protons from the matrix (P-phase) to the N-phase with a ~P/O ratio of 1.75 (P/O2 = 3.5).
Complex IV single step CIV, Tm Tm: Electron flow through Complex IV (cytochrome c oxidase) is measured in intact mitochondria after inhibiton of CIII by antimycin A, and addition of ascorbate (As) and the artificial substrate TMPD (Tm). Ascorbate has to be titrated first. It reduces TMPD, which further reduces cytochrome c, which is the substrate of CIV. Since CIV is a proton pump of the electron transfer system, the single step of CIV-linked respiration can be measured in different coupling states: Tm(L), Tm(P), and Tm(E). Measurement of CIV activity requires uncoupler titrations to eliminate any potential control by the phosphorylation system, and a cytochrome c test to avoid any limitation by cytochrome c release.
Coupled respiration Coupled 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 state 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) in any substrate control 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 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.
Critical oxygen pressure pc The critical oxygen pressure, pc, is defined as the partial oxygen pressure, pO2, below which aerobic catabolism (respiration or oxygen consumption) declines significantly. If anaerobic catabolism is activated simultaneously to compensate for lower aerobic ATP generation, then the limiting oxygen pressure, pl, is equal to the pc. In many cases, however, the pl is substantially lower than the pc.
Cross-linked respiratory states CLRS Coordinated respiratory SUIT protocols are designed to include cross-linked respiratory states, which are common to these protocols. Different SUIT protocols address a variety of respiratory control steps which cannot be accomodated in a single protocol. Cross-linked respiratory states are included in each individual coordinated protocol, such that these states can be considered as replicate measurements, which also allow for harmonization of data obtained with these different protocols.
DT-system DT The ADP-ATP phosphorylation system. See Phosphorylation system.
Diapause Diapause is a preprogrammed form of developmental arrest that allows animals to survive harsh environmental conditions and may also allow populations to synchronize periods of growth and reproduction with periods of optimal temperatures and adequate water and food. Diapause is endogenously controlled, and this dormancy typically begins well before conditions become too harsh to support normal growth and development [1,2]. » MiPNet article
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.
ETS capacity E E.jpg ETS capacity is the respiratory electron transfer system capacity, E, of mitochondria in the experimentally induced noncoupled state. The conditions for measurement and expression of respiration vary (oxygen flux in state E, JO2E or oxygen flow in state E, IO2E). If these conditions are defined and remain consistent within a given context, then the simple symbol E for respiratory state is used to substitute the more explicit expression for respiratory activity. In state E, the mt-membrane potential is almost fully collapsed and provides a reference state for flux control ratios. In intact mitochondria, the ETS capacity depends not only on the inner membrane-bound ETS (mETS, with respiratory Complexes CI to CIV, electron-transferring flavoprotein ETF, and glycerophosphate dehydrogenase) but also integrates transporters across the inner mt-membrane, the TCA cycle and other matrix dehydrogenases. Its experimental determination in mitochondrial preparations or intact cells requires the measurement of oxygen consumption in the presence of defined substrates and of an established uncoupler at optimum concentration. This optimum concentration is determined by stepwise titration of the uncoupler up to the concentration inducing maximum flux. » MiPNet article
ETS substrate types n.a.
SUIT-Catg NFSGpTm.jpg

ETS substrate types in mitochondrial SUIT protocols are types of reduced substrates feeding electrons into the electron transfer system (ETS) at different levels of mitochondrial pathways. Distinction of substrate types on the basis of four mt-pathway types provides the rationale for defining categories of SUIT protocols.

ETS substrates type 4 feed electrons into dehydrogenases and enzyme systems upstream of the type 3 pathway level. Electron transfer from type 4 substrates (N and F) converges at the N-junction and F-junction. Representative type N substrates are pyruvate, glutamate and malate, and also citrate, oxoglutarate and others. The corresponding dehydrogenases (PDH, GDH, MDH and mtME; IDH, OgDH) generate NADH, the substrate of Complex I (CI). Type F substrates are fatty acids involved in β-oxidation, generating (enzyme-bound) FADH2, the substrate of electron transferring flavoprotein (CETF). Succinate does not belong to the type 4 substrates, since FADH2 is the product of CII, whereas FADH2 is the substrate of CETF. Fatty acid oxidation (FAO) 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 type N 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 AcetylCo into the TCA cycle catalyzed by citrate synthase.

ETS substrates type 3 (NADH, FADH2, succinate, glycerophosphate) feed electrons into respiratory complexes directly upstream of the Q-junction. NADH is the substrate of Complex I (CI). FADH2 is the substrate of electron transferring flavoprotein (CETF) localized on the inner side of the inner mt-membrane. Succinate is the substrate of succinate dehydrogenase (SDH, CII) localized on the inner side of the inner mt-membrane. Glycerophosphate is the substrate of glycerophosphate dehydrogenase complex (CGpDH) localized on the outer face of the inner mt-membrane. Choline is the type 3 substrate of choline dehydrogenase.

ETS substrates type 2 (Coenzyme Q) feed electrons into Complex III (CIII) with further electron transfer downstream of the Q-junction.

ETS substrates type 1 may be artificial electron donors (e.g. TMPD, Tm) essentially bypassing the ETS, reducing cytochrome c and feeding electrons directly into the terminal electron acceptor, cytochrome c oxidase (CIV) or alternative oxidases (single enzymatic step).
ETS-competent substrate state ETS-competent substrate control state, see Substrate control state.
Electron transfer system ETS E.jpg The mitochondrial electron transfer system (ETS; synonymous with 'electron transport system') transfers electrons 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).
Excess E-P capacity ExP Excess E-P capacity The excess E-P capacity, ExP, is the difference of the ETS 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 approaches E at increasing uncoupling.
Excess E-R capacity ExR Excess E-R capacity The Excess E-R capacity, ExR, is the difference of ETS capacity and ROUTINE respiration, ExR = E-R. For further information, see Cell ergometry.
FAO FAO, F
F-junction
In the fatty acid oxidation or FAO-linked substrate state, one or several fatty acids 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 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 AcetylCo 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 AcetylCo into the TCA cycle catalyzed by citrate synthase.
Free ETS capacity ≈E Free ETS capacity The free ETS capacity, ≈E, is the ETS 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 ETS state, therefore, is partitioned into the free ETS 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 Free OXPHOS capacity 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 Free ROUTINE activity 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.
Glutamate alone G
G
G: Glutamate is a NADH-linked type 4 substrate. When supplied as the sole fuel substrate, G is transported by the electroneutral glutamate-/OH- exchanger, and is oxidised via mt glutamate dehydrogenase in the mitochondrial matrix.
Hyperthermia Hyperthermia in endotherms is a state of stressful up to lethal elevated body core temperature. In humans, the limit of hyperthermia (fever) is considered as >38.3 °C, compared to normothermia at a body temperature of 36.5 to 37.5 °C.
Hypothermia Hypothermia in endotherms is a state of stressful up to lethal low body core temperature. In humans, the limit of hypothermia is considered as 35 °C, compared to normothermia at a body temperature of 36.5 to 37.5 °C. Hypothermia is classified as mild (32–35 °C), moderate (28–32 °C), severe (20–28 °C), and profound (<20 °C).
Hypoxic hypox Hypoxia (hypox) is defined as the state when insufficient O2 is available for respiration.
Intact cells Ce Intact cells (Ce) are characterized by an intact cell membrane. Cell viability should be >95% for various experimental investigations, including cell respirometry. In contrast, the cell membrane of intact cells can be permeabilized selectively by mild detergents (digitonin), to obtain the mt-preparation of permeabilized cells used for cell ergometry.
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 ADP or 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 c50 or p50.
LEAK respiration L L.jpg LEAK 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 (theoretically, absence of inorganic phosphate presents an alternative), or after enzymatic inhibition of the phosphorylation system. 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 is higher than the LEAK component in state P (OXPHOS capacity). The conditions for measurement and expression of respiration vary (oxygen flux in state L, JO2L or oxygen flow in state L, IO2L). If these conditions are defined and remain consistent within a given context, then the simple symbol L for respiratory state can be used as a substitute for the more explicit expression for respiratory activity. » MiPNet article
LEAK state with ATP LT L.jpg LEAK state with ATP, LT, 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).
LEAK state with oligomycin LOmy L.jpg The LEAK state with Omy is a LEAK state induced by inhibition of ATP synthase by oligomycin (LOmy). ADP and ATP may or may not be present.
LEAK state without adenylates LN L.jpg 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 E.jpg 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 oxygen pressure pl The limiting oxygen pressure, pl, is defined as the partial oxygen pressure, pO2, below which anaerobic catabolism is activated to contribute to total ATP generation. The limiting oxygen pressure, pl, may be substantially lower than the critical oxygen pressure, pc, below which aerobic catabolism (respiration or oxygen consumption) declines significantly.
Malate alone M
M
M: Malate alone does not support respiration of mt-preparations if oxaloacetate cannot be metabolized further in the absence of a source of acetyl-CoA. Transport of oxaloacetate across the inner mt-membrane is restricted particularly in liver. Mitochondrial citrate and 2-oxoglutarate (α-ketoglutarate) are depleted by antiport with malate. Succinate is lost from the mitochondria through the dicarboxylate carrier. OXPHOS capacity with malate alone is only 1.3% of that with Pyruvate&Malate in isolated rat skeletal muscle mitochondria. Many mammalian and non-mammalian mitochondria have a mt-isoform of NADP+- or NAD(P)+-dependent malic enzyme (ME), the latter being particularly active in proliferating cells. Then malate alone can support high respiratory activities.
Mitochondrial membrane potential mtMP, Δψmt The mitochondrial membrane potential, mtMP, is the electric part of the protonmotive force, Δpmt.

Δψmt = Δpmt - ΔµH+ / F

mtMP or Δψmt 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
N-linked substrate state N
N-junction
NADH-linked substrates (CI-linked) are type N substrates of ETS-level 4, feeding electrons into the N-junction catalyzed by various mt-dehydrogenases. N-supported flux is induced in mt-preparations by addition of NADH-generating substrates individually or in combination: pyruvate, glutamate, malate, oxoglutarate, citrate, hydroxybutyrate. These type N substrates are (indirectly) linked to Complex I by the corresponding dehydrogenase-catalyzed reactions reducing NAD+ to NADH+H+. 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 pesence of type N substrates can be further suppressed by the CII-inhibitor malonic acid).
NS e-input NS, CI&II NS e-input (CI&II e-input) is electron input from a combination of N and S substrates through Complexes CI and CII simultaneously into the Q-junction corresponding to TCA cycle function in vivo, with convergent electron flow through the ETS. In mt-preparations, NS e-input requires addition not only of N (CI-linked) substrate (pyruvate&malate or glutamate&malate), but of S (succinate) simultaneously, since metabolite depletion in the absence of succinate prevents a significant stimulation of S-linked respiration. For more details, see: Additive effect of convergent electron flow.
NS-substrate state NS, CI&II
NS-substrate state
The NS-linked substrate state (flux in the NS-linked substrate state, NS; or Complex I&II, CI&II-linked substrate state) is induced in mt-preparations by addition of NADH-generating substrates (N-linked substrate state, or CI-linked pathway control) in combination with succinate (S- or CII-linked). Whereas NS expresses substrate control in terms of substrate types (N and S), CI&II defines the same concept in terms of the convergent pathway to the Q-junction (pathway control). NS is the abbreviation for the combination of N- or NADH-linked substrates (CI-linked) and S- or succinate-linked substrates (CII-linked). 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 E.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 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 (noncoupled) ETS 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 ETS capacity).
Normothermia Normothermia in endotherms is a state when body core temperature is regulated within standard limits. In humans, normothermia is considered as a body temperature of 36.4 to 37.8 °C. Normothermia, however, has a different definition in the context of ectotherms. » MiPNet article
OXPHOS capacity P P.jpg OXPHOS capacity (P) is the respiratory capacity of mitochondria in the ADP-activated state of oxidative phosphorylation, at saturating concentrations of ADP (compare State 3), 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). Oxygen consumption in the OXPHOS state, P, 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. 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. If conditions for measurement and expression of respiration vary, explicit symbols are used, expressing OXPHOS capacity as oxygen flux in state P, JO2P or as oxygen flow in state P, IO2P. If these conditions are defined and remain consistent within a given context, then the simple symbol P for respiratory state can be used to substitute the more explicit expression for respiratory activity.
Oligomycin Omy Oligomycin (Omy) is an inhibitor of ATP synthase by blocking its proton channel (Fo subunit), which is necessary for oxidative phosphorylation of ADP to ATP (energy production). The inhibition of ATP synthesis also inhibits respiration. In OXPHOS analysis, Omy is used to induce a LEAK state of respiration.
Oxidative phosphorylation OXPHOS P.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 and accompanied by an intrinsically uncoupled component of respiration. The OXPHOS state (P) of respiration provides a measure of OXPHOS capacity, which is frequently corrected for residual oxygen consumption (ROX).
Oxidative stress Oxidative stress results from an imbalance between pro-oxidants and antioxidants shifting the equilibrium in favor of the pro-oxidants. This process can be due by an increment in pro-oxidants, by a depletion of antioxidant systems or both. Oxidative stress generates oxidative damage of proteins, lipids and DNA.
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%.
PGMOct PGMOct PGMOct: Pyruvate & Glutamate & Malate & Octanoylcarnitine.

MitoPathway control: CI&FAO

SUIT protocols: SUIT-RP1, SUIT-RP2
PGMS PGMS
PGMS
PGMS: Pyruvate & Glutamate & Malate & Succinate.

MitoPathway control: CI&II


2-oxoglutarate is produced through the citric acid cycle from citrate by isocitrate dehydrogenase, from oxaloacetate and glutamate by the transaminase, and from glutamate by the glutamate dehydrogenase. If the 2-oxoglutarate carrier does not outcompete these sources of 2-oxoglutarate, then the TCA cycle operates in full circle with external pyruvate&malate&glutamate&succinate
PGMSOct PGMSOct PGMSOct: Pyruvate & Glutamate & Malate & Succinate & Octanoylcarnitine.

MitoPathway control: CI&II&FAO

SUIT protocol: SUIT-RP1, SUIT-RP2

This substrate combination supports convergent electron flow to the Q-junction.
PGMSOctGp PGMSOctGp PGMSOctGp: Pyruvate & Glutamate & Malate & Succinate & Octanoylcarnitine & Glycerophosphate.

MitoPathway control: CI&II&FAO&GpDH

SUIT protocol: SUIT-RP2

This substrate combination supports convergent electron flow to the Q-junction.
PM PM
PM
PM: Pyruvate & Malate.

MitoPathway control: CI

SUIT protocol: SUIT-RP1

Pyruvate (P) is oxidatively decarboxylated to acetyl-CoA and CO2, yielding NADH catalyzed by pyruvate dehydrogenase. Malate (M) is oxidized to oxaloacetate by mt-malate dehydrogenase located in the mitochondrial matrix. Condensation of oxaloacate with acetyl-CoA yields citrate (citrate synthase). 2-oxoglutarate (α-ketoglutarate) is formed from isocitrate (isocitrate dehydrogenase).
PMOct PMOct PMOct: Pyruvate & Malate & Octanoylcarnitine.

MitoPathway control: CI&FAO

SUIT protocol: SUIT-RP1, SUIT-RP1

This substrate combination supports CI-linked flux which is typically higher than FAO capacity. In SUIT-RP1, PMOct is induced after PM(E), to evaluate any additive effect of adding Oct. In SUIT-RP2, FAO OXPHOS capacity is measured first, testing for the effect of increasing malate concentration (compare Malate alone, and pyruvate is added to compare FAO as the background state with CI&FAO as the reference state.
Phosphorylation system DT
From Gnaiger 2014 MitoPathways
The phosphorylation system is the functional unit utilizing the protonmotive force to phosphorylate ADP (D) to ATP (T), and may be defined more specifically as the DT-phosphorylation system or DT-system. The DT-system consists of adenylate nucleotide translocase, phosphate carrier, and ATP synthase. Mitochondrial adenylate kinase, mt-creatine kinase and mt-hexokinase constitute extended components of the DT-phosphorylation system, controlling local AMP and ADP concentrations and forming metabolic channels. Since substrate-level phosphorylation is involved in the TCA-cycle, the mtDT system includes succinyl-CoA synthase (GDP to GTP or ADP to ATP).
ROUTINE respiration R R.jpg In the intact cell, ROUTINE respiration or ROUTINE activity 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). 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 stimulated or un-inhibited by a metabolic control variable, X, with high flux in relation to the background state, Y.
Residual oxygen consumption ROX ROX.jpg 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). Mitochondrial respiration is frequently corrected for ROX, then distinguishing ROX-corrected ROUTINE, LEAK, OXPHOS or ETS (R, L, P and E) from the corresponding apparent fluxes that have not been corrected for ROX (R´, L´, P´ and E´). When expressing ROX as a fraction of total respiration (flux control ratio), apparent flux 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 substrate control 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).
S S
Succinate
Succinate, an ETS-level 3 substrate; succinate-induced respiratory state, see Succinate alone.
SGp SGp SGp: Succinate & Glycerophosphate.

MitoPathway control: CII&GpDH; obtained as PGMSOctGp(Rot)

SUIT protocol: SUIT-RP1, SUIT-RP2

This pathway control state is obtained in the presence of CI-linked and FAO-linked substrates after inhibition of CI by rotenone, which simultaneously inhibits FAO.
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 ROX.jpg 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 P.jpg 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 E.jpg Noncoupled 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 noncoupled ETS capacity (E; State 3u) (Gnaiger 2009; Rasmussen and Rasmussen 2000).
State 4 LT L.jpg 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.
Static head L L.jpg 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 state Substrate control states are obtained in mitochondrial preparations (isolated mitochondria, permeabilized cells, permeabilized tissues, tissue homogenate) by depletion of endogenous substrates and addition of specific ETS substrate types to the mitochondrial respiration medium. Mitochondrial substrate control states have to be defined complementary to mitochondrial coupling control states. Coupling states (LEAK, OXPHOS, ETS) require electron transfer system competent substrate states, including oxygen supply. Categories of SUIT protocols are defined according to ETS substrate types. » MiPNet article
Succinate alone S
S
S: When succinate is added without rotenone, oxaloacetate is formed from malate by the action of malate dehydrogenase. Oxaloacetate accumulates and is a more potent competitive inhibitor of succinate dehydrogenase than malonate even at small concentration. Reverse electron flow from CII to CI is known to stimulate production of reactive oxygen species under these conditions to extremely high, nonphysiological levels. Addition of malate reduces superoxide production with succinate, probably due to a shift in the redox state and oxaloacetate inhibition of CII. Compare: Complex II-linked substrate state.
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