MitoPedia: SUIT

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MitoPedia

MitoPedia: SUIT

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

MitoFit
MitoPedia: SUIT is a component of OROBOROS support.
In the Library of SUIT protocols
» MitoPedia: SUIT - you are here: SUIT A, SUIT concept, and a list of all SUIT protocols.
»A: MitoPedia: SUIT A - these are SUIT protocols used for cohort studies.
»B: MitoPedia: SUIT B - these are published SUIT protocols considered less suitable for cohort studies.
»C: MitoPedia: SUIT C - these are SUIT protocols used for exploratory research.
See also:
» O2k-Protocols: O2k-Demo experiments


Communicated by Doerrier C, Plattner C, Gnaiger E 2016-05-13; edited 2016-11-08.
SUIT protocols

SUIT: Substrate-uncoupler-inhibitor-titration

  • SUIT protocols are applied to mitochondrial preparations and are categorized by (i) the SUIT category, (ii) A further distinction is provided in the SUIT name by listing in parentheses the N-type substrates applied, again independent of the sequence of titrations, e.g. NS(GM), NS(PM), FNSGp(PGM). (iii) A sequentially selected number is added, e.g. SUIT_FNS(PM)01 (see Figure).
  • Coupling control protocols (CCP) are a special case of simple SUIT protocols primarily applied to intact cells (ce), typically with a sequence of ROUTINE respiration (R), optionally oligomycin-induced LEAK respiration (L), uncoupler-stimulated ETS capacity (E), and inhibitor-induced residual oxygen consumption (ROX). Cell-membrane impermeable substrates (succinate, ADP) may be titrated to assess the fraction of permeabilized cells (pce). The name of a CCP starts with CCP optionally followed by the abbreviation for any substrate types titrated and a sequentially selected number, e.g. CCP(S)01. It is of basic importance to provide information on the composition of the respiration medium, particularly distinguishing media with exogenous substrates (culture media) and mt-respiration media (e.g. MiR05) excluding respiratory ce-substrates, when respiration is strictly based on endogenous ETS substrates.
  • Educational: The library of SUIT protocols was developed to introduce the basic concept by simple coupling/pathway control diagrams.
  • Practical: The SUIT protocols must meet the detailed practical requirements to provide a complete guide through the practical experiment, defining every titration step.
  • Compatible: The nomenclature must provide a unique term for each family of SUIT protocols and each particular variation within a family, compatibel with a database.
  • Fast: The terminology should help to recognize the essentially features of a SUIT protocol sufficiently fast, but detailed explanations are provided in the definitions, explanations and discussions.
  • SUIT sequence - sequence of titration steps: Each titration step on coupling control and pathway control starts with a sequential number, followed by the acronym of the substance titrated.
  • Quality control: Titration steps related to quality control (e.g. cytochrome c test) do not have a number, but start with "_", followed by the acronym of the previously titrated coupling/pathway control variable and by the substrated titrated for quality control in parentheses.
ROX-continued.jpg
indicates in SUIT protocols the option to extend the titrations by (1) inhibition of electron transfer (typically by antimycin A, Ama) to induce ROX, optionally followed by an assay of cytochrome c oxidase activity, with sequential titration of ascorbate and TMPD (AsTm) and azide (Azd) for evaluation of the chemical O2 background due to autoxidation reactions.


Library of SUIT protocols: SUIT A

SUIT familySystematic nameCoupling/pathway control diagramReference
CCP(S)02CCP(S)02_R,1SD,2Omy,3U-4Rot,5AmaCCP(S)02.jpgA Stadlmann 2006 Cell Biochem Biophys
CCP(S)03CCP(S)03_R,1U-2Rot,3S,4AmaCCP(S)03.jpgA CCP_RP1
CCP(S)04CCP(S)04_R,1Omy,2U-3Rot,4S,5AmaCCP(S)04.jpgA CCP_RP2
CCP01CCP01_R,1U-CCP 01.jpgA Steinlechner-Maran_1996_Am J Physiol Cell Physiol
CCP02CCP02_R,1Omy,2U-CCP 02.jpgA Huetter 2004 Biochem J
SUIT FNS(GM)01FNS(GM)01_1OctM,2D,3G,4S,5Rot,6Omy,7U-SUIT FNS(GM)01.jpgA Gnaiger 2015 Scand J Med Sci Sports
SUIT FNS(GM)02FNS(GM)02_1OctM,2D,3G,4S,5U,6Rot-SUIT FNS(GM)02.jpgA Pesta 2012 Methods Mol Biol
SUIT FNS(PGM)02FNS(PGM)02_1OctM,2D,3G,4P,5S,6U,7Rot-SUIT FNS(PGM)02.jpgA Schoepf 2016 FEBS J
SUIT FNS(PM)01FNS(PM)01_1OctM,2D,3P,4S,5U,6Rot-SUIT FNS(PM)01.jpgA linked to SUIT_FNSGp(PGM)02 - SUIT RP2 (human skeletal muscle)
SUIT FNSGp(PGM)01FNSGp(PGM)01_1PM,2D,3U,4G,5S,6Oct,7Rot,8Gp-SUIT-RP1A: SUIT_RP1 - MiPNet21.06 SUIT RP
SUIT FNSGp(PGM)02FNSGp(PGM)02_1D,2OctM,3P,4G,5S,6Gp,7U,8Rot-SUIT FNSGp(PGM)02.jpgA: SUIT_RP2 - MiPNet21.06 SUIT RP
SUIT N(PGM)01N(PGM)01_1PGM,2D,3U-SUIT N(PGM)01.jpgA Krumschnabel_2014_Methods Enzymol; linear segment in SUIT_NS(PGM)04
SUIT NS(PGM)02NS(PGM)02_1PM,2D,3G,4S,5U,6Rot-SUIT NS(PGM)02.jpgA Lemieux 2017 bioRxiv
SUIT NS(PGM)03NS(PGM)03_1GM,2D,3P,4S,5U,6Rot-SUIT NS(PGM)03.jpgA Lemieux 2017 bioRxiv
SUIT NS(PM)01NS(PM)01_1PM,2D,3U,4S,5Rot-SUIT NS(PM)01.jpgA linked to SUIT_FNSGp(PGM)01 - SUIT RP1 (human skeletal muscle)

SUIT states

TermAbbreviationDescription
Anaplerotic pathway control stateaAnaplerotic pathway control states are fuelled by single substrates which are transported into the mitochondrial matrix and increase the pool of intermediates of the tricarboxylic acid cycle. Malic enzyme (mtME), phosphoenopyruvate carboxykinase (PEPCK), propionyl-CoA carboxylase, and pyruvate carboxylase play important roles in anaplerosis. The glutamate anaplerotic pathway control state and malate anaplerotic pathway control state are the most important anaplerotic substrate control states (aN).
Complex I&II-linked substrate stateCI&II-linked, NSSee NS-pathway control state
Complex I-linked substrate stateCI-linked, NSee N-pathway control state
Complex II-linked substrate stateCII-linked, SRot, SSee S-pathway control state
Complex IV single stepCIVCIV: 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: L, P, and 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. Total oxygen uptake in the ascorbate&TMPD(&c) stimulated state (Tm) has to be corrected for chemical background oxygen consumption.
Coupling control stateCCSCoupling 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 pathway 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 pathway control state.
Fatty acid oxidation pathway control stateF, FAO
F-junction
In the fatty acid oxidation pathway control state (F, FAO), 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.
GM pathway control stateGM
GM
GM: Glutamate & Malate.

MitoPathway control state: N

When glutamate&malate are added to isolated mitochondria or permeabilized cells, glutamate and transaminase are responsible for the metabolism of oxaloacetate, comparable to the metabolism with acetyl-CoA and citrate synthase.
Glutamate anaplerotic pathway control stateG
G
G: Glutamate is an anaplerotic NADH-linked type 4 substrate (aN). When supplied as the sole fuel substrate in the glutamate pathway control state, G is transported by the electroneutral glutamate-/OH- exchanger, and is oxidised via mt-glutamate dehydrogenase in the mitochondrial matrix.
Glycerophosphate pathway control stateGp
Gp-pathway
The glycerophosphate pathway control state (Gp) is an ETS 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 system at the level of CoQ.
Malate anaplerotic pathway control stateM
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 (mtME), the latter being particularly active in proliferating cells. Then the anaplerotic pathway control state with malate alone (aN) supports high respiratory activities comparable to the NADH-linked pathway control states (N) with with pyruvate&malate or glutamate&malate substrate combinations (PM pathway control state, GM pathway control state).
NADH pathway control stateN
N-junction
The NADH pathway control state (N) is obtained by addition of NADH-linked substrates (CI-linked), feeding electrons into the N-junction catalyzed by various mt-dehydrogenases. N-supported flux is induced in mt-preparations by addition of NADH-generating substrate combinations of pyruvate (P), glutamate (G), malate (M), oxaloacetate (Oa), oxoglutarate (Og), citrate, hydroxybutyrate. These N-junction substrates are (indirectly) linked to Complex I by the corresponding dehydrogenase-catalyzed reactions reducing NAD+ to NADH+H+. The most commonly applied N-junction substrate combinations are: PM, GM, PGM. The malate anaplerotic pathway control state (M alone) is a special case related to malic enzyme (mtME). The glutamate anaplerotic pathway control state (aN; G alone) supports respiration through glutamate dehydrogenase (mtGDH). 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 ETS pathway level 4 is active.
NS-pathway control stateNS, CI&II
NS-pathway control
NS-pathway control is exerted in the NS-linked substrate state (flux in the NS-linked substrate state, NS; or Complex I&II, CI&II-linked substrate state). NS is induced in mt-preparations by addition of NADH-generating substrates (N-pathway control state, or CI-linked pathway control) in combination with succinate (S-pathway control state; 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.
OctPGM pathway control stateOctPGMOctPGM: Octanoylcarnitine & Pyruvate & Glutamate & Malate.

MitoPathway control state: FN

SUIT protocols: SUIT_FNSGp(PGM)01 - SUIT RP1, SUIT_FNSGp(PGM)01 - SUIT RP2
OctPGMS pathway control stateOctPGMSOctPGMS: Octanoylcarnitine & Pyruvate & Glutamate & Malate & Succinate.

MitoPathway control state: FNS

SUIT protocol: SUIT_FNSGp(PGM)01 - SUIT_RP1, SUIT_FNSGp(PGM)02 - SUIT RP2

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

MitoPathway control state: FNSGp

SUIT protocol: SUIT_FNSGp(PGM)02 - SUIT RP2

This substrate combination supports convergent electron flow to the Q-junction.
OctPM pathway control stateOctPMOctPM: Octanoylcarnitine & Pyruvate & Malate.

MitoPathway control state: FN

SUIT protocol: SUIT_FNSGp(PGM)01 - SUIT RP1, SUIT_FNSGp(PGM)02 - SUIT RP1

This substrate combination supports N-linked flux which is typically higher than FAO capacity (F/FN<0 in the OXPHOS state). 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 anaplerotic pathway control state, M alone), and pyruvate is added to compare FAO as the background state with FN as the reference state.
PGMS pathway control statePGMS
PGMS
PGMS: Pyruvate & Glutamate & Malate & Succinate.

MitoPathway control state: NS

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
PM pathway control statePM
PM
PM: Pyruvate & Malate.

MitoPathway control state: N

SUIT protocol: SUIT_FNSGp(PGM)01 - 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).
Pathway control statePCS, mtPCS
SUIT-catg FNSGpCIV.jpg

Pathway control states (synonymous with ETS substrate control 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. Mitochondrial pathway control states, mtPCS, have to be defined complementary to mitochondrial coupling control states. Coupling states (LEAK, OXPHOS, ETS) require electron transfer system competent substrate control states, including oxygen supply. Categories of SUIT protocols are defined according to mitochondrial pathway control states.

» MiPNet article
SGp-pathway control stateSGpSGp: Succinate & Glycerophosphate.

MitoPathway control state: SGp; obtained with OctPGMSGp(Rot)

SUIT protocol: SUIT_FNSGp(PGM)01 - SUIT RP1, SUIT_FNSGp(PGM)02 - 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.
Succinate control stateS
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, pathological levels. Addition of malate reduces superoxide production with succinate, probably due to a shift in the redox state and oxaloacetate inhibition of CII. Compare: S-pathway control state.
Succinate pathway control stateS
Succinate

The Succinate pathway control state (S) is achieved with succinate as the single substrate, at ETS-level pathway level 3: succinate-induced respiratory state; CII-linked; SRot. S supports electron flux through Complex II to CII-bound flavin adenine dinucleotide (FADH2) to Q. 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).

SUIT concept

TermAbbreviationDescription
Categories of SUIT protocolsSUIT-catg
SUIT-catg MitoPathway types.jpg

Categories of SUIT protocols group SUIT protocols according to all pathway control states involved in a protocol (controlling ETS pathway types; e.g. S, NS, FNS, FNSGp), independent of titrations of inhibitors which block the oxidation of specific substrate types. ROX states may or may not be included in a SUIT protocol, which does not change its category.

  • F - ETS-level 5: FADH2-linked substrates (FAO) with obligatory support by the N-linked pathway.
  • N - ETS-level 4: NADH-linked substrates (CI-linked).
  • S - ETS-level 3: Succinate (CII-linked).
  • Gp - ETS-level 3: Glycerophosphate (CGpDH-linked).
  • CIV - ETS-level 1: Artificial electron transfer susbstrate TMPD (Tm) maintained in a reduced state by ascorbate (As) and reducing cytochrome c as the substrate of CIV.
» MiPNet article
Cell respirationCell respiration channels metabolic fuels into the chemiosmotic coupling (bioenergetic) machinery of oxidative phosphorylation, being regulated by and regulating oxygen consumption (or consumption of an alternative final electron acceptor) and molecular redox states, ion gradients, mitochondrial (or microbial) membrane potential, the phosphorylation state of the ATP system, and heat dissipation in response to intrinsic and extrinsic energy demands. See also respirometry. In internal or cell respiration in contrast to fermentation, redox balance is maintained by the use of external electron acceptors, transported into the cell from the environment. The chemical potential from electron donors to electron acceptors is converted in the electron transfer system to generate a chemiosmotic potential that in turn drives ATP synthesis.
Coupling control protocolCCPA coupling control protocol, CCP, induces different coupling control states at constant substrate supply. In intact cells, the CCP can be applied by using membrane-permeable inhibitors of the phosphorylation system (e.g. oligomycin) and uncouplers (e.g. CCCP). 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. The term phosphorylation control protocol, PCP, has been introduced synonymous for CCP. » MiPNet article
Coupling control stateCCSCoupling 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 pathway 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 pathway control state.
Coupling/pathway control diagramCPCD
SUIT protocols
Coupling/pathway control diagrams illustrate the respiratory states obtained step-by-step in substrate-uncoupler-inhibitor titrations in a SUIT protocol. Each step (to the next state) is defined by an initial state and a metabolic control variable, X. The respiratory states are shown by boxes. X is usually the titrated substance in a SUIT protocol. If X (ADP, uncouplers, or inhibitors of the phosphorylation system, e.g. oligomycin) exerts coupling control, then a transition is induced between two coupling control states. If X (fuel substrates, e.g. pyruvate and succinate, or ETS inhibitors, e.g. rotenone) exerts pathway control, then a transition is induced between two pathway control states. The type of metabolic control (X) is shown by arrows linking two respiratory states, with vertical arrows indicating coupling control, and horizontal arrows indicating pathway control. Marks in DatLab define the section of an experimental trace in a given respiratory state (steady state). Events in DatLab define the titration of X inducing a transition in the SUIT protocol. The specific sequence of coupling control and pathway control steps defines the SUIT protocol pattern. The coupling/pathway control diagrams define the categories of SUIT protocols (see Figure).
Cross-linked respiratory statesCLRSCoordinated 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.
DatLab and SUIT protocolsThis is a brief summary of steps to be taken for performing a high-resolution respirometry experiment with SUIT protocols using the OROBOROS Oxygraph-2k and DatLab software. (1) Search for a specific SUIT protocol name (go to MitoPedia: SUIT). The list of MitoPedia SUIT protocols can be sorted by categories of SUIT protocols (sorting by SUIT protocol name), which is listed as the 'abbreviation' of the SUIT protocol name. (2) Copy the template for Mark names into your DatLab subdirectory: DatLab\APPDATA\MTEMPLAT. (3) Copy the DatLab-Analysis template for this SUIT protocol. (4) Follow the link to the corresponding publication or MiPNet communication, where the pdf file describing the SUIT protocol is available. (5) A DatLab demo file may be available providing an experimental example. After each sequential titration, a mark is set on the plot for flux or flow. After having set all marks, pull down the 'Mark names' menu, select the corresponding SUIT protocol for mark names, and rename all marks. The Mark names template also provides standard values of the titration volume preceding each mark. (6) Go to 'Mark statistics' [F2], copy to clipboard, and paste into the sample tab in the DatLab-Analysis template.
Example:
  • SUIT protocol name: SUIT_NS(GM)01
  • Mark names in DatLab: SUIT_NS(GM)01
  • DatLab-Analysis template: SUIT_NS(GM)01.xlsx
  • MiPNet communciation: MiPNet12.23 FibreRespiration
  • DatLab demo file: MiPNet12.23 FibreRespiration.DLD
ETS substrate typesn.a.See Pathway control state
F-junction
F-junction
The F-junction is a junction for convergent electron flow in the electron transfer system (ETS) from fatty acids through fatty acyl CoA dehydrogenase (reduced form FADH2) to electron transferring flavoprotein (CETF), and further transfer through the Q-junction to Complex III (CIII). The concept of the F-junction and N-junction provides a basis for defining categories of SUIT protocols. Fatty acid oxidation, in the F-pathway control state, not only depends on electron transfer through the F-junction (which is typically rate-limiting) but simultaneously generates NADH and thus depends on N-junction throughput. Hence FAO can be inhibited completely by inhibition of Complex I (CI). In addition and independent of this source of NADH, the N-junction substrate malate is required as a co-substrate for FAO in mt-preparations, since accumulation of AcetylCoA inhibits FAO in the absence of malate. Malate is oxidized in a reaction catalyzed by malate dehydrogenase to oxaloacetate (yielding NADH), which then stimulates the entry of AcetylCoA into the TCA cycle catalyzed by citrate synthase.
Flux control factorFCFFlux control factors express the control of respiration by a metabolic control variable, X, as a fractional change of flux from YX to ZX, normalized for ZX. ZX is the reference state with high (stimulated or un-inhibited) flux; YX is the background state at low flux, upon which X acts.
jX = (ZX-YX)/ZX = 1-YX/ZX

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

» MiPNet article
Flux control ratioFCRFlux control ratios (FCR), are ratios of oxygen flux in different respiratory control states, normalized for maximum flux in a common reference state, to obtain theoretical lower and upper limits of 0.0 and 1.0 (0% and 100%). For a given protocol or set of respiratory protocols, flux control ratios provide a fingerprint of coupling and substrate control independent of (i) mt-content in cells or tissues, (ii) purification in preparations of isolated mitochondria, and (iii) assay conditions for determination of tissue mass or mt-markers external to a respiratory protocol (CS, protein, stereology, etc.). FCR obtained from a single respirometric incubation with sequential titrations (sequential protocol; SUIT protocol) provide an internal normalization, expressing respiratory control independent of mitochondrial content and thus independent of a marker for mitochondrial amount. FCR obtained from separate (parallel) protocols depend on equal distribution of subsamples obtained from a homogenous mt-preparation or determination of a common mitochondrial marker.
Harmonized SUIT protocolsH-SUITHarmonized SUIT protocols (H-SUIT) are designed to include cross-linked respiratory states. When performing harmonized SUIT protocols in parallel, measurements of cross-linked respiratory states can be statistically evaluated as replicates across protocols. Additional information is obtained on respiratory coupling and substrate control by including respiratory states that are not common (not cross-linked) across the harmonized protocols.
Metabolic control variableXA metabolic control variable, X, causes the transition between a background state, YX, and a reference state, ZX. X may be a stimulator or activator of flux, inducing the step change from background to reference steady state (Y to Z). Alternatively, X may be an inhibitor of flux, absent in the reference state but present in the background state (step change from Z to Y).
MitoFit protocolsMitoFit protocols are moderated by the MitoFit moderators (MitoFit team), either as protocols with direct reference to publications available to the scientific communicty, or protocols additionally described and made available in Bioblast with full information on authors (including contact details), author contribuitons, and editor (moderator) in charge. This is part of the MitoFit Quality Control System for establishing a comprehensive MitoFit data repository, which will require global input and cooperation.
N-junction
N-junction
The N-junction is a junction for convergent electron flow in the electron transfer system (ETS) from type N substrates (further details »N-pathway control state) through the mt-NADH pool to Complex I (CI), and further transfer through the Q-junction to Complex III (CIII). Representative type N substrates are pyruvate (P), glutamate (G) and malate (M). The corresponding dehydrogenases (PDH, GDH, MDH) and some additional TCA cycle dehydrogenases (isocitrate dehydrogenase, oxoglutarate dehydrogenase generate NADH, the substrate of Complex I (CI). The concept of the N-junction and F-junction provides a basis for defining categories of SUIT protocols based on pathway control states.
NS e-inputNS, CI&IINS e-input or the NS-pathway control state is electron input from a combination of substrates for the N-pathway control state and S-pathway control state through Complexes CI and CII simultaneously into the Q-junction. NS e-input corresponds to TCA cycle function in vivo, with convergent electron flow through the ETS. In mt-preparations, NS e-input requires addition not only of NADH- (N-) linked substrates (pyruvate&malate or glutamate&malate), but of succinate (S) 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.
OXPHOS capacityPP.jpg OXPHOS capacity (P) is the respiratory capacity of mitochondria in the ADP-activated state of oxidative phosphorylation, at saturating concentrations of ADP (possibly in contrast to State 3), inorganic phosphate, oxygen, and defined reduced substrates. » MiPNet article
Pathway control statePCS, mtPCS
SUIT-catg FNSGpCIV.jpg

Pathway control states (synonymous with ETS substrate control 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. Mitochondrial pathway control states, mtPCS, have to be defined complementary to mitochondrial coupling control states. Coupling states (LEAK, OXPHOS, ETS) require electron transfer system competent substrate control states, including oxygen supply. Categories of SUIT protocols are defined according to mitochondrial pathway control states.

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Phosphorylation systemDT
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).
Physiological pathway control stateSee Pathway control state.
Q-junction
Q-junction
The Q-junction is a junction for convergent electron flow in the electron transfer system (ETS) from type N substrates and mt-matix dehydrogenases through Complex I (CI), from type F substrates and FA oxidation through electron-transferring flavoprotein complex (CETF), from succinate (S) through Complex II (CII), from glycoreophosphate (Gp) through glycerophosphate dehydrogenase complex (CGpDH), from choline through choline dehydrogenase, from dihydro-orotate through dihydro-orotate dehydrogenase, and other enzyme complexes into the Q-cycle (ubiquinol/ubiquinone), and further downstream to Complex III (CIII) and CIV. The concept of the Q-junction, with the N-junction and F-junction upstream, provides the rationale for defining pathway control states and categories of SUIT protocols.
Respiratory stateRespiratory states of mitochondrial preparations and intact cells are defined in the current literature in many ways and with a diversity of terms. Mitochondrial respiratory states must be defined in terms of both, the coupling control state and the pathway control state.
SUITSUITSUIT is the abbreviation for Substrate-Uncoupler-Inhibitor Titration. SUIT protocols are used with mt-preparations to study respiratory control in a sequence of coupling and substrates states induced by multiple titrations within a single experimental assay. Further details: Substrate-uncoupler-inhibitor titration, MitoPedia: SUIT.
SUIT protocol librarySUITsThe Substrate-uncoupler-inhibitor titration (SUIT) protocol library contains classes of SUIT protocols with coupling and substrate control defined for mitochondrial preparations.
SUIT protocol namesSUITp-Names
SUIT protocols

The short name of a SUIT protocol starts with (i) the SUIT category which shows the pathway control states (ETS pathway types; e.g. N, S, NS, FNS, FNSGp), independent of the actual sequence of titrations. (ii) A further distinction is provided in the SUIT name by listing in parentheses the substrates applied in the N-pathway control states, again independent of the sequence of titrations, e.g. NS(GM), NS(PM), FNSGp(PGM). (iii) A sequentially selected number is added, e.g. SUIT_FNS(PM)01 (see Coupling/pathway control diagram).

The systematic name of a SUIT protocol starts with the SUIT category, followed by an underline dash and the sequence of titration steps (mark names, #X, separated by a comma). The Marks define the section of a respiratory state in the SUIT protocol. The Mark name contains the sequential number and the metabolic control variable, X. The metabolic control variable is the name of the preceding SUIT event. The MitoPedia list of SUIT protocols can be sorted by the short name or the systematic name (hence by SUIT protocol category. The SUIT protocol pattern is best illustrated by a coupling/pathway control diagram.
SUIT protocol patternSUITp-PatternThe SUIT protocol pattern describes the type of the sequence of coupling and substrate control steps in a SUIT protocol, which may be liner, orthogonal, or diametral.
SUIT reference protocolSUIT RPThe substrate-uncoupler-inhibitor titration (SUIT) reference protocol, SUIT RP, provides a common baseline for comparison of mitochondrial respiratory control in a large variety of species, tissues and cell types, mt-preparations and laboratories, for establishing a database on comparative mitochondrial phyisology. The SUIT RP consists of two harmonized SUIT protocols (SUIT_FNSGp(PGM)01 - SUIT RP1 and SUIT_FNSGp(PGM)02 - SUIT RP2). These are coordinated such that they can be statistically evaluated as replicate measurements of cross-linked respiratory states, while additional information is obtained when the two protocols are conducted in parallel. Therefore, these harmonized SUIT protocols are complementary with their focus on specific respiratory coupling and pathway control aspects, extending previous strategies for respirometrc OXPHOS analysis.
SUIT_RP1: SUIT_FNSGp(PGM)01 - FNSGp(PGM)01_D(c)-CIV: FNSGp(PGM)01_1PM,2D(c),3U,4G,5S,6Oct,7Rot,8Gp,9Ama,10Tm,11Azd
SUIT_RP2: SUIT_FNSGp(PGM)02 - FNSGp(PGM)02_M(c)-CIV: FNSGp(PGM)02_1D(M.1),2Oct,3M2(c),4P,5G,6S,7Gp,8U,9Rot,10Ama,11Tm,12Azd
Substrate control stateSee Pathway control state
Substrate-uncoupler-inhibitor titrationSUITMitochondrial Substrate-uncoupler-inhibitor titration (SUIT) protocols are used with mitochondrial preparations to study respiratory control in a sequence of coupling and substrates states induced by multiple titrations within a single experimental assay.

Library of SUIT protocols: all SUIT protocols

SUIT familySystematic nameCoupling/pathway control diagramReference
CCP(S)01CCP(S)01_R,1S,2U-3Rot,4AmaCCP(S)01.jpgB Steinlechner-Maran 1997 Transplantation
CCP(S)02CCP(S)02_R,1SD,2Omy,3U-4Rot,5AmaCCP(S)02.jpgA Stadlmann 2006 Cell Biochem Biophys
CCP(S)03CCP(S)03_R,1U-2Rot,3S,4AmaCCP(S)03.jpgA CCP_RP1
CCP(S)04CCP(S)04_R,1Omy,2U-3Rot,4S,5AmaCCP(S)04.jpgA CCP_RP2
CCP01CCP01_R,1U-CCP 01.jpgA Steinlechner-Maran_1996_Am J Physiol Cell Physiol
CCP02CCP02_R,1Omy,2U-CCP 02.jpgA Huetter 2004 Biochem J
SUIT FNS(GM)01FNS(GM)01_1OctM,2D,3G,4S,5Rot,6Omy,7U-SUIT FNS(GM)01.jpgA Gnaiger 2015 Scand J Med Sci Sports
SUIT FNS(GM)02FNS(GM)02_1OctM,2D,3G,4S,5U,6Rot-SUIT FNS(GM)02.jpgA Pesta 2012 Methods Mol Biol
SUIT FNS(PGM)01FNS(PGM)01_1PalM,2D,3Oct,4P,5G,6U,7S,8Rot-SUIT FNS(PGM)01.jpgB Lemieux 2011 Int J Biochem Cell Biol
SUIT FNS(PGM)02FNS(PGM)02_1OctM,2D,3G,4P,5S,6U,7Rot-SUIT FNS(PGM)02.jpgA Schoepf 2016 FEBS J
SUIT FNS(PGM)03FNS(PGM)03_1OctM,2D,3PG,4S,5U,6Rot-SUIT FNS(PGM)03.jpgC MiPNet18.13 IOC84 Alaska
SUIT FNS(PM)01FNS(PM)01_1OctM,2D,3P,4S,5U,6Rot-SUIT FNS(PM)01.jpgA linked to SUIT_FNSGp(PGM)02 - SUIT RP2 (human skeletal muscle)
SUIT FNSGp(PGM)01FNSGp(PGM)01_1PM,2D,3U,4G,5S,6Oct,7Rot,8Gp-SUIT-RP1A: SUIT_RP1 - MiPNet21.06 SUIT RP
SUIT FNSGp(PGM)02FNSGp(PGM)02_1D,2OctM,3P,4G,5S,6Gp,7U,8Rot-SUIT FNSGp(PGM)02.jpgA: SUIT_RP2 - MiPNet21.06 SUIT RP
SUIT N(PGM)01N(PGM)01_1PGM,2D,3U-SUIT N(PGM)01.jpgA Krumschnabel_2014_Methods Enzymol; linear segment in SUIT_NS(PGM)04
SUIT NS(GM)01NS(GM)01_1GM,2D,3S,4U,5Rot-SUIT NS(GM)01.jpgB MiPNet12.23 FibreRespiration
SUIT NS(PGM)01NS(PGM)01_1PM,2D,3G,4U,5S,6Rot-SUIT NS(PGM)01.jpgB Lemieux 2011 Int J Biochem Cell Biol
SUIT NS(PGM)02NS(PGM)02_1PM,2D,3G,4S,5U,6Rot-SUIT NS(PGM)02.jpgA Lemieux 2017 bioRxiv
SUIT NS(PGM)03NS(PGM)03_1GM,2D,3P,4S,5U,6Rot-SUIT NS(PGM)03.jpgA Lemieux 2017 bioRxiv
SUIT NS(PGM)04NS(PGM)04_1PGM,2D,3U,4S,5Rot-SUIT NS(PGM)04.jpgC MiPNet18.13 IOC84 Alaska
SUIT NS(PGM)05NS(PGM)05_1PGM,2D,3S,4U,5Rot-SUIT NS(PGM)05.jpgC MiPNet18.13 IOC84 Alaska
SUIT NS(PGM)06NS(PGM)05_1PGM,2D,3S,4Rot,5U-SUIT NS(PGM)06.jpgC
SUIT NS(PM)01NS(PM)01_1PM,2D,3U,4S,5Rot-SUIT NS(PM)01.jpgA linked to SUIT_FNSGp(PGM)01 - SUIT RP1 (human skeletal muscle)