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
Substrate-uncoupler-inhibitor titration, SUIT
MitoPedia: SUIT is a component of Oroboros support.
Updated 2019-10-09 by Doerrier C, Gnaiger E.
Attention: future in construction (2019-10-09). We develop a concept of SUIT protocols compatible with a mt-database. 
Template NextGen-O2k.jpg


MitoPedia: SUIT


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» Introduction to SUIT protocols
SUIT protocols

Introduction to SUIT protocols

SUIT: Substrate-uncoupler-inhibitor-titration

  • Coupling control protocols (CCP) are a special case of SUIT protocols primarily applied to living cells (ce; ceCCP), typically with a sequence of ROUTINE respiration (R), optionally oligomycin-induced LEAK respiration (L), uncoupler-stimulated ET-capacity (E), and inhibitor-induced residual oxygen consumption (Rox). Cell-membrane permeable substrates (glucose, pyruvate) may be applied to evaluate their effect on cell physiology, and cell-membrane impermeable substrates (succinate, ADP) are titrated as a cell viability test to assess the fraction of dead cells (dce) with permeable cell membranes and functional mitochondria. It is important to provide information on the composition of the respiration medium, particularly distinguishing media with exogenous substrates and high Ca2+ concentration (culture media) and mt-respiration media with Ca2+ chelators (e.g. MiR05) excluding respiratory substrates, when respiration is strictly based on endogenous ET-pathway substrates.
  • Educational: The library of SUIT protocols is 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 titration experiment, defining every titration step (see DL-Protocols).
  • Compatible: The systematic name of a SUIT protocol must be unique for each variation within a category of SUIT protocols, compatible 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. Some respiratory states specifically require multiple titrations of the same substance for evaluation of the optimum concentration. Such multiple titrations are indicated by an asterisc following the acronym of the substance.
  • 5U* - stepwise titration of an uncoupler to measure ET-capacity as the noncoupled state of maximum flux at optimum uncoupler concentration.
  • Quality control: Titration steps related to quality control (e.g. cytochrome c test) may or may not be included in a selected state of a SUIT protocol, are not considered in the categories of SUIT protocols, and do not have a new number in the name of the SUIT protocol but maintain the number of the particular coupling/pathway control state.


  • Flexibility in DL-Protocols: Fixed sequence of events and marks can be changed (Skip/Added) in a SUIT protocol by the user (to be provided in the next DatLab version). Editions of concentrations and titration volumes of injections in a specific DL-Protocol are already available as user-specific DL-Protocol [File]\Export\DL-Protocol User (*.DLPU).


  • ROX-continued.png
    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.


  • O2k-Application: Displays the O2k-Applications of a specific SUIT protocol.
SUIT-nomenclature O2k-Applications.png
  • Modular approach: Select your DLP protocol from our archive of recommended SUIT-A library. The modular approach is designed to make easier to resolve your scientific question using protocols that guide you step by step.



Questions.jpg


Click to expand or collaps


Library of SUIT protocols

MitoPedia: SUIT

SUIT A Respirometry: recommended protocols

»A: MitoPedia: SUIT A - SUIT protocols used for cohort studies, available as DL-Protocols with DatLab 7. SUIT A protocols have a unique D### code: e.g. SUIT-008 O2 pfi D014).
SUIT numberShort nameDescription/ReferenceCoupling/pathway control diagram
SUIT-001RP1Doerrier 2018 Methods Mol BiolSUIT-001
SUIT-002RP2A: Doerrier 2018 Methods Mol Biol1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot-.png
SUIT-003CCP-ceA: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdfCe1;ce2(Omy);ce3U-.png Ce5S;1Dig;1c-.png
SUIT-004RP1-short1PM;2D;3U;4S;5Rot.png
SUIT-005RP2-shortA: when malate-anaplerotic activity is zero1OctM;2D;3P;4S;5U;6Rot-.png
SUIT-006CCP-mtprepA: Coupling control protocol, mtprep1X;2D;2c;3Omy;4U;5Ama.png
SUIT-007Glutamate anaplerosisA: Glutamate anaplerotic pathway1G;2D;3M;4U-.png
SUIT-008PM+G+S_OXPHOS+Rot_ETA: Additivity between the N- and S-pathway in the Q-junction1PM;2D;3G;4S;5U;6Rot.png
SUIT-010Digitonin testA: Optimization of digitonin concentration for pceRespirometric test of optimum digitonin concentration
SUIT-011GM+S_OXPHOS+Rot_ETA: Maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway controlSUIT-011
SUIT-012PM+G_OXPHOSA: Coupling control (L- P- E) with NADH-linked substrates (PM and PGM)1PM;2D;3G;4U-.png
SUIT-014GM+P+S_OXPHOS+Rot_ETA: Cells or tissue types that display a preference for GM over PM to support NADH-linked respiration1GM;2D;3P;4S;5U;6Rot-.png
SUIT-015F+G+P+S_OXPHOS+Rot_ETA: F-pathway in LEAK state and OXPHOS state1OctM;2D;3G;4P;5S;6U;7Rot-.png
SUIT-016F+G+S+Rot_OXPHOS+OmyA: F-pathway in LEAK state and OXPHOS state1OctM;2D;3G;4S;5Rot;6Omy;7U-.png
SUIT-017F+G+S_OXPHOS+Rot_ETA:1OctM;2D;2c;3G;4S;5U;6Rot-.png
SUIT-019Pal+Oct+P+G_OXPHOS+S+Rot_ETA:1PalM;2D;3Oct;4P;5G;6U;7S;8Rot-.png
SUIT-020PM+G+S+Rot_OXPHOS+OmyA: simultaneous determination of O2 flux and mt-membrane potentialBioblast pdf1PM;2D;3G;4S;5Rot;6Omy;7U-.png
SUIT-021OXPHOS (GM+S+Rot+Omy)A: simultaneous determination of O2 flux and mt-membrane potential1GM;2D;3S;4Rot;5Omy;6U-.png
SUIT-022AOX (ce CN+SHAM)A: Determination of the respiration due to the alternative oxidase pathwayCe1;ce2KCN;ce3SHAM.v2.png
SUIT-023AOX-ce SHAM+CNA: Determination of respiration through the CIII-CIV pathwayCe1;ce2SHAM;ce3KCN.png
SUIT-024ATPase (PM)A: Determination of the presence of ATPases in mitochondrial preparations.Ce1;1Dig;1PM;2T;2D;3Omy-.png
SUIT-025OXPHOS (F+M+P+G+S+Rot) 1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Rot-.png
SUIT-027Malate anaplerosisA: Malate anaplerotic pathway1M;2D;3M;4P;5G-.png


MitoPedia: SUIT

SUIT A FluoRespirometry: recommended protocols

»A: MitoPedia: SUIT A - these are SUIT protocols used for cohort studies, and are available as DL-Protocols with DatLab 7. SUIT A protocols have a unique D## code: e.g. SUIT-008 O2 pfi D14).
SUIT numberDescription/ReferenceCoupling/pathway control diagram
SUIT-006A: Coupling control protocol, mtprep1X;2D;2c;3Omy;4U;5Ama.png
SUIT-013ASUIT013 AmR ce D023.png
SUIT-018A: Short protocol for oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenateSUIT-018
SUIT-020A: simultaneous determination of O2 flux and mt-membrane potentialBioblast pdf1PM;2D;3G;4S;5Rot;6Omy;7U-.png
SUIT-021A: simultaneous determination of O2 flux and mt-membrane potential1GM;2D;3S;4Rot;5Omy;6U-.png
SUIT-026A Short protocol to study RET-related H2O2 production1S;2Rot;3D;4Ama.png
MitoPedia: SUIT

SUIT A: all recommended protocols

» A: MitoPedia: SUIT A - SUIT protocols used for cohort studies
MitoPedia: SUIT

SUIT B: published protocols

» B: MitoPedia: SUIT B - these are published SUIT protocols, not considered as level A.
MitoPedia: SUIT

SUIT C: exploratory protocols

» C: MitoPedia: SUIT C - these are SUIT protocols used for exploratory research.


All SUIT protocols

MitoPedia: SUIT


Click to expand or collaps
» Library of SUIT protocols: all SUIT protocols
SUIT codeO2k-ApplicationCoupling/pathway control diagramSUIT numberDescription/Reference
1OctM;2D;3PG;4S;5U;6Rot-O21OctM;2D;3PG;4S;5U;6Rot-.pngC MiPNet18.13 IOC84 Alaska
1PGM;2D;3S;4Rot;5U-O21PGM;2D;3S;4Rot;5U-.pngC
1PGM;2D;3S;4U;5Rot-O21PGM;2D;3S;4U;5Rot-.pngC MiPNet18.13 IOC84 Alaska
1PGM;2D;3U;4S;5Rot-O21PGM;2D;3U;4S;5Rot-.pngC MiPNet18.13 IOC84 Alaska
1PM;2D;3G;4U;5S;6Rot-O21PM;2D;3G;4U;5S;6Rot-.pngB Lemieux 2011 Int J Biochem Cell Biol
SUIT-001SUIT-001Doerrier 2018 Methods Mol Biol
SUIT-001 O2 ce-pce D003O2SUIT-RP1D003_ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11AzdA - SUIT-001
SUIT-001 O2 ce-pce D004O2SUIT-RP1 for PBMCs and PLTsD004_ce1;1Dig;1PM;2D;2c;3U;4G;5S;(6Oct);7Rot;8Gp;9Ama;10AsTm;11AzdA - SUIT-001
SUIT-001 O2 mt D001O2SUIT-RP1D001_1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11AzdSUIT-001 - SUIT reference protocol Reference protocol RP1 for isolated mitochondria, tissue homogenate and permeabilized cells
SUIT-001 O2 pfi D002O2SUIT-RP1D002_1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11AzdSUIT-001
SUIT-0021D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot-.pngA: Doerrier 2018 Methods Mol Biol
SUIT-002 O2 ce-pce D007O2SUIT-RP2D007_ce1;1Dig;1D;2M;3Oct;3c;4M;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13AzdA - SUIT-002
SUIT-002 O2 ce-pce D007aO2SUIT-RP2 for PBMCs and PLTsD007a_ce1;1Dig;1D;2M;3Oct;3c;4M;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13AzdA - SUIT-002
SUIT-002 O2 mt D005O21D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.pngD005_1D;2M;3Oct;3c;4M;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13AzdA - SUIT-002
SUIT-002 O2 pfi D006O2SUIT-RP2D006_1D;2M;3Oct;3c;4M;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13AzdA -SUIT-002
SUIT-003Ce1;ce2(Omy);ce3U-.png Ce5S;1Dig;1c-.pngA: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
SUIT-003 AmR ce D017AmRCe1;ce2P;ce3Omy;ce4U;ce5Rot;ce6S;ce7Ama.pngD017_ce1;ce2P;ce3Omy;ce4U;ce5Rot;ce6S;ce7AmaB: CCP(P) and cell membrane permeability test with succinate - SUIT-003
SUIT-003 AmR ce D058AmRCe1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama.pngD058_ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5AmaB: Coupling control in intact cells with oligomycin to study the inhibitory effect of the AmR assay components on the respiration ; ce: non-permeabilized cells -SUIT-003
SUIT-003 AmR ce D059AmRCe1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama.pngD059_ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5AmaB: Experimental control for SUIT-003 AmR ce D058
SUIT-003 Ce1;ce1P;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4AzdO2Ce1;ce1P;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4Azd.pngC SUIT-003 Coupling control protocol and respirometric cell viability test (CCV protocol) without oligomycin.
SUIT-003 Ce1;ce1SD;ce2Omy;ce3U-O2Ce1;ce1SD;ce2Omy;ce3U;ce4Rot;ce5Ama.pngB SUIT-003 Stadlmann 2006 Cell Biochem Biophys
SUIT-003 Ce1;ce1SD;ce3U;ce4Rot;ce5AmaO2Ce1;ce1SD;ce3U;ce4Rot;ce5Ama.pngB SUIT-003 CCP(S)
SUIT-003 Ce1;ce2SD;ce3Omy;ce4U-O2Ce1;ce2SD;ce3Omy;ce4U;ce5Rot;ce6Ama.pngB SUIT-003 Stadlmann 2006 Cell Biochem Biophys
SUIT-003 Ce1;ce2SD;ce3U;ce4Rot;ce5AmaO2Ce1;ce2SD;ce3U;ce4Rot;ce5Ama.pngB Steinlechner-Maran 1997 Transplantation
SUIT-003 Ce1;ce2U;ce3Rot;ce4S;ce5AmaO2Ce1;ce2U;ce3Rot;ce4S;ce5Ama.pngB SUIT-003 CCP(S)
SUIT-003 Ce1;ce3U;ce4Rot;ce5S;ce6AmaO2Ce1;ce3U;ce4Rot;ce5S;ce6Ama.pngB SUIT-003 CCP(S)
SUIT-003 O2 ce D009O2Ce1;ce2Omy;ce3U-.pngD009_ce1;ce2Omy;ce3U;ce4Rot;ce5AmaA: - SUIT-003
SUIT-003 O2 ce D012O2Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama.pngD012_ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5AmaB - SUIT-003
SUIT-003 O2 ce D028O2Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama.pngD028_ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6AmaB SUIT-003 coupling control protocol with succinate for permeability test
SUIT-003 O2 ce D037O2Ce1;ce1Glc;ce2(Omy);ce3U;ce4Ama.pngD037_ce1;ce1Glc;(ce2Omy);ce3U;ce4AmaA Coupling control protocol and Crabtree effect - SUIT-003
SUIT-003 O2 ce D038O2Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.pngD038_ce1;(ce2Omy);ce3U;ce3Glc;ce3'U;ce4AmaA Coupling control protocol and Crabtree effect test -SUIT-003
SUIT-003 O2 ce D039O2Ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama.jpgD039_ce1;(ce2Omy);ce3U;ce4Rot;ce5AmaA: Coupling control in intact cells without oligomycin; ce: non-permeabilized cells -SUIT-003
SUIT-003 O2 ce D050O2Ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama.pngD050_ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5AmaA: - Coupling control protocol for living cells with plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) - SUIT-003
SUIT-003 O2 ce D060O2Ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama.pngD060_ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7AmaB - coupling control protocol for living cells with cell permeable compounds MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv -SUIT-003
SUIT-003 O2 ce D061O2Ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama.pngD061_ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7AmaB Carrier control for the coupling control protocol for living cells with cell permeable compounds MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv - SUIT-003 O2 ce D060
SUIT-003 O2 ce D062O2Ce1;ce1DMSO;(ce2Omy);ce3U;ce4Rot;ce5Ama.pngD062_ce1;ce1DMSO;(ce2Omy);ce3U;ce4Rot;ce5AmaA: Carrier control for the Coupling control protocol for living cells with plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) - SUIT-003 O2 ce D050
SUIT-003 O2 ce-pce D013O2Ce1;ce1P;ce2Omy;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4Azd.pngD013_ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce3M;ce4Rot;ce5S;1Dig;1U;1c;2Ama;3AsTm;4AzdC - SUIT-003
SUIT-003 O2 ce-pce D018O2Ce1;ce1P;ce2Omy;ce3U;ce4Glc;ce5Rot;ce6S;1Dig;1U;1c;2Ama;3AsTm;4Azd.pngD018_ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4AzdC SUIT-003 Second version of the Coupling control protocol and respirometric cell viability test (CCV protocol)
SUIT-003 O2 ce-pce D020O2Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd.pngD020_ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4AzdA Coupling control protocol and respirometric cell viability test; ce: living cells, pce: permeabilized cells - SUIT-003
SUIT-0041PM;2D;3U;4S;5Rot.png
SUIT-004 O2 pfi D010O21PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd.pngD010_1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8AzdSUIT-004
SUIT-0051OctM;2D;3P;4S;5U;6Rot-.pngA: when malate-anaplerotic activity is zero
SUIT-005 O2 pfi D011O21OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd.pngD011_1OctM;2D;3P;4S;5U;6Rot;7Ama;8AsTm;9AzdA: - SUIT-005
SUIT-0061X;2D;2c;3Omy;4U;5Ama.pngA: Coupling control protocol, mtprep
SUIT-006 AmR mt D048AmR1PM;2D;3Omy;4U;5Ama.pngD048_1PM;2D;3Omy;4U;5AmaA: short protocol for simultaneous determination of O2 flux and H2O2 in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-006
SUIT-006 Fluo mt D034Fluo1PM;2D;3Omy;4U;5Ama.pngD034_1PM;2D;3Omy;4U;5AmaA: short protocol for simultaneous determination of O2 flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) -SUIT-006
SUIT-006 MgG mt D055MgG1PM;2D;3Cat;4U;5Ama.pngD055_1PM;2D;3Cat;4U;5AmaA: short coupling control protocol for simultaneous determination of O2 flux and mitochondrial ATP production in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) -SUIT-006
SUIT-006 O2 ce-pce D029O2Ce1;1Dig;1PM;2D;2c;3Omy;4U;5Ama.pngD029_ce1;1Dig;1PM;2D;2c;3Omy;4U;5AmaA to analyse the coupling control on permeabilized cells in N(PM) pathway control state- SUIT-006
SUIT-006 O2 mt D022O21SRot;2D;2c;3(Omy);4U;5Ama.pngD022_1SRot;2D;2c;(3Omy);4U;5AmaA - SUIT-006 - Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)
SUIT-006 O2 mt D047O21PM;2D;2c;3Omy;4U;5Ama.pngD047_1PM;2D;2c;3Omy;4U;5AmaA SUIT-006 - is a coupling control protocol in the N-pathway for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k chamber.
SUIT-0071G;2D;3M;4U-.pngA: Glutamate anaplerotic pathway
SUIT-007 O2 ce-pce D030O2Ce1;1Dig;1G;2D;2c;3M;4U;5Ama.pngD030_ce1;1Dig;1G;2D;2c;3M;4U;5AmaA pce: permeabilized cells - SUIT-007
SUIT-0081PM;2D;3G;4S;5U;6Rot.pngA: Additivity between the N- and S-pathway in the Q-junction
SUIT-008 O2 ce-pce D025O2Ce1;1Dig;1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.pngD025_ce1;1Dig;1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9AzdA: for cells-permeabilized cells Lemieux 2017 Sci Rep - SUIT-008
SUIT-008 O2 mt D026O21PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.pngD026_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9AzdA: SUIT protocol for mt; mt: isolated mitochondria and tissue homogenate - SUIT-008
SUIT-008 O2 pce D25O21PM;2D;3G;4S;5U;6Rot-.pngC25_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9AzdB: SUIT 8 for permeabilized cells Lemieux 2017 Sci Rep
SUIT-008 O2 pfi D014O21PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.pngD014_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9AzdA - Lemieux 2017 Sci Rep - SUIT-008
SUIT-009 AmR ce-pce D019SUIT9D019_ce1;1Dig;1S;2D;3P;4Rot;5AmaB: short protocol for H2O2 (AmR) in ce: non-permeabilized cells, pce: permeabilized cells -SUIT-009
SUIT-009 AmR mt D021AmRSUIT-009D021_1S;2D;3P;4Rot;5AmaB: short protocol for H2O2 (AmR) in mitochondrial preparations; mt: isolated mitochondria, tissue homogenate and permeabilized cells - SUIT-009
SUIT-009 O2 ce-pce D016SUIT-009D016_ce1;1Dig;1S;2D;3P;4Rot;5AmaB - SUIT-009
SUIT-009 O2 mt D015SUIT9D015_1S;2D;3P;4Rot;5AmaB - SUIT-009
SUIT-010Respirometric test of optimum digitonin concentrationA: Optimization of digitonin concentration for pce
SUIT-010 O2 ce-pce D008O2Respirometric test of optimum digitonin concentrationD008_ce1;ce2Rot;ce3S;ce4D;1Dig;1cA -SUIT-010
SUIT-011SUIT-011A: Maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control
SUIT-011 O2 pfi D024O21GM;2D;2c;3S;4U;5Rot;6Ama.pngD024_1GM;2D;2c;3S;4U;5Rot;6AmaA pfi: permeabilized fibers - SUIT-011
SUIT-0121PM;2D;3G;4U-.pngA: Coupling control (L- P- E) with NADH-linked substrates (PM and PGM)
SUIT-012 O2 ce-pce D052O2Ce1;1Dig;1PM;2D;2c;3G;4U;5Ama.pngD052_ce1;1Dig;1PM;2D;2c;3G;4U;5AmaA Linear coupling control with NADH-linked substrates for pce - SUIT-012
SUIT-012 O2 mt D027O21PM;2D;2c;3G;4U;5Ama.pngD027_1PM;2D;2c;3G;4U;5AmaA Linear coupling control with NADH-linked substrates for isolated mitochondria, tissue homogenate and permeabilized cells- SUIT-012
SUIT-013SUIT013 AmR ce D023.pngA
SUIT-013 AmR ce D023AmRSUIT013 AmR ce D023.pngD023_ce1A SUIT-013
SUIT-0141GM;2D;3P;4S;5U;6Rot-.pngA: Cells or tissue types that display a preference for GM over PM to support NADH-linked respiration
SUIT-014 O2 pfi D042O21GM;2D;2c;3P;4S;5U;6Rot;7Ama.pngD042_1GM;2D;3P;4S;5U;6Rot;7AmaA SUIT-014
SUIT-0151OctM;2D;3G;4P;5S;6U;7Rot-.pngA: F-pathway in LEAK state and OXPHOS state
SUIT-015 O2 pti D043O21OctM;2D;3G;4P;5S;6U;7Rot;8Ama.pngD043_1OctM;2D;3G;4P;5S;6U;7Rot;8AmaA pti: permeabilized tissue- SUIT-015
SUIT-0161OctM;2D;3G;4S;5Rot;6Omy;7U-.pngA: F-pathway in LEAK state and OXPHOS state
SUIT-016 O2 pfi D044O21OctM;2D;3G;4S;5Rot;6Omy;7U;7c;8Ama.pngD044_1OctM;2D;3G;4S;5Rot;6Omy;7U;8AmaA pfi: permeabilized fibers- SUIT-016
SUIT-0171OctM;2D;2c;3G;4S;5U;6Rot-.pngA:
SUIT-017 O2 mt D046O21OctM;2D;2c;3G;4S;5U;6Rot;7Ama.pngD046_1OctM;2D;3G;3c;4S;5U;6Rot;7AmaA: SUIT-017
SUIT-017 O2 pfi D049O21OctM;2D;2c;3G;4S;5U;6Rot;7Ama.pngD049_1OctM;2D;2c;3G;4S;5U;6Rot;7AmaA: SUIT-017
SUIT-018SUIT-018A: Short protocol for oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenate
SUIT-018 AmR mt D031AmR1GMS;2D;3Ama.pngD031_1GMS;2D;3AmaA -SUIT-018
SUIT-018 AmR mt D040AmRSUIT-018D040_1GMS;2D;3AmaA: simultaneous determination of O2 and H2O2 flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) at high oxygen concentration (tissue hyperoxia)-SUIT-018
SUIT-018 AmR mt D041AmR1GMS;2D;3Ama.pngD041_1GMS;2D;3AmaA -SUIT-018
SUIT-018 O2 mt D054O21GMS;2D;2c;3Ama.pngD054_1GMS;2D;3AmaB: SUIT-018
SUIT-0191PalM;2D;3Oct;4P;5G;6U;7S;8Rot-.pngA:
SUIT-019 O2 pfi D045O21PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot;9Ama.pngD045_1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot;9AmaA SUIT-019
SUIT-0201PM;2D;3G;4S;5Rot;6Omy;7U-.pngA: simultaneous determination of O2 flux and mt-membrane potentialBioblast pdf
SUIT-020 Fluo mt D033Fluo1PM;2D;3G;4S;5Rot;6Omy;7U;8Ama.pngD033_1PM;2D;3G;4S;5Rot;6Omy;7U;8AmaA:short protocol for simultaneous determination of O2 flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria, permeabilized cells, and tissue homogenate)- SUIT-020
SUIT-020 O2 mt D032O21PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama.pngD032_1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8AmaA: short protocol for determination of O2 flux in mitochondrial preparations (isolated mitochondria, permeabilized cells, and tissue homogenate)-SUIT-020
SUIT-0211GM;2D;3S;4Rot;5Omy;6U-.pngA: simultaneous determination of O2 flux and mt-membrane potential
SUIT-021 Fluo mt D036Fluo1GM;2D;3S;4Rot;5Omy;6U;7Ama.pngD036_1GM;2D;3S;4Rot;5Omy;6U;7AmaA: short protocol for simultaneous determination of O2 flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria, permeabilized cells, and tissue homogenate)-SUIT-021
SUIT-021 O2 mt D035O21GM;2D;2c;3S;4Rot;5Omy;6U;7Ama.pngD035_1GM;2D;2c;3S;4Rot;5Omy;6U;7AmaA:short protocol for simultaneous determination of O2 flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria, permabilized cells, and tissue homogenate)-SUIT-021
SUIT-022Ce1;ce2KCN;ce3SHAM.v2.pngA: Determination of the respiration due to the alternative oxidase pathway
SUIT-022 O2 ce D051O2Ce1;ce2KCN;ce3SHAM.v2.png Ce1;ce2KCN;ce3SHAM.pngD051_ce1;ce2KCN;ce3SHAMA Determination of the respiration due to the alternative oxidase pathway -SUIT-022
SUIT-023Ce1;ce2SHAM;ce3KCN.pngA: Determination of respiration through the CIII-CIV pathway
SUIT-023 O2 ce D053O2Ce1;ce2SHAM;ce3KCN.pngD053_ce1;ce2SHAM;ce3KCNA Determination of the respiration due to the cytochrome c oxidase pathway -SUIT-023
SUIT-024Ce1;1Dig;1PM;2T;2D;3Omy-.pngA: Determination of the presence of ATPases in mitochondrial preparations.
SUIT-024 O2 ce-pce D056O2Ce1;1Dig;1PM;2T;2D;3Omy;4Ama.pngD056_ce1;1Dig;1PM;2T;2D;3Omy;4AmaDetermination of the presence of ATPases in mitochondrial preparations - SUIT-024
SUIT-0251D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Rot-.png
SUIT-025 O2 mt D057O21D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Rot;9Ama.pngD057_1D;2M;3Oct;3c;4M;5P;6G;7S;8Rot;9Ama - SUIT-025
SUIT-0261S;2Rot;3D;4Ama.pngA Short protocol to study RET-related H2O2 production
SUIT-026 AmR mt D064AmRSUIT-026 AmR mt D064.pngD064_1S;2Rot;3D;4AmaA - SUIT-026
SUIT-026 O2 mt D063O21S;2Rot;3D;3c;4Ama.pngD063_1S;2Rot;3D;3c;4AmaB - SUIT-026
SUIT-0271M;2D;3M;4P;5G-.pngA: Malate anaplerotic pathway
SUIT-027 O2 ce-pce D065O2Ce1;1Dig;1M;2D;3M;4P;5G;6Ama.pngD065_ce1;1Dig;1M;2D;3M;4P;5GA - SUIT-027
SUITbrowserUse the SUITbrowser to find the best substrate-uncoupler-inhibitor-titration (SUIT) protocol for your research questions.
Open the SUITbrowser: http://suitbrowser.oroboros.at/
Each SUIT protocol receives a score from 0 to 3 in different categories that correspond to a research question (e.g. information on coupling control, information on fatty acid oxidation, among others). This score is set depending on the information which one can get from each SUIT protocol (3: this SUIT protocol is ideal, highly informative for the specific question; 2: information provided; 1: information provided, but is not a SUIT protocol mainly focused for the study of this question; 0: the specific information/question is not provided by the SUIT protocol). It is possible to choose multiple research questions at a time, to find the best protocols that address this combination of problems.

Glossary

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» List of SUIT states

List of 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 stateNSSee NS-pathway control state (previous: CI&II-linked)
Complex I-linked substrate stateNSee N-pathway control state (previous: CI-linked)
Complex II-linked substrate stateSRot, SSee S-pathway control state (previous: CII-linked)
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-pathway, CIV-linked respiration (L, P, and E) can be measured in different coupling states. 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 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.
Electron transfer-pathway stateET-pathway state
SUIT-catg FNSGpCIV.jpg

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

» MiPNet article
FNFN
F-junction
FN is induced in mt-preparations by addition of 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.
FNSFNS
F-junction
FNS 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) 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.
FNSGpFNSGp
Convergent electron flow

MitoPathway control state: FNSGp

Octanoylcarnitine or Palmitoylcarnitine & Pyruvate &/or Glutamate & Malate & Succinate & Glycerophosphate.

SUIT protocol: SUIT-002

This substrate combination supports convergent electron flow to the Q-junction.
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 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.
GM pathway control stateGM
GM
GM: Glutamate & Malate.

MitoPathway control state: NADH Electron transfer-pathway state

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.
GMSGMS
GMS
GMS: Glutamate & Malate & Succinate.

MitoPathway control: CI&II

Transaminase catalyzes the reaction from oxaloacetate to 2-oxoglutarate, which then establishes a cycle without generation of citrate. OXPHOS is higher with GS (CI&II) compared to GM (CI) or SRot (CII). This documents an additive effect of convergent CI&II electron flow to the Q-junction, with consistent results obtained with permeabilized muscle fibres and isolated mitochondria (Gnaiger 2009).
Glutamate anaplerotic pathway control stateG
G
G: Glutamate is an anaplerotic NADH-linked type 4 substrate (N). 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. The G-pathway plays an important role in glutaminolysis.
Glycerophosphate pathway control stateGp
Gp-pathway
The glycerophosphate pathway control state (Gp) is an 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-respirationLL.jpg EAK-respiration or LEAK oxygen flux, L, compensating for proton leak, proton slip, cation cycling and electron leak, is a dissipative component of respiration which is not available for performing biochemical work and thus related to heat production. LEAK-respiration is measured in the LEAK state, in the presence of reducing substrate(s), but absence of ADP - abbreviated as L(n) (theoretically, absence of inorganic phosphate presents an alternative), or after enzymatic inhibition of the phosphorylation system, which can be reached with the use of oligomycin - abbreviated as L(Omy). The LEAK state is the non-phosphorylating resting state of intrinsic uncoupled or dyscoupled respiration when oxygen flux is maintained mainly to compensate for the proton leak at a high chemiosmotic potential, when ATP synthase is not active. In this non-phosphorylating resting state, the electrochemical proton gradient is increased to a maximum, exerting feedback control by depressing oxygen flux to a level determined mainly by the proton leak and the H+/O2 ratio. In this state of maximum protonmotive force, LEAK-respiration, L, is higher than the LEAK component of OXPHOS capacity, P. The conditions for measurement and expression of respiration vary (oxygen flux in the LEAK state, JO2L, or oxygen flow, IO2L). If these conditions are defined and remain consistent within a given context, then the simple symbol L for respiratory rate can be used as a substitute for the more explicit expression for respiratory activity. » MiPNet article
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 pyruvate&malate or glutamate&malate substrate combinations (PM pathway control state, GM pathway control state).
NADH Electron transfer-pathway stateN
N-junction
The NADH electron transfer-pathway state (N) is obtained by addition of NADH-linked substrates (CI-linked), feeding electrons into the N-junction catalyzed by various mt-dehydrogenases. N-supported flux is induced in mt-preparations by addition of NADH-generating substrate combinations of pyruvate (P), glutamate (G), malate (M), oxaloacetate (Oa), oxoglutarate (Og), citrate, hydroxybutyrate. These N-junction substrates are (indirectly) linked to Complex I by the corresponding dehydrogenase-catalyzed reactions reducing NAD+ to NADH+H+. The most commonly applied N-junction substrate combinations are: PM, GM, PGM. The malate anaplerotic pathway control state (M alone) is a special case related to malic enzyme (mtME). The glutamate anaplerotic pathway control state (G alone) supports respiration through glutamate dehydrogenase (mtGDH). Oxidation of tetrahydrofolate is a NAD(P)H linked pathwaynwith formation of formate. In mt-preparations, succinate dehydrogenase (SDH; CII) is largely substrate-limited in N-linked respiration, due to metabolite depletion into the incubation medium. The residual involvement of S-linked respiration in the N-pathway control state can be further suppressed by the CII-inhibitor malonic acid). In the N-pathway control state ET pathway level 4 is active.
NS-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-OXPHOS capacity provides an estimate of physiologically relevant maximum mitochondrial respiratory capacity. NS is induced in mt-preparations by addition of 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.
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
OctGMOctGMOctGM: Octanoylcarnitine & Glutamate & Malate.

MitoPathway control state: FN

SUIT protocols: SUIT-015, SUIT-016, SUIT-017
OctGMSOctGMSOctGMS: Octanoylcarnitine &Glutamate & Malate& Succinate.

MitoPathway control state: FNS

SUIT protocols: SUIT-016, SUIT-017
OctM pathway control stateOctMOctM: Octanoylcarnitine & Malate.

MitoPathway control state: F

SUIT protocols: SUIT-002, SUIT-015, SUIT-016, SUIT-017

Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the FAO-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) FAO-pathway could be overestimated due to a contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. High concentration of malate, typically 2 mM, saturates the F-pathway.
OctPGM pathway control stateOctPGMOctPGM: Octanoylcarnitine & Pyruvate & Glutamate & Malate.

MitoPathway control state: FN

SUIT protocols: SUIT-002

This substrate combination supports N-linked flux which is typically higher than FAO capacity (F/FN<1 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 and glutamate is added to compare FAO as the background state with FN as the reference state.
OctPGMS pathway control stateOctPGMSOctPGMS: Octanoylcarnitine & Pyruvate & Glutamate & Malate & Succinate.

MitoPathway control state: FNS

SUIT protocol: SUIT-001, SUIT-002, SUIT-015

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-002

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-002, SUIT-005

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.
OctPMSOctPMSOctPMS: Octanoylcarnitine & Pyruvate & Malate & Succinate.

MitoPathway control state: FNS

SUIT protocol: SUIT-005
PGMPGM
PGM
PGM: Pyruvate & Glutamate & Malate.

MitoPathway control state: NADH Electron transfer-pathway state

SUIT protocol: SUIT-001, SUIT-012, SUIT-014

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). Glutamate&malate is a substrate combination supporting an N-linked pathway control state, when glutamate is transported into the mt-matrix via the glutamate-aspartate carrier and reacts with oxaloacetate in the transaminase reaction to form aspartate and oxoglutarate. Glutamate as the sole substrate is transported by the electroneutral glutamate-/OH- exchanger, and is oxidized in the mitochondrial matrix by glutamate dehydrogenase to α-ketoglutarate ( 2-oxoglutarate), representing the glutamate anaplerotic pathway control state. 2-oxoglutarate (α-ketoglutarate) is formed from isocitrate (isocitrate dehydrogenase, from oxaloacetate and glutamate by the transaminase, and from glutamate by the glutamate dehydrogenase.
PGMS pathway control statePGMS
PGMS
PGMS: Pyruvate & Glutamate & Malate & Succinate.

MitoPathway control state: NS-pathway control state

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: 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp- - 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).
PMSPMS
PMS
PMS: Pyruvate & Malate & Succinate.

MitoPathway control: CI&II

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). This documents an additive effect of convergent CI&II electron flow to the Q-junction, with consistent results obtained with permeabilized muscle fibres and isolated mitochondria (Gnaiger 2009).
PalMPalMPalM: Palmitoylcarnitine & Malate.

MitoPathway control state: Fatty acid oxidation pathway control state

SUIT protocols: SUIT-019
PalOctMPalOctMPalOctM: Palmitoylcarnitine & Octanoylcarnitine & Malate.

MitoPathway control state: Fatty acid oxidation pathway control state

SUIT protocols: SUIT-019
PalOctPGMPalOctPGMPalOctPGM: Palmitoylcarnitine & Octanoylcarnitine & Pyruvate & Glutamate & Malate.

MitoPathway control state: FN

SUIT protocols: SUIT-019
PalOctPGMSPalOctPGMSPalOctPGMS: Palmitoylcarnitine & Octanoylcarnitine & Pyruvate & Glutamate & Malate & Succinate.

MitoPathway control state: FNS

SUIT protocols: SUIT-019
PalOctPMPalOctPMPalOctPM: Palmitoylcarnitine & Octanoylcarnitine & Pyruvate & Malate.

MitoPathway control state: FN

SUIT protocols: SUIT-019
SGp-pathway control stateSGpSGp: Succinate & Glycerophosphate.

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

SUIT protocol: SUIT-001 and ((SUIT-002
Succinate-pathwayS, SRot
Succinate

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).
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Click to expand or collaps
» SUIT concept

SUIT concept

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

Categories of SUIT protocols group SUIT protocols according to all substrate types involved in a protocol (F, N, S, Gp), independent of the sequence of titrations of substrates and inhibitors which define the Electron transfer-pathway states. The N-type substrates are listed in parentheses, independent of the sequence of titrations. ROX states may or may not be included in a SUIT protocol, which does not change its category. Similarly, the CIV assay may or may not be added at the end of a SUIT protocol, without effect on the category of a SUIT protocol.

  • F - ET-pathway-level 5: FADH2-linked substrates (FAO) with obligatory support by the N-linked pathway.
  • N - ET-pathway-level 4: NADH-linked substrates (CI-linked).
  • S - ET-pathway-level 3: Succinate (CII-linked).
  • Gp - ET-pathway-level 3: Glycerophosphate (CGpDH-linked).
  • Y(X)- In the SUIT general protocols Y makes reference to the ET-pathway state and X to the combination os substrates added for the corresponding pathway.
» MiPNet article
Cell respirationCell respiration channels metabolic fuels into the chemiosmotic coupling (bioenergetic) machinery of oxidative phosphorylation, being regulated by and regulating oxygen consumption (or consumption of an alternative final electron acceptor) and molecular redox states, ion gradients, mitochondrial (or microbial) membrane potential, the phosphorylation state of the ATP system, and heat dissipation in response to intrinsic and extrinsic energy demands. See also respirometry. In internal or cell respiration in contrast to fermentation, redox balance is maintained by the use of external electron acceptors, transported into the cell from the environment. The chemical potential from electron donors to electron acceptors is converted in the Electron transfer-pathway to generate a chemiosmotic potential that in turn drives ATP synthesis.
Coupling control protocolCCPA coupling control protocol, CCP, induces different coupling control states at a constant electron transfer-pathway state. Residual oxygen consumption (Rox) is finally evaluated for Rox correction of flux. The CCP may be extended, when further respiratory states (e.g. cell viability test; CIV assay) are added to the coupling control module consisting of three coupling control states. The term phosphorylation control protocol, PCP, has been introduced synonymous for CCP. » MiPNet article
Coupling control stateCCSCoupling control states are defined in mitochondrial preparations (isolated mitochondria, permeabilized cells, permeabilized tissues, homogenates) as LEAK, OXPHOS, and ET-pathway states, with corresponding respiration rates (L, P, E) in any electron transfer-pathway state which is competent for electron transfer. These coupling states are induced by application of specific inhibitors of the phosphorylation system, titration of ADP and uncouplers. In living cells, the coupling control states are LEAK, ROUTINE, and ET-pathway states of respiration (L, R, E), using membrane-permeable inhibitors of the phosphorylation system (e.g. oligomycin) and uncouplers (e.g. CCCP). Coupling control protocols induce these coupling control states sequentially at a constant electron transfer-pathway state.
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 ET-pathway inhibitors, e.g. rotenone) exerts pathway control, then a transition is induced between two Electron transfer-pathway 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 define the section of an experimental trace in a given respiratory state (steady state). Events 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.
D-numberD###D number is the unique code given for each SUIT protocol. In the same SUIT protocol family (SUIT-###), there might be different protocols, specifically designed for different sample type (e.g., different mitochondrial preparations) or for different applications (e.g., O2, AmR, Fluo, MgG). Since the use of different kinds of sample or application may result in slightly different steps, each protocol receives a different D-number.
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 Oroboros O2k 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-011
  • Mark names in DatLab: 1GM;2D;2c;3S;4U;5Rot-
  • DatLab-Analysis template: SUIT_NS(GM)01.xlsx
  • MiPNet communciation: MiPNet12.23 FibreRespiration
  • DatLab demo file: MiPNet12.23 FibreRespiration.DLD
ET-pathway substrate typesn.a.See Electron transfer-pathway state
Electron transfer-pathway stateET-pathway state
SUIT-catg FNSGpCIV.jpg

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

» MiPNet article
F-junction
F-junction
The F-junction is a junction for convergent electron flow in the electron transfer-pathway (ET-pathway) from fatty acids through fatty acyl CoA dehydrogenase (reduced form FADH2) to electron transferring flavoprotein (CETF), and further transfer through the Q-junction to Complex III (CIII). The concept of the F-junction and N-junction provides a basis for defining categories of SUIT protocols. Fatty acid oxidation, in the F-pathway control state, not only depends on electron transfer through the F-junction (which is typically rate-limiting) but simultaneously generates NADH and thus depends on N-junction throughput. Hence FAO can be inhibited completely by inhibition of Complex I (CI). In addition and independent of this source of NADH, the N-junction substrate malate is required as a co-substrate for FAO in mt-preparations, since accumulation of AcetylCoA inhibits FAO in the absence of malate. Malate is oxidized in a reaction catalyzed by malate dehydrogenase to oxaloacetate (yielding NADH), which then stimulates the entry of AcetylCoA into the TCA cycle catalyzed by citrate synthase.
Flux control factorFCFFlux control factors express the control of respiration by a metabolic control variable, X, as a fractional change of flux from YX to ZX, normalized for ZX. ZX is the reference state with high (stimulated or un-inhibited) flux; YX is the background state at low flux, upon which X acts.
ΔjX = (ZX-YX)/ZX = 1-YX/ZX

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

» MiPNet article
Flux control ratioFCRFlux control ratios (FCR), are ratios of oxygen flux in different respiratory control states, normalized for maximum flux in a common reference state, to obtain theoretical lower and upper limits of 0.0 and 1.0 (0% and 100%). For a given protocol or set of respiratory protocols, flux control ratios provide a fingerprint of coupling and substrate control independent of (i) mt-content in cells or tissues, (ii) purification in preparations of isolated mitochondria, and (iii) assay conditions for determination of tissue mass or mt-markers external to a respiratory protocol (CS, protein, stereology, etc.). FCR obtained from a single respirometric incubation with sequential titrations (sequential protocol; SUIT protocol) provide an internal normalization, expressing respiratory control independent of mitochondrial content and thus independent of a marker for mitochondrial amount. FCR obtained from separate (parallel) protocols depend on equal distribution of subsamples obtained from a homogenous mt-preparation or determination of a common mitochondrial marker.
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 contributions, 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-pathway (ET-pathway) from type N substrates (further details »N-pathway control state) through the mt-NADH pool to Complex I (CI), and further transfer through the Q-junction to Complex III (CIII). Representative type N substrates are pyruvate (P), glutamate (G) and malate (M). The corresponding dehydrogenases (PDH, GDH, MDH) and some additional TCA cycle dehydrogenases (isocitrate dehydrogenase, oxoglutarate dehydrogenase generate NADH, the substrate of Complex I (CI). The concept of the N-junction and F-junction provides a basis for defining categories of SUIT protocols based on Electron transfer-pathway states.
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 ET-pathway. 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
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 phosphorylation system or P»-system. The P»-system consists of adenine nucleotide translocase, phosphate carrier, and ATP synthase. Mitochondrial adenylate kinase, mt-creatine kinase and mt-hexokinase constitute extended components of the P»-system, controlling local AMP and ADP concentrations and forming metabolic channels. Since substrate-level phosphorylation is involved in the TCA-cycle, the P»-system includes succinyl-CoA ligase (GDP to GTP or ADP to ATP).
Physiological pathway control stateSee Electron transfer-pathway state.
Q-junction
Q-junction
The Q-junction is a junction for convergent electron flow in the Electron transfer-pathway (ET-pathway) from type N substrates and mt-matix dehydrogenases through Complex I (CI), from type F substrates and FA oxidation through electron-transferring flavoprotein complex (CETF), from succinate (S) through Complex II (CII), from glycerophosphate (Gp) through glycerophosphate dehydrogenase complex (CGpDH), from choline through choline dehydrogenase, from dihydro-orotate through dihydro-orotate dehydrogenase, and other enzyme complexes into the Q-cycle (ubiquinol/ubiquinone), and further downstream to Complex III (CIII) and Complex IV (CIV). The concept of the Q-junction, with the N-junction and F-junction upstream, provides the rationale for defining Electron transfer-pathway states and categories of SUIT protocols.
Respiratory stateRespiratory states of mitochondrial preparations and intact cells are defined in the current literature in many ways and with a diversity of terms. Mitochondrial respiratory states must be defined in terms of both, the coupling control state and the electron transfer-pathway state.
SUITSUITSUIT is the abbreviation for Substrate-Uncoupler-Inhibitor Titration. SUIT protocols are used with mt-preparations to study respiratory control in a sequence of coupling and pathway control states induced by multiple titrations within a single experimental assay.
SUIT protocol librarySUITsThe Substrate-uncoupler-inhibitor titration (SUIT) protocol library contains a sequential list of SUIT protocols (D001, D002, ..) with links to the specific SUIT pages. Classes of SUIT protocols are explained with coupling and substrate control defined for mitochondrial preparations.
SUIT protocol namesSUITp-Names
SUIT protocols

The SUIT protocol name starts with (i) the SUIT category which shows the Electron transfer-pathway states (ET 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 (1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp- - SUIT RP1 and 1D;2OctM;3P;4G;5S;6Gp;7U;8Rot- - 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: 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp- - 1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10Tm;11Azd
SUIT_RP2: 1D;2OctM;3P;4G;5S;6Gp;7U;8Rot- - 1D;2OctM;2c;3P;4G;5S;6Gp;7U;8Rot;9Ama;10Tm;11Azd
SUITbrowserUse the SUITbrowser to find the best substrate-uncoupler-inhibitor-titration (SUIT) protocol for your research questions.
Open the SUITbrowser: http://suitbrowser.oroboros.at/
Each SUIT protocol receives a score from 0 to 3 in different categories that correspond to a research question (e.g. information on coupling control, information on fatty acid oxidation, among others). This score is set depending on the information which one can get from each SUIT protocol (3: this SUIT protocol is ideal, highly informative for the specific question; 2: information provided; 1: information provided, but is not a SUIT protocol mainly focused for the study of this question; 0: the specific information/question is not provided by the SUIT protocol). It is possible to choose multiple research questions at a time, to find the best protocols that address this combination of problems.
Substrate control stateSee Electron transfer-pathway 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.


See also:
» O2k-Demo experiments