Doerrier 2018 Methods Mol Biol

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Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70.

» PMID: 29850993

Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Meszaros AT, Gnaiger E (2018) Methods Mol Biol

Abstract: Protocols for high-resolution respirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria and tissue homogenates offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques, small needle biopsies of muscle, and mitochondrial preparation methods. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation (OXPHOS) improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling control and electron transfer pathway states. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The noncoupled state of maximum respiration is experimentally induced by titration of established uncouplers (CCCP, FCCP, DNP), to collapse the protonmotive force across the mitochondrial inner membrane and measure the electron transfer capacity (ET; open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between non-phosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ET capacity. If OXPHOS capacity (maximally ADP stimulated O2 flux) is less than ET capacity, the phosphorylation pathway contributes to flux control. Physiological substrate combinations supporting the NADH&succinate-pathway are required to reconstitute tricarboxylic acid cycle function. This supports maximum ET and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. ET-pathways with electron entry separately through NADH (pyruvate&malate or glutamate&malate) or succinate (succinate&rotenone) restricts ET capacity and artificially enhances flux control upstream of the Q-cycle, providing diagnostic information on specific ET-pathway branches. O2 concentration is maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental O2 limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background O2 flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in high-resolution respirometry.


Bioblast editor: Gnaiger E O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck Oroboros


Labels: MiParea: Respiration, Instruments;methods 


Organism: Human, Mouse, Rat, Saccharomyces cerevisiae  Tissue;cell: Heart, Skeletal muscle, Endothelial;epithelial;mesothelial cell, Blood cells, HEK, Platelet  Preparation: Intact cells, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria 

Regulation: Oxygen kinetics  Coupling state: LEAK, ROUTINE, OXPHOS, ET  Pathway: F, N, S, Gp, CIV, NS, Other combinations, ROX  HRR: Oxygraph-2k, TIP2k, O2k-Protocol 

MitoPathways, MitoFitPublication, SUIT-001, SUIT-001 O2 mt D001, SUIT-001 O2 pfi D002, SUIT-001 O2 ce-pce D003, SUIT-001 O2 ce-pce D004, SUIT-002, SUIT-002 O2 mt D005, SUIT-002 O2 pfi D006, SUIT-002 O2 ce-pce D007, SUIT-002 O2 ce-pce D007a, SUIT-010, SUIT-010 O2 pce D008, MitoEAGLEPublication, SUIT-010, SUIT-010 O2 ce-pce D050, SUIT-010 O2 ce-pce D008 

RP1 and RP2 in mt-preparations

SUIT RP1:1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd
SUIT RP2:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

RP1&RP2.png Harmonization between RP1 and RP2