Pesta 2012 Methods Mol Biol

Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.

» PMID: 22057559; Bioblast pdf

Pesta D, Gnaiger E (2012) Methods Mol Biol

Abstract: Protocols for high-resolution respirometry of intact cells, permeabilized cells, and permeabilized muscle fibres offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques and small needle biopsies of muscle. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation 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 protols with stepwise modulation of coupling and substrate control. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The non-coupled state of maximum respiration is experimentally induced by titration of established uncouplers (FCCP, DNP), to collapse the proton gradient across the mitochondrial inner membrane and measure the capacity of the electron transfer system (ETS, 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 ETS capacity. If OXPHOS capacity (maximally ADP stimulated oxygen flux) is less than ETS capacity, the phosphorylation system contributes to flux control. Physiological Complex I+II substrate combinations support maximum ETS and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. Substrate control with electron entry separately through Complex I (pyruvate+malate or glutamate+malate) or Complex II (succinate+rotenone) restricts ETS capacity and artificially enhances flux control upstream of the Q-cycle, providing diagnostic information on specific branches of the ETS. Oxygen levels are maintained above air saturation in protocols with permeabilized muscle fibres to avoid experimental oxygen 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 oxygen flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in high-resolution respirometry. • Keywords: Substrate-uncoupler-inhibitor titration, Human vastus lateralis, Needle biopsy, HEK, HPMC, HUVEC, Fibroblasts, Routine respiration, Oxidative phosphorylation, Q-junction, Pyruvate, Glutamate, Malate, Succinate, Leak, Coupling control, Uncoupling, Oxygraph, Oxygen flux, Residual oxygen consumption, Instrumental background

• O2k-Network Lab: AT_Innsbruck_Gnaiger E, AT Innsbruck MitoCom

Labels: MiParea: Respiration, Instruments;methods, mt-Biogenesis;mt-density, Comparative MiP;environmental MiP, Exercise physiology;nutrition;life style, mt-Medicine 

Stress:Hypoxia  Organism: Human  Tissue;cell: Skeletal muscle  Preparation: Intact cells, Permeabilized cells, Permeabilized tissue  Enzyme: Marker Enzyme"Marker Enzyme" is not in the list (Adenine nucleotide translocase, Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase, Inner mt-membrane transporter, Marker enzyme, Supercomplex, TCA cycle and matrix dehydrogenases, ...) of allowed values for the "Enzyme" property.  Regulation: ADP, Coupling efficiency;uncoupling, Cyt c, Flux control, O2"O2" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Substrate, Threshold;excess capacity  Coupling state: LEAK, ROUTINE, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. 

HRR: Oxygraph-2k, TIP2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

• Referred to in Gnaiger_2012_MitoPathways, Chapter 1.

Correction

• page 9: add 20 µl instead of 21 µl saponin stock solution
• Fig. 7a: 'Myothiazol' should be corrected to 'Myxothiazol'.

Bioblast Alert

• The most popular O2k-Publication on Bioblast of the year 2013.
• Bioblast alert 2011 (4)