Difference between revisions of "Uncoupler titrations"

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high-resolution terminology - matching measurements at high-resolution

Uncoupler titrations

Description

In uncoupler titrations various uncouplers, such as CCCP, FCCP or DNP are applied to uncouple mitochondrial electron transfer from phosphorylation (ATP synthase, ANT and phosphate transporter), particularly with the aim to obtain the noncoupled ET-state with optimum uncoupler concentration at maximum oxygen flux.

MitoPedia concepts: "MitoFit Quality Control System" is not in the list (MiP concept, Respiratory state, Respiratory control ratio, SUIT concept, SUIT protocol, SUIT A, SUIT B, SUIT C, SUIT state, Recommended, ...) of allowed values for the "MitoPedia concept" property. MitoFit Quality Control System"MitoFit Quality Control System" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property.

MitoPedia methods: Respirometry

MitoPedia O2k and high-resolution respirometry: O2k-Open Support

MitoPedia topics: Uncoupler

Uncoupler titrations in HRR

DatLab settings

Recommendation: Set "Slope smoothing" to 20 for performing and analyzing experiments with biological sample particularly for uncoupler titrations.

Optimum uncoupler concentration

Stepwise titrations of an uncoupler is necessary to achive the optimum concentration for obtaining maximum flux as a measure ET-capacity (noncoupled respiration). It is important to avoid inhibition of respiration by too high uncoupler concentrations. The underlying mechanism for the latter is not clear.

The optimum concentration of an uncoupler has to be determined for every biological system. It varies with incubation medium, sample concentration, pharmacological treatment (with or without oligomycin), and pathophysiological state (e.g. induction of apoptosis). A single dose of uncoupler usually leads to an artefact in the estimation of maximum flux or Electron transfer-pathway capacity (for discussion, see Artefacts by single dose uncoupling).

The optimum uncoupler (CCCP, FCCP, DNP) concentration for the noncoupled state varies over a large concentration range, depending on the medium ('binding' of uncoupler), type and concentration of sample. This is true for various uncouplers, such as CCCP, FCCP and DNP (Steinlechner-Maran 1996 Am J Physiol Cell Physiol). To evaluate the optimum concentration, an uncoupler titration has to be performed initially. For subsequent application series, we recommend a few titrations starting close to optimum concentration (Huetter_2004_Biochem J, Pesta 2012 Methods Mol Biol). Optimum CCCP or FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.

See Steinlechner-Maran et al (1996) for a comparison of uncoupler titrations with FCCP and DNP from the ROUTINE state to the ET state of cell respiration.

Coupling control protocol

Uncoupler titrations after inhibition of respiration by oligomycin in coupling control protocols with living cells yield the sequence of ROUTINE respiration, LEAK respiration and ET-capacity, followed by inhibition to ROX (Huetter 2004 Biochem J, Gnaiger 2008 POS). The highest accuracy of uncoupler titrations is achieved by titrations with the TIP2k at high concentrations of the stock solution (Gnaiger 2008 POS. Increasing the concentration in small steps, most accurately titrated by the TIP2k, is recommended (0.5 or 0.25 µM steps or even smaller).

Bioblast links: Uncoupling - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Specific

» Artefacts by single dose uncoupling

» ATP synthase

» CCCP

» Coupling-control protocol

» DNP

» Dyscoupled respiration

» FCCP

» Is respiration uncoupled - noncoupled - dyscoupled?

» Noncoupled respiration: Discussion

» Uncoupler

» Uncoupled respiration - see » Noncoupled respiration

» Uncoupling proteins

» Uncoupling protein 1

» Uncoupler titrations - Optimum uncoupler concentration

Respiratory states and control ratios

» Biochemical coupling efficiency

» Coupling-control state

» Electron-transfer-pathway state

» Electron-transfer pathway

ET capacity

» E-L coupling efficiency

» Flux control efficiency

» Flux control ratio

» LEAK-control ratio

» LEAK respiration

» Noncoupled respiration

» OXPHOS

» OXPHOS capacity; » State 3

» OXPHOS-control ratio, P/E ratio

» Respiratory acceptor control ratio

» ROUTINE-control ratio

» ROUTINE respiration

» ROUTINE state

» State 3u

» State 4

» Uncoupling-control ratio UCR

Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1
Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002

General (alphabetical order)

» Adenine nucleotide translocase

» Adenylates

» Electron transfer pathway

» Mitochondrial preparations

» mt-membrane potential

» Oxygen flux

» Phosphorylation system

» Proton leak

» Proton slip

» TIP2k

Other keyword lists

» Template:Keywords: Force and membrane potential

References

Fontana-Ayoub M, Fasching M, Gnaiger E (2016) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(18):1-10. - »Bioblast link«
Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. - »Bioblast link«
Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28. - »Bioblast link«
Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58. - »Bioblast link«
Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. - »Bioblast link«

@@ Line 25: / Line 25: @@
 :::: The optimum concentration of an uncoupler has to be determined for every biological system. It varies with incubation medium, sample concentration, pharmacological treatment (with or without oligomycin), and pathophysiological state (e.g. induction of apoptosis). A single dose of uncoupler usually leads to an artefact in the estimation of maximum flux or Electron transfer-pathway capacity (for discussion, see [[Talk:Rogers_2011_PlosOne#Uncoupled_flux_does_not_reflect_electron_transfer_system_capacity|Artefacts by single dose uncoupling]]).
-:::: The optimum uncoupler (CCCP, FCCP, DNP) concentration for the noncoupled state varies over a large concentration range, depending on the medium ('binding' of uncoupler), type and concentration of sample.  This is true for various uncouplers, such as CCCP, FCCP and DNP. <ref> Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. [[Steinlechner-Maran_1996_Am J Physiol Cell Physiol |»Bioblast link«]]</ref>  To evaluate the optimum concentration, an uncoupler titration has to be performed initially. For subsequent application series, we recommend a few titrations starting close to optimum concentration. <ref>Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28. [[Huetter_2004_Biochem J |»Bioblast link«]]</ref>,<ref>Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58. [[Pesta 2012 Methods Mol Biol |»Bioblast link«]]</ref>  Optimum CCCP or FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.
+:::: The optimum uncoupler (CCCP, FCCP, DNP) concentration for the noncoupled state varies over a large concentration range, depending on the medium ('binding' of uncoupler), type and concentration of sample.  This is true for various uncouplers, such as CCCP, FCCP and DNP ([[Steinlechner-Maran 1996 Am J Physiol Cell Physiol]]).  To evaluate the optimum concentration, an uncoupler titration has to be performed initially. For subsequent application series, we recommend a few titrations starting close to optimum concentration ([[Huetter_2004_Biochem J]], [[Pesta 2012 Methods Mol Biol]]). Optimum CCCP or FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.
-:::: See Steinlechner-Maran et al for a comparison of uncoupler titrations with [[FCCP]] and [[DNP]] from the [[ROUTINE state]] to the [[ET-capacity|ET state]] of cell respiration. <ref> Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. [[Steinlechner-Maran_1996_Am J Physiol Cell Physiol |»Bioblast link«]]</ref>
+:::: See [[Steinlechner-Maran_1996_Am J Physiol Cell Physiol |Steinlechner-Maran et al (1996)]] for a comparison of uncoupler titrations with [[FCCP]] and [[DNP]] from the [[ROUTINE state]] to the [[ET-capacity|ET state]] of cell respiration.
 === Coupling control protocol ===
-:::: Uncoupler titrations after inhibition of respiration by oligomycin in [[coupling control protocol]]s with living cells yield the sequence of [[ROUTINE respiration]], [[LEAK respiration]] and [[ET-capacity]], followed by inhibition to ROX ([[Huetter 2004 Biochem J]], [[Gnaiger 2008 POS]]). The highest accuracy of uncoupler titrations is achieved by titrations with the [[TIP2k]] at high concentrations of the stock solution. <ref>Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. [[Gnaiger_2008_POS |»Bioblast link«]]</ref> Increasing the concentration in small steps, most accurately titrated by the [[TIP2k]], is recommended (0.5 or 0.25 µM steps or even smaller).
+:::: Uncoupler titrations after inhibition of respiration by oligomycin in [[coupling control protocol]]s with living cells yield the sequence of [[ROUTINE respiration]], [[LEAK respiration]] and [[ET-capacity]], followed by inhibition to ROX ([[Huetter 2004 Biochem J]], [[Gnaiger 2008 POS]]). The highest accuracy of uncoupler titrations is achieved by titrations with the [[TIP2k]] at high concentrations of the stock solution ([[Gnaiger 2008 POS]]. Increasing the concentration in small steps, most accurately titrated by the [[TIP2k]], is recommended (0.5 or 0.25 µM steps or even smaller).
+{{Keywords: Uncoupling}}
 == References ==
-::::# Fontana-Ayoub M, Fasching M, Gnaiger E (2014) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(17):1-9. - [[MiPNet03.02 Chemicals-Media |»Bioblast link«]]
+::::# Fontana-Ayoub M, Fasching M, Gnaiger E (2016) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(18):1-10. - [[MiPNet03.02 Chemicals-Media |»Bioblast link«]]
 ::::# Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. - [[Gnaiger_2008_POS |»Bioblast link«]]
 ::::# Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28. - [[Huetter_2004_Biochem J |»Bioblast link«]]
+::::# Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58. - [[Pesta 2012 Methods Mol Biol |»Bioblast link«]]
+::::# Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. - [[Steinlechner-Maran_1996_Am J Physiol Cell Physiol |»Bioblast link«]]