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Difference between revisions of "Flux / Slope"

From Bioblast
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Look for MiPNets and take information from [[DatLab-Analysis templates]]
Look for MiPNets and take information from [[DatLab-Analysis templates]]


=== H2O2 ===
=== H<sub>2</sub>O<sub>2</sub> ===
:::: Further information about hydrogen peroxide detection using Amplex UltraRed assay could be  found under the  
:::: Further information about hydrogen peroxide detection using Amplex UltraRed assay could be  found under the  
  ''In preparation'' Add a link to [[Amplex UltraRed]]
  ''In preparation'' Add a link to [[Amplex UltraRed]]
==== How to Analyse with DatLab 7.4 ====
==== How to Analyse with DatLab 7.4 ====
::::* MiPNet24.10 Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4. Mitochondr Physiol Network 24.10(01):1-5.
 
::::* Instruction to the Excel template for H<sub>2</sub>O<sub>2</sub> flux analysis using Amplex UltraRed assay in MiR05-Kit: [[MiPNet24.10 H2O2 flux analysis| MiPNet24.10 Hydrogen peroxide flux analysis]]
::::* The calculations applied in the Excel template are provided here complying with Oroboros transparency policy:
 
::::» [[MiPNet20.14]]????
::::» [[MiPNet20.14]]????



Revision as of 12:32, 1 August 2019


high-resolution terminology - matching measurements at high-resolution


Flux / Slope

Description

Flux / Slope is the pull-down menu in DatLab for (1) normalization of flux (chamber volume-specific flux, sample-specific flux or flow, or flux control ratios), (2) flux baseline correction, (3) Instrumental background oxygen flux, and (4) flux smoothing, selection of the scaling factor, and stoichiometric normalization using a stoichiometric coefficient. A Savitzky-Golay smoothing filter is used in DatLab as a basis of calculating the time derivative (Flux / Slope) of the signal (oxygen, fluorescence, ..).

For each signal channel, the signal for the measured substance X is typically calibrated as an amount of substance concentration, cX [µM = nmol/mL]. The signal of the potentiometric channel, however, is primarily expressed logarithmically as pX=-log(cX/c°) and then transformed to cX. The slope is calculated as the change of concentration over time, dcX/dt [nmol/(s · mL)]. In a chemical reaction, the change of substance X is stoichiometrically related to the changes of all other substrates and products involved in the reaction. If the stoichiometry of the reaction is normalized for substance X, then its stoichiometric coefficient is unity and νX equals 1 if the substance is a product formed in the reaction, but νX equals -1 if the substance is a substrate consumed in the reaction. Oxygen is formed in photosynthesis and νX=1 when expressing photosynthesis as oxygen flux. Oxygen is consumed in aerobic respiration and νX=-1 when expressing respiration as oxygen flux.

Abbreviation: J

Flux-Slope-menu.png
Contributed by Cardoso LH, Iglesias-Gonzalez J & Gnaiger Erich 2019-07-30

Slope configuration menu

Slope configuration in DatLab 7

Slope

In an ideally closed chamber, the external fluxes are zero, thus that the concentration changes are exclusively due to (internal) transformations, which are chemical reactions. Then respiratory flux expressed per unit of chamber volume can be calculated from the slope of oxygen concentration over time (Gnaiger 2014),
 Eq(1):  X Slope neg = dcX/dt · νX-1 · SF  | Units: [pmol/(s·mL)]
For the oxygen channel, X=O2, cO2 is the oxygen conentration [nmol/mL = µmol/L = µM], dcO2/dt is the (positive) slope of oxygen concentration over time [nmol/(s · mL)], νO2-1 = -1 is the stoichiometric coefficient for the reaction of oxygen consumption (oxygen is removed in the chemical reaction, thus the stoichiometric coefficient is negative, expressing oxygen flux as the negative slope; Gnaiger 1993), and SF=1,000 is the scaling factor (converting units for the amount of oxygen from nmol to pmol).

Flux baseline correction

If the sample itself introduces a constant flux in a baseline state (indicated as subscript 0), which is not considered to represent the metabolic reaction, then a flux baseline correction (bc) may be applied by subtracting the baseline flux, JV,0, from the total flux:
Eq(2b):  JV,X(bc) = JV,X - JV,0  |  Units: [pmol/(s·mL)]

O2 background correction

In an experimentally closed chamber, external fluxes due to diffusion or convection into or out of the chamber are minimized, ideally to zero. Unavoidable external fluxes are corrected for, as are side reactions due to the measuring system, such as oxygen consumption by the oxygen sensor or chemical background effects due to autoxidation of chemical components added to the medium. These external fluxes and side reactions of the experimental system are lumped together in the background flux, J°V,X. A Instrumental background oxygen flux is applied to the total slope, to obtain the metabolic flux, JV,X, describing the experimental reaction under investigation:
Eq(2a):  JV,X = dcX/dt · νX-1 · SF - J°V,X  | Units: [pmol/(s·mL)]
J°V,O2 is the volume-specific background oxygen flux.

Normalization

Eqs(1) and (2) describe the dynamics of X per unit volume.
Eq(2a):  JV,X = X Slope neg - J°V,X  |  Units: [pmol/(s·mL)] 
Eq(2b):  JV,X(bc) = JV,X - JV,0  |  Units: [pmol/(s·mL)]
Normalization for the amount or concentration of sample yields metabolic flow per number of cells, IX, or specific metabolic flux per mass, Jm,X (unstructured analysis), or per mt-content (per mt-marker) in structured analysis (Gnaiger 2014).
Eq(3a): IX = JV,X/(106 cells/mL)  |  Units: [pmol/(s·106 cells)] = [amol/(s·cell)] 
Eq(3b): IX(bc) = (JV,X - JV,0)/(106 cells/mL)  |  Units: [pmol/(s·106 cells)] = [amol/(s·cell)]
Eq(4a): Jm,X = JV,X/(mg/mL)  |  Units: [pmol/(s·mg)]
Eq(4b): Jm,X(bc) = (JV,X - JV,0)/(mg/mL)  |  Units: [pmol/(s·mg)]

Flux Control Ratio

Flux control ratios are dimensionless, using a reference flux for normalization. The reference flux is determined in a metabolic reference state (1) as internal mt-marker (Gnaiger 2014),
Eq(5a): FCR = JV,X/JV,1
Eq(5b): FCR(bc) = (JV,X - JV,0)/(JV,1 - JV,0)

Smoothing, noise, and sample stability

Recorded data: The default data recording interval is 2 s, applied with most SUIT protocols. The larger the data recording interval, the lower is the time resolution and the larger is the smoothing of flux.
Smoothing in Flux / Slope: The default setting of Slope smoothing is 40 data points, i.e., 40 data points or an interval of 80 s at a data recording interval of 2 s are used for calculation of a non-linear fit, from which the slope is calculated for the current data point. 40 is used for O2 calibration and instrumental O2 background tests. 20 is recommended for data recording and analysis in typical SUIT experiments. When a larger noise is observed particularly in experiments with permeabilized muscle fibres, and increase to 25 displays more representative traces.
Flux may decline over time, due to (1) loss of respiratory capacity within a respiratory state when the properties of the sample are not stable, (2) reversible oxygen limitation in the closed chamber, (3) an overshoot directly after re-oxygenation, or (4) a slow response to an inhibitor. Flux may increase over time due to (1) a slow response to the addition of ADP or a fuel substrate, or (2) an undershoot observed in rare cases after re-oxygenation. An extended time interval is required for recording flux in a particular respiratory state, if (1) flux increases over time (this may take >30 min, with a mark set at the final steady-state), and (2) flux shows high levels of noise (then 10 min may be required for setting a mark).

Flux analysis

In a SUIT protocol each titration can be considered as a transition between a background state and a reference state, expressed as a flux control factor, FCF. A sequence of steady-state states can be analysed in terms of flux control ratios, FCR, corrected for a common baseline, and normalized for flux in a common reference state. If a steady-state (constant) flux is not reached between sequential respiratory states induced by titrations, corrections for dynamic changes of flux as a function of time may be applied, else the conditions for steady-state analysis break down. In such cases, step analysis with continuous evaluation of a change of flux (second-time derivative) may provide the only reliable information. In reality, some noise or bias is included in steady-state analysis above a threshold level, below which such data sets would be excluded from the steady-state analysis.

O2

How to Analyse with DatLab 7.4

» MiPNet24.06 DatLab7.4 O2 AnalysisTempl====> Put the pdf link

Previous versions of DatLab

Look for MiPNets and take information from DatLab-Analysis templates

H2O2

Further information about hydrogen peroxide detection using Amplex UltraRed assay could be found under the
In preparation Add a link to Amplex UltraRed

How to Analyse with DatLab 7.4

  • Instruction to the Excel template for H2O2 flux analysis using Amplex UltraRed assay in MiR05-Kit: MiPNet24.10 Hydrogen peroxide flux analysis
  • The calculations applied in the Excel template are provided here complying with Oroboros transparency policy:
» MiPNet20.14????

References

  • Gnaiger E (1993) Nonequilibrium thermodynamics of energy transformations. Pure Appl Chem 65:1983-2002. - »Bioblast link«
  • Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. OROBOROS MiPNet Publications, Innsbruck:80 pp. - »Bioblast link«
  • Press WH, Teukolsky SA (1990) Savitzky-Golay smoothing filters. Computers in Physics Nov/Dec 1990:669-72. - »Bioblast link«

From DatLab

Further links

DatLab links to this page from

MitoPedia methods: Respirometry, Fluorometry 


MitoPedia O2k and high-resolution respirometry: DatLab