Difference between revisions of "Talk:Hydrogenion flux"

Latest revision as of 14:04, 5 December 2020

For critical evaluation

The measurement of H⁺ flux alone is not sufficient to determine, if the origin of H⁺ is the glycolysis or other sources. For example, the carbon dioxide formed during the mitochondrial respiration acts as a net source of H⁺ into the media and as consequence has to be taken into account. During the oxidation of the glucose, we have two main metabolic pathways involved and both have a net effect over the H⁺ flux:

H2CO3 is not fully dissociated. Therefore, there is not a simple and constant stoichiometry between bicarbonate and H⁺ production.

Proton flux versus ECAR

The extracellular acidification rate (ECAR) is the change of pH in the incubation medium over time and can only be measured in a closed system (why?). pH is the negative decadic logarithm of proton activity which is, in diluted solutions, in close approximation to the negative decadic logarithm of proton concentration.

Thus, measured changes in pH over time (ECAR) must be transformed from the logarithmic to the linear scale to obtain proton flux.

Therefore, ECAR is of interest in relation to acidification issues in the incubation buffer or culture medium but must not be confused with the physiologically relevant metabolic proton flux.

H⁺ flux and glycolysis

Measurement of proton flux and glycolysis are related under specifically controlled conditions. Such conditions must be carefully evaluated, may require modifications of protocols, must be corrected for acid-base reactions unrelated to glycolysis and thus need data analysis beyond reporting changes of pH.

Glycolysis is the degradation of glucose to pyruvate. Depending on the subsequent metabolism of pyruvate, glycolysis is indirectly related to H⁺ flux. Pyruvate can either be converted to lactate, catalyzed by lactate dehydrogenase in the cytosol, or converted to Acetyl-CoA catalysed by pyruvate dehydrogenase), feeding into the TCA cycle (mitochondria). The catabolism of pyruvate can have an impact on proton flux as illustrated by the following equations:

As we can observe, the production of protons per molecule of glucose is three times higher in the mitochondria via dissolution of carbone dioxide than by conversion of pyruvate to lactate in the cytosol. Furthermore, the equations illustrate that the measurement of proton flux alone is not sufficient to determine the origin of protons. Experimental settings can help to estimate the main source of proton production, e.g. by inhibition of OXPHOS (link to SUIT protocols).

To accurately measure proton flux induced by biological sample, the buffering capacity of the medium has to be small but still sufficient to keep the pH in the desired range for a limited period of time. Furthermore, the buffering capacity has to be determined (MiPNet23.15 O2k-pH ISE-Module) and taken into account when proton flux is calculated from the measured changes in pH. An alternative approach is to use buffers with very low buffering capacity and keep the pH value inside the desired limits by a pH-Stat. Here, proton flux can be calculated either by changes in pH over time (previous calculation of buffering capacity of the medium required) or by the amount of injected base via pH Stat. MiPNet23.15 O2k-pH ISE-Module

Measurement of proton flux with the O2k-pH ISE-Module

The Oroboros O2k supports the modular O2k-MultiSensor extension for recording potentiometric (voltage) signals simultaneously with the oxygen signals in both O2k-chambers. Potentiometric measurements result in a voltage signal (pX) which is typically a linear function of the logarithm of the activity (concentration) of the substance of interest (the analyte). A calibrated pH electrode displays the negative decadic logarithm of the H⁺ ion activity (potentia hydrogenii) and thus got its name “pH electrode”.

Applications

The majority of novel applications will address aerobic or anaerobic glycolysis in intact cells, using the measurement of proton flux as an indirect but continuous record of lactate production and corresponding acidification of the medium, while simultaneously monitoring oxygen concentration and oxygen consumption. For this application, specific experimental settings are required to leave lactic acid production as the dominant mechanism of acidification.

The pH electrode in the O2k can also be used in conjunction with a study of mitochondrial permeability transition (e.g. SE_Lund_Elmer E).

For simultaneous measurement of O₂ and pH, we refer to the classical literature on bioenergetics and the discovery of the chemiosmotic coupling mechanism, the quantification of H⁺/O₂ stoichiometric ratios for proton pumping (Peter Mitchell).

O2k signal and output

O2k signal: The O2k-pH ISE-Module is operated through the pX channel of the O2k, with electric potential (volt [V]) as the primary and raw signal
O2k output: type I and II

Compare measurement of pH with the pH electrode and ratiometric fluorometric methods

» Carboxy SNARF 1

» HPTS

Bioblast links: pH and protons - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

pH and protons

» pH

» hydrogen ion H⁺

» hydron H⁺

» hydronium ion H₃O⁺

» hydride H^-

» proton p⁺

» pH buffering capacity

» proton flux

» proton pump versus hydrogen ion pump

» proton leak

» proton slip

» protonmotive force

O2k-pH

» O2k-Catalogue: O2k-pH ISE-Module

» O2k-Manual pH electrode: MiPNet23.15 O2k-pH ISE-Module

» O2k-SOP: MiPNet08.16 pH calibration

» File:PH-Calibration-List.xls

» NextGen-O2k, ratiometric: Carboxy SNARF 1

» NextGen-O2k, ratiometric: HPTS

» pH calibration buffers

O2k-Publications

» O2k-Publications: O2k-pH ISE-Module

HRFR - general

» O2k-Manual: MiPNet22.11 O2k-FluoRespirometer manual

» O2k signals and output

» O2k-SOP: MiPNet14.06 Instrumental O2 background

» MiPNet19.18A O2k-Series G: Start

» ESD

» O2k configuration

» O2k control

» O2k-FluoRespirometer

» O2k-Main Unit#O2k-Series

» Titration-Injection microPump

» Compare: O2k-TPP+_ISE-Module

DatLab

» DatLab 7

» MiPNet26.06 DatLab 7: Guide

» DatLab 6

» MiPNet19.18C DatLab 6: Guide

» MiPNet19.18D O2k-Series G and DatLab 6: Calibration

» Reference layouts for DatLab graphs

References

MiPNet23.15 O2k-pH ISE-Module

MiPNet24.06 Oxygen flux analysis - DatLab 7.4

MiPNet12.10 TIP2k-manual

MiPNet15.08 TPP electrode

MiPNet22.11 O2k-FluoRespirometer manual

» MitoPedia O2k and high-resolution respirometry: O2k hardware

» MitoPedia O2k and high-resolution respirometry: DatLab

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

MitoPedia concepts: MiP concept

MitoPedia methods: Respirometry

@@ Line 1: / Line 1: @@
-{{MitoPedia
+__TOC__
-|abbr=''J''<sub>H</sub><small>+</small>
-|description=Volume-specific '''proton flux''' is measured in a closed system as the time derivative of proton concentration, expressed in units [pmol·s<sup>-1</sup>·mL<sup>-1</sup>]. Proton flux can be measured in an open system at steady state, when any acidification of the medium is compensated by external supply of an equivalent amount of base. The extracellular acidification rate (ECAR) is the change of pH in the incubation medium over time, which is zero at steady state. Volume-specific proton flux is comparable to volume-specific [[oxygen flux]] [pmol·s<sup>-1</sup>·mL<sup>-1</sup>], which is the (negative) time derivative of oxygen concentration measured in a closed system, corrected for instrumental and chemical background.
-[[pH]] is the negative logarithm of proton activity. Therefore, ECAR is of interest in relation to acidification issues in the incubation buffer or culture medium. The physiologically relevant metabolic proton flux, however, must not be confused with ECAR.
+== For critical evaluation ==
-|info=[[Gnaiger 2014 MitoPathways]]
+:::: The measurement of H<sup>+</sup> flux alone is not sufficient to determine, if the origin of H<sup>+</sup> is the glycolysis or other sources. For example, the carbon dioxide formed during the mitochondrial respiration acts as a net source of H<sup>+</sup> into the media and as consequence has to be taken into account. During the oxidation of the glucose, we have two main metabolic pathways involved and both have a net effect over the H<sup>+</sup> flux:
-}}
+:::: H2CO3 is not fully dissociated. Therefore, there is not a simple and constant stoichiometry between bicarbonate and H<sup>+</sup> production.
-__TOC__
-== Proton flux versus glycolytic flux ==
-::::# Measured changes in pH over time (ECAR) must be transformed from the logarithmic scale to the linear scale of proton flux.
-::::# Measurement of extracellular proton flux and glycolytic flux are related under specifically controlled conditions. Such conditions must be carefully evaluated, may require modifications of protocols, and must be corrected for acid-base reactions unrelated to glycolytic flux.
-::::# The measurement of proton flux alone is not sufficient to determine if the origin of the protons is the glycolysis or other sources. For example, the carbon dioxide formed during the mitochondrial respiration acts as a net donor of protons into the media and as consequence has to be taken into account. During the oxidation of the glucose, we have two main metabolic pathways involved and both have a net effect over the proton flux:
 [[File:Proton production Metabolic pathways.png|center|400ppx]]
 :[[File:Lactate.png|center|400ppx]]
 :[[File:Carbon dioxide.png|center|400ppx]]
-:::: As we can observe, the production of protons by oxidative phosphorylation is three times higher than the one produced by the glycolysis per molecule of glucose. However, the chemical rate of production could be used to determine which is the main source of protons in our sample under specific conditions.
-* We have also to take into account the pka for the point of equilibrium of the most common weak acids that will be formed during both processes:
+== Proton flux versus ECAR ==
+:::: The extracellular acidification rate (ECAR) is the change of pH in the incubation medium over time and can only be measured in a closed system (why?). pH is the negative decadic logarithm of proton activity which is, in diluted solutions, in close approximation to the negative decadic logarithm of proton concentration.
+:::: Thus, measured changes in pH over time (ECAR) must be transformed from the logarithmic to the linear scale to obtain proton flux.
+Therefore, ECAR is of interest in relation to acidification issues in the incubation buffer or culture medium but must not be confused with the physiologically relevant metabolic proton flux.
+== H<sup>+</sup> flux and glycolysis ==
+:::: Measurement of proton flux and glycolysis are related under specifically controlled conditions. Such conditions must be carefully evaluated, may require modifications of protocols, must be corrected for acid-base reactions unrelated to glycolysis and thus need data analysis beyond reporting changes of pH.
+:::: Glycolysis is the degradation of glucose to pyruvate. Depending on the subsequent metabolism of pyruvate, glycolysis is indirectly related to H<sup>+</sup> flux. Pyruvate can either be converted to lactate, catalyzed by [[lactate dehydrogenase]] in the cytosol, or converted to Acetyl-CoA catalysed by [[pyruvate dehydrogenase]]), feeding into the [[Tricarboxylic acid cycle | TCA cycle]] (mitochondria). The catabolism of pyruvate can have an impact on proton flux as illustrated by the following equations:
-== Additional resources ==
-» O2k-Manual: [[MiPNet23.15 O2k-pH ISE-Module]]
-» O2k-SOP: [[MiPNet08.16 pH calibration]]
+[[File:Proton_production.png]]
-»{{MitoPedia O2k and high-resolution respirometry
-|mitopedia O2k and high-resolution respirometry=O2k hardware
-}}
-{{Technical support integrated}}
-== O2k signal and output ==
-:::# [[O2k signals and output#Signal of the O2k and add-on modules |O2k signal]]: The [[O2k-pH ISE-Module]] is operated through the pX channel of the O2k, with electric potential (volt [V]) as the primary and raw signal
-:::# [[O2k signals and output#O2k output |O2k output]]: type I and II
-== pH changes versus glycolytic flux ==
-:::: Measurement of extracellular proton flux and glycolytic flux are related under specifically conrolled conditions. Such conditions must be carefully evaluated, may require modifications of protocols, and need data analysis beyond reporting changes of pH.
-::::* The extracellular acidification rate (ECAR) is the change of pH over time, which may be of interest in relation to acidification problems in a culture medium or incubation buffer. [[pH]] is the negative logarithm of proton activity. Comparable to volume-specific [[oxygen flux] [pmol·s<sup>-1</sup>·mL<sup>-1</sup>]], which is the (negative) time derivative of oxygen concentration measured in a closed system, volume-specific [[proton flux]] is the time derivative of proton concentration, expressed in units [pmol·s<sup>-1</sup>·mL<sup>-1</sup>]]. The physiologically relevant metabolic proton flux, therefore, must not be confused with ECAR.
+As we can observe, the production of protons per molecule of glucose is three times higher in the mitochondria via dissolution of carbone dioxide  than by conversion of pyruvate to lactate in the cytosol. Furthermore, the equations illustrate that the measurement of proton flux alone is not sufficient to determine the origin of protons. Experimental settings can help to estimate the main source of proton production, e.g. by inhibition of [[Oxidative phosphorylation | OXPHOS]] (link to SUIT protocols).
-::::» [[Proton flux]]
-::::* To accurately measure biologically induced changes in pH, the buffering capacity of the medium has to be small. This may be addressed either by using or preparing media with a buffering capacity that is low but still sufficient to keep the pH in the desired range for a limited period of time. An alternative approach is to use buffers with very low buffering capacity and keep the pH value inside the desired limits by a [[O2k-pH_ISE-Module#pH-Stat|pH-Stat]].
+To accurately measure proton flux induced by biological sample, the buffering capacity of the medium has to be small but still sufficient to keep the pH in the desired range for a limited period of time. Furthermore, the buffering capacity has to be determined ([[MiPNet23.15 O2k-pH ISE-Module]]) and taken into account when proton flux is calculated from the measured changes in pH. An alternative approach is to use buffers with very low buffering capacity and keep the pH value inside the desired limits by a [[O2k-pH_ISE-Module#pH-Stat|pH-Stat]]. Here, proton flux can be calculated either by changes in pH over time (previous calculation of buffering capacity of the medium required) or by the amount of injected base via pH Stat. [[MiPNet23.15 O2k-pH ISE-Module]]
+== Measurement of proton flux with the O2k-pH ISE-Module ==
-== Compare measurement of pH with the pH electrode and ratiometric fluorometric methods (NextGen-O2k) ==
+:::: The [[Oroboros O2k]] supports the modular O2k-MultiSensor extension for recording potentiometric (voltage) signals simultaneously with the oxygen signals in both O2k-chambers. Potentiometric measurements result in a voltage signal ('''pX''') which is  typically a linear function of the logarithm of the activity  (concentration) of the substance of interest (the ''analyte''). A  calibrated pH electrode displays the negative decadic logarithm of the H<sup>+</sup> ion activity (potentia hydrogenii) and thus got its name “pH electrode”.
-::::» [[Carboxy SNARF 1]]
-::::» [[HPTS]]
+== Applications  ==
-== O2k-Manual ==
+:::: The majority of novel applications will address aerobic or anaerobic glycolysis in intact cells, using the measurement of proton flux as an indirect but continuous record of lactate production and corresponding acidification of the medium, while simultaneously monitoring oxygen concentration and oxygen consumption. For this application, specific experimental settings are required to leave lactic acid production as the dominant mechanism of acidification.
-=== Introduction and scope ===
-:::: The [[Oroboros O2k]] supports the modular O2k-MultiSensor extension for recording potentiometric (voltage) signals simultaneously with the oxygen signals in both O2k-chambers. Potentiometric measurements result in a voltage signal ('''pX''') which is  typically a linear function of the logarithm of the activity  (concentration) of the substance of interest (the ''analyte''). A  calibrated pH electrode displays the negative decadic logarithm of the H<sup>+</sup> ion activity (potentia hydrogenii) and thus got its name “pH electrode”. Using a pH / reference electrode module,the extracellular proton flux can either be calculated by changes in pH over time (previous calculation of buffering capacity of the medium required) or by the amount of injected base via pH Stat. MiPNet 16.0x
+:::: The pH electrode in the O2k can also be used in conjunction with a study of mitochondrial permeability transition (e.g. [[SE_Lund_Elmer E]]).
+:::: For simultaneous measurement of O<sub>2</sub> and pH, we refer to the classical literature on bioenergetics and the discovery of the chemiosmotic coupling mechanism, the quantification of H<sup>+</sup>/O<sub>2</sub> stoichiometric ratios for proton pumping (Peter Mitchell).
+== O2k signal and output ==
+:::# [[O2k signals and output#Signal of the O2k and add-on modules |O2k signal]]: The [[O2k-pH ISE-Module]] is operated through the pX channel of the O2k, with electric potential (volt [V]) as the primary and raw signal
+:::# [[O2k signals and output#O2k output |O2k output]]: type I and II
+== Compare measurement of pH with the pH electrode and ratiometric fluorometric methods ==
+::::» [[Carboxy SNARF 1]]
+::::» [[HPTS]]
+{{Keywords: pH}}
+== References ==
+::::* [[MiPNet08.16 pH calibration]]
+::::* [[MiPNet15.03 O2k-MultiSensor-ISE]]
+::::* [[MiPNet23.15 O2k-pH ISE-Module]]
+::::* [[MiPNet24.06 Oxygen flux analysis - DatLab 7.4]]
+::::* [[MiPNet12.10 TIP2k-manual]]
+::::* [[MiPNet15.08 TPP electrode]]
-=== DatLab analysis ===
+::::* [[MiPNet22.11 O2k-FluoRespirometer manual]]
-==== [[Image:Px referencelayouts.png|right|600 px]] pX reference layouts ====
-:::: '''Graph  layout''': Four [[Layout for DatLab graphs|reference layouts]] are available in [[DatLab 7]] based on the recorded pX  signal:
-:::::*'''01 Potentiometric'''
-:::::*'''02a TPP_calibration'''
-:::::*'''02b TPP_with_O2flux'''
-:::::*'''02c TPP_calibrated_with_O2flux'''
-:::: These layouts can be selected in [Layout / Reference layouts / O2 & pX].
-:::: '''Reference layouts''' can be modified and saved as user-defined layouts, see [[MitoPedia: DatLab]].
-:::: [[Image:O2kcontrol px.PNG|400 px|right]]
+»{{MitoPedia O2k and high-resolution respirometry
-==== pX settings  ====
+|mitopedia O2k and high-resolution respirometry=O2k hardware
-:::: In the '''[[O2k configuration]] window''' the pX channel can be activated and a label for the inserted pX electrode can be entered for documentation purposes.
-:::: '''Gain''' and '''Offset voltage [mV]''' for the pX channel can be set in the '''[[O2k control]] window''' [F7], tab: '''Potentiometric, pX'''. The gain influences the  "pX Raw Signal” recorded in DatLab. Therefore, a gain of 1 will give the  same voltage [V] as would be measured with any multimeter between  reference and measuring electrode.
-==== pX calibration ====
-:::: From  the Calibration menu the '''pX Calibration window''' window [Potentiometric, pX] is opened. This feature allows for a simple two-point  linear calibration of pX (or –pX) as a function of recorded voltage,  using data ranges marked for calibration [Select marks] or known pX values, see [[MiPNet19.18D O2k-Series G and DatLab 6: Calibration| O2k-calibration]] and [[MiPNet19.18D O2k-Series G and DatLab 6: Calibration| pH calibration]].
-:::: '''Calibrations  for different signal types: '''There is only one set of calibration  values for each pX channel, irrespective of the connected electrode. If a  pX channel was calibrated for a pH electrode, these values will  initially also be used to calculate the calibrated signal when the pH  electrode is exchanged for a TPP<sup>+</sup> electrode. Even  when observing only the raw (not the calibrated) signal, the time  derivative (Slope pX) will be calculated from the calibrated signal,  which might lead to confusion when the time derivative is used to access  stability or signal drift. It is, therefore, suggested to set the  calibrated signal to the raw signal whenever the raw signal is to be  used as the primary data source.
-:::: [[Image:PX calibration window.JPG| right|500 px]] Calibration values from other files can be imported with '''Copy from file''' in the pX calibration window.
-== Specifications ==
-:::: Specifications provided by OROBOROS INSTRUMENTS for quality control of pH electrodes:
-::::* Drift (after 45 min stabilization, integrated over 5 minutes, 37 °C, 2 mM buffering capacity): <= 20 µpH/s.
-== pH-Stat ==
-:::: TIP2k setups in the DatLab template file '''DLTemplates_pH.dlt''' and Spreadsheet (e.g. Excel) templates for determining proton form base injections are available for download: [[File:DLTemplates.dlt| here]]. Please note that of course the more straightforward calculation of proton flows from the measured pH slope is also possible while operating in pH-Stat mode!
-== Applications  ==
-:::: For simultaneous measurement of O2 and pH, we refer to the classical literature on bioenergetics and the discovery of the chemiosmotic coupling mechanism, the quantification of H+/O2 stoichiometric ratios for proton pumping (Peter Mitchell). Other groups (e.g. [[SE_Lund_Elmer E]]) have used the pH electrode in the O2k in conjunction with a study of mitochondrial permeability transition.
-:::: The majority of novel applications will address the problem of aerobic glycolysis in intact cells, using the measurement of proton production as an indirect but continuous record of lactate production and corresponding acidification of the medium, while simultaneously monitoring oxygen concentration and oxygen consumption. In a well buffered culture medium, the pH change is extremely small relative to the amount of protons (lactic acid) produced, hence a low-buffering capacity medium needs to be applied. A titration of acid (lactic acid or HCl) into the low-buffering capacity medium yields the pH-dependent buffering capacity (Delta H+ added/Delta H+ measured by the pH electrode). Under various metabolic conditions, lactic acid production is the dominant mechanism causing acidification, hence the pH measurement is a good indirect indicator of aerobic glycolysis.
-== Protocols ==
-:::* [[SUIT-003 O2 ce D067]]
-{{Template:SUIT text D067}}
-== Demo Experiment with simulated proton flow ==
-[[File:PH PS demofile 6.png|500px]]
-:::: Medium: imidazole buffered medium, see above
-::::# Calibration steps (for calculating buffering capacity): 30, 90, 150 HCl pmol/(s mL): TIP 1 mmol/L HCl, pump speed 0.06, 0.18, 0.3 µL/s
-::::# Simulated proton flow 30,90,150 pmol HCl/(s mL) in pH stat mode: The pH value was held inside narrow limits by using the TIP in pH stat mode (100 mM KOH, Tip set up similar to the one included in DLTemplates_pH.dlt available from http://www.oroboros.at/index.php?id=ph-oxygen. The proton flow was simulated using a second TIP
-{{Keywords: pH}}
-== References ==
-::::* '''O2k-Manual''': [[Media:MiPNet19.18 O2k-Core Manual.pdf|Contents: O2k-Core Manual.pdf]]
-{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Manual]] [[Additional label::O2k-Core]]
-| mainlabel=Chapter
-|?Has title=Section
-|?Was published in year=Last update
-|format=broadtable
-|limit=500
-|sort=
-|order=ascending
-|offset=0
 }}
+»{{MitoPedia O2k and high-resolution respirometry
-[[Image:Titration-Injection-microPump.jpg|150px|left|link=http://www.bioblast.at/index.php?title=TIP2k-Module]]
+|mitopedia O2k and high-resolution respirometry=DatLab
-::::* '''TIP2k-Manual***
-::::» [[O2k-Catalogue: TIP2k]]
-::::» [[O2k-Publications: TIP2k]]
-{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Manual]] [[Instrument and method::TIP2k]]
-| mainlabel=Chapter
-|?Has title=Section
-|?Was published in year=Last update
-|format=broadtable
-|limit=500
-|sort=
-|order=ascending
-|offset=0
 }}
+{{Technical support integrated}}
-{{Keywords: pH}}
 {{MitoPedia concepts
@@ Line 172: / Line 87: @@
 }}
 {{MitoPedia methods
-|mitopedia method=Respirometry, Fluorimetry
+|mitopedia method=Respirometry
 }}