Hydrogenion flux

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Hydrogenion flux

Description

Volume-specific hydrogenion flux or H+ flux is measured in a closed system as the time derivative of H+ concentration, expressed in units [pmol·s-1·mL-1]. H+ 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 H+ flux is comparable to volume-specific oxygen flux [pmol·s-1·mL-1], 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 hydrogen ion activity. Therefore, ECAR is of interest in relation to acidification issues in the incubation buffer or culture medium. The physiologically relevant metabolic H+ flux, however, must not be confused with ECAR.

Abbreviation: JH+

Reference: Gnaiger 2020 BEC MitoPathways

H+ flux versus glycolytic flux

  1. Measured changes in pH over time (ECAR) must be transformed from the logarithmic scale to the linear scale of H+ flux.
  2. Measurement of extracellular H+ production 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.


Additional resources

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

» O2k-SOP: MiPNet08.16 pH calibration

» MitoPedia O2k and high-resolution respirometry: O2k hardware 



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O2k-Open Support


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



O2k signal and output

  1. 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
  2. O2k output: type I and II


pH changes versus glycolytic flux

Measurement of extracellular H+ production 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 H+ activity. Comparable to volume-specific [[oxygen flux] [pmol·s-1·mL-1]], which is the (negative) time derivative of oxygen concentration measured in a closed system, volume-specific H+ flux]] is the time derivative of H+ concentration, expressed in units [pmol·s-1·mL-1]]. The physiologically relevant metabolic H+ flux, therefore, must not be confused with ECAR.
  • Very small buffering capacity is required: 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 pH-Stat.




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