Volume-specific proton flux is measured in a closed system as the time derivative of proton concentration, expressed in units [pmol·s-1·mL-1]. 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-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 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.
Reference: Gnaiger 2014 MitoPathways
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 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.
- 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:
- As we can observe, the production of protons by the 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 determinant 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:
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pH changes versus glycolytic flux
- Measurement of extracellular proton 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 proton 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 proton flux is the time derivative of proton concentration, expressed in units [pmol·s-1·mL-1]]. The physiologically relevant metabolic proton flux, therefore, must not be confused with ECAR.
- » Proton flux
- 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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