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]. It 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. Proton flux can also 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 (pH stat).
Reference: Gnaiger 2014 MitoPathways
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. 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.
Proton 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 proton flux. Pyruvate can either be converted to lactate, catalysed 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”.
- 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
Compare measurement of pH with the pH electrode and ratiometric fluorometric methods
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