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| Using the pH probe in the O2k to measure proton production requires some modifications of standard protocols and in fact a certain degree of method development. Some of the encountered challenges are
| | #REDIRECT [[O2k-pH ISE-Module]] |
| * very small buffering capacity required
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| * calculation of proton flux from the observed parameters
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| == pH-Stat==
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| One approach we have developed is to use the [[TIP]] to run in a "pH stat" mode, i.e. keeping the pH constant by a feedback controlled automatic injection of base. Besides keeping the pH in the desired range this can actually be used to determine proton flow from the amount of base injected, circumventing the determination of buffering capacity, see below. The "pH-Stat" allows using very weakly buffered media (2 mM buffering substances) and might even make buffering obsolete.
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| The feedback modus of the [[TIP]] can be used in two ways to achieve a pH-Stat modus:
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| * pH-Stat_strict: This program keeps the pH value strictly between user defined upper and lower limits. The difference between the upper and lower limit will determine the time between injections depending on the current proton flow. Therefore, the time between injections may vary drastically with changing proton fluxes.
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| * pH-Stat_interval: This program adjusts the pH value in certain time intervals back to the upper limit. The difference between the upper and lower limit is set extremely small but a defined minimum pause between injections of e.g. 180 s is defined. Therefore, usually a base injection will be done every 3 minutes and the pH value will oscillate between the upper limit and some (proton flux dependent) lower limit. The lower limit set in the program has no significance because the minimum pause time will not have elapsed when the lower limit is met.
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| While the pH-Stat_strict is necessary to keep the pH value in a precisely defined range the pH-Stat_interval program ensures defined periods undisturbed by any injection of base. Such periods are necessary for the calculation of proton flow from the observed pH change and theoretically also for measuring respiration (however, if a 100 mM KOH the disturbance of the oxygen signal by the small amounts of KOH added was usually very small).
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| Both mentioned TIP setups can be found in the DatLab template file DLTemplates_pH.
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| == Calculating Proton Flow ==
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| === From the Change of the pH Signal===
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| To calculate the actual proton flow in the system from the observed change in the pH signal the buffering capacity of the medium has to be determination of before the introduction of sample.
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| This is best achieved by setting up the [[TIP]] with HCl in the syringes and simulate the expected proton production of the sample by setting an appropriate flow rate for the TIP. In the simplest implementation just one flow rate is used for about 5 minutes. From the first time derivative of the pH signal the buffering capacity can be calculated. Afterwards the TIP has to be re-fitted with syringes containing KOH (NaOH) for using the pH-Stat during the actual biological experiment. The observed rates of pH changes during the biological experiment (pX slope/ first time derivative of pH signal) can then be directly converted to Proton flow values using the buffering capacity determined before. This is shown in the spreadsheet file "pH Stat Template Buffer Capacity_one_point"
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| In a more sophisticated approach a multiple point calibration is done: Several proton flows are simulated before the experiment, a linear regression between set proton flow and observed pH slope is done and the regression parameters are used to calculate proton flows from the pX slopes observed during the biological experiment.
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| These approaches assume a linear relationship between pH change and introduced protons. This is an approximation that is only valid for very small pH changes. In other words the buffering capacity has to be constant during the entire experiment.
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| === From the Amount of Injected Base===
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| The limitations mentioned above can be overcome by using the amount of base necessary in the pH-Stat approach to hold the pH value constant. If the pH values at the beginning and at the end of a time interval are identical then the proton floe during this time interval can be directly calculated from the amount of base injected to keep the pH value constant. While this method does not assume a constant buffering capacity during the entire experiment there are some drawbacks:
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| * Usually only the "pH-Stat-strict" approach will assure the identical pH values at the beginning and the end of a time period necessary for this approach (This could possibly be overcome by more advanced data analysis).
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| * Only one value for proton flow for each period between injections is calculated in contrast to the continuous recording facilitated by the first mentioned method.
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| * The injected volumes have to be read out form TIP events. This can be partially automated in a spreadsheet template (pH Stat Template Injected Volume) but is more tedious than the method using the buffering capacity
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| * Initial trials with simulated proton flows indicted this method to be slightly less precise than the method using the buffering capacity.
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| A potential compromise would be to use the method based on buffering capacity for routine calculations but check selection (late) phases of the experiment with the method based on added base volume to detect any significant changes in buffering capacity during the experiment.
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| == Potential Applications ==
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| On the simultaneous measurement of O2 and pH, we may refer to the
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| classical literature on bioenergetics and the discovery of the
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| chemiosmotic coupling mechanism, the quantification of H+/O2
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| stoichiometric ratios for proton pumping (Peter Mitchell). Other groups (e.g. Eskil Elmer -
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| http://www.oroboros.at/index.php?id=mipnet-sweden#c1588) have used the pH
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| electrode in the O2k in conjunction with a study of mitochondrial
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| permeability transition.
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| The majority of novel applications will address the problem of aerobic
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| glycolysis in intact cells, using the measurement of proton production as
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| an indirect but continuous record of lactate production and corresponding
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| acidification of the medium, while simultaneously monitoring oxygen
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| concentration and oxygen consumption. In a well buffered culture medium,
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| the pH change is extremely small relative to the amount of protons (lactic
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| acid) produced, hence a low-buffering capacity medium needs to be applied.
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| A titration of acid (lactic acid or HCl) into the low-buffering capacity
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| medium yields the pH-dependent buffering capacity (Delta H+ added/Delta H+
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| measured by the pH electrode). Under various metabolic conditions, lactic
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| acid production is the dominant mechanism causing acidification, hence the
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| pH measurement is a good indirect indicator of aerobic glycolysis.
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| {{#set:Technical service=pH}}
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| {{Technical service}}
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