Separation of Respiratory and Glycolytic Proton Fluxes

Separation of total extracellular acidification into respiratory proton production rate (PPR_resp) and glycolytic proton production rate (PPR_glyc) was carried out using Eq 1 as described in (5), with the same assumptions about substrate oxidation and substrate identity.
$\begin{array}{l}\text{Glycolytic rate}={\text{PPR}}_{\text{glyc}}\\ =\left(\frac{{\text{ECAR}}_{\text{tot}}}{\text{BP}}\right)-({\text{OCR}}_{\text{tot}}-{\text{OCR}}_{\text{rot}/\text{myx}})(\text{max}{\text{H}}^{+}/{\text{O}}_2)(\frac{{10}^{\text{pH}-{\text{pK}}_1}}{1+{10}^{\text{pH}-{\text{pK}}_1}})\end{array}$
where ECAR = extracellular acidification rate (mpH/min), tot = total, BP = buffering power (mpH/pmol H⁺), OCR = oxygen consumption rate (pmol O₂/min), OCR_rot/myx = non-mitochondrial OCR remaining after complete inhibition of mitochondrial electron transport, max H⁺/O₂ = the maximum H⁺ released to the medium per O₂ consumed (and CO₂ generated) by respiration, (see [5 (link)]), and K₁ = the combined equilibrium constant of CO₂ hydration and H₂CO₃ dissociation to HCO₃^- + H⁺. The overall pK for CO_2(aq) + H₂O → HCO₃^- + H⁺ = 6.093 at 37°C ([18 ], p. 45). The spreadsheet used for these calculations in [6 (link)] incorporates Eq 1, enabling experimental data (starting pH, buffering power, maximum H⁺/O₂, oxygen consumption rate, and total extracellular acidification rate) to be entered and proton production rate to be calculated. This spreadsheet is available for download [6 (link)].
For this calculation, we assumed that all of the CO₂ produced remained in the XF24 wells [5 (link)], and that the cells used only the supplied glucose, which was completely oxidized. For complete oxidation of glucose, 1 CO₂ is produced for each O₂ consumed (i.e., the respiratory quotient, RQ, = 1), and a maximum of 1 H⁺ is generated by the hydration and dissociation of each CO₂, giving a maximum H⁺/O₂ ratio of 1. We assumed that prior to substrate addition the cells oxidized mixed endogenous substrates, primarily glycogen. Glycogen oxidation also has maximum H⁺/O₂ of 1, and we therefore assumed an overall RQ of 1 and maximum H⁺/O₂ ratio of 1 for pre-substrate-addition metabolism [5 (link)]. The separation of total extracellular acidification into respiratory and glycolytic proton production rates is accurate to the extent that these assumptions are correct; if, for example, substrate oxidation was incomplete and a significant fraction of the carbon was incorporated into molecules more reduced than CO₂ (such as organic acids, proteins or nucleic acids), use of the maximum H⁺/O₂ value would overestimate glycolytic rate. If pre-substrate-addition metabolism was primarily of substrates whose RQ is less than 1, such as fatty acids, using an RQ of 1 would underestimate glycolytic rate. However, these assumptions can easily be assessed for internal consistency by post-hoc measurement of lactate produced during the experiment; under the conditions used here for C2C12 myoblasts, measured lactate production agreed quantitatively with the amounts expected from calculated glycolytic rates after correction for respiratory proton production [5 (link)], suggesting that the assumptions were essentially correct.