The contributions of respiration and glycolysis to extracellular acid production

Background: The rate at which cells acidify the extracellular medium is frequently used to report glycolytic rate, with the implicit assumption that conversion of uncharged glucose or glycogen to lactate(-) +H+ is the only significant source of acidification. However, another potential source of extracellular protons is the production of CO2 during substrate oxidation: CO2 is hydrated to H2CO3, which then dissociates to HCO3- + H+. Methods: O-2 consumption and pH were monitored in a popular platform for measuring extracellular acidification (the Seahorse XF Analyzer). Results: We found that CO2 produced during respiration caused almost stoichiometric release of H+ into the medium. With C2C12 myoblasts given glucose, respiration-derived CO2 contributed 34% of the total extracellular acidification. When glucose was omitted or replaced by palmitate or pyruvate, this value was 67-100%. Analysis of primary cells, cancer cell lines, stem cell lines, and isolated synaptosomes revealed contributions of CO2-produced acidification that were usually substantial, ranging from 3% to 100% of the total acidification rate. Conclusion: Measurement of glycolytic rate using extracellular acidification requires differentiation between respiratory and glycolytic acid production. General significance: The data presented here demonstrate the importance of this correction when extracellular acidification is used for quantitative measurement of glycolytic flux to lactate. We describe a simple way to correct the measured extracellular acidification rate for respiratory acid production, using simultaneous measurement of oxygen consumption rate. Summary statement: Extracellular acidification is often assumed to result solely from glycolytic lactate production, but respiratory CO2 also contributes. We demonstrate that extracellular acidification by myoblasts given glucose is 66% glycolytic and 34% respiratory and describe a method to differentiate these sources. (C) 2014 Elsevier B.V. All rights reserved.