The contributions of respiration and glycolysis to extracellular acid production

Biochim Biophys Acta. 2015 Feb;1847(2):171-181. doi: 10.1016/j.bbabio.2014.10.005. Epub 2014 Oct 27.


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: O2 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.

Keywords: Bicarbonate; Carbon dioxide; Extracellular acidification rate; Extracellular flux; Glycolysis; Oxygen consumption rate.

MeSH terms

  • Animals
  • Carbon Dioxide / metabolism
  • Cells, Cultured
  • Glycolysis*
  • Hydrogen-Ion Concentration
  • Lactic Acid / metabolism
  • Mice
  • Oxygen Consumption*
  • Rats


  • Carbon Dioxide
  • Lactic Acid