Shona A Mookerjee1, Renata L S Goncalves2, Akos A Gerencser3, David G Nicholls2, Martin D Brand3. 1. College of Pharmacy, Touro University California, 1310 Club Drive, Vallejo, CA 94592, USA; Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945, USA. Electronic address: shona.mookerjee@tu.edu. 2. Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945, USA. 3. College of Pharmacy, Touro University California, 1310 Club Drive, Vallejo, CA 94592, USA; Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945, USA.
Abstract
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.
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.
Authors: Charles E Hammond; Craig Beeson; Giovanni Suarez; Richard M Peek; Steffen Backert; Adam J Smolka Journal: Am J Physiol Gastrointest Liver Physiol Date: 2015-06-04 Impact factor: 4.052
Authors: Hehai Pan; Adam Strickland; Vedavathi Madhu; Zariel I Johnson; Saswati N Chand; Jonathan R Brody; Andrzej Fertala; Zhaomin Zheng; Irving M Shapiro; Makarand V Risbud Journal: Matrix Biol Date: 2018-08-07 Impact factor: 11.583