Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Abstract

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis 1 . Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate 2 , the same is not true for extracellular acid production and its relationship to glycolytic rate [3][4][5][6] . Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO 2 , produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate -+ H + and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO 2 , hydration to H 2 CO 3 and dissociation to HCO 3 -+ H + is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6 . Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

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“…However, following mitochondrial inhibition with oligomycin, glycolytic reserve capacity was increased 28% in NAD + ‐treated cells ( P < .05; Figure 2K), suggesting hESC glycolytic activity was being actively suppressed by NAD + treatment. To further explore this metabolic change, the contributions from glycolytic and mitochondrial metabolism to extracellular acidification were calculated . Control hESCs displayed a basal glycolytic:oxidative contribution of ~74:26 (Figure 2L).…”

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“…B) Relative glycolytic and mitochondrial ATP production rates were calculated using the data generated in Fig. 1A as described (Mookerjee and Brand, 2015). Data are mean ± SD from 20 samples from 2 independent experiments.…”