Simultaneous measurement of the TCA cycle and respiration in isolated mitochondria and intact cells with the XF24‐3 Analyzer

Abstract

Measuring mitochondrial dysfunction is increasingly important in the study of neurodegenerative and cardiovascular disease, metabolic syndrome, diabetes, cancer, and aging. Mitochondrial function is usually measured by oxygen consumption rates using respirometry methods. However, a critical aspect of mitochondrial function may be overlooked when only O2 consumption is employed: the tricarboxylic acid (TCA) or Krebs cycle, a central metabolic pathway. Measuring the TCA cycle function requires monitoring the flux of an additional analyte, such as carbon dioxide (CO2). The reported method describes simultaneous monitoring of carbon dioxide evolution rates (CDER) and oxygen consumption rates (OCR) using isolated mitochondria and intact cells, in real‐time, using the XF24‐3 Analyzer. This technology employs fluorescent sensors specific for CO2 and O2, which operate reversibly, and reveal details of mitochondrial respiration and TCA cycle activities. Results indicate that O2 consumption and CO2 evolution may be monitored simultaneously, and that data agrees with attributes of TCA cycle and mitochondrial function obtained by other methods. Differential rates of O2 and CO2 flux can be identified relative to substrate utilization and interdependency among the TCA cycle, electron transport, and oxidative phosphorylation systems. Support for this research is provided by Seahorse Bioscience.