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Simultaneous measurement of the TCA cycle and respiration in isolated mitochondria and intact cells with the XF24‐3 Analyzer
Author(s) -
Rogers George W.,
Jogunoori Smitha P.,
Munkholm Christiane,
Neilson Andy,
Ferrick David A.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb210
Subject(s) - citric acid cycle , oxidative phosphorylation , mitochondrion , flux (metallurgy) , respiration , oxygen , biochemistry , chemistry , bioenergetics , carbon dioxide , metabolism , tricarboxylic acid , biophysics , microbiology and biotechnology , biology , anatomy , organic chemistry
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 O 2 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 (CO 2 ). 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 CO 2 and O 2 , which operate reversibly, and reveal details of mitochondrial respiration and TCA cycle activities. Results indicate that O 2 consumption and CO 2 evolution may be monitored simultaneously, and that data agrees with attributes of TCA cycle and mitochondrial function obtained by other methods. Differential rates of O 2 and CO 2 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.