Measurement of Fatty Acid Oxidation by High-Resolution Respirometry: Special Considerations for Analysis of Skeletal and Cardiac Muscle and Adipose Tissue

Abstract

High-resolution respirometry is a state-of-the-art approach for the quantitation of mitochondrial function. Isolated mitochondria, cultured cells, or tissues/fibers are suspended in oxygenated respiration medium within a closed chamber and substrates or inhibitors added in a stepwise manner. The dissolved oxygen concentration decreases as aerobic metabolism in the specimen proceeds, recorded by an oxygen sensor within the chamber to give a quantifiable measure of oxygen consumption by the sample. Measuring oxygen consumption using a variety of respiratory substrates or respiratory complex-targeted inhibitors enables multiple respiratory pathways to be interrogated to determine the functional capacity of the mitochondria in real time. Using a substrate-uncoupler-inhibitor titration (SUIT) protocol, we have developed a method which makes use of differing chain length fatty acids to derive a measure of fatty acid-stimulated respiration through β-oxidation in a variety of tissue types including skeletal and cardiac muscles and brown and white adipose tissues. This report provides technical details of the protocol, and the adaptations employed, to generate robust analysis of mitochondrial fatty acid β-oxidation.