Cardoso 2023 BPS2023 San Diego
Event: BPS2023 San Diego US
Respirometry of mitochondrial preparations is applied for functional diagnosis of mitochondrial defects. Sequential titrations of substrates, inhibitors, and uncouplers of electron transfer (ET) and phosphorylation systems allow assessment of ET pathways and coupling in oxidative phosphorylation. We developed reference protocols for diagnosis of mitochondrial respiratory function in health and disease.
Malate is critical for analysis of fatty acid oxidation (FAO), to prevent inhibition by accumulating acetyl-CoA. Malate kinetics of ADP-stimulated respiration was performed by high-resolution respirometry in absence and presence of FA. In permeabilized HEK 293T cells, where mitochondrial malic enzyme is expressed, malate alone (2 mM but not 0.1 mM) stimulated respiration up to NADH-linked capacity, without further increase by pyruvate. This leads to overestimation of FAO when octanoylcarnitine and 2 mM malate are used. Without malate-anaplerotic capacity in mouse heart, 0.1 mM malate was sufficient to stimulate maximum FAO with octanoylcarnitine. This distinct kinetics provides the key to measure FAO compared to NADH-linked respiration in mitochondrial preparations from mouse brain with malate-anaplerotic activity and low ratio of FAO- to NADH-linked OXPHOS capacity, and heart without anaplerosis and high FAO/NADH respiratory ratio.
This protocol was extended by titration of pyruvate and glutamate to stimulate NADH production in the mitochondrial matrix, then succinate and glycerophosphate, thus investigating convergent CoQ-reducing pathways related to carbohydrate metabolism. Finally, the uncoupler CCCP was titrated stepwise up to maximum ET capacity, followed by rotenone to inhibit CI and thus FAO, and antimycin A to inhibit CIII for quantifying residual oxygen consumption.
This titration protocol was standardized as a reference for bioenergetic profiling of various sample preparations from different tissues and cell types, providing a general diagnostic tool.
Affiliations and support
- Oroboros Instruments GmbH, Austria
- Supported by the European Union’s Horizon 2020 research and innovation program Grant 857394.
List of abbreviations, terms and definitions - MitoPedia
Labels: MiParea: Respiration, Instruments;methods
Tissue;cell: Heart, Nervous system
Regulation: Fatty acid